ObjectiveTo observe the correlation between referred hyperaesthesia and acupoint sensitization in kidney diseases patients and to investigate the rule of regional sensitized point distribution in rats.MethodsA total of 158 kidney-related diseases patients from 5 hospitals in China were recruited in the present study. The tenderness was palpated along the chest， back， shoulder， upper limbs， lower limbs，etc. by specially-assigned researcher in each hospital. In animal experiments， experimental group rats（n=8） received an injection of 10% mustard oil into subcortical region of the right kidney to trigger renal inflammation， while the control group rats（n=9） received an injection of saline solution into subcortical region of the right kidney. All the rats accepted tail venous injection of 5% Evans blue for examining the distribution of the blue dye exudation spots at the body surface.ResultsThe tenderness spots were found at the abdomen， waist， buttock， lower limbs and primarily concentrated in the T9—L4 and S2 spinal segments. The location of referred pain overlaps with acupoints. In experimental rats， cutaneous exudation points were distributed across the abdomen， lumbar-dorsal region， and sacral area， corresponding to the dermatomal regions innervated by the T7—L5 spinal segments.ConclusionIn the case of kidney diseases， a regular “referred sensitization” response frequently occurs in the dermatomere area innervated by the corresponding T8—L1 segments and adjacent nerve distribution areas.
Objective To investigate the regulatory effect of u201CJiajiu201D (EX-B2) electroacupuncture (EA) on the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome in the lumbar spinal cord and to explore its mechanism in improving limb dysfunction in amyotrophic lateral sclerosis (ALS) mice. Methods Fifty-four mice carrying the human superoxide dismutase 1 G93A (hSOD1G93A) gene were randomly divided into model, Jiaji EA, and inhibitor groups, with 18 mice per group. Eighteen mice not carrying the hSOD1G93A gene served as wild group. At 60 d of age, the Jiaji EA group was given EA at L1u2014L2 and L5u2014L6 EX-B2 points with continuous wave, 1 mA, 2 Hz. The needles retained for 20 min, twice every week. The inhibitor group received intraperitoneal injection of the NLRP3 inhibitor (10 mg/kg, twice every week). All treatments were administered for 4 consecutive weeks. The survival period of mice in each group was observed to assess disease progression; the rotarod test was conducted to evaluate motor coordination and limb motor function; HE staining was used to observe pathological changes in the gastrocnemius, and muscle atrophy was assessed by measuring the cross-sectional area of the gastrocnemius fiber; Nissl staining was performed to evaluate the number of motor neurons in the lumbar spinal cord anterior horns; the protein expressions of NLRP3, Caspase-1, apoptosis-associated speck-like protein (ASC), interleukin-1u03B2 (IL-1u03B2), interleukin-18 (IL-18), and tumor necrosis factor-u03B1 (TNF-u03B1) in the lumbar spinal cord were detected by Western blot; the mRNA expressions of NLRP3, Caspase-1, ASC, IL-1u03B2, IL-18, and TNF-u03B1 were detected by real-time quantitative PCR. Results Compared with the wild group, mice in the model group exhibited shortened survival time (Pu0026lt;0.01); reduced time spent on the rotating rod starting from the 13th week of age (Pu0026lt;0.05); significantly shorter and rounded gastrocnemius fibers with enlarged inter-fiber spaces, nuclear translocation and leakage. The cross-sectional area of the gastrocnemius fibers was reduced (Pu0026lt;0.01); the number of motor neurons in the spinal anterior horns was decreased (Pu0026lt;0.01); protein and mRNA expressions of NLRP3, Caspase-1, ASC, IL-1u03B2, IL-18, and TNF-u03B1 were increased in the lumbar spinal cord (Pu0026lt;0.01). Compared with the model group, survival time was prolonged in the Jiaji EA and inhibitor groups (Pu0026lt;0.01); rotarod time was prolonged starting from the 13th week of age (Pu0026lt;0.05); gastrocnemius fibers exhibited more regular margins and larger morphology, with reduced inter-fiber spaces, and reduced number of rounded fibers and improvement in nuclear translocation and leakage; cross-sectional areas of gastrocnemius fiber were increased (Pu0026lt; 0.01); the numbers of motor neurons in the spinal anterior horns were increased (Pu0026lt;0.01); protein and mRNA expressions of NLRP3, Caspase-1, ASC, IL-1u03B2, IL-18, and TNF-u03B1 decreased in the lumbar spinal cord (Pu0026lt;0.05, Pu0026lt; 0.01). Compared with the inhibitor group, mice in the Jiaji EA group exhibited prolonged rotarod time from the 17th week of age (Pu0026lt;0.05); the cross-sectional area of the gastrocnemius fiber increased (Pu0026lt;0.05); protein and mRNA expressions of NLRP3, Caspase-1 and IL-18 in the lumbar spinal cord were higher (Pu0026lt;0.05, Pu0026lt;0.01), while protein and mRNA expressions of TNF-u03B1 were lower (Pu0026lt;0.05, Pu0026lt;0.01). Conclusion EX-B2 EA improves limb function and prolongs survival in ALS mice, with its underlying mechanism potentially involving the alleviation of neuroinflammation through inhibition of NLRP3 inflammasome activation.
Objective To observe the effect of electroacupuncture (EA) at u201CZusanliu201D (ST36) on colonic mucosa injury, expressions of nuclear factor u03BAB (NF-u03BAB), and Aquaporin 8 (AQP8) in 5-fluorouracil (5-FU)-treated mice with transplanted colorectal cancer (CRC), so as to explore its mechanisms underlying improvement of colonic mucosal injury. Methods A colorectal cancer cell strain CT26 was subcutaneously injected into BALB/c mice to establish an animal model of transplanted CRC. When the tumor grew to 100u2014300 mm3, the mice were randomly divided into the tumor-bearing model, chemotherapy, and chemotherapy+EA groups, with 6 mice in each group. Another 6 normal BALB/c mice were used as the normal group. The mice of the chemotherapy group and the chemotherapy+EA group received intraperitoneal injection of 5-FU solution (50 mg/kg, 0.01 mL/g), once daily for 5 d. For mice of the chemotherapy + EA group, EA (2 Hz, 1u20142 mA) was applied to bilateral ST36 for 5 min immediately after chemotherapy. During the experiment, the mouse daily Disease Activity Index (DAI) score was determined for assessing the disease severity. The tumor volume was measured once daily. At the end of the experiment, the mice were sacrificed to measure the tumor weight and colonic length. Histopathological changes of the colon tissue were observed after H.E. staining. The serum contents of diamine oxidase (DAO) and D-lactate (D-LA) were detected using ELISA. The immunoactivity of colonic AQP8 was detected using immunohistochemistry, the immunofluorescence intensity of colonic NF-u03BAB was detected using immunofluorescence staining for assessing the nuclear translocation status, and the protein expression levels of colonic NF-u03BAB and AQP8 were detected using Western blot. Results Compared with the normal group, the tumor-bearing model group had no significant changes in the DAI score, contents of serum DAO and D-LA, immunoactivity, immunofluorescence intensity and protein expression levels of colonic NF-u03BAB and AQP8. In comparison with the tumor-bearing model group, the DAI score in chemotherapy group, and serum DAO and D-LA contents, nuclear translocation and protein expression levels of NF-u03BAB in the chemotherapy group were considerably increased (Pu0026lt;0.05), while the colonic length, tumor volume, tumor weight, and the immunoactivity level of AQP8 were strikingly decreased (Pu0026lt;0.05). Comparison between the chemotherapy and chemotherapy+EA groups showed that the DAI score, serum DAO and D-LA contents, nuclear translocation and protein expression levels of NF-u03BAB in the chemotherapy+EA group were significantly lower than those in the chemotherapy group (Pu0026lt;0.05), while the immunoactivity level of AQP8 in the chemotherapy+EA group was notably higher than that in the chemotherapy group (Pu0026lt;0.05), and no significant differences were found between the chemotherapy and chemotherapy+EA groups in the levels of colonic length, tumor volume and mass. H. E. staining showed that the colonic tissue structure in the tumor-bearing group had no abnormal changes, and that in the chemotherapy group displayed pathological injury, including shortening and thickening of intestinal villi, destruction of glandular structure, increased shedding of goblet cells, infiltration of inflammatory cells, and enlargement of cell nuclei. Compared with the chemotherapy group, the colonic structure in the chemotherapy+EA group was improved, including increase in the villi length, and reduction in the disordered arrangement of the tissue cells and infiltration of inflammatory cells. Conclusion EA of ST36 can mitigate intestinal mucosal damage to maintain intestinal barrier function in 5-FU-treated mice with transplanted CRC, which may be related with its functions in inhibiting the excessive activation of the NF-u03BAB signaling pathway, up-regulating the expression levels of AQP8 protein, and reducing DAO and D-LA release.
ObjectiveTo explore the effects of acupoint catgut embedding on intestinal microbiota and inflammatory response in simple obese rats and its underlying mechanism.MethodsSD rats were fed with high‑fat diet to establish simple obesity models， and the model rats were randomly divided into model group， orlistat group， catgut embedding group， AMPK activator （AICAR） group， and catgut embedding + AMPK inhibitor （Compound C） group， with 8 rats in each group. Another 8 SD rats were set as the control group. In the catgut embedding group， catgut embedding was performed at acupoints “Zhongwan” （CV12）， “Guanyuan” （CV4）， “Zusanli” （ST36）， and “Fenglong” （ST40）， once a week for 4 consecutive weeks. Rats in the orlistat group received intragastric administration of orlistat at 0.048 g/kg once daily for 4 weeks. Rats in the AICAR group were given tail‑vein injection of AICAR at 50 mg/kg once daily for 4 weeks. Rats in the catgut embedding + Compound C group received catgut embedding combined with tail‑vein injection of Compound C at 250 μg/kg. Body weight and body length were measured and Lee’s index was calculated. Fasting plasma glucose （FPG） and fasting insulin （FINS） were detected snd the insulin resistance index was calculated. Serum levels of total cholesterol （TC）， triglyceride （TG）， low‑density lipoprotein‑cholesterol （LDL‑C）， and high‑density lipoprotein‑cholesterol （HDL‑C） were determined by biochemical analyzer. Serum contents of tumor necrosis factor‑α （TNF‑α）， interleukin‑6 （IL‑6）， lipopolysaccharide （LPS）， and interleukin‑10 （IL‑10） were measured by ELISA. Pathological changes of colon tissues were observed by HE staining. Immunofluorescence was used to detect the positive expression of Occludin and Claudin‑1 in colon tissue. The abundance of intestinal bacterial genera was determined by PCR. Western blot was applied to detect the protein expression of AMP‑activated protein kinase （AMPK）/sirtuin‑1 （SIRT1）/peroxisome proliferator‑activated receptor‑γ coactivator‑1α （PGC‑1α） pathway in colon and adipose tissues.ResultsCompared with the control group， the model group showed significantly increased body weight， Lee’s index， serum TC， TG， LDL‑C， TNF‑α， IL‑6， LPS， FPG， FINS， insulin resistance index， as well as the abundances of Enterococcus and Desulfovibrio （P<0.05）； while serum IL‑10 and HDL‑C levels， the abundances of Bifidobacterium， Lactobacillus， and Akkermansia， the fluorescence intensity of Occludin and Claudin‑1 in colon tissues， the p‑AMPK/AMPK ratio， and the protein levels of SIRT1 and PGC‑1α in colon and adipose tissues were decreased （P<0.05）. Compared with the model group， the orlistat group， catgut embedding group and AICAR group exhibited significantly decreased body weight， Lee’s index， serum TC， TG， LDL‑C， TNF‑α， IL‑6， LPS， FPG， FINS， insulin resistance index， and the abundances of Enterococcus and Desulfovibrio （P<0.05）； whereas serum IL‑10 and HDL‑C levels， the abundances of Bifidobacterium， Lactobacillus， Akkermansia， the fluorescence intensity of Occludin and Claudin‑1 in colon tissues， the p‑AMPK/AMPK ratio， and the protein levels of SIRT1 and PGC‑1α in colon and adipose tissues were elevated （P<0.05）. Compared with the catgut embedding group， the catgut embedding + Compound C group had significantly higher body weight， Lee’s index， serum TC， TG， LDL‑C， TNF‑α， IL‑6， LPS， FPG， FINS， insulin resistance index， and abundances of Enterococcus and Desulfovibrio （P<0.05）； while serum IL‑10 and HDL‑C levels， the abundances of Bifidobacterium， Lactobacillus， Akkermansia， colon Occludin and Claudin‑1 fluorescence intensity， p‑AMPK/AMPK ratio， and SIRT1 and PGC‑1α protein expression in colon and adipose tissues were reduced （P<0.05）.ConclusionAcupoint catgut embedding can alleviate systemic low‑grade inflammatory response， regulate blood lipid metabolism， modulate the abundance of beneficial intestinal bacteria， and repair intestinal barrier function in simple obese rats. Its mechanism may be related to the AMPK/SIRT1/PGC‑1α signaling pathway.
ObjectiveTo observe the effects of moxibustion at “Shenque” （CV8） and “Guanyuan” （CV4） on gut microbiota， intestinal barrier and blood–brain barrier （BBB）， peripheral and central neuroinflammation， and 5-hydroxytryptamine （5-HT）ergic neurons in the dorsal raphe nucleus （DRN） in a mouse model of chronic fatigue syndrome （CFS） based on the gut–brain axis theory， so as to explore the underlying mechanisms of moxibustion in treating CFS.MethodsMale C57BL/6 mice were randomly divided into control group， model group， and moxibustion group， with 10 mice in each group. The CFS model was established by combining exhaustive swimming with food restriction. Concurrently with modeling， the moxibustion group received moxibustion at CV8 and CV4 once daily， 10 min per session， for 28 consecutive days. After treatment， fatigue-like behaviors were evaluated by open field test， forced swimming test， and tail suspension test. Gut microbiota diversity and composition were analyzed by 16S rDNA sequencing. Colonic mRNA expression of tight junction proteins zonula occludens-1 （ZO-1） and Occludin was detected by real-time quantitative PCR. Serum levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） were measured by multiplex flow cytometry； serum lipopolysaccharide （LPS） and S100 calcium-binding protein β （S100β） levels were determined by ELISA. Immunofluorescence was used to detect positive expression of ionized calcium-binding adapter molecule 1 （Iba-1） and IL-1β， as well as the number of 5-HTergic neurons in the DRN. Spearman correlation analysis was performed to assess the associations between gut microbiota and various gut–brain axis parameters.ResultsCompared with the control group， the model group showed prolonged immobility time in the open field， tail suspension， and forced swimming tests （P<0.05， P<0.01）； increased α-diversity indices （Chao and Shannon） of gut microbiota （P<0.01， P<0.05）； β-diversity analysis revealed a clear spatial separation between model and control groups. The model group also exhibited decreased abundance of Lactobacillus （P<0.001）， and increased abundances of Spirochaetes， Treponema， Clostridium vadinBB60 group， Ruminococcus， and Desulfovibrionaceae （P<0.05， P<0.01， P<0.001）； reduced colonic ZO-1 and Occludin mRNA expression （P<0.01， P<0.05）； elevated serum levels of LPS， IL-6， TNF-α， and S100β （P<0.05， P<0.01）； increased Iba-1 and IL-1β positive expression in the DRN （P<0.001， P<0.01）， and an increased number of 5-HTergic neurons （P<0.01）. Compared with the model group， all the above parameters were reversed in the moxibustion group （P<0.01， P<0.05， P<0.001）. Correlation analysis revealed that the abundance of Lactobacillus was significantly negatively correlated with the number of 5-HTergic neurons in the DRN （ρ=-0.900， P<0.001）， and was also significantly correlated with behavioral improvement， intestinal barrier repair， and alleviation of inflammatory responses （P<0.05， P<0.01， P<0.001）.ConclusionMoxibustion can significantly improve fatigue-like behaviors in CFS mice， and its mechanism may involve regulation of the gut–brain axis， i.e.， through modulating gut microbiota， repairing the intestinal barrier and BBB， alleviating peripheral and central neuroinflammation， thereby inhibiting the aberrant increase of 5-HTergic neurons in the DRN and restoring peripheral–central homeostasis.
ObjectiveTo explore the response characteristics of whole-brain c-Fos signals in brain regions associated with acupuncture and moxibustion analgesia， and to identify potential targets for revealing differences in the central regulatory mechanisms underlying their analgesic effects.MethodsC57BL/6J mice were randomly divided into sham， model， acupuncture， and moxibustion groups， with 8 mice in each group. Neuropathic pain was induced by spared nerve injury （SNI） of the sciatic nerve. The acupuncture and moxibustion groups received electroacupuncture and moxibustion interventions respectively for 30 min each day at “Zusanli” （ST36） for 7 consecutive days. Mechanical and thermal pain thresholds were measured to assess analgesic efficacy. Whole-brain c-Fos expression was detected by immunofluorescence. Bioinformatics methods were employed to analyze the number of analgesia-responsive brain regions， shared and specific regions， functional brain networks， and hub nodes based on c-Fos expression levels.ResultsAfter the final treatment， compared with the sham group， the model group showed significantly decreased mechanical and thermal pain thresholds （P<0.001）. Compared with the model group， both acupuncture and moxibustion groups showed significantly increased thresholds （P<0.01， P<0.001）， with the acupuncture group exhibiting significantly higher thresholds than the moxibustion group （P<0.05）. Whole-brain c-Fos signals were significantly increased in the model group compared with the sham group， while both treatment groups showed significant reductions relative to the model group. A total of 48， 6 and 13 brain regions presented significant alterations in c-Fos signals in the SNI group， acupuncture group and moxibustion group， respectively. In the acupuncture group， the significantly altered brain regions mainly included the primary cingulate cortex area 1 （Cg1R，L）， paraventricular nucleus of the hypothalamus （PVNR）， primary somatosensory cortex hindlimb region （S1HLL）， basolateral amygdala （BLAL）， magnocellular part of the paraventricular hypothalamic nucleus （PaLML）， and left posterior paraventricular hypothalamic nucleus （PaPoL） （P<0.05， P<0.01）. For the moxibustion group， the prominent brain regions with changed c-Fos signals were the locus coeruleus （LCR，L）， ateral hypothalamus （LHR，L）， ventral part of the medial geniculate nucleus （MGVL）， right and left paraventricular nucleus of the hypothalamus （PVNR，L）， S1HLL， Cg1R，L， BLAR，L， basomedial amygdala （BMAL）， substantia nigra （SNR）， and mediolateral secondary visual cortex （V2MLR） （P<0.05， P<0.01， P<0.001）. Within the analgesic brain functional networks of acupuncture and moxibustion， the primary somatosensory cortex （S1R）， dorsomedial thalamic nucleus （DenL） and dorsomedial hypothalamic nucleus （DMR） served as common significantly responsive nodes. S1， Dorsal Endopiriform Nucleus （Den） and central amygdala （CeCL） were identified as nodes with strong functional connectivity in both networks. In addition， the cornu ammonis 2 region of the hippocampus （CA2R） and CeC represented the nodes with the highest connectivity degree in the acupuncture network and moxibustion network， respectively.ConclusionAcupuncture shows superior analgesic effects to moxibustion in the SNI model. Both interventions likely exert analgesia primarily through negative activation across multiple brain regions. Cg1， PVN， S1HL， and BLA may be shared response regions； PaPo and PaLM may be specific to acupuncture； and LC， LH， MGV， SN， BMA， and V2ML may be specific to moxibustion. S1， Den， and CeC may represent shared functional network pathways， while CA2 and CeC may serve as core hubs in the acupuncture and moxibustion analgesic brain networks， respectively.
ObjectiveTo compare the differences in interventional effects among three stimulation modalities—electroacupuncture （EA） at single acupoint “Zusanli” （ST36）， single acupoint “Sanyinjiao” （SP6）， and their combination （ST36 + SP6）， on behavioral and histological outcomes in a mouse model of irritable bowel syndrome （IBS）， and to examine the differential neuronal responses in the peripheral and central nervous systems under these modalities， so as to provide experimental evidence for exploring the neural mechanisms underlying acupoint combination stimulation in IBS treatment.MethodsAn IBS model was established in male C57BL/6J mice by intrarectal instillation of Zymosan. The mice were randomly divided into blank control group， model group， ST36 group， SP6 group， and acupoint combination group （n=20 per group except n=18 for the model group）. After 7 consecutive days of EA stimulation （2 Hz/100 Hz， 1 mA， 15 min per session， once daily）， abdominal mechanical hyperalgesia （referred pain） was assessed using von Frey filaments， motor function by rotarod test， and 3D spontaneous fine behavioral characteristics by an AI-based unsupervised 3D behavioral analysis system. Colon tissue structure and inflammatory levels were observed by HE staining， and colonic inflammatory cytokine expression was detected by Western blot， to evaluate the symptom-relieving effects of single versus combined acupoint stimulation. For neural tracing， retrograde viral tracers were injected into ST36 and SP6 to label dorsal root ganglion （DRG） neurons innervating these acupoints in C57BL/6J mice. In vivo two-photon calcium imaging was performed to record DRG neuronal responses to different EA modalities （1 mA， 1 min） in Thy1-GCaMP6s mice， and activity-dependent neuronal labeling was applied using transgenic TRAP2；Ai14 mice to identify central neurons activated by EA （2 Hz/100 Hz， 1 mA， 15 min per session）， aiming to reveal peripheral–central neuronal response differences between single and combined acupoint stimulation.ResultsCompared with the blank control group， the model group showed increased abdominal mechanical withdrawal frequency and area under the curve （P<0.001， P<0.05）， decreased motor function （latency to fall and rotarod speed at fall， P<0.01）， aggravated colonic tissue damage and inflammatory responses （P<0.001）， elevated expression of IL-1β and TNF-α （P<0.001）， and altered spontaneous behavioral patterns （P<0.001， P<0.01， P<0.05）. All three EA modalities significantly alleviated abdominal mechanical hyperalgesia in IBS mice （P<0.001， P<0.05， P<0.01）， with the combined stimulation showing a larger effective interventional window than either single acupoint. Compared with single-acupoint stimulation， combined stimulation also more effectively restored colonic tissue damage， reduced TNF-α expression （P<0.05）， and shortened the duration of spontaneous arching episodes and altered the transition patterns among different spontaneous behavioral actions （P<0.01， P<0.05）. DRG neurons exhibited highly heterogeneous responses to the three stimulation modalities， which could be classified into 3 major categories and 7 subcategories based on their firing characteristics. Furthermore， combined stimulation activated a greater number of DRG neurons than single acupoint stimulation （P<0.05）， and the neuronal calcium signal intensity was also higher （P<0.05）. Finally， significant differences were observed in the number of responsive neurons across different brain regions under the three modalities. The activated neuronal populations evoked by ST36， SP6， and combined stimulation showed maximal overlap in the paraventricular nucleus of the hypothalamus （PVN）， whereas the overlap was minimal in the insular cortex （IC）， suggesting that IC neuronal activation requires the synergistic effect of combined acupoint stimulation.ConclusionCombined stimulation at ST36 and SP6 produces superior therapeutic effects over single acupoint stimulation in ameliorating abdominal mechanical hyperalgesia， colonic structural damage， inflammatory status， and spontaneous behavioral changes induced by visceral pain in IBS model mice， and this advantage may arise from the synergistic integration of peripheral and central neuronal responses to different EA signals.
Acupuncture， as a typical representative of non-drug therapy， regulates central functions by activating the peripheral nervous system， and has become a leading hotspot in the field of neuromodulation. Focusing on the peripheral-cerebral mechanism of acupuncture effects， this article systematically reviews the cutting-edge interdisciplinary advancements in acupuncture research and modern life sciences from three dimensions： neuro-immune anti-inflammatory actions， target organ function regulation， and “one acupoint affecting multiple organs” intervention for comorbidities. To analyze the key issues， clarify academic viewpoints， and trigger thoughts and discussions， the following are the main points： 1） Based on the established understanding that acupuncture stimulation activates autonomic nerve pathways such as the brainstem vagus-adrenal and spinal cord sympathetic-splenic axes， to exert anti-inflammatory effects， it has also been revealed that some vagus nerves can directly traverse and form synaptic connections with catecholaminergic neurons in the celiac ganglion via collateral branches， rather than relaying through relay neurons. It further seeks to clarify the hierarchical relationship among the catecholamine anti-inflammatory， cholinergic anti-inflammatory， sympathetic/parasympathetic nerve pathways， particularly paying attention to the relationship between the concentration-dependent receptor affinity of norepinephrine in the inflammatory microenvironment and its association with acupuncture effects. 2） Current researches have demonstrated that acupuncture regulates the homeostasis of the body and internal organs by rebalancing the sympathetic-parasympathetic system at multiple levels， identifying the “off-target” phenomenon caused by the loss of response of tissue cells and receptors under pathological conditions is a key factor affecting the “last mile” of the acupuncture effect. 3） Taking the convergence of different visceral input information mediated by the intrinsic structures of the spinal cord， as well as the occurrence of comorbidity in the interactive sensitization of the pelvic and sacral organs as an example， results of modern research have provided possible explanations for the cross-segment’s and same-segment’s “one acupoint affecting multiple organs” and “multiple acupoints affecting one organ” effects of acupuncture. In summary， this article aims to clarify how acupuncture regulates the body’s homeostasis through different peripheral-cerebral autonomic nervous pathways and levels， and offers a review of existing research from four perspectives to stimulate new insights and foster intellectual exchange.
ObjectiveTo establish a mouse model of trigeminal neuropathic pain induced by chronic constriction injury of the infraorbital nerve （CION）， screen the optimal frequency of electroacupuncture （EA） for trigeminal neuralgia （TN）， so as to explore the peripheral-central interaction mechanism of the “Hegu” （LI 4）-ventral posterior nucleus （VP）-trigeminal ganglion （TG） pathway.MethodsMale C57BL/6J mice were used to establish the TN model via the CION method. EA （2 Hz / 100 Hz / 2 /100 Hz） was applied to bilateral LI 4. Mechanical withdrawal thresholds （MWTs） of the vibrissal pad were measured using von-Frey filaments. Anxiety-like behaviors were assessed by the elevated plus maze （EPM） test， and depression-like behaviors by the tail suspension （TS） test and sucrose preference （SP） test. Neuronal activity in eight brain regions， including the dorsal raphe nucleus （DRN）， VP， and spinal trigeminal nucleus caudalis （Sp5C）， was evaluated by c-Fos immunofluorescence. Double immunofluorescence labeling was used to detect co-expression of c-Fos with 5-hydroxytryptamine （5-HT）， glutamate （Glu）， or γ-aminobutyric acid （GABA） in the DRN to identify activated neuron subtypes. Neural connectivity between “Hegu”-dorsal root ganglion （DRG）-spinal dorsal horn （DH）-VP and TG-Sp5C-VP was traced using chemical or viral tracers.ResultsCompared with the normal group， the MTs of mice were significantly decreased in the model group （P<0.0001）. The MTs in the 2 Hz， 100 Hz， and 2 Hz/100 Hz EA groups were all increased （P<0.05， P<0.001）， and the analgesic effect of the 2 Hz/100 Hz EA group was superior to that of the 2 Hz and 100 Hz EA groups （P<0.05， P<0.01）. In the EPM test， the time spent in the open arms was significantly decreased in the model group （P<0.0001）， while it was increased in the 100 Hz EA and 2 Hz/100 Hz EA groups （P<0.01， P<0.0001）. Moreover， the time spent in the open arms was longer in the 2 Hz/100 Hz EA group than in the 2 Hz EA group （P<0.01）. In the TS test， the immobility time was increased in the model group （P<0.0001）， whereas EA reversed this change （P<0.0001）. The 2 Hz/100 Hz EA group showed a more significant improvement in depression-like behavior than the 2 Hz and 100 Hz EA groups （P<0.05）. In the SP test， the sucrose preference was decreased in the model group （P<0.0001）， whereas EA reversed this change （P<0.001， P<0.0001）. The 2 Hz/100 Hz EA group showed a more significant improvement in depression-like behavior than the 2 Hz and 100 Hz EA groups （P<0.01， P<0.05）. Tracing results showed that after injection of a chemical tracer into LI 4， tracer signals were observed in the C6 and C7 DRG. After injection of a retrograde trans-multisynaptic virus into the VP， viral expression was observed in the DH， Sp5C， and TG. Immunofluorescence results showed that neuronal activity in the Sp5C， VP， ventral posteromedial nucleus （VPM）， parafascicular nucleus （PaF）， and DRN was increased in the model group （P<0.01， P<0.0001， P<0.05）， while EA reversed these changes （P<0.0001， P<0.01， P<0.001）. Compared with the normal group， the model group showed decreased 5-HT neuronal activity （P<0.0001） and increased Glu and GABA neuronal activities （P<0.0001， P<0.05）， all of which were reversed by 2 Hz/100 Hz EA （P<0.0001， P<0.05）.ConclusionBehavioral results show that 2 Hz/100 Hz EA is superior to single-frequency stimulation in improving mechanical hypersensitivity and associated negative affective behaviors in CION model mice， thus it is selected as the optimal intervention frequency for subsequent central response research. Under CION conditions， abnormal activation was observed in the Sp5C， VP， VPM， PaF， and DRN， accompanied by decreased activity of 5-HT neurons and increased activities of GABA and Glu neurons in the DRN， 2 Hz/100 Hz EA can reverse these phenomena. The tracing results indicated the existence of neuroanatomical connections of the LI 4-DRG-DH-VP and TG-Sp5C-VP pathways. These findings provide objective evidence for investigating the LI 4-VP-TG response mechanism underlying EA intervention in trigeminal neuralgia.
ObjectiveTo investigate the role of tyrosine hydroxylase （TH）-positive neurons in the rostral ventrolateral medulla （RVLM） in the effects of electroacupuncture （EA） on intestinal dysmotility in mice with functional constipation （FC）.MethodsEighteen C57BL/6J mice were randomly assigned to the normal， model， and EA groups， with 6 mice in each group. FC model was established by oral administration of loperamide hydrochloride. Mice in the EA group received EA at Tianshu （ST25） and Shangjuxu （ST37）， 5 days per treatment course for 2 courses. After intervention， The defecation status of mice in each group was observed， and the fecal water content was determined， the time for the first black stool excretion was recorded， the small-intestinal propulsion rate was measured. Serum and colonic TH levels were determined by ELISA. TH/c-Fos co-expression in the RVLM was assessed by immunofluorescence staining. Subsequently， 12 C57BL/6J mice were randomly divided into the control virus+clozapine-N-oxide （CNO） group and inhibitory virus+CNO group， with 6 mice per group. Another 18 C57BL/6J mice were randomly assigned to the control virus+CNO group， EA group， and excitatory-virus+CNO+EA group， with 6 mice per group. The corresponding viral vectors were injected into the RVLM， followed by intraperitoneal administration of CNO （1 mg/kg） 21 d after viral expression. Mice in the EA and excitatory-virus+CNO+EA groups received EA treatment as described above. Following completion of EA treatment， fecal parameters and intestinal motility were assessed as described above. Finally， rAAV-hSyn-GCaMP6s virus was injected into the RVLM of 18 mice. After 21 days of viral transduction， mice were randomly assigned to normal control， model control， and model+EA groups （6 mice per group）. Mice in the model+EA group received EA treatment as described above. Post-EA intervention， multichannel fiber photometry was employed to record calcium signal dynamics in RVLM neurons.ResultsNeuronal fibers in the RVLM formed synaptic connections with the proximal colon. Compared with the normal group， the model group exhibited prolonged first black stool expulsion time， reduced total fecal pellet count， decreased fecal water content and small intestinal transit rate （P<0.001， P<0.01）， and elevated TH levels in serum and colon （P<0.01）. TH and c-Fos expression， as well as TH/c-Fos co-expression in the RVLM， were all significantly increased （P<0.001）. Compared with the model group， the EA group showed shortened first black stool expulsion time increased total fecal pellet count， elevated fecal water content and small intestinal transit rate （P<0.001， P<0.01）， and reduced TH levels in serum and colon （P<0.01）. TH and c-Fos expression， as well as TH/c-Fos co-expression in the RVLM， were all significantly decreased （P<0.001）. Compared with the control virus+CNO group， the inhibitory-virus+CNO group exhibited shortened first black stool expulsion time， increased total fecal pellet count， and elevated fecal water content and small intestinal transit rate （P<0.001， P<0.01）. Compared with the control virus+CNO group， the EA group showed shortened first black stool expulsion time， increased total fecal pellet count， elevated fecal water content and small intestinal transit rate （P<0.001， P<0.01）. Compared with the EA group， the excitatory virus+CNO+EA group showed prolonged first black stool expulsion time， reduced total fecal pellet count， and decreased fecal water content and small intestinal transit rate （P<0.001， P<0.01）. Compared with the normal control group， the model control group exhibited significantly increased Ca2+ activity in the RVLM （P<0.001）， whereas EA significantly reduced RVLM Ca2+ activity compared with the model control group （P<0.001）.ConclusionElectroacupuncture at Tianshu （ST25） and Shangjuxu （ST37） may improve intestinal dysmotility in FC mice by suppressing the activity of TH-positive neurons in the RVLM.
ObjectiveTo investigate the mechanism of electroacupuncture （EA） in improving inflammatory injury in chronic obstructive pulmonary disease （COPD） from the perspective of pulmonary airway epithelial cell senescence and to analyze its correlation with the cyclic GMP-AMP synthase-stimulator of interferon genes （cGAS-STING） signaling pathway.MethodsTwenty-four specific pathogen-free male Sprague-Dawley （SD） rats were randomly divided into a blank group， a model group， an EA group， and a non-acupoint EA group， with 6 rats in each group. A COPD rat model was established by intratracheal instillation of lipopolysaccharide （LPS） combined with cigarette smoke exposure. LPS （1 mg/mL， 0.2 mL each time） was administered on days 1， 15， 30， and 45， while cigarette smoke exposure （20 cigarettes/time， 1 h/time） was performed daily for 60 days. After modeling， rats in the EA group received EA stimulation at bilateral "Feishu" （BL13） and "Zusanli" （ST36） using sparse-dense waves （4 Hz/ 20 Hz） with an intensity of 1–3 mA for 20 min once daily for 14 consecutive days. Rats in the non-acupoint EA group received the same stimulation at non-meridian and non-acupoint sites at the tail root. Rats in the blank and model groups were only restrained for the same duration. After intervention， pulmonary function was evaluated， hematoxylin-eosin （HE） staining was performed to observe pathological changes and inflammatory scores of lung tissues， and enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， interleukin-10 （IL-10）， and transforming growth factor-β1 （TGF-β1） in bronchoalveolar lavage fluid （BALF）. Senescence-associated β-galactosidase （SA-β-Gal） staining and immunofluorescence were used to assess cellular senescence and DNA damage， respectively. Western blot was used to detect the protein expression of cyclin-dependent kinase inhibitor 2A （P16）， cyclin-dependent inhibitor 21 （P21）， cGAS， stimulator of interferon genes （STING）， and phosphorylated STING （p-STING）. Quantitative real-time polymerase chain reaction （qPCR） was used to determine the mRNA expression levels of P16， P21， cGAS， and STING.ResultsCompared with the blank group， the model group showed significantly decreased forced expiratory volume in 0.3 seconds （FEV0.3）， forced vital capacity （FVC）， and FEV0.3/FVC ratio （P0.01）. The airway epithelial structure of the lung tissue was damaged， accompanied by increased peribronchial inflammatory cell infiltration and significantly elevated inflammation scores （P0.01）. The levels of TNF-α， IL-1β， IL-6， and TGF-β1 in BALF were significantly increased （P0.01）， whereas the level of IL-10 was significantly decreased （P0.01）. The proportion of SA-β-Gal-positive area in the pulmonary airway epithelium was increased， and the fluorescence intensity of phosphorylated histone H2AX （γH2AX） was enhanced. In addition， the protein expression levels of P16， P21， cGAS， total STING， and p-STING in the lung tissue were significantly increased （P0.01）. The mRNA expression levels of P16， P21， cGAS， and STING were also significantly increased （P0.01）. Compared with the model group， the EA group exhibited significant improvements in FEV0.3， FVC， and FEV0.3/FVC ratio （P0.01）. Lung histopathological injury was alleviated， accompanied by reduced inflammatory cell infiltration and significantly decreased inflammation scores （P0.05）. The levels of TNF-α， IL-1β， IL-6， and TGF-β1 in BALF were significantly reduced （P0.01）， whereas the level of IL-10 was significantly increased （P0.01）. The proportion of SA-β-Gal-positive area was reduced， the fluorescence intensity of γH2AX was decreased， and the protein expression levels of P16， P21， cGAS， total STING， and p-STING were significantly downregulated （P0.01）. The mRNA expression levels of P16， P21， cGAS， and STING were also significantly decreased （P0.01）. No significant improvement was observed in the non-acupoint EA group （P0.05）.ConclusionEA may alleviate DNA damage and cellular senescence in pulmonary airway epithelial tissues by inhibiting abnormal activation of the cGAS-STING signaling pathway， thereby improving inflammatory status and exerting protective effects on COPD.
ObjectiveThis study utilized surface electromyography （EMG） detection methods to evaluate the therapeutic value of the combination of needle retention and traction technique with electroacupuncture in treating patients with refractory facial paralysis.MethodsSixty eligible subjects were randomly divided into an observation group and a control group， each consisting of 30 cases. The control group received conventional acupuncture at the affected side's facial points such as Yangbai， Yuyao， Qianzheng， Qianliao， Sibai， Jiachexia， Dicang， and Taiyang. After needle insertion， the treatment was stopped once the qi sensation was achieved without any special manipulation. Then， electroacupuncture was applied to the pairs of Yangbai and Taiyang， Dicang and Jiachexia， and Qianliao and Sibai. The observation group， on the basis of the above acupuncture treatment， received needle retention and traction along the paths from Yangbai to Yuyao， Qianliao to Dicang， and Jiachexia to Dicang. The same electroacupuncture parameters were used in both groups. Each treatment session lasted for 30 minutes， and the treatment was conducted five times a week for a total of 28 days. The changes in the integrated electromyography （IEMG） values of the frontalis， zygomaticus， and orbicularis oris muscles during voluntary contractions before and after treatment were recorded using surface EMG. The House-Brackmann （H-B） facial nerve function grading scale and the Sunnybrook assessment system scores were also compared to comprehensively evaluate the clinical efficacy of the intervention.ResultsAfter treatment， the IEMG values of the three target muscles in both groups significantly increased compared to those before treatment （P 0.05）， and the observation group's values were significantly higher than those of the control group （P0.05）. Both groups showed significant improvements in H-B grading and Sunnybrook scores after treatment （P0.05）， with the observation group demonstrating better functional recovery than the control group （P0.05）. In terms of overall efficacy， the effective rate of the observation group was 90.00% （27/30）， while that of the control group was 73.33% （22/30）， and the difference between the two groups was statistically significant （P0.05）.ConclusionThe combined application of stuck-needle lifting manipulation and electroacupuncture stimulation can significantly enhance the therapeutic benefits for refractory facial paralysis， demonstrating a positive effect on alleviating patients' clinical signs and symptoms.
Objective To observe the effect of electroacupuncture(EA)on the homeostasis of Th17/Treg cells and the expressions of key proteins of the janus kinase 1/signal transducer and activator of transcription 3 signaling pathway in myocardial tissue of rats with myocardial ischemia(MI),so as to explore its mechanisms underlying amelioration of inflammatory injury following MI.Methods SD rats were randomly divided into control,model,and EA groups,with 6 rats in each group.The MI model was established by subcutaneous injection of isoproterenol hydrochloride(5 mg ∙ kg-1 ∙ d-1),once daily for 7 consecutive days.After successful modeling,the rats of EA group received EA stimulation(2 Hz/10 Hz,2 to 3 mA)of unilateral"Neiguan"(PC6)and"Zusanli"(ST36)for 20 min,once daily for 21 d.The standard limb lead II electrocardiogram(ECG)was recorded to analyze the height of ST and amplitude of T wave for assessing the degree of ischemic myocardial injury.The hematoxylin-eosin(H.E.)staining was used to observe histopathological changes in the myocardial tissue,and the Masson staining performed to examine the collagen deposition of the myocardial cells.The concentrations of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-17,and IL-10 in the serum and myocardial tissue were detected using enzyme-linked immunosorbent assays(ELISA),and the proportions of Th17 cells and Treg cells in the myocardial tissue analyzed using flow cytometry.The expression levels of signal transducers and activators of transcription 3(STAT3),phosphorylated(p)-STAT3,forkhead box protein 3(Foxp3),Janus kinase 1(JAK1),retinoic acid-related orphan nuclear receptor-α(RORα),suppressor of cytokine signaling 3(SOCS3)proteins in the myocardial tissue were detected using Western blot.Results Compared with the control group,the model group had a significant increase in the ST height and T-wave amplitude,myocardial collagen volume fraction(CVF),TNF-α and IL-17 contents in the serum and myocardial tissue,myocardial Th17/Treg ratio,and expression levels of myocardial JAK1,STAT3,p-STAT3,and RORα proteins(P<0.01,P<0.05),and a striking decrease in the proportion of Treg cells,contents of myocardial and serum IL-10,and expression levels of myocardial FOXP3 and SOCS3(P<0.01,P<0.05).In contrast to the model group,both the increase and the decrease of the indexes mentioned above were reversed in the EA group(P<0.05,P<0.01).H.E.staining showed necrosis and dissolution of a large number of myocardial cells,with increased cell spacing and blurred boundaries,inflammatory infiltration and fibrous tissue hyperplasia in the model group,which was relatively and significantly milder in the degree of myocardial injury in the EA group.Conclusion EA at PC6 and ST36 can improve myocardial inflammation injury in rats with MI,which may be related to its function in regulating the homeostasis of Th17/Treg cells in myocardial tissue through JAK1/STAT3 signaling,balancing the secretion of pro-inflammatory factors TNF-α,IL-17 and anti-inflammatory factor IL-10.
Objective To observe the effect of acupuncture at the acupoint pair u201CHouxiu201D (SI3) and u201CHuantiaou201D (GB30) on the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway and autophagy of nucleus pulposus cells in a rat model of intervertebral disc degeneration (IDD), so as to explore its underlying mechanism in delaying IDD. Methods A total of 36 male SD rats were randomly assigned to sham operation, model and acupoint pair groups, with 12 rats in each group. The IDD model was established by annulus fibrosus puncture. After modeling, rats of the acupoint pair group received acupuncture stimulation of bilateral SI3 and GB30, with the acupuncture needles retained for 20 min, once daily for 14 consecutive days. Before and after modeling and after the intervention, the mechanical withdrawal reflex threshold (mechanic pain threshold) of the right foot was measured using VonFrey filaments, and the thermal pain threshold measured by using a thermal pain tester. The morphological characteristics of the intervertebral disc tissue were observed by H.E. staining. The contents of type u2161 collagen (Collagen u2161), Aggrecan, matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 13 (MMP13), and transcription factor sex-determining region Y-box protein 9 (SOX9) in the nucleus pulposus tissue were detected by ELISA. The mRNA relative expression levels of SOX9 and MMP13 in the nucleus pulposus tissue were detected by real-time fluorescence quantitative PCR. The positive expression of microtubule-associated protein 1 light chain 3 (LC3) in the nucleus pulposus tissue was detected by immunofluorescence staining. The expression levels of PI3K/AKT/mTOR signaling pathway-related proteins and LC3-u2161, Beclin1, and chaperone 1 (p62) in the nucleus pulposus tissue were detected by Western blot. Results After modeling, in contrast to the sham operation group, the model group showed a striking decrease in the mechanical and thermal pain thresholds on day 7 and 14, contents of Aggrecan, collagen u2161 and SOX9, and the expression levels of SOX9 mRNA and p62 protein, ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR in the nucleus pulposus tissue (Pu0026lt;0.001, Pu0026lt;0.01), and a notable increase in the contents of MMP3 and MMP13, and MMP13 mRNA expression level, LC3 immunofluorescence intensity, and protein expressions of LC3-u2161 and Beclin 1 (Pu0026lt;0.001). Under light microscope, the nucleus pulposus cells in the model group was relatively small in the number and discorded in the distribution, with a large number of vacuoles and chaotic matrix arrangement, and the lumbar intervertebral discs showed obvious degeneration, and decrease in the height. In comparison with the model group, both the decrease and increase of the indexes mentioned above were all reversed in the acupoint pair group (Pu0026lt;0.001, Pu0026lt;0.01, Pu0026lt;0.05). The results of H.E. stain displayed that in the acupoint pair group, the arrangement of the nucleus pulposus cells was more regular, and the number of vacuoles was reduced. Conclusion Acupuncture of acupoint pair SI3 and GB30 can mitigate pain and regulate the autophagy process of lumbar intervertebral disc nucleus cells, reduce the degree of degradation of cytoplasmic matrix, and thereby delay the progression of IDD, which may be associated with its function in activating PI3K/AKT/mTOR signaling pathway in IDD rats.
ObjectiveThis study aimed to investigate the modulatory effects of eye-acupuncture on the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） signaling pathway and to elucidate its therapeutic efficacy and underlying molecular mechanisms in vascular cognitive impairment （VCI）.MethodsMale SD rats were randomly assigned to six experimental groups： sham-operated control， VCI model， Nrf2 agonist （TBHQ）， eye-acupuncture， eye-acupuncture + Nrf2 inhibitor （ML385）， and Nrf2 inhibitor alone. VCI was induced via modified bilateral permanent common carotid artery occlusion （2-VO）. Following successful model establishment， rats in the eye-acupuncture group received daily 30-minute acupuncture stimulation at four periocular points—“Heart Area”， “Liver Area”， “Kidney Area” and “Upper Zone”—for four consecutive weeks. The agonist and inhibitor groups received daily intraperitoneal injections of TBHQ （25 mg/kg） or ML385 （20 mg/kg）， respectively. In the eye-acupuncture + inhibitor group， ML385 （20 mg/kg） was administered 30 minutes prior to each acupuncture session. Cognitive function was assessed using the Morris water maze test and novel object recognition task. Hippocampal histopathology was evaluated by hematoxylin–eosin （HE） and Nissl staining. Serum levels of interleukin-1β （IL-1β） and IL-10 were quantified by enzyme-linked immunosorbent assay （ELISA）. Microglial polarization status was determined via immunofluorescent double-labeling for ionized calcium-binding adapter molecule 1 （Iba-1）， cluster of differentiation 86 （CD86）， and 206 （CD206）. Protein and mRNA expression levels of Nrf2， HO-1， inducible nitric oxide synthase （iNOS）， and arginase-1 （Arg-1） in hippocampal tissue were analyzed by Western blotting and quantitative real-time polymerase chain reaction （qRT-PCR）， respectively.ResultCompared with the sham operation group， the escape latency of rats in the model group increased， and the numbers of Iba-1， CD86 and CD206 positive cells， the positive cell rates of CD86/Iba-1 and CD206/Iba-1， the concentrations of serum IL-1β and IL-10， the expressions of iNOS， Arg-1 protein and mRNA increased （P < 0.01）； the number of times crossing the original platform， the time spent in the target quadrant， the new object recognition index， and the expressions of Nrf2， HO-1 protein and mRNA decreased （P < 0.01）； the arrangement of neurons in the hippocampus became loose， the shapes were irregular， and necrotic cells appeared， with a reduction in Nissl bodies. Compared with the model group， the time spent in the target quadrant， the number of times crossing the original platform， the new object recognition index， the concentration of IL-10， the number of CD206 positive cells， the positive cell rate of CD206/Iba-1， and the expressions of Nrf2， HO-1， Arg-1 protein and mRNA in the eye-acupuncture group and the agonist group increased （P < 0.01）； the escape latency， the concentration of IL-1β， the number of Iba-1 and CD86 positive cells， the positive cell rate of CD86/Iba-1， the protein and mRNA expressions of iNOS decreased （P < 0.01）； the arrangement of hippocampal neurons became more regular， the morphology tended to be normal， and the number of neurons and Nissl bodies increased； the expression in the inhibitor group was opposite （P < 0.05， P < 0.01）. Compared with the eye-acupuncture group， the eye-acupuncture + inhibitor group had a decrease in the time spent in the target quadrant， the number of times crossing the original platform， the new object recognition index， the concentration of IL-10， the number of CD206 positive cells， the positive cell rate of CD206/Iba-1， and the expressions of Nrf2， HO-1， Arg-1 protein and mRNA （P < 0.01）； the escape latency， the concentration of IL-1β， the number of Iba-1 and CD86 positive cells， the positive cell rate of CD86/Iba-1， the protein and mRNA expressions of iNOS increased （P < 0.05， P < 0.01）； the arrangement of hippocampal neurons became irregular， necrosis increased， and the number of Nissl bodies decreased.ConclusionEye-acupuncture ameliorates VCI by activating the Nrf2/HO-1 signaling pathway， thereby promoting microglial polarization toward the neuroprotective M2 phenotype， mitigating neuroinflammation， and preserving hippocampal neuronal structure and function.
Objective To observe the role of leptin receptor (LepR) in the dorsal vagal complex (DVC) in electroacupuncture (EA)-mediated regulation of food intake, so as to explore its central mechanism underlying improvement of metabolic disorders. Methods Ten C57BLKS/JGpt mice were randomly selected as the normal group, and the remaining mice were fed with high-fat diet (HFD) to establish a obesity model. Successfully modeled mice were randomly divided into the HFD group and EA group, with 10 mice in each group. The EA group received EA stimulation (2 mA, 2 Hz/15 Hz) at bilateral u201CZusanliu201D (ST36) for 20 min, once daily, 6 times per week, for 5 weeks. The body weight, food intake, liver tissue wet weight, inguinal white adipose tissue (iWAT) weight, epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT) weight, and blood glucose levels were measured. The serum insulin and leptin contents were measured using ELISA. Immunofluorescence staining was used to detect the co-expression of LepR and proto-oncogene c-Fos protein (c-Fos), the expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the nucleus tractus solitarii (NTS) and dorsal motor nucleus of the vagus nerve (DMV), and the co-expression of choline acetyltransferase (ChAT) and c-Fos in DMV. Results Compared with the normal group, the HFD group showed a significant increase in the body weight, liver and adipose (iWAT, eWAT and BAT) wet weight, serum insulin and leptin contents, fasting blood glucose level, and glucose tolerance area under the curve (AUC, Pu0026lt;0.01), the number of LepR and c-Fos co-expression neurons in the DMV and NTS, p-STAT3 positive and ChAT and c-Fos co-expression neurons in the DMV were remarkably decreased (Pu0026lt;0.05, Pu0026lt;0.01). In comparison with the HFD group, the body weight, food intake, liver and adipose wet weight, serum leptin content, fasting blood glucose, and glucose tolerance AUC were significantly decreased in the EA group (Pu0026lt;0.01), while the number of LepR and c-Fos co-expression neurons in the DMV and NTS, p-STAT3 positive and ChAT and c-Fos co-expression neurons in the DMV were notably increased in the EA group (Pu0026lt;0.05, Pu0026lt;0.01). No significant differences were observed in the random blood glucose content and p-STAT3 positive expression in the NTS among the three groups and in the serum level of insulin after EA. Conclusion EA at ST36 can inhibit food intake and reduce body weight in obesity mice, which may be associated with restoration of LepR-ralated signaling in the DVC and improvement of cholinergic vagal efferent neuronal activity in the DMV.
Objective To observe the effect of electroacupuncture (EA) at u201CHeguu201D (LI4) and u201CQuchiu201D (LI11) on skin inflammatory injury, itching, and poly(ADP-ribose) polymerase 1 (PARP1)-dependent cell death (Parthanatos) in mice with atopic dermatitis (AD) based on the protease-activated receptor 2 (PAR2)/transient receptor potential vanilloid 3 (TRPV3) pathway, and to explore the possible mechanism of EA in the treatment of AD. Methods Thirty-six male BALB/c mice were randomly divided into a normal group, a model group, and an EA group, with 12 mice in each group. The AD model was established by topical application of calcipotriol (MC903) solution. After successful modeling, the EA group received EA at bilateral LI4 and LI11 once daily, 30 min per session, for 7 consecutive days. The scoring atopic dermatitis (SCORAD) index and scratching frequency were recorded before and after intervention in each group. Histopathological morphology of skin tissue was observed by HE staining. Cell apoptosis in skin tissue was detected by TUNEL assay. The protein expressions of PAR2, TRPV3, interleukin (IL)-33, IL-31, IL-13, PARP1, mitochondrial apoptosis-inducing factor 1 (AIFM1), and macrophage migration inhibitory factor (MIF) in skin tissue were measured by Western blot. The mRNA expression levels of PAR2, TRPV3, inflammatory factors (IL-33, IL-31, IL-13), and Parthanatos-related genes (PAR2, TRPV3, PARP1, AIFM1, MIF) in skin tissue were detected by real-time quantitative PCR. The expression of PARP1 in skin tissue was determined by immunofluorescence. Results Compared with the normal group, the model group showed significantly increased SCORAD score (Pu0026lt;0.01), elevated scratching frequency (Pu0026lt;0.01), higher skin cell apoptosis rate (Pu0026lt;0.01), up-regulated protein and mRNA expressions of PAR2, TRPV3, inflammatory factors (IL-33, IL-31, IL-13), and Parthanatos-related factors PARP1, AIFM1, MIF (Pu0026lt;0.01, Pu0026lt;0.05), as well as enhanced PARP1 fluorescence intensity (Pu0026lt;0.01). Compared with the model group, the EA group exhibited decreased SCORAD score (Pu0026lt;0.01), reduced scratching frequency (Pu0026lt;0.01), lower skin cell apoptosis rate (Pu0026lt;0.01), down-regulated protein and mRNA expressions of PAR2, TRPV3, inflammatory factors (IL-33, IL-31, IL-13), and Parthanatos-related factors PARP1, AIFM1, MIF (Pu0026lt;0.05, Pu0026lt;0.01), and weakened PARP1 fluorescence intensity (Pu0026lt;0.01). HE staining showed severe skin lesions, marked epidermal hyperplasia and massive inflammatory cell infiltration in the model group, whereas the EA group displayed relatively intact epidermal structure, significantly alleviated structural damage, and less inflammatory cell infiltration. Conclusion EA at LI4 and LI11 can alleviate skin inflammatory injury and itching in AD mice, and its mechanism may be related to down-regulating the PAR2/TRPV3 pathway and inhibiting Parthanatos in skin cells.
Objective To observe the effect of electroacupuncture (EA) on motor function and skeletal muscle morphology in SAMP8 mice, and to explore the mechanisms of EA improving motor dysfunction in Alzheimeru2019s disease from the peroxisome proliferator-activated receptor u03B3 coactivator 1u03B1 (PGC-1u03B1)/fibronectin type u2162 domain-containing protein 5 (FNDC5)/brain-derived neurotrophic factor (BDNF) pathway. Methods SAMP8 mice were randomly divided into model and EA groups, with 9 mice in each group. Nine anti-aging SAMR1 mice were used as control group. In the EA group, u201CBaihuiu201D(GV20), u201CDazhuiu201D(GV14) and u201CShenshuu201D(BL23) were needled with EA for 20 min every day, 8 d as a course of treatment, with an interval of 2 d between courses. A total of 3 courses was proceeded. The motor function of mice was detected by grasping test, swimming test, suspension test and hind limb clamping test. The morphological structure of gastrocnemius muscle was observed by HE staining. The positive expressions of PGC-1u03B1, FNDC5 and BDNF in gastrocnemius muscle were observed by immunohistochemical staining. The expression levels of PGC-1u03B1, FNDC5 and BDNF mRNAs in gastrocnemius muscle and cerebral motor cortex were detected by real-time fluorescence quantitative PCR. The expression levels of PGC-1u03B1, FNDC5 and BDNF proteins in gastrocnemius muscle and cerebral motor cortex were detected by Western blot. Results Compared with the control group, the peak grip strength, average and maximum swimming speed, and suspension test score of the model group were significantly decreased (Pu0026lt;0.01), and the hind limb clamping test score was significantly increased (Pu0026lt;0.01). The gastrocnemius muscle fibers were loose and irregularly arranged, the spacing was widened and the cytoplasmic staining was uneven. The positive expression area ratios of PGC-1u03B1, FNDC5 and BDNF in gastrocnemius muscle were significantly decreased (Pu0026lt;0.01), and the relative expression levels of PGC-1u03B1, FNDC5 and BDNF mRNAs and proteins in gastrocnemius muscle and cerebral motor cortex were significantly decreased (Pu0026lt;0.01). Compared with the model group, the peak grip strength, average and maximum swimming speed, and suspension test score of the EA group were significantly increased (Pu0026lt;0.01), and the hind limb clamping test score was significantly decreased (Pu0026lt;0.01). The muscle fibers were intact and arranged regularly, the spacing was narrowed, and the cytoplasmic staining was uniform.The positive expression area ratios of PGC-1u03B1, FNDC5 and BDNF were significantly increased (Pu0026lt;0.01), and the relative expression levels of PGC-1u03B1, FNDC5 and BDNF mRNAs and proteins in gastrocnemius muscle and cerebral motor cortex were significantly increased (Pu0026lt;0.01, Pu0026lt;0.05). The correlation analysis results of PGC-1u03B1, FNDC5 and BDNF mRNA expression in gastrocnemius and cerebral motor cortex showed a highly positive correlation (ru0026gt;0.70, Pu0026lt;0.01). Conclusion EA can improve motor dysfunction and skeletal muscle morphology in SAMP8 mice, and its mechanism may be related to up-regulation of PGC-1u03B1, FNDC5 and BDNF expressions in gastrocnemius and cerebral motor cortex.
ObjectiveTo investigate the therapeutic effect of electroacupuncture（EA） on mice with recurrent psoriasis， so as to explore its mechanism from the perspective of memory T cell differentiation and the tumor necrosis factor receptor-associated factor 2 （TRAF2）/nuclear factor-κB （NF-κB） signaling pathway.MethodsBALB/c mice were divided into batches for establishment of recurrent psoriasis model and mechanism verification experiments. The recurrence experiment included blank control group， model group， EA group， methotrexate（MTX） group， and EA + MTX group. The verification experiment additionally set up EA + NF-κB inhibitor group and EA + NF-κB agonist group. The recurrent psoriasis model was induced by topical imiquimod （IMQ） application on the back skin. EA was applied at “Quchi” （LI11） and “Zusanli” （ST36） with sparse-dense wave， frequency of 2 Hz/30 Hz and intensity of 1 mA， once daily for 7 consecutive days， 15 min each time. Lesion changes were observed and psoriasis area and severity index （PASI） scores were evaluated. HE staining was adopted to observe histopathological alterations of lesional skin tissues. ELISA was used to detect the contents of interleukin （IL）-6， IL-17A， IL-15 and tumor necrosis factor-α （TNF-α） in lesional skin tissues. Real-time quantitative PCR was performed to detect mRNA expressions of IL-15， C-X-C motif chemokine ligand （CXCL） 9， CXCL10 and C-X-C chemokine receptor 3 （CXCR3） in lesional skin tissue. Flow cytometry was applied to detect the proportions of CD8+ tissue-resident memory T cells （TRM） and CD4+ TRM in skin lesions. Western blot was used to test protein expression of phosphorylated NF-κB p65 （p-NF-κB p65）， NF-κB p52， phosphorylated TRAF2 （p-TRAF2）， and phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK） in lesional skin tissues.ResultsCompared with the blank control group， the model group exhibited significantly increased PASI score， contents of IL-6， TNF-α and IL-17A， proportions of CD8+ TRM and CD4+ TRM， mRNA expression of IL-15， CXCL9， CXCL10， CXCR3， as well as protein expressions of p-TRAF2， p-NF-κB p65 and NF-κB p52 （P<0.01， P<0.05）. Compared with the model group， all above indicators were markedly reversed in the EA group and EA + MTX group （P<0.01， P<0.05）. Except for the proportion of CD4+ TRM and IL-17A， the remaining indicators were significantly lower in the two EA-related groups than those in the MTX group （P<0.01， P<0.05）. Compared with the EA group， the EA + MTX group showed further decreased proportions of CD8+CD103+ TRM， down-regulated mRNA expressions of CXCL9， CXCL10 and reduced p-NF-κB p65 protein expression （P<0.01，P<0.05）. In verification experiments， compared with the model group， the EA group and EA + NF-κB inhibitor group presented obvious improvements in skin lesions， inflammatory factors， proportions of CD8+CD103+ TRM and CD4+ TRM， protein expressions of p-TRAF2， p-NF-κB p65， and mRNA levels of CXCL9， CXCL10， CXCR3 （P<0.05， P<0.01）. Compared with the EA group， these indicators were further decreased in the EA + inhibitor group （P<0.01， P<0.05）； in the EA + agonist group， the contents of IL-6， TNF-α， IL-17A， IL-15， proportion of CD8+CD103+ TRM， and protein expressions of p-TRAF2， p-NF-κB p65 were significantly elevated （P<0.01， P<0.05）.ConclusionEA can effectively alleviate symptoms of recurrent psoriasis. Its underlying mechanism may be related to inhibiting the activation of the TRAF2/NF-κB signaling pathway and specifically down-regulating the level of CD8+ memory T cells.
