OBJECTIVES:To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) on colonic mucosa injury, expressions of nuclear factor κB (NF-κB), 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 100-300 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, 1-2 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-κB was detected using immunofluorescence staining for assessing the nuclear translocation status, and the protein expression levels of colonic NF-κB 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-κB 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-κB in the chemotherapy group were considerably increased (P<0.05), while the colonic length, tumor volume, tumor weight, and the immunoactivity level of AQP8 were strikingly decreased (P<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-κB in the chemotherapy+EA group were significantly lower than those in the chemotherapy group (P<0.05), while the immunoactivity level of AQP8 in the chemotherapy+EA group was notably higher than that in the chemotherapy group (P<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. CONCLUSIONS: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-κB signaling pathway, up-regulating the expression levels of AQP8 protein, and reducing DAO and D-LA release.
OBJECTIVES:To investigate the effect of electroacupuncture (EA) on hepatic autophagy in obese mice by regulating the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/unc-51 like autophagy activating kinase 1 (ULK1) signaling pathway. METHODS:C57BL/6J mice were randomly divided into normal group, model group and EA group, with 8 mice in each group. The obese mice model was established by feeding high-fat diet. Mice in the EA group received EA at bilateral "Zusanli"(ST36) and "Tianshu"(ST25) for 30 min, 5 times a week for 4 consecutive weeks. The Lee's index and body mass of mice in each group were observed before and after treatment；fasting serum glucose was measured；the insulin tolerance test (ITT) was performed, and the area under the curve (AUC) was calculated；the levels of fasting insulin, serum low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triacylglycerol (TG) were detected by ELISA, and the insulin resistance index was calculated；the mass of mesenteric white adipose tissue (mWAT) and liver tissue was weighed；the morphological changes of mWAT and liver tissue were observed by HE staining；the lipid deposition in the liver tissue was observed by Oil Red O staining；the ultrastructure of liver tissue was observed by transmission electron microscopy；the protein expressions of p-AMPK/AMPK, p-mTOR/mTOR, ULK1, microtubule-associated protein 1 light chain 3 type Ⅱ (LC3- Ⅱ ), autophagy-related 5 (Atg5), autophagy-related 7 (Atg7), and sequestosome 1 (SQSTM1/p62) in liver tissue were detected by Western blot；the mRNA expressions of AMPK, mTOR, ULK1, LC3-Ⅱ, Atg5, Atg7, and p62 in liver tissue were detected by quantitative real-time PCR. RESULTS:Compared with the normal group, the model group showed significant increases in Lee's index, body mass, fasting blood glucose, fasting insulin, insulin resistance index, and ITT-AUC (P<0.01)；increased serum TG, TC, and LDL-C levels (P<0.01)；decreased serum HDL-C level (P<0.01)；increased mWAT and liver mass (P<0.01)；enlarged adipocytes with reduced cell number per unit area in mWAT；swollen and disorganized hepatocytes with numerous lipid droplets and vacuoles；extensive orange-red lipid deposition；mitochondrial shrinkage and reduced autophagic vacuoles；decreased AMPK mRNA and p-AMPK/AMPK protein expression (P<0.01)；increased mTOR mRNA and p-mTOR/mTOR protein expressions (P<0.01)；decreased mRNA and protein expressions of ULK1, LC3-Ⅱ, Atg5, and Atg7 (P<0.01)；and increased mRNA and protein expression of p62 (P<0.01). Compared with the model group, the EA group showedsignificant reductions in Lee's index, body mass, fasting blood glucose, fasting insulin, insulin resistance index, and ITT-AUC (P<0.01)；decreased serum TG, TC, and LDL-C levels (P<0.01)；increased serum HDL-C level (P<0.01)；reduced mWAT and liver mass (P<0.01, P<0.05)；decreased adipocyte diameter and increased cell number per unit area in mWAT；no obvious lipid droplets or vacuoles in hepatocytes；reduced orange-red lipid deposition；normal mitochondrial structure, increased autophagic vacuoles, and occasional autophagosomes and autolysosomes；increased AMPK mRNA and p-AMPK/AMPK protein expressions (P<0.01)；decreased mTOR mRNA and p-mTOR/mTOR protein expressions (P<0.01)；increased mRNA and protein expressions of ULK1, LC3-Ⅱ, Atg5, and Atg7 (P<0.01)；and decreased mRNA and protein expressions of p62 (P<0.01). CONCLUSIONS:EA at ST36 and ST25 can promote hepatic autophagy in obese mice, and its mechanism may be related to the regulation of the AMPK/mTOR/ULK1 signaling pathway.
Objective:Piper nigrum is a medicinal and edible spice that is popular worldwide due to its unique spicy flavor, and is considered as a folk medicine for treating digestive system diseases. Piperine (PIP) is a bioactive alkaloid derived from P. nigrum, with remarkable hepatoprotective efficacy. Hepatic fibrosis represents a critical phase in the advancement of distinct chronic liver conditions towards cirrhosis, and currently lacks effective therapeutic drugs. The study aims to investigate the hepatoprotective functions of PIP and the underlying mechanism of PIP in combating hepatic fibrosis. Methods:C57BL/6J mice with hepatic fibrosis were induced by thioacetamide (TAA) and subsequently subjected to treatment with PIP or curcumin. Immortalized rat hepatic stellate cells (HSCs) were stimulated with transforming growth factor-β (TGF-β), followed by culture with PIP or niclosamide [signal transducer and activator of transcription 3 (STAT3) inhibitor], respectively. Human hepatic stellate cell line LX-2 were activated by TGF-β and transfected with specific small interfering RNA (siRNA) to silence STAT3 gene, and afterwards cultured with PIP. The mouse AML-12 cells and macrophage-like murine cells Raw 264.7 were stimulated with lipopolysaccharide (LPS), followed by culture with PIP. Results:In vivo, PIP reduced serum transaminase levels, collagen deposition, and decreased the excessive accumulation of extracellular matrix (ECM), comprising α-smooth muscle actin (α-SMA), collagen type I (Collagen I), and tissue inhibitor of metalloproteinases-1 (TIMP-1)/matrix metalloproteinase 13 (MMP13) ratio expressions. PIP inhibited inflammatory cytokines release and myeloperoxidase (MPO) expression, including interleukin-1 receptor type 1 (IL-1R1), cysteine-aspartic acid protease-1 (Caspase-1), and IL-6. PIP specifically inhibited STAT3 phosphorylation at Ser727, downregulated phosphorylated janus kinase 2 (p-JAK2), and upregulated suppressor of cytokine signaling 3 (SOCS3) and protein inhibitor of activated STAT 1/3 (PIAS1/3) expressions. In vitro, PIP inhibited ECM deposition and inflammatory cytokines release in activated HSCs. PIP selectively suppressed p-STAT3 at Ser727 without affecting Tyr705, and modulated p-JAK2, SOCS3, and PIAS1/3 expressions in activated HSCs, function as a STAT3 inhibitor. PIP inhibited LPS‑induced M1 polarization of RAW 264.7 macrophages. PIP decreased Cleaved‑Caspase 3 expression and apoptotic cells in LPS‑induced AML‑12 hepatocytes. STAT3 deficiency amplified regulation of PIP on α-SMA and p-JAK2, as well as its upregulation on SOCS3 and PIAS1/3 in activated LX-2 cells. Conclusion:PIP improved hepatic fibrosis via inhibiting ECM excessive deposition and inflammatory secretion. Switching STAT3 phosphorylation at Ser727 might be the underlying targets for PIP against hepatic fibrosis, which provide an effective candidate and therapeutical strategy for hepatic fibrosis.
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.
OBJECTIVES:To explore the potential mechanism by which acupuncture alleviates sleep deprivation (SD)-induced ovarian dysfunction in mice via regulating the cyclic adenosine monophosphate (cAMP) signaling pathway based on transcriptomics. METHODS:Female C57BL/6 mice with regular estrous cycles were randomly divided into blank group, SD group and acupuncture group, with 15 mice in each group. Mice in the blank group maintained a normal light-dark schedule for 6 weeks；mice in the SD group and acupuncture group were subjected to 20-hour SD daily from 16：00 to 12：00 the next day for 6 consecutive weeks. Starting from the 4th week, mice in the acupuncture group received acupuncture treatment for 20 min per session at non-SD time points, 6 d a week for 3 consecutive weeks. The estrous cycle changes of mice were observed via vaginal exfoliative cell smear staining. Body weight, ovarian wet weight were measured and ovarian index was calculated. Hematoxylin-eosin (HE) staining was adopted to detect ovarian tissue morphology. ELISA was used to detect serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH), estradiol (E2), as well as ovarian tissue cAMP content. Transcriptomic technology was applied to screen differentially expressed genes (DEGs) in ovarian tissues of mice in each group followed by functional enrichment analysis. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions of follicle-stimulating hormone receptor (FSHR), cAMP response element-binding protein 1 (CREB1), protein kinase A catalytic subunit α (PRKACA) and adenylate cyclase 5 (ADCY5), respectively. RESULTS:After treatment, compared with the blank group, the rate of estrous cycle disorder was significantly elevated in the SD group (P<0.01)；body weight, ovarian wet weight and ovarian index were markedly decreased (P<0.01)；the morphological structure of ovarian tissue was damaged with increased atretic follicles；serum FSH and LH levels were up-regulated (P<0.01), while AMH and E2 levels were down-regulated (P<0.01). Compared with the SD group, the acupuncture group exhibited a reduced rate of estrous cycle disorder (P<0.01)；increased body weight, ovarian wet weight and ovarian index (P<0.01)；ameliorated pathological ovarian structure and fewer atretic follicles；decreased serum FSH and LH levels (P<0.01), and elevated serum AMH and E2 levels (P<0.01). A total of 173 intersecting DEGs with a callback trend were identified via transcriptomic sequencing. Gene ontology analysis revealed that these intersecting DEGs were mainly involved in biological processes such as response to other organisms, localized in cellular components including extracellular region, and associated with molecular functions like 2' -5' -oligoadenylate synthetase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis demonstrated that the intersecting DEGs were primarily enriched in the cAMP signaling pathway. Gene set enrichment analysis indicated that the cAMP signaling pathway was suppressed in the SD group but activated after acupuncture intervention. Compared with the blank group, the SD group showed significantly reduced ovarian cAMP content, mRNA expressions of FSHR, CREB1, ADCY5 and PRKACA, protein levels of FSHR, ADCY5, PRKACA, as well as p-CREB1/CREB1 ratio(P<0.01). Relative to the SD group, the acupuncture group presented increased ovarian cAMP content, up-regulated mRNA expressions of FSHR, CREB1, ADCY5 and PRKACA, elevated protein levels of FSHR, ADCY5, PRKACA, and an increased p-CREB1/CREB1 ratio (P<0.01, P<0.05). CONCLUSIONS:Acupuncture can ameliorate SD-induced ovarian dysfunction in mice, and its therapeutic mechanism may be related to regulating the expressions of key molecules including CREB1, FSHR, ADCY5 and PRKACA in the cAMP signaling pathway.
OBJECTIVES:To investigate the effect of electroacupuncture (EA) on central sensitization in rats with non-erosive reflux disease (NERD) of liver-stomach disharmony pattern, so as to explore its underlying mechanisms in improving NERD. METHODS:Forty male SD rats were randomly divided into control, model, EA, and medication (western medicine) groups (n=10 per group). The NERD model of liver-stomach disharmony pattern was established by intraperitoneal injection of ovalbumin plus aluminum hydroxide adjuvant (basic sensitization) combined with chronic tail-pinching stress stimulation. Rats in the EA group received EA stimulation (2 Hz, a strength that could induce a slight tremor of the limbs) at "Zhiyang" (GV9), "Shendao" (GV11), "Dazhui" (GV14), and "Baihui" (GV20) for 30 min, once daily for 2 consecutive weeks. Rats of the medication group received gavage of omeprazole (1.8 mg/kg) once daily for 2 weeks. The remaining 2 groups were administered an equal volume of distilled water. Before and after the intervention, the sucrose preference test was performed, and the mechanical withdrawal threshold (MWT) was measured using von Frey filaments. After the intervention, the abdominal withdrawal reflex (AWR) test was conducted, followed by an esophageal acid perfusion test. After perfusion, the rats were euthanized for tissue collection. The number of synaptic vesicles in the spinal cord dorsal horns and the intercellular space of the esophageal mucosal epithelium were observed by transmission electron microscopy (TEM). The contents of serum gastrin (GAS) and motilin (MTL) were measured by ELISA. The protein expression levels of nerve growth factor (NGF), tropomyosin receptor kinase A (TrkA), glutamate receptor AMPA type subunit 1 (GluA1), and postsynaptic density protein 95 (PSD95) in the spinal cord tissue were detected by Western blot, and the mRNA expression levels of NGF and TrkA in the spinal cord were measured by real-time quantitative PCR. The positive expression of GluA1 and PSD95 in the spinal cord was also assessed by immunofluorescence staining. RESULTS:Compared with the control group, the model group showed a significant decrease in the sucrose preference rate, MWT, body weight, visceral pain threshold, and serum GAS and MTL contents (P<0.01), and a significant increase in the AWR score of 40, 60, and 80 mmHg (P<0.01), number of spinal synaptic vesicles, expression levels of NGF, TrkA, GluA1 and PSD95 proteins, and NGF and TrkA mRNAs, as well as the immunofluorescence intensity of GluA1 and PSD95 (P<0.01). Following the intervention, the modeling induced decrease and increase of all the indexes (except for GluA1 protein expression) mentioned above in the EA group, and the modeling induced decreased levels of sucrose preference rate, MWT, body weight, visceral pain threshold, serum MTL, and the increased levels of the expression of NGF, TrkA, GluA1 and PSD95 proteins and TrkA mRNA in the medication group were reversed (P<0.01, P<0.05). The effect of EA was obviously superior to medication in up-regulating the levels of MWT and visceral pain threshold (P<0.01). Results of TEM showed that in comparison with the control group, the number of synaptic vesicles in the spinal cord was increased, and the intercellular spaces of esophageal mucosal epithelial cells were apparently widened in the model group, while after EA intervention, the number of synaptic vesicles was significantly decreased, and the intercellular spaces of esophageal mucosal epithelial cells tended to recover. CONCLUSIONS:Electroacupuncture can alleviate visceral hypersensitivity and reduce central sensitization in rats with NERD of the liver-stomach disharmony type, which may be associated with its function in inhibiting NGF/TrkA signaling pathway of spinal cord.
OBJECTIVES:To investigate the clinical efficacy of separated moxibustion in the treatment of rheumatoid arthritis (RA) and related negative emotions based on gut microbiota, so as to explore its potential mechanism of action. METHODS:A total of 70 RA patients were randomly divided into a control group (n=35, 2 cases dropped off, 3 cases were excluded) and an observation group (n=35, 3 cases dropped off, 2 cases were excluded), and 30 healthy participants who underwent physical examination during the same period were randomly enrolled as the normal group. The control group was given conventional drug therapy;the observation group was additionally treated with separated moxibustion at bilateral Zusanli (ST36), Shenshu (BL23) and Ashi points on the basis of the control group, once every other day, 3 times a week, for 5 consecutive weeks. The scores of disease activity score in 28 joints (DAS28), visual analogue scale (VAS) for pain, morning stiffness, gastrointestinal symptom rating scale (GSRS), self-rating anxiety scale (SAS), and self-rating depression scale (SDS) were compared between the control group and observation group before and after treatment. 16S ribosomal RNA (rRNA) gene sequencing was used to detect the composition structure and relative abundance of gut microbiota in the 3 groups before and after treatment. ELISA was adopted to measure the serum contents of lipopolysaccharide (LPS), lipopolysaccharide-binding protein (LBP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), 5-hydroxytryptamine (5-HT), and insulin-like growth factor-1 (IGF-1) in the control and observation groups before and after treatment. RESULTS:Compared with the baseline in the same group, the scores of DAS28, VAS, GSRS, SAS, SDS, as well as serum contents of LPS, LBP, TNF-α, IL-1β and IL-6 were significantly decreased in both the control and observation groups after treatment (P<0.05, P<0.01), and the reductions in the observation group were more significant than those in the control group (P<0.05, P<0.01). In contrast, morning stiffness score was significantly decreased, and serum contents of 5-HT and IGF-1 were significantly increased in the observation group after treatment compared with baseline and those in the control group after treatment (P<0.05, P<0.01). Before treatment, compared with the normal group at the same time point, the α -diversity of gut microbiota (Chao1, Ace, Sobs, Shannon indices) and the abundances of beneficial bacteria (Bacteroidota, Faecalibacterium, Bacteroides, Bifidobacterium) in the observation and control groups were significantly lower (P<0.01), while the Firmicutes/Bacteroidota (F/B) ratio and the abundances of opportunistic pathogenic bacteria (Firmicutes, Prevotella, Proteobacteria, Actinobacteriota, Escherichia-Shigella, Klebsiella) were significantly higher (P<0.01). Microbiota clustering analysis showed significant differences between the observation/control groups and the normal group. After treatment, all the above indicators were improved in observation/control groups, and the observation group showed significantly better outcomes in increasing α -diversity, restoring beneficial bacteria abundance, and reducing F/B ratio and pathogenic bacteria abundance than the control group (P<0.01, P<0.05). CONCLUSIONS:Separated moxibustion combined with conventional drugs exerts superior clinical efficacy to monotherapy with conventional drugs in relieving joint pain, improving gastrointestinal symptoms, and alleviating anxiety and depression in RA patients. Its mechanism may be associated with regulating gut microbiota diversity, optimizing microbiota structure, reducing inflammatory factor levels, and improving neurotransmitter metabolism.
Objective To observe the effect of electroacupuncture(EA)intervention on the interleukin-4(IL-4)/Janus kinase 1(JAK1)/signal transducer and activator of transcription 6(STAT6)signaling pathway in colon tissue of chronic ulcerative colitis(UC)mouse model,in order to explore the potential mechanism of EA in promoting M2 macrophage polarization and improving intestinal mucosal barrier injury in UC mice.Methods A total of 56 eight-week-old SPF BALB/c mice(28 males and 28 females)were stratified by sex and randomly assigned to blank group(15 mice)and modeling group(41 mice).3%DSS solution was used to induce the chronic UC model.The successfully induced mice were further divided into model,EA+11B11,and EA groups.Finally,12 mice were included in each group.Mice in the EA+11B11 group received intraperitoneal injection of IL-4 inhibitor 11B11(10 mg/kg)before EA,and mice in the EA group were treated with EA(10 Hz/50 Hz,1 mA)at"Guanyuan"(CV4),"Tianshu"(ST25),"Zusanli"(ST36)and"Shangjuxu"(ST37)for 20 min.The treatments were performed once daily for 14 consecutive days.After the intervention,the following aspects were evaluated.① Symptom improvement:including disease activity index(DAI)score,macroscopic morphology score in colon,and hematoxylin-eosin(HE)staining to observe the pathological changes in colon tissue;②Structure and function of intestinal mucosal barrier:including the observation of colonic tight junction structure by transmission electron microscopy,detection of intestinal leakage by in vivo imaging with fluorescein isothiocyanate-4D(FITC-4D),detection of the positive expressions of tight junction protein ZO-1 and occludin in colon tissue by immunofluorescence staining;③ Activation of IL-4/JAK1/STAT6 signaling pathway:detection of the relative expression levels of IL-4,p-JAK1/JAK1,and p-STAT6/STAT6 proteins in colon tissue by Western blot;④ Markers of M2 macrophage polarization:detection of the CD206+/CD86+ratio in mesenteric lymph nodes,and CD206 and arginase-1(Arg-1)positive expressions in colon tissue by flow cytometry and immunofluorescence staining,respectively.Results Compared with the blank group,mice in the model group showed significantly increased DAI score and macroscopic morphology score(P<0.05),disorganized colonic mucosal structure,severe intestinal fluorescence leakage,significantly decreased positive expressions of ZO-1 and occludin(P<0.05),reduced relative expression levels of IL-4,p-JAK1/JAK1,and p-STAT6/STAT6 proteins(P<0.05),as well as decreased CD206+/CD86+ratio and positive expressions of CD206 and Arg-1(P<0.05).Compared with the model group,the EA group exhibited reduced DAI and macroscopic morphology scores(P<0.05),improved colonic mucosal structure,and alleviated intestinal fluorescence leakage.The positive expressions of ZO-1 and occludin,and relative expression levels of IL-4,p-JAK1/JAK1,and p-STAT6/STAT6 proteins were significantly increased(P<0.05),along with elevated CD206+/CD86+ratio,and CD206 and Arg-1 positive expressions(P<0.05).Compared with the EA group,the EA+11B11 group showed higher DAI and macroscopic morphology scores(P<0.05),disrupted colonic mucosal structure,obvious intestinal fluorescence leakage,decreased positive expressions of ZO-1 and occludin,and relative protein expression levels of IL-4,p-JAK1/JAK1,and p-STAT6/STAT6(P<0.05),as well as reduced CD206+/CD86+ratio,CD206 and Arg-1 positive expressions(P<0.05).Conclusion EA can promote the repair of colonic mucosal barrier in mice with chronic UC and alleviate symptoms such as diarrhea and hematochezia,which may be associated with the activation of IL-4/JAK1/STAT6 signaling pathway and the inducing of M2 macrophage polarization in colon tissue.
Obesity is a chronic metabolic syndrome,and unhealthy lifestyles contribute to a continuous rise in its prevalence.As a non-pharmacological intervention with mild adverse reactions,electroacupuncture has achieved favorable therapeutic effects on obesity and its complications in recent years.This paper reviews studies on the mechanisms of electroacupuncture for obesity over the past decade.Electroacupuncture exerts weight-reducing effects via multiple targets and pathways,including regulating appetite-related neurons and neuropeptides in the hypothalamus,facilitating browning of white adipose tissue and lipid metabolism modulation,maintaining intestinal flora homeostasis,alleviating inflammatory responses and improving insulin resistance.By summarizing research advances in relevant mechanisms,this review aims to provide novel theoretical evidence and therapeutic strategies for electroacupuncture in the treatment of obesity and associated disorders.
Vascular dementia(VD)is a common cognitive impairment syndrome,for which there is currently no effective treatment.As a distinctive acupuncture therapy founded by Professor Jin Rui,the intelligence three-needling(Zhisanzhen,a specific set of 3 acupuncture points)selects the acupoints Shenting(GV24)and bilateral Benshen(GB13),and has shown significant clinical efficacy in improving cognitive function in patients with vascular dementia.This article systematically reviewed the research progress of intelligence three-needling in treating vascular dementia from both clinical and mechanistic perspectives:(1)Clinically,intelligence three-needling,applied via manual acupuncture or electroacupuncture,either alone or in combination with medication and rehabilitation training,can effectively improve patients'scores on the Mini-Mental State Examination and Hasegawa Dementia Scale,as well as their daily living abilities.(2)Mechanistically,intelligence three-needling exerts multi-target and multi-pathway neuroprotective effects,including reducing neurotoxic substances such as homocysteine and β-amyloid,inhibiting neuroinflammatory responses,modulating synaptic plasticity-related proteins,improving cerebral blood flow perfusion,and promoting neuronal repair and functional recovery by regulating the Eph/ephrin signaling pathway and GABAergic system.
OBJECTIVES:To observe the effect of "Tiaoshen Tongluo"(mind regulation and meridian-collateral dredging) electroacupuncture (EA) on the nuclear factor erythroid 2-related factor 2 (Nrf2)/triggering receptor expressed on myeloid cells 2 (TREM2) signaling pathway and hippocampal microglial polarization in rats with learning-memory impairment induced by middle cerebral artery occlusion/reperfusion (MCAO/R). METHODS:Male SD rats were randomly divided into 7 d sham group (n=10), 14 d sham group (n=10), and pre-model group. The MCAO/R model was established by Koizumi intraluminal filament MCAO method. Rats meeting the criteria of learning-memory impairment due to MCAO/R, identified by the Morris water maze tests, were randomly assigned to 7 d model, 7 d EA, 14 d model and 14 d EA groups, with 10 rats in each group. Rats of the EA groups received "Tiaoshen Tongluo" EA stimulation at "Baihui" (GV20), "Yintang" (GV24+), and bilateral "Taichong" (LR3) and "Hegu" (LI4) for 10 min per session, once daily, for 7 or 14 consecutive days. The modified neurological severity score (mNSS) was used to assess the neurological deficits before and after the intervention. Morris water maze test was conducted to evaluate learning and memory abilities. HE staining was used to observe histopathological changes, and Nissl staining was applied to examine neuronal morphology and Nissl bodies in the hippocampal CA1 region. Immunofluorescence double staining was employed to determine the proportions of CD68+/Iba1+ (M1 type) and CD206+/Iba1+ (M2 type) positive cells in the hippocampal tissue. Western blot was used to measure the relative protein expression levels of Nrf2, TREM2, DNAX activator protein 12(DAP12), tumor necrosis factor-α(TNF-α) and transforming growth factor-β(TGF-β) in the hippocampal tissue. RESULTS:Compared with the 7 d and 14 d sham groups, the 7 d and 14 d model groups had a significant increase in the mNSS (P<0.01), and an obvious down-regulation in expression levels of hippocampal Nrf2, TREM2, and DAP12 proteins (P<0.05, P<0.01), and loose arrangement of neurons and reduction in the number of Nissl bodies in the hippocampal CA1 region. In contrast to the 14 d sham group, the 14 d model group had a notable increase in the escape latency, the number of CD68+/Iba1+ dual labeled cells, and the expression level of TNF-α in the hippocampus (P<0.05, P<0.01), and a striking decrease in the number of original platform crossings and time spent in the target quadrant (P<0.01). Compared with the 7 d and 14 d model groups, the 7 d and 14 d EA groups exhibited reduction of mNSS and TNF-α expressions (P<0.01, P<0.05), up-regulation of expression levels of Nrf2 and TREM2 proteins (P<0.05, P<0.01), alleviated arrangement of CA1 neurons, and increased Nissl bodies. Compared with the 14 d model group, the 14 d EA group showed a decrease in the escape latency and the number of CD68+/Iba1+ dual labeled cells (P<0.01), and an increase in the number of original platform crossings and time spent in the target quadrant, number of CD206+/Iba1+ dual labeled cells, and the expression of DAP12 and TGF-β proteins(P<0.05, P<0.01). Compared with the 7 d EA group, the 14 d EA group showed a further reduction in mNSS score (P<0.05), and a further up-regulation of expression of TREM2 protein (P<0.05), a more compact neuronal arrangement in the CA1 region with the morphological structure being close to the normal. CONCLUSIONS:"Tiaoshen Tongluo" EA can improve learning-memory ability in MCAO/R rats, which may be associated with its functions in activating the Nrf2/TREM2 signaling pathway, inhibiting microglial M1 polarization and promoting M2 polarization. In addition, the therapeutic effect of 14 d EA is better than that of 7 d EA.
Alzheimer’s disease （AD） is a common neurodegenerative disorder， and brain energy metabolism disorders are closely related to the onset of AD. Acupuncture-moxibustion is one of the effective treatments for AD， which can significantly improve AD symptoms and slow down the progression of the disease. The mechanism of its action has also been continuously studied. This article summarizes the relevant research on acupuncture-moxibustion regulating brain energy metabolism to improve AD. The results show that acupuncture-moxibustion mainly improves AD brain energy metabolism disorders from the following aspects： 1） regulating glucose metabolism disorders （promoting glucose transport， increasing glucose uptake and utilization， and regulating glycolytic activity in the brain）， 2） regulating mitochondrial structure and dysfunction （improving mitochondrial structure and dynamics， enhancing electron transfer chain activity and ATP production， inhibiting abnormal opening of mitochondrial permeability transition pores）， 3） improving insulin resistance and damage to the insulin signaling pathway， and 4） restoring amino acid and lipid metabolic imbalance. It plays a role in neuroprotection and delaying the progression of the disease.
OBJECTIVES:To investigate the effects of herbal cake-separated moxibustion combined with atorvastatin on lipid metabolism, aortic pathological structure, plaque deposition, and integrin/ yes-associated protein (YAP)/ c-Jun N-terminal kinase (JNK) signaling pathway in ApoE-/- atherosclerosis (AS) mice, and to explore its preventive and therapeutic mechanisms for AS. METHODS:Male C57BL/6J mice were used as the blank group, and male ApoE-/- mice were randomly divided into the model group, medicine group, herbal cake-separated moxibustion group, and combined treatment group, with 9 mice per group. High-fat diet was used to establish the AS model. The herbal cake-separated moxibustion group received moxibustion at "Danzhong" (CV17) and "Shenque" (CV8) acupoints, once every other day, 3 times a week. The medicine group received atorvastatin calcium tablet via gavage, 3 mg·kg-1·d-1, once daily. The combined treatment group received an integrated approach that combined the interventions of the medicine group and herbal cake-separated moxibustion group. All groups were treated for 8 weeks. Body weight were observed before and after treatment. HE staining was used to observe aortic pathological morphology, and Oil Red O staining to observe aortic lipid plaque area. Biochemical analysis was used to detect serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. Serum vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) levels were detected by ELISA, and serum nitric oxide (NO) level was detected by colorimetric assay. Western blot was used to detect protein expression levels of integrin αVβ3, YAP, phosphorylated (p)-YAP, p-JNK1/2, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in the aorta. RESULTS:Compared to the blank group, the model group exhibited increased body weight (P<0.01)；thickened aortic intima, foam cells and lipid deposition, hypertrophic edema of smooth muscle cells, thinning of elastic fibers, widened endothelial cell gaps, and rupture of elastic membranes；significantly increased arterial lipid plaque area (P<0.01)；elevated serum TC, TG, LDL-C, IL-6, IL-8, TNF-α, ET-1, and VEGF levels (P<0.01), while HDL-C and NO levels decreased (P<0.01)；increased protein expression of integrin αVβ3, YAP, p-JNK1/2, ICAM-1, and VCAM-1 (P<0.01), and reduced p-YAP protein expression (P<0.01) in the aorta. Compared to the model group, the medicine group, herbal cake-separated moxibustion group, and combined treatment group showed reduced body weight (P<0.01), with a significantly lower weight change difference than that of the model group (P<0.01)；more regular aortic lumen structure, thinner intima, reduced foam cells and lipid deposition, alleviated smooth muscle cell edema, hypertrophy, and inflammatory infiltration；all other indicators also showed significant improvement compared to the model group (P<0.01, P<0.05). Compared to the herbal cake-separated moxibustion group, the medicine group and combined treatment group exhibited reduced plaque area (P<0.05, P<0.01), increased serum NO level (P<0.01), decreased LDL-C, ET-1, and VEGF levels (P<0.01), and reduced protein expressions of p-JNK1/2, ICAM-1, and VCAM-1 in the aorta (P<0.05, P<0.01)；the medicine group also showed decreased serum TC, TG, IL-6, IL-8, and TNF-α levels (P<0.01), increased HDL-C level (P<0.01), reduced integrin αVβ3 and YAP protein expressions in the aorta (P<0.01, P<0.05), and increased p-YAP protein expression (P<0.05). Compared to the medicine group and the herbal cake-separated moxibustion group, the combined treatment group exhibited a greater reduction in body weight (P<0.05, P<0.01), decreased serum levels of TC, TG, IL-6, IL-8, and TNF-α, as well as reduced expressions of integrin αVβ3 and YAP proteins in the aorta (P<0.01, P<0.05), elevated serum HDL-C level (P<0.01), and increased p-YAP protein expression (P<0.01). CONCLUSIONS:Herbal cake-separated moxibustion combined with atorvastatin exerts synergistic effects, ameliorates serum lipid profiles, mitigates endothelial dysfunction of aorta, and attenuates inflammatory responses in AS mice, potentially via modulation of the integrin/YAP/JNK signaling pathway in the aorta.
OBJECTIVES:To investigate the effect of prior acupuncture experience on the clinical efficacy of acupuncture for discogenic sciatica. METHODS:A secondary analysis was conducted on a multicenter, randomized controlled trial. A total of 216 patients with discogenic sciatica were randomly divided into an acupuncture group and a sham acupuncture group, with 108 patients in each group. The acupuncture group received a semi-standardized treatment protocol based on meridian pattern differentiation for acupoint selection, and manual manipulation was applied to achieve the deqi sensation. The sham acupuncture group received needling at non-acupoints without manipulation or the pursuit of deqi. Each session lasted for 30 min. The treatment continued for 4 weeks, with 3 sessions per week for the first 2 weeks and 2 sessions per week for the subsequent 2 weeks. The visual analog scale (VAS) score for leg pain and the Oswestry disability index (ODI) score for lumbar function were recorded at baseline and after 4 weeks of treatment. The differences in VAS and ODI scores from baseline to week 4 were used as the primary outcome measures to evaluate the efficacy. Subgroup analysis were performed to compare the efficacy between the acupuncture and sham acupuncture groups based on the presence or absence of previous acupuncture experience. RESULTS:Among the 216 patients, 81 had prior acupuncture experience and 135 had no prior acupuncture experience. In the subgroup with prior acupuncture experience or without previous acupuncture experience, the changes of VAS score for leg pain and the ODI score for lumbar function in the acupuncture group were significantly higher than those in the sham acupuncture group after 4 weeks of intervention (P<0.001, P<0.01). Interaction analysis showed that there was no interaction effect between prior acupuncture experience and intervention factors on the changes in VAS and ODI scores. CONCLUSIONS:Acupuncture has a favorable effect on improving leg pain and lumbar dysfunction in patients with discogenic sciatica, regardless of prior acupuncture experience.
The occurrence and development of pain-related emotions are closely associated with structural and functional alterations in central neural circuits. Acupuncture can effectively alleviate pain-related emotions and reduce pain-associated negative emotions such as anxiety, depression and pain-aversive memory, and its mechanism is tightly correlated with the regulatory effect of acupuncture on the functions of specific neural circuits. This article sorts out the pain-related emotion-associated neural circuits modulated by acupuncture, and summarizes the relevant circuits as follows： the anxiety-related circuits include rostral anterior cingulate cortex glutamatergic neurons → thalamus (rACCGlu→Th), rostral anterior cingulate cortex glutamatergic neurons → dorsal raphe nucleus (rACCGlu→DRN), rostral anterior cingulate cortex glutamatergic neurons → ventrolateral periaqueductal gray (rACCGlu→vlPAG), basolateral amygdala CaMKⅡ neurons → rostral anterior cingulate cortex (BLACaMKⅡ→rACC), and ventral tegmental area dopaminergic neurons → nucleus accumbens (VTADA→NAc)； the depression-related circuit is infralimbic cortex CaMKⅡα+ neurons → basolateral amygdala (ILCaMKⅡα+→BLA)； the circuit related to pain-aversive memory and reward is infralimbic cortex glutamatergic neurons → nucleus accumbens(ILGlu→NAc). Among these, acupuncture may exert analgesic and anxiolytic effects by inhibiting the rACCGlu→Th and rACCGlu→vlPAG circuits, and activating the rACCGlu→DRN, BLACaMKⅡ→rACC and VTADA→NAc circuits； produce analgesic and antidepressant effects by suppressing the ILCaMKⅡα+→BLA circuit； and achieve analgesic effects as well as regulate pain-aversive memory and reward by activating the ILGlu→NAc circuit. This review provides a reference for the research and application of neural circuit mechanisms underlying acupuncture treatment of pain-related emotions.
OBJECTIVES:To investigate the effect of heat-sensitive moxibustion at Lianquan (CV23) combined with the Mendelsohn maneuver in the treatment of post-stroke dysphagia. METHODS:A total of 105 patients with post-stroke dysphagia from January 2024 to February 2025 were selected and divided into three groups by random number table method, with 35 cases in each group. Control group 1 received the Mendelsohn maneuver, control group 2 received heat-sensitive moxibustion at CV23, and the observation group received the Mendelsohn maneuver combined with heat-sensitive moxibustion at CV23. All groups were treated continuously for 4 weeks. The clinical efficacy, lateral pharyngeal wall movement, mobility of the hyolaryngeal complex, surface electromyography (sEMG) signals, swallowing function including water swallowing test (WST), standardized swallowing assessment (SSA), and videofluoroscopic swallowing study (VFSS), swallowing quality of life questionnaire (SWAL-QOL), nutritional status including serum levels of prealbumin (PA), albumin (ALB), transferrin (TRF), as well as complications and adverse reactions were compared among the three groups. RESULTS:The total effective rate of the observation group was 94.29%, which was higher than 71.43% in control group 1 and 68.57% in control group 2 (P<0.05). After 4 weeks of treatment, the lateral pharyngeal wall movement, superior and anterior displacement of the hyoid bone, superior and anterior displacement of the thyroid cartilage, maximum amplitude of sEMG, VFSS score, SWAL-QOL score, and serum levels of ALB, TRF, and PA in the observation group, control group 1, and control group 2 were all increased compared with before treatment (P<0.05), and those in the observation group were higher than those in control group 1 and control group 2 (P<0.05). The swallowing duration of sEMG, WST grade, and SSA score were decreased compared with before treatment in the 3 groups (P<0.05), and those in the observation group were lower than those in control group 1 and control group 2 (P<0.05). During intervention, only 1 patient in control group 2 had small blisters at the moxibustion site, and no significant abnormalities were found in general vital signs and routine examinations among the three groups before and after intervention. At 3-month follow-up, the complication rate in the observation group (5.71%) was lower than that in control group 1 (25.71%) and control group 2 (31.43%, P<0.05). CONCLUSIONS:The Mendelsohn maneuver combined with heat-sensitive moxibustion at CV23 can effectively activate the pharyngeal constrictor muscles, improve sEMG signals, promote the recovery of swallowing function in patients with post-stroke dysphagia, and exert positive effects in reducing related complications, improving nutritional status and quality of life.
OBJECTIVES:To investigate the mechanism of electroacupuncture (EA) regulating glycolipid metabolism and improving obesity by activating farnesoid X receptor (FXR)/takeda G protein-coupled receptor 5 (TGR5)/glucagon-like peptide-1 (GLP-1) and FXR/fibroblast growth factor 15 (FGF15) signaling pathways mediated by intestinal FXR in obese mice induced by high-fat diet. METHODS:Fifty SPF male C57BL/6 mice were randomly divided into a blank group (n=8) and a pre-model group (n=42). The obese mice model was established by high-fat diet. Twenty-four obese mice were randomly divided into the model group, the EA group and the sham EA group, with 8 mice in each group. In the EA group, bilateral "Zusanli" (ST36) and "Tianshu" (ST25) were electroacupunctured for 30 min each time. In the sham EA group, ST36 and ST25 were selected to be shallowly pricked to the subcutaneous, and the EA instrument was connected without electricity. Both groups received interventions once every other day, 3 times a week for 4 weeks. The body weight and Lee's index of mice in each group were recorded before and after intervention. The contents of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), fasting blood glucose (FBG) and fasting insulin (FINS) were detected, and the homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated. The morphological changes of intestinal tissue in each group were observed by HE staining. The expression of FXR, TGR5, FGF15 and GLP-1 proteins and mRNAs in intestinal tissue of mice was detected by Western blot and fluorescence quantitative PCR separately. RESULTS:Compared with the blank group, the body weight, Lee's index, serum TG, TC, LDL-C, FBG, FINS contents and HOMA-IR were increased (P<0.01), the intestinal mucosal tissue was significantly damaged, the mucosal epithelial cells were damaged, shed, and arranged in disorder, there was obvious inflammatory cell infiltration in the stroma, and the expression levels of FXR, TGR5, GLP-1, FGF15 proteins and mRNAs in the ileum tissue were significantly decreased (P<0.01) in the model group. Compared with the model group, the body weight was decreased (P<0.01) in mice of the EA and sham EA groups；the Lee's index, serum TG, TC, LDL-C, FBG, FINS contents and HOMA-IR were decreased (P<0.01), the degree of pathological changes in the ileum tissue was significantly lighter, and the expression levels of FXR, TGR5, GLP-1, FGF15 proteins and mRNAs in the ileum tissue were significantly increased (P<0.01) in mice of the EA group；the expression level of TGR5 mRNA was increased (P<0.01) in the sham EA group. Compared with the EA group, all indicators were reversed (P<0.01) in the sham EA group. CONCLUSIONS:EA can significantly improve the body weight and Lee's index of obese mice, reduce blood lipid content, and improve glucose metabolism and insulin resistance related indicators. The mechanism may be achieved through FXR/TGR5/GLP-1 and FXR/FGF15 signaling pathways.
OBJECTIVES:Based on the vessel-collateral theory, "qi deficiency and blood stasis leading to collateral obstruction and accumulation", electroacupuncture was delivered in patients with ventricular remodeling after percutaneous coronary intervention (PCI) for acute ST-segment elevation myocardial infarction (STEMI), and the clinical efficacy was evaluated. METHODS:A prospective randomized controlled design was adopted. Eighty patients with cardiac insufficiency after STEMI-PCI (qi deficiency and blood stasis) were enrolled and randomly assigned to a control group (40 cases, with 2 cases dropped out) and an electroacupuncture group (40 cases, with 2 cases dropped out). In the control group, the standardized western medication was administered. In the electroacupuncture group, on the basis of the regimen as the control group, electroacupuncture was operated at Neiguan (PC6), Danzhong (CV17), Shenmen (HT7), Qihai (CV6), Xuehai (SP10) and Zusanli (ST36) with dense-disperse wave, at a frequency of 2 Hz/100 Hz, 30 min per session, once every other day, for 8 consecutive weeks. Three-dimensional speckle tracking echocardiography was used to detect cardiac function indicators:left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS), and cardiac structural indicators:left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) in both groups before and after treatment. ELISA was used to detect the serum levels of cardiac fibrosis markers:soluble suppression of tumorigenicity 2 (sST2) and galectin-3 (Gal-3);immunoturbidimetry was used to detect the heart failure marker:N-terminal pro-B-type natriuretic peptide (NT-proBNP). Changes in traditional Chinese medicine (TCM) syndrome scores were compared and the safety was evaluated in the two groups. RESULTS:Compared with the control group, the electroacupuncture group showed the increase in LVEF and absolute value of GLS (P<0.01, P<0.001) and the decrease in LVEDV and LVESV (P<0.01, P<0.001) after treatment;serum fibrosis markers (sST2, Gal-3) levels were reduced (P<0.05, P<0.01), the serum heart failure marker (NT-proBNP) level was decreased (P<0.001), and the scores of TCM symptoms were improved (P<0.01, P<0.001). Gompared with before treatment, both groups showed improvement (P<0.001) in the above indicators after treatment. No serious adverse event was reported. CONCLUSIONS:Electroacupuncture may conduce to ameliorating cardiac functions and ventricular remodeling indicators in patients after STEMI-PCI, which is probably related to the modulation of cardiac fibrosis markers in serum.
Objective To explore the mechanism of hyperalgesia priming(HP)and evaluate the intervention effect of electroacupuncture(EA)on pain transition.Methods Fifty-nine male C57 mice were used for 4 separate experiments.In the first experiment,the mice were divided into a control group,a sham sensitization group,and a sensitization group,with 5 mice per group.In the second experiment,the mice were divided into a sham sensitization group,a sensitization group,and a sensitization+EA group,with 5 mice per group.The third experiment consisted of a sensitization group and a sensitization+minocycline group,with 7 mice per group.In the fourth experiment,the mice were divided into a sensitization group,a sensitization+EA+PBS group,and a sensitization+EA+colony-stimulating factor(CSF)group,with 5 mice per group.A HP mouse model was established via consecutive intraplantar injections of carrageenan and prostaglandin E2.EA was applied to bilateral"Zusanli"(ST36)and"Kunlun"(BL60)for 30 min,once daily for 8 d.Mechanical paw withdrawal thresholds(PWTs)were measured at different time points to assess pain transition and the effect of EA.Immunofluorescence was used to detect the activation of microglia marker(Iba1)and astrocyte marker(GFAP)in the spinal cord dorsal horn.Intrathecal injection of minocycline was also administered,and its impacts on Iba1 activation and PWTs were tested.After the experiment,skeleton and Sholl analyses were performed to analyze the morphology of microglia.To further examine the role of CSF-1,the factor was administered intrathecally to assess its influence on the effects of EA.Results In the pain transition mouse model,the PWTs at various time points decreased significantly(P<0.01).After modeling,the microglia was activated(P<0.01),but the astrocyte was not.EA increased the PWTs in pain transition mice(P<0.01)and suppressed microglia activation(P<0.01).Intrathecal minocycline administration inhibited microglia activation,reduced Iba1 expression in the spinal cord dorsal horn(P<0.01),and effectively reversed the PWTs reduction(P<0.01).Subsequent intrathecal CSF-1 injection promoted microglia activation and blocked the analgesic effect of EA in the model mice(P<0.01).Conclusion Microglia activation is critical in pain transition.EA can alleviate hyperalgesia in HP mice by inhibiting microglia activation.
