Objective To observe the effect of electroacupuncture (EA) on the cognitive ability, hypoxia-inducible factor-1 alpha (HIF-1u03B1)/6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) signaling pathway and glycolytic function in the hippocampus of Alzheimeru2019s disease (AD) model mice, so as to elucidate its potential mechanism underlying improvement of cognitive function. Methods Male APP/PS1 transgenic mice were used as the AD model and randomly divided into model, EA and EA plus dimethyloxaloylglycine (DMOG, antagonist of u03B1-ketoglutarate cofactor and inhibitor of HIF prolylhydroxylase) groups (n=6 each). Age-matched C57BL/6J mice served as the control group (n=6). EA stimulation (2 Hz, 1 mA) was applied to bilateral u201CShenshuu201D(BL23) and u201CZusanliu201D(ST36), acupuncture was applied to u201CBaihuiu201D (GV20) for 20 min, once every other day for 4 weeks. The mice in the EA+DMOG group was received intraperitoneal injection of DMOG (50 mg/kg) solution on the basis of EA starting from the 4th week. The mouseu2019s cognitive function was evaluated by using Morris water maze and novel object recognition tests. Hematoxylin-eosin (H.E.) staining and immunohistochemistry were used to observe hippocampal neuronal morphology and u03B2-amyloid (Au03B2)1-42 deposition. The contents of pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) in the hippocampal tissue were assayed using ELISA, and the activity of hippocampal hexokinase (HK) and content of lactic acid were detected using biochemical methods. Western blot was employed to detect the expression levels of hippocampal HIF-1u03B1 and PFKFB3 protein, and real-time quantitative PCR was employed to detect the expression levels of hippocampal HIF-1u03B1, PFKFB3, HK, PKM2, and LDHA mRNAs. The immunofluorescence histochemistry technique was used to measure the fluorescence intensity of hippocampal LDHA. Results Compared with the control group, the model group showed significantly longer escape latency, fewer platform crossings and lower novel object recognition index (Pu0026lt;0.01). After EA intervention, the escape latency was evidently shorter, the platform crossings and the novel object recognition index were apparently increased in the EA group (Pu0026lt;0.01), suggesting an improvement of the recognition ability and learning-memory ability. Following administration of DMOG, the effects of EA in shortening the escape latency, and increasing the number of platform crossings and novel object recognition index were eliminated (Pu0026lt;0.01, Pu0026lt;0.05, compared with the EA group). In contrast to the control group, the model group had a considerable increase in the optical density of Au03B21-42, contents of PKM2, LDHA, lactate, and HK activity, expression levels of HIF-1u03B1 and PFKFB3 proteins and mRNAs, and HK, PKM2, LDHA mRNAs, and LDHA fluorescence density (Pu0026lt;0.01). Compared with the model group, the increased levels of Au03B21-42 optical density, contents of PKM2, LDHA, lactate and HK activity, expression levels of HIF-1u03B1 and PFKFB3 proteins and mRNAs, and HK, PKM2, LDHA mRNAs, and LDHA fluorescence density were all reversed in the EA group (Pu0026lt;0.01). In comparison with the EA group, the decreased levels of Au03B21-42 optical density, contents of PKM2, LDHA, lactate, and HK activity, expression levels of HIF-1u03B1 and PFKFB3 proteins and mRNAs, and HK, PKM2, LDHA mRNAs, and LDHA fluorescence intensity were reversed by EA+DMOG (Pu0026lt;0.01, Pu0026lt;0.05). HE staining showed swollen or disintegrated neuronal cell bodies in the hippocampal CA1 region, deepened nuclear staining and expanded intercellular space in the model group. In comparison with the model group, the EA group (not the EA+DMOG group) displayed relatively intact nuclei of neurons, mitigated swelling of cell body and reduction in the infiltration of inflammatory cells and uniform distribution of cells in the CA1 region. Conclusion EA can significantly improve the cognitive function in AD model mice, which may be related to its function in inhibiting the hippocampal HIF-1u03B1/PFKFB3 signaling pathway to reduce glycolytic enzyme activity and abnormal lactate accumulation.
Objective To investigate the ameliorative effect of acupuncture at u201CNeiguanu201D (PC6) on behavioral deficits, hippocampal neuronal damage, and neuroinflammation in young rats with valproic acid (VPA)-induced autism spectrum disorder (ASD). Methods Pregnant SD rats were randomly divided into a control group and a model group. The ASD model was established by a single intraperitoneal injection of VPA (600 mg/kg) on gestational day 12.5. After birth, the offspring young rats were then randomly divided into control (CON), ASD model (model), sham-acupuncture (ASD+sham-Acu), acupuncture (ASD+Acu), solvent (DMSO) control (ASD+DMSO), and positive drug bumetanide (ASD+Bu) groups (n=9 in each group). Manual acupuncture (uniform reinforcing-reducing manipulation) was applied to bilateral PC6 of newborn rats for 30 min, once daily, 6 d a week, for 15 d (from the 7th day to the 21st day after birth). After acupuncture intervention, behavioral tests including the Morris water maze test, three-chamber social test, and self-grooming test were conducted. The hippocampal tissue was collected for observing changes of neuronal morphology and dendritic spine density by using hematoxylin-eosin and Golgi staining. The expression levels of synapse-related proteins postsynaptic density protein 95 (PSD95), glutamate decarboxylase 67 (GAD67), vesicular GABA transporter (VGAT) and pyroptosis-related proteins NOD-like receptor protein 3 (NLRP3), gasdermin domain-N-terminal (GSDMD-NT) and cleaved Caspase-1 in the hippocampal tissue were detected by Western blot. The levels of hippocampal interleukin (IL)-1u03B2, IL-18 and activity of LDH were measured by enzyme-linked immunosorbent assay. The level of hippocampal reactive oxygen species (ROS) were detected by using commercial kits. The activity of hippocampal Iba1-positive cell (microglia) was assessed by immunofluorescence staining, and the cell apoptosis was detected by TdT-mediated dUTP nick-end labeling staining. Results Compared with the CON group, the model group showed a significant increase in the escape latency of water maze test, self-grooming times, LDH activity, ROS level, IL-1u03B2 and IL-18 contents, expression of PSD95, NLRP3, ratios of GSDMD-NT/GSDMD and cleaved Caspase-1/pro-Caspase-1, number of Iba1-positive cells and the apoptosis rate (Pu0026lt;0.01, Pu0026lt;0.001), and a considerable decrease in the body mass, number of platform crossings and time spent in the target quadrant, social index and social preference, number of dendritic spine, and the expressions of GAD67 and VGAT proteins (Pu0026lt;0.05, Pu0026lt; 0.01, Pu0026lt;0.001). In comparison with the model group, both the increased and decreased levels of all the indexes mentioned above were reversed in the ASD+Acu and ASD+Bu groups (Pu0026lt;0.05, Pu0026lt;0.01, Pu0026lt;0.001), and the effects of ASD+Acu were evidently superior to those of ASD+Bu in up-regulating the expressions of GAD67 and VGAT proteins (Pu0026lt;0.05), but inferior to those of ASD+Bu in shortening the escape latency, reducing ROS level, and down-regulating the protein expressions of PSD95 and NLRP3, ratio of cleaved Caspase-1/pro-Caspase-1, and apoptosis rate (Pu0026lt; 0.05, Pu0026lt;0.01, Pu0026lt;0.001). The effects of acupuncture were evidently better than those of the sham acupuncture in increasing the body mass and number of platform crossings, shortening the escape latency, prolonging the time spent in the target quadrant, increasing the social index and social preference, reducing the self-grooming times, elevating the number of dendritic spine, and the expressions of GAD67 and VGAT proteins (Pu0026lt;0.05, Pu0026lt;0.01, Pu0026lt;0.001), and in down-regulating the LDH activity and ROS levels, IL-1u03B2 and IL-18 contents, and the expressions of PSD95 and NLRP3, ratios of GSDMD-NT/GSDMD and cleaved Caspase-1/pro-Caspase-1, Iba1-positive cell number, and the apoptosis rate (Pu0026lt;0.05, Pu0026lt;0.01, Pu0026lt;0.001). Similarly, in comparison with the ASD+DMSO group, the ASD+Bu group showed a significant improvement in the aforementioned behavioral and molecular biological indexes (Pu0026lt;0.05, Pu0026lt;0.01, Pu0026lt;0.001). Conclusion Acupuncture at PC6 can improve social deficits, repetitive stereotyped behavior and cognitive impairment in VPA-induced ASD-like young rats, which may be related to its functions in alleviating hippocampal neuronal damage, regulating the balance of synaptic proteins, and inhibiting oxidative stress and NLRP3 inflammasome-mediated neuroinflammation.
Objective To 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-u03BAB (NF-u03BAB) signaling pathway. Methods BALB/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-u03BAB inhibitor group and EA + NF-u03BAB agonist group. The recurrent psoriasis model was induced by topical imiquimod (IMQ) application on the back skin. EA was applied at u201CQuchiu201D (LI11) and u201CZusanliu201D (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 and tumor necrosis factor u03B1 (TNF-u03B1) 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-u03BAB p65 (p-NF-u03BAB p65), NF-u03BAB p52, phosphorylated TRAF2 (p-TRAF2), and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in lesional skin tissues. Results Compared with the blank control group, the model group exhibited significantly increased PASI score, contents of IL-6, TNF-u03B1 and IL-17A, proportions of CD8+ TRM and CD4+ TRM, mRNA expressions of IL-15, CXCL9, CXCL10, CXCR3, as well as protein expressions of p-TRAF2, p-NF-u03BAB p65 and NF-u03BAB p52 (Pu0026lt;0.01, Pu0026lt;0.05). Compared with the model group, all above indicators were markedly reversed in the EA group and EA + MTX group (Pu0026lt;0.01, Pu0026lt;0.05, except for the proportion of CD4+ TRM). 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 (Pu0026lt;0.01, Pu0026lt;0.05). Compared with the EA group, the EA + MTX group showed further decreased proportion of CD8+CD103+ TRM, down-regulated mRNA expressions of CXCL9, CXCL10 and reduced p-NF-u03BAB p65 protein expression (Pu0026lt;0.01, Pu0026lt;0.05). In verification experiments, compared with the model group, the EA group and EA + NF-u03BAB 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-u03BAB p65, and mRNA levels of CXCL9, CXCL10, CXCR3 (Pu0026lt;0.01, Pu0026lt;0.05). Compared with the EA group, these indicators (except for PASI score) were further decreased in the EA + inhibitor group (Pu0026lt;0.01, Pu0026lt;0.05)uFF1B in the EA + agonist group, all these indicators were significantly elevated (Pu0026lt;0.01, Pu0026lt;0.05). Conclusion EA can effectively alleviate symptoms of recurrent psoriasis. Its underlying mechanism may be related to inhibiting the activation of the TRAF2/NF-u03BAB signaling pathway and specifically down-regulating the level of CD8+ memory T cells.
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.
