[Cathepsin-B involved in effect of electroacupuncture by inhibiting the activation of NLRP3 inflammasome in rats with acute gouty arthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on Cathepsin-B in the synovium of the knee joint of acute gouty arthritis(AGA) rats, so as to explore the mechanism of EA in the treatment of AGA. METHODS: A total of 60 male Wistar rats were randomly divided into normal control,model, medication and EA groups, with 15 rats in each group. Rat model of AGA was established by injection of 0.2 mL sodium urate crystal suspension into the left knee joint cavity. The rats in the medication group were treated with colchicine by gavage(0.3 mg·kg-1·d-1), and the rats in the EA group were treated with EA at the left "Sanyinjiao" (SP6) and "Zusanli"(ST36) for 10 min each time, once a day for a week. The Coderre gait grading standard was used to score the gait of rats. The pathological morphology of synovial tissue of the left knee joint was observed by H.E. staining. The expression levels of Cathepsin-B protein and Nod-like receptor pyrin domain 3(NLRP3), apoptosis-associated speck-like protein containing CARD (ASC),Caspase-1, interleukin-1ß(IL-1ß) and IL-18 mRNAs were detected by Western blot and real-time fluorescence quantitative PCR, respectively. RESULTS: Compared with the normal control group, the degree of synovitis infiltration in the model group was more serious. And the gait score，the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3,ASC,Caspase-1, IL-1ß,IL-18 were significantly increased (P<0.01).After the interventions, the degree of inflammatory infiltration was mild, The gait score, the protein expression level of Cathepsin-B and the mRNA expression levels of NLRP3 and ASC,Caspase-1，IL-1ß,IL-18 were significantly decreased in both medication and EA groups in contrast to the model group (P<0.01, P<0.05). Compared with medication group, the mRNA expression levels of Caspase-1 and IL-18 in the EA group were increased (P<0.05). CONCLUSION: EA may inhibit the activation of NLRP3 inflammasome by reducing the activity of Cathepsin-B in the synovium of the knee joint, so as to treat AGA.

[Effect of electroacupuncture combined with Donepezil on learning-memory ability and expression of hippocampal ß-amyloid clearance-related genes in SAMP8 mice].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) combined with Donepezil on learning-memory ability and gene expression of ß-amyloid (Aß) clearance-related factors in the hippocampus in senescence-accelerated mouse prone 8 (SAMP8) mice, so as to explore their synthetic effect in improving dementia of Alzheimer's disease (AD)．. METHODS: Male SAMP8 mice (30-week-old) were randomly divided into model, medication and EA＋medication groups (n＝6 mice in each group), and other 6 senescence-resistant 1 (SAMR1) mice were used as the control group. Mice of the medication and EA＋medication group received gavage of Donepezil (1.3 mg•kg－1•d－1) once daily for 4 weeks. EA (2 Hz, 1 mA) was applied to "Baihui"(GV20) and "Yintang" (EX-HN3) for 15 min, once daily, 6 days a week for 4 weeks for rats in the EA＋medication group. The Morris water maze (MWM) task (including place navigation tests and space exploration trials) was used to assess the mouse's learning-memory ability. Histopathological changes of hippocampus tissue were observed by H．E. staining. The expression levels of matrix metalloprotein 9 (MMP-9), low density lipoprotein receptor-related protein-1 (LRP-1), P-glycoprotein (Pgp, an important drug transporter responsible for multidrug resistance), Claudin-5 (a component of tight junction strands that serves as a physical barrier to prevent solutes and water from passing freely through the paracellular space between epithelial or endothelial cell sheets of blood-brain barrier, BBB) and Aß mRNAs of the hippocampus tissue were detected by quantitative real-time PCR. RESULTS: Compared with the control group, the average escape latency of place navigation tests, and the expression levels of MMP-9 and Aß mRNAs were significantly increased (P<0.01), and the number of platform quadrant-crossing times of space exploration trials, and the expression levels of LRP-1, Pgp and Claudin-5 mRNAs considerably decreased in the model group (P<0.01). After the intervention, the learning-memory ability was significantly improved in the medication and EA＋medication groups (P<0.01，P<0.05), the expression levels of Aß mRNAs in the medication and EA＋medication groups and MMP-9 mRNA in the EA＋medication group were obviously down-regulated (P<0.01), and those of LRP-1 and Pgp mRNAs in the medication and EA＋medication groups and Claudin-5 mRNA in the EA＋medication group were remarkably up-regulated (P<0.05, P<0.01). The therapeutic effect of EA＋medication was apparently superior to that of simple medication in shortening the escape latency (P<0.05，P<0.01) and in down-regulating the expression of MMP-9 and Aß mRNAs(P<0.01), and in increasing the number of platform quadrant-crossing times(P<0.01), and expression levels of LRP-1, Pgp and Claudin-5 mRNAs (P<0.01). H．E. staining showed scatted and loose arrangement of neurons in the hippocampus, with reduction of number of cell layers and unclear nucleoli, which was relatively milder in the medication and EA＋medication groups. CONCLUSION: EA can enhance the effect of Donepezil in improving learning-memory ability in AD mice possibly by regulating expression of MMP-9, LRP-1, Pgp and Claudin-5 mRNAs and strengthening the effect of Donepezil in transporting Aß via BBB.

[Effect of electroacupuncture combined with Schwann cell transplantation on limb locomotor ability, regional remyelination and expression of spinal CD4 and CD8 proteins in compressive spinal injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with transplantation of Schwann cells (SCs) on limb locomotor, myelin sheath repair and expression of CD4 and CD8 in compressed spinal cord injury (CSCI) rats, so as to explore its mechanisms underlying improvement of CSCI. METHODS: A total of 45 female SD rats were randomly divided into normal control, model, EA, Schwann cell (SC) transplantation, and EA＋SC transplantation groups (n＝9 rats in each group). The CSCI model was established by laminectomy at T12-L2 and clip compression. Rats of the SC transplantation group accepted injection of the cultured SC suspension (2×106/6 µL) into the central, upper and lower sites of the injured spinal cord (5 mm in depth) 7-8 days after CSCI modeling. EA (2 Hz) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP6) for 10 min, once daily and 6 days a week for 3 weeks. The Basso, Beattie and Bresnahan locomotor rating scale (BBB scale) was used to evaluate the function state of CSCI. Morphological changes of the regional injured tissue were observed under light microscope after H．E. staining. The myelin sheath repair state and survival of SCs were detected by Luxol fast blue (LFB) staining and immunofluorescence histochemistry, and the expression of CD4, CD8 and P0 of the injured spinal cord was detected by Western blot. RESULTS: Compared with the normal control group, the BBB scores at the time-points of 0 d, and 1, 2, and 3 weeks were significantly decreased in the model group (P<0.001), and those of the EA＋SC transplantation group at the 2nd and 3rd week were significantly higher than those of the model group (P<0.05). No significant changes of BBB scores were found after EA and SC transplantation relevant to the model group (P>0.05). LFB staining showed a disordered arrangement of the nerve fibers in the white matter, myelinociasis and obvious decrease of the medullated fibers in the model group, and these situations were relatively milder in both EA and SC transplantation groups and obviously milder in the EA＋SC transplantation group. H．E. staining displayed that the structure of the injured region of the spinal cord was incomplete, accompanied with a large number of defect cavities and neuronal karyopyknosis in the model group, while the structure was relatively clear, with an increase of the normal neurons and fewer neuronal karyopyknosis in the EA＋SC transplantation group. Compared with the normal control group, MBP in the model group was significantly decreased (P<0.001)，and P0 was significantly increased (P<0.001). Compared with the model group, the expressions of MBP and P0 were significantly increased in the EA, SC transplantation, and EA＋SC transplantation groups (P<0.01, P<0.001), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (P<0.001). The average immunofluorescence intensity of Hoechst33342-labeled SCs was significantly higher in the EA＋SC transplantation group than in the SC transplantation group (P<0.05). After CSCI, the expression levels of spinal CD4, CD8 and P0 proteins had no significant changes in comparison with the normal control group (P>0.05), while after the intervention and in comparison with the model group, the expression levels of P0 protein were significantly increased in the EA, SC transplantation and EA＋SC transplantation groups (P<0.05), and was significantly higher in the EA＋SC transplantation group than in both EA and SC transplantation groups (P<0.05). The expression levels of CD4 and CD8 proteins were significantly lower in the EA＋SC transplantation group than in the SC transplantation group (P<0.05)．. CONCLUSION: EA＋SCs transplantation can improve the locomotor function in CSCI rats, which may be related to its effects in increasing the survival of transplanted SCs to promote the remyelination and in reducing the immune rejecting reaction.

[Therapeutic effect of massage on denervated skeletal muscle atrophy in rats and its mechanism].

OBJECTIVE: To investigate the therapeutic effects of massage on denervated skeletal muscle atrophy in rats and its mechanism. METHODS: Forty-eight male SD rats were randomly divided into model group (n=24) and massage group (n=24). Gastrocnemius muscle atrophy model was established by transecting the right tibial nerve of rat. On the second day after operation, the gastrocnemius muscle of the rats in the massage group was given manual intervention and the model group was not intervened. Six rats were sacrificed at the four time points of 0 d, 7 d, 14 d and 21 d. The gastrocnemius of the rats were obtained and measured the wet mass ratio after weighing. Cross-sectional area and diameter of the muscle fiber were measured after HE staining. The relative expressions of miR-23a, Akt, MuRF1 and MAFbx mRNA were tested with qPCR. RESULTS: Compared with 0 d, the wet weight ratio, cross-sectional area and diameter of gastrocnemius muscle showed a progressive decline in the model group and massage group. The wet weight ratio, cross-sectional area and diameter of gastrocnemius muscle in the massage group were higher than those in the model group on 7 d, 14 d and 21 d (P＜0.05, P＜0.01). Compared with 0 d, the expressions of MuRF1, MAFbx and Akt mRNA were increased first and then were decreased in the model group and massage group. The expression of MuRF1 mRNA in massage group was lower than that in model group on 7 d and 21 d (P＜0.05, P＜0.01). The expression of MAFbx mRNA in massage group was lower than that in model group on 7 d, 14 d and 21 d (P＜0.01, P＜0.05, P＜0.01). The expression of Akt mRNA in massage group was higher than that in model group on 7 d, 14 d and 21 d (P＜0.05, P＜0.01). Compared with 0 d, the expression of miR-23a mRNA was increased in the model group and massage group on 21 d, and the expression of miR-23a mRNA in massage group was higher than that in model group (P＜ 0.05). CONCLUSION: Massage can delay the atrophy of denervated skeletal muscle. The mechanism may be related to up-regulation of the expression of miR-23a and Akt mRNA, down-regulation of the expressions of MuRF1 and MAFbx mRNA, inhibition of protein degradation rate, and reduction of skeletal muscle protein degradation.

[Electroacupuncture improves denervated gastrocnemius atrophy by regulating expression of fork-head protein 3A，muscle atrophy F-box and myogenic differentiation antigen proteins in sciatic nerve injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on morphological changes of denervated gastrocnemius(GS) and the expression of fork-head protein(FOXO3A), muscle atrophy F-box(MAFbx)and myogenic differentiation antigen (Myod1) in sciatic nerve injury rats, so as to reveal its mechanism underlying improvement of myoatrophy. METHODS: Eighteen male Sprague-Dawley rats were randomly divided into sham operation, model and EA groups (n＝6 per group). The model of gastrocnemius atrophy was established by crushing the right sciatic nerve. Then, EA (2 Hz) was applied to the right "Zusanli" (ST36) and "Huantiao" (GB30) for 10 min, once a day for 14 successive days. The wet weight of the GS on both sides was weighted to calculate the wet weight ratio (the injured side /the healthy side), and the cross-sectional area (CSA) and diameter of GS fibers were measured after H．E. staining. The expressions of FOXO3A, MAFbx and Myod1 protein and mRNA in the GS tissue were tested using Western blot and fluorescence quantitative PCR, separately. RESULTS: Following modeling, the GS wet weight ratio, CSA and fiber diameter were smaller in the model group than those in the sham group (P<0.01), and were significantly higher in the EA group than in the model group (P<0.01). H．E. staining showed that the GS fibers became smaller and the myocyte got round in the model group, while the GS fibers were bigger and the myocyte was relatively regular in morphology in the EA group. After modeling, the expression levels of FOXO3A, MAFbx and Myod1 mRNA and protein were evidently higher in the model group (P<0.01); Moreover, after EA treatment, modeling-induced increasing of expression levels of FOXO3A and MAFbx mRNA and protein were revised (P<0.01), while the increased expression level of Myod1 was further up-regulated relavant to that in the model group (P<0.01)．. CONCLUSION: EA of ST36 and GB30 can suppress the up-regulated expression of FOXO3A and MAFbx mRNA and protein and further promote the expression of Myod1 mRNA and protein in the GS tissue in rats with denervated GS atrophy, which may contribute to its function in relieving the myoatrophy, promoting the skeletal muscle protein hydrolysis and differentiation of satellite cells.

[Effect of electroacupuncture on amyotrophia and expression of myogenic differentiation-related genes of gastrocnemius in rats with chronic constriction injury of sciatic nerve].

OBJECTIVE: To explore the effect of electroacupuncture(EA) on amyotrophia and expression of paired box7(Pax7), myogenic differentiation antigen-1 (Myod1), myogenin (Myog), myosin heavy chain- Ⅱa (Myh2), myosin heavy chain-Ⅱx (Myh1) and myosin heavy chain-Ⅰ (Myh7) of denervated gastrocnemius in rats with chronic constriction injury (CCI) of the right sciatic nerve, so as to explore its mechanisms underlying postponing development of amyotrophia. METHODS: Sixty-six SD adult male rats were randomly divided into sham operation (sham) group (n＝24), model group (n＝24) and EA group (n＝18). The denervated muscle (gastrocnemius) atrophy model was established by CCI of the right sciatic nerve. EA (2 Hz，1.0 mA) was applied to the right "Zusanli"(ST36) and "Huantiao"(GB30) for 10 min, once a day, six times a week and for 1, 2 and 4 weeks, respectively. After complete dissection of bilateral gastrocnemius muscles, their wet weight levels were measured and the ratio of wet weight (＝that of the operation side/that of the non-operation side) was calculated, and the cross-sectional area (CSA) and diameter of the gastronemius were detected after fixation in 4% paraformaldehyde, sectioning, and H．E. staining. The expression levels of Pax7, Myod1, Myog, Myh2, Myh1 and Myh7 mRNAs in the gastrocnemius tissue after 3 weeks of modeling were detected with quantitative real time-PCR (qPCR). RESULTS: After 1 week of modeling, the ratios of wet weight of gastrocnemius and the CSA and fiber diameter at the 2nd, 3rd and 5th week were significantly smaller in the model group than in the sham group (P<0.05). The expression levels of Myod1 and Myog mRNAs were significantly up-regulated (P<0.01), and those of Myh2, Myh1 and Myh7 considerably down-regulated in the model group compared with the sham group (P<0.05, P<0.01). No significant changes were found in the expression levels of Pax7 mRNA after modeling and EA intervention (P>0.05)．Following EA intervention, the CSA and diameterof the gastronemius were obviously increased (P<0.05), and the expression levels of Myod1, Myog and Myh7 further markedly or remarkably up-regulated in comparison with the model group (P<0.05, P<0.01). No significant changes were found in the ratio of wet weight of gastrocnemius at the 3 time-points, and the expression levels of Myh2 mRNA and Myh1 mRNA in the EA group relevant to the model group after 3 weeks of modeling (P>0.05). CONCLUSION: EA treatment may delay gastrocnemius atrophy in CCI rats, which is possibly associated with its effects in up-regulating the expression of Myod1, Myog and Myh7 mRNAs to control the differentiation of the satellite cells and the muscle fiber type transformation.

[Effect of Electroacupuncture on Synovial M 1/M 2 Macrophage Polarization in Rats with Acute Gouty Arthritis].

OBJECTIVE: To explore the effect of electroacupuncture (EA) on the expression of synovial AMP-activated protein kinase (AMPK) protein α， arginase-1 mRNA, nitric oxide synthase 2 (NOS 2) mRNA, NOD-like receptor protein 3 (NLRP 3) mRNA, and interleukin-1 ß (IL-1 ß) mRNA in acute gouty arthritis (AGA) rats, so as to explore its mechanisms underlying improvement of AGA via M 1/M 2 macrophage polarization. METHODS: Male Wistar rats were randomly divided into normal control, model, medication (colchicine) and EA groups (n＝15 rats in each group). The AGA model was established by injection of sodium urate crystal (MSU) suspension (0.2 mL) into the articular cavity of the left knee. The rats of the normal control group received articular injection of normal saline (0.2 mL) of the left knee, and those of the medication group were treated by gavage of the colchicine (0.3 mg•kg－1•d－1) once daily for 7 days. EA (2 Hz/10 Hz, 1.0 mA) was applied to "Zusanli"(ST 36) and "Sanyinjiao" (SP 6) of the left hind limb for 10 min, once daily for 7 days. The inflammatory conditions of the synovial membrane tissue of the left knee joint were observed by H．E. staining. The expression levels of phosphorylated AMPKα (p-AMPKα) protein, and arginase-1 (a maker of M 2 macrophages) mRNA, NOS 2 (a maker of M 1 macrophages) mRNA, NLRP 3 mRNA, and IL-1 ß mRNA in the knee joint synovial tissue were detected by Western blot and quantitative real-time PCR, respectively. RESULTS: Compared with the normal group, the inflammatory cell infiltration of the synovial tissue was more severe, the expression of p-AMPKα protein was significantly decreased (P<0.01), and the expression levels of arginase-1, NOS 2, IL-1 ß and NLRP 3 mRNAs were considerably increased in the model group (P<0.01). The expression levels of p-AMPKα protein and arginase-1 mRNA were significantly up-regulated, and those of NOS 2, IL-1 ß and NLRP 3 mRNAs obviously down-regulated in both EA and medication groups relevant to the model group (P<0.01, P<0.05), suggesting an increase of M 2 macrophage and a decrease of M 1 macrophage activity after EA. No significant differences were found between the EA and medication groups in up-regulating p-AMPKα expression and in down-regulating NOS 2, IL-1 ß and NLRP 3 mRNA expression (P>0.05), except higher up-regulation of arginase-1 mRNA in the medication group (P<0.05)．. CONCLUSION: EA intervention can up-regulate the expression of arginase-1 mRNA and p-AMPKα protein, and down-regulate the expression of NOS 2, IL-1 ß and NLRP 3 mRNAs in synovial tissues in AGA rats, which may contribute to its anti-inflammatory effect by promoting conversion of macrophages from M 1 pro-inflammatory phenotype to M 2 anti-inflammatory phenotype.