[The anti-inflammatory effect of electroacupuncture in mice with spinal cord injury and molecular mechanism based on transcriptome sequencing technology].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on neural function and spinal cord pathological morphology in spinal cord injury(SCI) mice and investigate the anti-inflammatory molecular mechanism of EA on SCI mice from the aspects of gene by using bioinformatics. METHODS: Seventy-two female C57BL/6 mice were randomized into sham operation, model and EA groups, with 24 mice in each group. The SCI model was established by clamping the spinal cord with a serrefine after laminectomy at the 1st lumbar vertebra(L1). EA(1.5 Hz/7.5 Hz, 1.0 mA) was applied to bilateral "Jiaji"(EX-B2) and "Zusanli"(ST36) for 10 min, once a day for 14 consecutive days. Basso Mouse Scale(BMS) score was used to assess the hindlimb locomotor function of mice. Histopathological changes of the injured area of the spinal cord were determined by HE staining. The spinal cord RNA was sequenced by using RNA-Seq technology. The bioinformatic analysis was then performed to detect the diffe-rential genes between groups, and the function classification and the involved pathways were enriched. The mRNA and protein expressions of differential genes were detected and verified by using qRT-PCR and Western blot. RESULTS: Compared with the sham operation group, BMS score of the model group was significantly decreased(P<0.05), while that of EA group was increased relevant to the model group (P<0.05). HE staining showed loose and disordered structure and arrangement, cavitation, more inflammatory infiltration, nucleus pycnosis, and neuronal necrosis in the model group, which was alleviated in the EA group. Compared with the sham operation group, 565 differential genes were detected in the model group, including 545 up-regulated and 20 down-regulated, while 41 were detected between the EA and the model group, including 2 up-regulated and 39 down-regulated in the EA group. Fifteen genes that were all up-regulated after modeling and down-regulated after EA intervention were detected by using Venn plot, which are Retn, Adipoq, Myh1, Actn2, Pck1, Klhl41, Fabp4, Hspb7, Myot, Ankrd2, Hrc, Cox6a2, Obscn, Col2a1, Mybpc1, and 3 inflammation-related genes(Fabp4, Adipoq and Pck1) were finally acquired. The 15 differential genes were annotated into main biological processes, cell composition and molecular function in the GO function classification analysis. The 15 differential genes were then enriched into different KEGG pathways, including the peroxisome proliferatorsactivated receptor (PPAR) signaling pathway, Adipocytokine signaling pathway. The mRNA and protein expressions of Fabp4, Adipoq and Pck1 in spinal cord detected by qRT-PCR and Western blot were significantly increased in the model group (P<0.001, P<0.01), while these were significantly decreased in the EA group relevant to the model group(P<0.001, P<0.01, P<0.05). CONCLUSION: EA can promote the repair of nerve function and improve inflammatory infiltration in SCI mice. The mechanism may be closely related to the down-regulation of inflammatory factors Fabp4, Adipoq and Pck1 expression, and the regulation of PPAR and Adipocytokine signaling pathways.

Therapeutic effects and central mechanism of acupuncture and moxibustion for treating functional dyspepsia: study protocol for an fMRI-based randomized controlled trial.

BACKGROUND: Functional dyspepsia (FD) is one of the most common functional gastrointestinal disorders, with a high prevalence and significant influence on the quality of life (QoL). Either acupuncture or moxibustion is effective for dyspepsia, which is confirmed by both ancient documents and modern research. However, the therapeutic advantage and underlying mechanism between acupuncture and moxibustion for FD remain unclear. METHODS: This randomized controlled fMRI trial aims to (i) evaluate the therapeutic advantages of acupuncture and moxibustion treatment for FD, (ii) investigate the similarities and differences in cerebral activity elicited by acupuncture and moxibustion, and (iii) analyze the possible correlations between brain responses and clinical variables thus to explore the potential central mechanism of acupuncture and moxibustion for treating FD. Ninety-two FD patients will be randomly assigned to either the acupuncture group or the moxibustion group in a 1:1 ratio. Twenty sessions of acupuncture or moxibustion treatment over 4 weeks will be performed on each patient. The short form Leeds Dyspepsia Questionnaire, the Nepean Dyspepsia Index, etc., are used to evaluate the therapeutic effects. The heart rate variability will be analyzed to investigate the autonomic nerve function. Thirty-six FD patients in each group will be randomly selected for the fMRI scan to detect cerebral activity changes. DISCUSSION: We expect the results will deepen our knowledge on the clinical value and underlying mechanism of acupuncture and moxibustion and provide a reference for a better selection of interventions for treating FD. TRIAL REGISTRATION: Chinese Clinical Trial Registry ( www.chictr.org.cn ) ChiCTR2100049496. Registered on 2 August 2021.

[Effect of electroacupuncture combined with Schwann cell transplantation on remyelination of axons and neuregulin1 expression in rats with compressed spinal injury].

OBJECTIVE: To explore the effect of electroacupuncture (EA) combined with Schwann cell (SC) transplantation (SCT) on remyelination of axons and neuregulin (Nrg1) in rats with compressed spinal cord injury(CSCI),so as to explore the mechanism of EA and SCT underlying improvement of CSCI. METHODS: SD female rats were randomly divided into normal, mo-del, EA, SCT, and EA+ SCT groups (n=40 per group). A self-developed model of spinal compressed injury was adopted in this study. Rats of the model group were administrated laminectomy without treatment. Rats in the EA group were administrated EA stimulation at "Dazhui"(GV14), "Mingmen"(GV7), bilateral "Zusanli" (ST36) and "Taixi" (KI3) on the second day post-surgery for 10 min. Rats in the SC group were administrated SCT at 1 week post-surgery, and in the EA+SC group were given EA stimulation combined with SCT. The injured spinal cord tissue was obtained 0, 2, 4 and 8 weeks after compressed spinal injury. The functional recovery was assessed by Basso-Beattie-Bresnahan (BBB) score. The survivals and migration of SC after transplantation, myelination were observed by immunofluorescence. The ultrastructure of myelin in injured site was observed by transmission electron microscope，and the expression levels of glial fibrillary acidic protein (GFAP), protein zero(P0), and Nrg1 and Nrg1-ntf (cleavage protein of Nrg1) proteins of the spinal cord were detected by Western blot. RESULTS: Compared with the normal group, BBB scores in the model group was significantly decreased（P<0.05）,nervous fibers were demyelinated, numbers of normal and newborn myelination were decreased(P<0.05)，expression of P0 was significantly increased (P<0.05)，expression of GFAP was significantly increased(P<0.05)，and the expression levels of Nrg1 and Nrg1-ntf proteins were decreased(P<0.05). In comparison with the model group, the BBB scores in the EA, SCT and EA+SCT groups were significantly increased（P<0.05,P<0.01）, demyelination was improved, numbers of normal and newborn myelinations were increased(P<0.05,P<0.01)，expressions of P0 were significantly increased (P<0.05,P<0.01)，expressions of GFAP were significantly decreased(P<0.05)，and the expression levels of Nrg1 and Nrg1-ntf proteins were increased (P<0.05, P<0.01).The differences were most significant in the EA+SCT group among the three groups. CONCLUSION: EA can improve the locomotor function in CSCI rats, which may be rela-ted to its functions in promoting the survival and migration of transplanted SC and remyelination, and increasing the expressions of Nrg1 and its cleavage protein after SC transplantation.

[Comparative analysis of electroacupuncture, metformin and their combination on cognitive function and senile plaques of cerebral cortex and hippocampus in APP/PS1 mice].

OBJECTIVE: To compare the effect of electroacupuncture (EA), metformin and EA plus metformin on the cognitive ability and senile plaques (SPs) in cerebral cortex and hippocampus of Alzheimer's disease (AD) mice, so as to explore a better treatment method for AD. METHODS: Twenty-four male APP/PS1 mice were randomly divided into model, metformin (medication), EA and EA+medication groups, with 6 mice in each group. Other 6 male wild C57 mice were used as the control group. EA (2 Hz, 1.0 mA) was applied to "Baihui" (GV20) and "Shenshu" (BL23) for 15 min, once a day, for 4 weeks, with 1 day's off every week. The mice of the medication group received gavage of metformin (300 mg·kg-1·d-1) once a day for 4 weeks. Morris water maze tests were used to assess the cognitive function of mice. H.E. staining was used to observe the histopathological changes of neurons in the cortex and hippocampus. Immunohistochemical method was used to observe the cerebral cortex and hippocampal SPs. The expression levels of SPs formation-related proteins: ß-site amyloid precursor protein cleaving enzyme 1(ßACE1) and insulin-degrading enzyme (IDE) in the cortex and hippocampus were detected by Western blot. RESULTS: Compared with the control group, the escape latency, number of SPs and the expression of ßACE1 in the cortex and hippocampus were ob-viously increased (P<0.01), and the times of platform quadrant crossing and the expression of IDE protein were markedly decreased in the model group (P<0.01). In comparison with the model group, the escape latency, and the number of SPs and expression of ßACE1 proteins in the cortex and hippocampus in the 3 treatment groups were significantly down-regulated (P<0.01), while the times of platform quadrant crossing, and the expression of IDE protein in both cortex and hippocampus of the three treatment groups were considerably up-regulated (P<0.01). Comparison among the three treatment groups showed that the therapeutic effect of EA+medication was significantly superior to that of medication and simple EA in down-regulating the escape latency, the number of SPs and expression of ßACE1 in the cortex and hippocampus (P<0.01), and in up-regulating the times of the platform quadrant crossing, and expression of IDE protein in both cortex and hippocampus (P<0.01). No significant differences were found between the simple medication and simple EA in all the indexes mentioned above (P>0.05). CONCLUSION: EA, metformin and EA plus metformin can improve cognitive ability and relieve SP formation in cerebral cortex and hippocampus in AD mice, which may be associated with their functions in down-regulating the expression of ßACE1 and up-regulating the expression of IDE. The therapeutic effects of EA plus metformin are apparently better than those of simple EA and simple metformin.

[Effects of electroacupuncture on autophagy factors and liver lipid metabolism in rapidly aging mice].

Objective: To study the effects of electroacupuncture on the expressions of autophagy-related factors LC3-Ⅱ, Beclin1, Atg7, and P62 in the liver of rapidly aging (senescence accelerated mouse/prone8,SAMP8) mice, and to explore the mechanisms of electroacupuncture to improve liver lipid metabolism in mice. Methods: Thirty-week-old male SAMP8 mice were randomly divided into model group, drug group, and electroacupuncture group, with 7 mice in each group. Seven anti-rapid aging SAMR1 mice of the same age were used as the control group. The animals in the control group and the model group were bred routinely for 2 weeks without any intervention; the drug group was treated with intraperitoneal injection of rapamycin at the dose of 10 mg·kg-1·d-1, once a day, 6 consecutive days a week; the electroacupuncture group was given "Shenshu" and "Taichong" Electroacupuncture at point(15 minutes a day, 6 consecutive days a week). The serum lipid metabolism and liver lipid deposition of mice were detected, the distribution of liver autophagy body, the protein and mRNA expressions of liver LC3 - Ⅱ, Beclin1, Atg7 and P62 were determined. Results: Compared with the control group, the total cholesterol (TC), triglycerides (TG), and low density lipoprotein (LDL) of the model group were increased significantly(P＜0.01). In the model group, lipid droplet deposition was obvious, autophagosomes were decreased, the protein and mRNA expression levels of autophagy- related factors LC3-Ⅱ, Beclin1 and Atg7 were decreased significantly (P＜0.01), while the protein and mRNA expressions of P62 were increased significantly (P＜0.01). Compared with the model group, the serum contents of TG, TC, and LDL of the mice in the electroacupuncture group and the drug group were decreased significantly (P＜0.01), lipid droplet deposition was reduced, autophagosomes were increased, the protein and mRNA expression levels of LC3 -Ⅱ, Beclin1 and Atg7 were increased significantly(P＜0.01), and the protein and mRNA expression levels of P62 were decreased significantly(P＜0.01). The protein and mRNA expression levels of Beclin1 and Atg7 in the liver of the electroacupuncture group were not significantly different from the drug group (P＞0.05). Conclusion: Electroacupuncture can alleviate liver lipid metabolism disorders, which may be related to the regulation of the expressions of liver autophagy related factors LC3-Ⅱ, Beclin1, Atg7, and P62, thereby promoting liver autophagy in SAMP8 mice.

[Influence of electroacupuncture on locomotor function and expression of spinal HMGB1 and TLR4 in mice with spinal cord injury].

OBJECTIVE: To observe the effect of electroacupuncture(EA)on locomotor activity and the expression of high-mobility group box-1(HMGB1) and Toll-like receptor 4(TLR4) in mice with spinal cord injury(SCI), so as to explore its mechanisms underlying improvement of SCI at the acute stage. METHODS: Forty-eight female C57BL/6 mice were equally randomized into 3 groups: sham operation, model and EA. The SCI model was established by clamping the spinal cord with a serrefine after laminectomy at the 1st lumbar vertebra(L1). EA (1.5 Hz/7.5 Hz, 1.0 mA) was applied to "Jiaji"(EXH-B2) for 10 min, once a day for 5 and 14 days, separately. The hindlimb locomotor function was assessed by Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB). Histopathological changes of the injured area of the spinal cord were determined by H.E. staining. The expression levels of spinal HMGB1, TLR4, ionized calcium binding adaptor molecule 1(Iba1) proteins were detected by Western blot, and the Iba1-positive microglial cells and HMGB1 and Iba1 co-labelled microglia were displayed by immunofluorescence staining. RESULTS: After SCI, the BBB scores on day 5 and 14 were obviously decreased (P<0.05), and the expression of HMGB1 on day 14, TLR4 on day 5 and 14, the number of Iba1-positive microglia as well as the co-expressed HMGB1/Iba1-positive microglia on day 5 and 14 were significantly increased in the model group relevant to the sham operation group (P<0.05, P<0.01). In the EA intervention group, SCI-induced reduction of BBB scores on day 5 and 14, and increases of the expression of HMGB1 and Iba1 on day 14, and TLR4 on day 5 and 14, and the number of Iba1-positive cells as well as the co-expressed HMGB1/Iba1-positive microglia on day 14 were reversed relevant to the model group (P<0.05,P<0.01). H.E. staining showed a structural disorder with lots of cavities, severe inflammatory infiltration with a large quantity of inflammatory cells, and a reduction of normal neurons in the injured spinal cord tissue in the model group， which was relatively milder, with lower activation of microglia in the EA group. CONCLUSION: EA can significantly improve locomotor function in SCI mice, which is associated with its effects in suppressing the expression of inflammatory factors such as HMGB1, TLR4, Iba1 and the over-activation of microglia.

[Effects of electroacupuncture at "Jiaji"(EX-B2) on autophagy and endoplasmic reticulum stress in spinal cord injury mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Jiaji" (EX-B2) on the levels of autophagy and endoplasmic reticulum stress in mice with spinal cord injury (SCI), so as to explore its mechanism underlying improvement of SCI. METHODS: A total of 60 female C57BL/6 mice were randomly divided into sham operation, model and EA groups， which were further divided into 7 d and 14 d subgroups (10 mice in each subgroup). The SCI model was established by pressing the exposed spinal cord (L1) with a vascular clamp for 15 s. EA was applied to bilateral EX-B2 3 h after modeling, once a day for 7 and 14 d, respectively. Basso Mouse Scale(BMS) for locomotion was used to evaluate hindlimb motor function on day 7 and 14 after SCI. H.E. staining was used to observe histopathologic changes of the injured spinal cord tissue, and Western blot employed to detect the expression of glucose regulatory protein-78 (GRP78), Caspase-12, microtubule-associated protein light chain 3 II (LC-II) and P62(also known as sqstm1/Sequestome1) proteins. Immunofluorescence staining was used to detect the immunoacti-vities of spinal CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP, an endoplasmic reticulum stress-inducible protein) and P62. RESULTS: On the 7th and 14th day after SCI, the BMS scores and expression levels of LC3II protein were significantly down-regulated (P<0.05), and the expression levels of P62, GRP78 and Caspase-12 proteins, the immunoactivities of CHOP and P62 were all significantly up-regulated on both day 7 and 14 in the model group than in the sham operation group (P<0.05).Compared with the model group, the BMS scores and the expression levels of LC3II protein were significantly increased on both day 7 and 14 (P<0.05), while the expression levels of P62, GRP78 and Caspase-12 proteins, and the immunoactivities of CHOP and P62 were obviously decreased on day 7 and 14 in the EA group (P<0.05). Outcomes of H.E. stain showed that the cells with nuclei pyknosis and swelling and the necrotic cells appeared in the model group, which was relatively fewer in the EA group. CONCLUSION: EA of EX-B2 can improve the locomotor function in SCI mice, which may be related to its effects in up-regulating the expression of LC3II (to promote cell autophagy), and down-regulating the expression of P62, GRP78, Caspase-12 and CHOP proteins (to inhibit endoplasmic reticulum stress) in the spinal cord tissue.

[Electroacupuncture improves locomotor function by regulating expression of inflammation and oxidative stress-related proteins in mice with spinal cord injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of apolipoprotein E (ApoE) and related proteins of inflammation and anti-oxidative stress in spinal cord in mice with spinal cord injury (SCI), so as to explore its mechanisms underlying function repair. METHODS: Thirty-six female C57BL/6 mice were equally randomized into 3 groups: sham operation, model and EA. The SCI model was established by clamping the spinal cord for 25 s with a serrefine after laminectomy of the 1st lumbar vertebra (L1). EA (1.5 Hz/7.5 Hz, 1.0 mA) was applied to bila-teral "Zusanli" (ST36) and "Sanyinjiao" (SP6) for 10 min, once a day for 7 days. The hindlimb locomotor function was assessed according to the state of the range of motion, coordination, claw gesture of the hind leg ankle-joint, trunk stabi-lity and the tail posture by using Basso Mouse Scale(BMS). The histopathological changes of the injured area of the spinal cord were determined by H.E. staining. The expression levels of ApoE, phosphorylated nuclear transcription factor-κB(p-NF-κB), interleukin 1 beta(IL-1ß), phosphorylated extracellular regulatory protein kinase(p-ERK1/2), extracellular regulatory protein kinase(ERK1/2), nuclear factor erythroid 2-related factor 2(Nrf2) and heme oxidase-1(HO-1) in the spinal cord were detected by Western blot, and the glial fibrillary acidic protein (GFAP)-positive astrocytes were displayed by immunofluorescence staining. RESULTS: After modeling, the BMS scores were significantly decreased in the model group compared with the sham operation group (P<0.05). Following EA, the BMS scores were markedly increased in the EA group relevant to the model group (P<0.05), suggesting an improvement of the hindlimb locomotor function. H.E. stain showed structural disorder with lots of cavities, severe inflammatory infiltration with large quantity of inflammatory cells, and apparent reduction of normal neurons in the injured spinal cord tissue of model group, which was milder in the EA group. The expression levels of ApoE, p-NF-κB, IL-1ß, p-ERK1/2 (not ERK1/2), Nrf2 and HO-1 were significantly increased in the model group than those in the sham operation group (P<0.05). Compared with the model group, the expression levels of ApoE, p-ERK1/2, Nrf2 and HO-1 were further notably up-regulated (P<0.05), and those of p-NF-κB and IL-1ß proteins obviously down-regulated in the EA group (P<0.05). Immunoflorescence staining showed that the number of GFAP-positive cells was apparently increased in the model group compared with the sham operation group and observably decreased in the EA group relevant to the model group (P<0.05). CONCLUSION: EA can significantly improve locomotor function in SCI mice, which is associated with its effects in reducing inflammation, oxi-dative stress reactions and reactive astrocyte proliferation via up-regulating expression of ApoE, p-ERK1/2, and Nrf2/HO-1 (antioxidant pathway) and inhibiting IL-1ß and NF-κB expression.

[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.

[Effect of electroacupuncture of acupoints on the healthy limb (opposing needling) on acute skeletal muscle contusion in rats].

OBJECTIVE: To observe the therapeutic effect of electroacupuncture (EA) of "Zusanli" (ST36) and "Ashi"-point on the healthy side (opposing needling) on muscular injury and expression of myogenin (myoG) and fast myosin skeletal heavy chain (Fast MyHC) proteins in the gastrocnemius muscle (GM) tissues in skeletal muscle contusion rats，so as to explore its mechanism underlying improvement of skeletal muscle injury. METHODS: A total of 54 male SD rats were divided into normal control (n ＝ 6)，model (n＝24) and opposing needling (EA, n＝24) groups. The latter two groups were further randomized into 3, 5, 7 and 14 d subgroups (n＝6 per subgroup). The skeletal muscle contusion model of the hind-limb was established by using a self-made striking device. EA (1 Hz/3 Hz，1－2 mA) was applied to ST36 and "Ashi"-point on the uninjured side of the hind-limb for 15 min every time, once a day for 3, 5, 7 and 14 days, respectively. The injured GM was harvested on the 3rd, 5th, 7th and 14th day after muscular contusion. The morphological changes of the injured GM and the mean cross-sectional areas (CSAs) of the neonatal muscle cells were observed by microscope after H．E. staining. The immunoactivity of desmin protein (myogenic marker protein of myoblast cell) of GM was detected by immunofluorescence stain on the 7th day after injury, and the expression levels of myoG (on the 3rd and 5th day after injury) and fast MyHC protein of GM tissues (on the 7thand 14th day after injury) were detected by Western blot. RESULTS: H．E. staining of GS tissue showed fewer neuronal myocytes with disordered arrangement at different sizes, and appearance of some collagenous fibers among the mesenchyme on day 7 and 14 after muscular contusion, which was relatively milder in the EA group. In the EA group, the CSA values of the neonatal muscle cells were significantly larger than those in the model group on the day 7th (P<0.05), 14th (P<0.001) after injury. On day 7 after muscular contusion, the desmin was found to express on the cellular membrane of GM in the normal control group, while in the model group, the desmin expressed mainly in the cellular plasma in the model group, and on the cellular membrane of neonatal myocytes in the EA group, respectively. The desmin positive myocytes showed disordered arrangement and different sizes after muscular contusion, whereas the situations of the EA group were close to those of the normal control group. Desmin expression was up-regulated in the EA group compared with the model group which was not significant difference (P>0.05). On the 3rd and 5th day after muscular contusion, the expression level of myoG protein was significantly up-regulated in the model group compared with the normal control group (P<0.001), and significantly up-regulated in the EA group than that in the model group (P<0.001). On the 7th and14th day after contusion, the expression level of fast MyHC protein was significantly down-regulated in the model group relevant to the normal control group (P<0.001), and markedly up-regulated in the EA group relevant to the model group (P<0.01)．. CONCLUSION: EA of ST36 and "Ashi"-point on the contralateral limb can up-regulate the expression of myoG and fast MyHC proteins of GM in acute skeletal muscle contusion rats, which may contribute to its effect in promoting the repair of skeletal muscle injury.

[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.

Curcumin Ameliorates Memory Deficits by Enhancing Lactate Content and MCT2 Expression in APP/PS1 Transgenic Mouse Model of Alzheimer's Disease.

Curcumin is a natural product with several anti-Alzheimer's disease (AD) neuroprotective properties. This study aimed to investigate the effects of curcumin on memory deficits, lactate content, and monocarboxylate transporter 2 (MCT2) in APP/PS1 mouse model of AD. APP/PS1 transgenic mice and wild-type (WT) C57BL/6J mice were used in the present study. Spatial learning and memory of the mice was detected using Morris water-maze test. Cerebral cortex and hippocampus lactate contents were detected using lactate assay. MCT2 expression in the cerebral cortex and hippocampus was examined by immunohistochemistry and Western blotting. Results showed that spatial learning and memory deficits were improved in curcumin-treated APP/PS1 mouse group compared with those in APP/PS1 mice group. Brain lactate content and MCT2 protein level were increased in curcumin-treated APP/PS1 mice than in APP/PS1 mice. In summary, our findings indicate that curcumin could ameliorate memory impairments in APP/PS1 mouse model of AD. This phenomenon may be at least partially due to its improving effect on the lactate content and MCT2 protein expression in the brain. Anat Rec, 302:332-338, 2019. © 2018 Wiley Periodicals, Inc.

[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.

[Changes in expression of autophagy-related factors during acute contusion repair of skeletal muscle].

OBJECTIVES: To explore the possible biological mechanism of skeletal muscle contusion repair through researching the changes in expression of autophagy-related genes and proteins in SD rats with acute skeletal muscle contusion. METHODS: Six rats were randomly selected as the control group from 30 male SD rats, acute skeletal muscle contusion model were established in the remaining 24 rats with self-made hitter, then the model rats were randomly divided into 4 groups (3 d, 5 d, 7 d, 14 d groups, n=6). On the 3rd, 5th, 7th and 14th day after injury, injured gastrocnemius of each group was harvested. The morphological and the ultra-microstructure changes of gastrocnemius after injury were observed by HE staining and transmission electron microscope (TEM) respectively. The relative protein levels of (LC3-Ⅱ) and P62 of each group were observed by Western blot. The relative mRNA levels of atg7, atg10, atg12, atg16L1 of each group were observed by RTPCR. RESULTS: The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5th day after injury, new muscle fibers were clearly observed in 7 d group. The results of TEM showed that, compared with the control group, oncotic mitochondria could be clearly seen in the 3 d, 5 d, 7 d groups. Also, the Z line changed from disappearing to drift thickening, sarcoplasmic reticulum dilatation gradually improved, there was no evident difference between the 14 d group and the control group, suggesting that the damage has preliminarily healed. The results of Western blot showed that the expressions of LC3-Ⅱand P62 were increased at first and then decreased. The expression of LC3-Ⅱwas markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3rd day after injury (P<0. 01), and returned to normal level on the 14th day. The results of RT-PCR showed that the expression of atg10 mRNA in the natural recovery group of 3 d, 5 d, 7 d, 14 d was firstly decreased and then increased, the atg10 mRNA was markedly down-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0. 01). The expression of atg7, atg12, atg16L1 mRNA was generally increased at first and then decreased, it was markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (P<0.01, P<0.05, P<0.01). CONCLUSIONS: The above results indicate that the autophagy is involved in repair of skeletal muscle injury by its autophagyrelated factors,regularly changes after contusion, and the rate of damage repair may be related to the level of autophagy.

[The Effect of Electroacupuncture Combined with Treadmill Training on Skeletal Muscle Contusion in Rats].

OBJECTIVE: To study the effect and mechanism of electroacupuncture(EA)combined with treadmill training in rats with skeletal muscle contusion. METHODS: A total of sixty Sprague Dawley rats were randomly divided into normal,model, EA, treadmill, and combination groups (n=12/group). The self-made striking device was used to establish skeletal muscle contusion model. EA was applied at "Zusanli" (ST 36) and Ashi point in the EA group. Treadmill was applied to train rats in the treadmill group. Rats in the combination group were received the above two methods. All the treatment was given once a day until the 3rd and 14th days after injury. The cross-sectional area and diameter of neonatal gastrocnemius muscle cells 14 days after injury were observed by HE staining. The expression levels of mammalian target of rapamycil (mTOR), myogenin (myoG) in gastrocnemius 3 days after injury and gastrocnemius mTOR, Fast myosin skeletal heavy chain (Fast MyHC) 14 days after injury were observed by Western blot. RESULTS: The cross-sectional area and diameter of neonatal muscle gastrocnemius cells in the model group were significantly lower than those in the normal group (P<0.01), and those in all the intervention groups were significantly higher than those of the model group (P<0.05), with better results in the combination group compared with those in the EA and treadmill groups (P<0.05). On the 3rd day after injury, the expressions of mTOR and myoG proteins in the model group were significantly higher compared with those in the normal group (P<0.01), and those in all the intervention groups were up-regulated in comparison with the model group (all P<0.01). The mTOR and myoG proteins in the treadmill group were lower than those in the EA group (P<0.01). The above two indexes in the combination group were higher than those in the EA and treadmill groups (P<0.05, P<0.01). On the 14th day after injury, the expression of mTOR was up-regulated in the model group compared with that in the normal group, but without significant difference (P<0.05), while the expression of Fast MyHC decreased (P<0.01). The expressions of mTOR and Fast MyHC in all the intervention groups increased compared with those in the model group (P<0.05, P<0.01). The expressions of mTOR and Fast MyHC proteins in the treadmill group were significantly down-regulated compared with those in the EA group (P<0.05, P<0.01). The two indexes in the combination group were higher than those in the other two intervention groups (P<0.01). CONCLUSIONS: EA combined with treadmill training can improve the myogenic differentiation and maturation, alleviate the injury of skeletal muscle, which may be related to its effect of increasing the expression of mTOR protein and positively regulating myoG and Fast MyHC proteins.

[Effects of Electroacupuncture on Apoptosis-related Protein Expression of Gastrocnemius Muscle Cells in Rats with Denervated Sciatic Nerve].

OBJECTIVE: To observe the anti-apoptosis effect of electroacupuncture (EA) on gastrocnemius muscle cells in rats with denervated sciatic nerve, so as to explore its possible mechanisms underlying delaying atrophy of skeletal muscle. METHODS: Sixty-three male Sprague-Dawley rats were randomly divided into sham operation group, model group, and EA group, and then further divided into 3 subgroups in each group(n=7/subgroup). Gastrocnemius muscle atrophy model was established by transecting the sciatic nerve of rat. EA (5 Hz, 1.5 mA) was applied to "Zusanli" (ST 36) and "Chengshan" (BL 57) acupoints at the affected side for 10 min, once a day for 1, 2, 3 weeks, respectively. The gastrocnemius muscles were sampled on the 7th, 14th, 21th d after modeling, separately. The apoptotic cells were detected by TUNEL staining. Bcl-2, Bax, Cyt-C and Caspase-3 protein expressions were determined by Western blot. RT-PCR was used to check Caspase-3 gene expression. RESULTS: Compared with the sham operation group, the cell apoptotic index in gastrocnemius was markedly higher, gastrocnemius Bcl-2 protein expression was markedly down-regulated, and gastrocnemius Bax, Cyt-C and Caspase-3 protein expressions were considerably up-regulated in the model group at all time-points (P<0.05). Compared with the model group, the cell apoptotic indexes in gastrocnemius were significantly lower in the three EA subgroups (P<0.05). Bcl-2 protein expressions were markedly up-regulated while Bax, Cyt-C and Caspase-3 protein expressions were significantly down-regulated in the EA group 14, 21 days after modeling compared with the corresponding model subgroups (P<0.05). The changes of Caspase-3 mRNA expression levels of gastrocnemius in all groups were similar to those of Caspase-3 protein expressions. CONCLUSIONS: Electroacupuncture intervention can effectively increase Bcl-2 expression and decrease Bax, Cyt-C and Caspase-3 expression in gastrocnemius muscle, and consequently reduce the apoptosis of muscle cell, which may contribute to its effect in delaying the skeletal muscle atrophy of denervated sciatic nerve.

[Effects of Electroacupuncture on Adaptive Hypertrophy of Gastrocnemius and Proliferation and Differentiation of Muscle Satellite Cells in Rats].

OBJECTIVE: To explore the effects and mechanism of different duration electroacupuncture (EA) on adaptive hypertrophy of gastrocnemius and the expressions of proliferation cell nuclear antigen (PCNA), paired box transcription factor 7 (Pax 7), myogenic differentiation antigen (MyoD 1), myogenin (MyoG), myosin heavy chain-Ⅰ(Myh 7), transforming growth factor-ß 1 (TGF-ß 1), Smad 3 in rats. METHODS: Twenty-four SD adult male rats were randomly divided into normal, two-week EA (A 2), four-week EA (A 4) and eight-week EA (A 8) groups, 6 rats in each group. Except the normal group, EA was applied at right "Zusanli" (ST 36) and "Huantiao" (GB 30) for 10 min in the other three groups, separately for 2 weeks, 4 weeks and 8 weeks, once a day, 6 times a week. The ratio of gastrocnemius wet weight was calculated after EA. The cross-sectional area and diameter of muscle fiber were measured after HE staining. The expression of PCNA protein was detected by immunofluorescence. The relative expressions of Pax 7, MyoD 1, MyoG, Myh 7, TGF-ß 1 and Smad 3 mRNA were tested with real-time PCR. RESULTS: The expressions of PCNA protein were obviously higher in the A 2 and A 4 groups than that in the normal group. The expression of genes in the EA intervention groups showed a trend of increasing first and then decreasing except Smad 3 mRNA. The relative expressions of Pax 7 and TGF-ß 1 mRNAs reached their peaks in the A 2 group compared with those in the normal group (P<0.01). The ratio of gastrocnemius wet weight, cross-sectional area and diameter of muscle fiber, and the relative expressions of MyoD 1, MyoG, Myh 7 mRNAs reached their peaks in the A 4 group (P<0.01), while the relative expression of Smad 3 mRNA showed a trend of decreasing first and then increasing and reached its bottom at the 4th week (P<0.01). CONCLUSIONS: EA at "Zusanli" (ST 36) and "Huantiao" (GB 30) may induce adaptive hypertrophy of gastrocnemius in rats by up-regulating the expressions of Pax 7, PCNA, TGF-ß 1, MyoD 1, MyoG, Myh 7 and down-regulating the expression of Smad 3, promoting the proliferation of skeletal satellite cells, myogenic differentiation, myotubes terminal differentiation, increasing the wet weight, fiber cross-sectional area and diameter of gastrocnemius.

Internalization of aquaporin-4 after collagenase-induced intracerebral hemorrhage.

Brain edema formation following intracerebral hemorrhage (ICH) appears to be related with aquaporin-4 (AQP4), which is critically involved in brain volume homeostasis and water balance. Despite its importance, the regulation of AQP4 expression involved in transmembrane water movements still remains rudimentary. Many studies suggest that the internalization of several membrane-bound proteins, including AQP4, may occur with or without lysosomal degradation. Previously, we investigated the internalization of AQP4 in retinal ischemic-reperfusion model. Here, we test the hypothesis that AQP4 is internalized post-ICH and then degraded in the lysosome. The results demonstrated that both AQP4 and the mannose-6-phosphate receptor (MPR) co-localized in perihematomal region at 6 hr post-ICH. In addition, AQP4 and lysosomal-associated membrane protein 1 (LAMP1) also co-localized in perihematomal region, with co-expression increasing followed by a gradual decrease at different time windows post-ICH (6, 12, 24, 48, and 72 hr). After ICH, the Evans blue leakage happened very early at 1 hr and the brain swelling occurred at 3 hr. Moreover, we also found the AQP4 mRNA and AQP4 protein were increased post-ICH. These results suggest that AQP4 is internalized and the lysosome is involved in degrading the internalized AQP4 post-ICH. Both the AQP4 internalization and lysosomal degradation may provide biophysical insights regarding the potential of new treatments for brain edema.

Loss of AQP4 polarized localization with loss of ß-dystroglycan immunoreactivity may induce brain edema following intracerebral hemorrhage.

The aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular and subpial membrane domains of astrocytes where it is concentrated. These membranes form the interface between the neuropil and the extracellular liquid spaces. The brain-selective deletion of the dystroglycan (DG) gene causes a disorganization of AQP4 on the astroglial endfeet. First, we analyzed the expression of AQP4, ß-DG in the brain following intracerebral hemorrhage (ICH) and correlated AQP4 expression with the expression pattern of the ß-DG, which is a component of dystrophin-dystroglycan complex (DDC). Besides, the vessels ultrastructure and brain water content were investigated at different time points post-ICH (day 1, day 3, day 7). We found that AQP4 polarity was disturbed in parallel with the loss of ß-DG in the perihematomal area post-ICH. At day 1 post-ICH, brain edema was obvious and the damage of vascular ultrastructure was the most severe. These results suggest a role for ß-DG in targeting and stabilizing AQP4 channel in astrocytic cells, which may be critical for water homeostasis in brain.