[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.

[Effect of electroacupuncture on hepatocyte autophagy and oxidative stress in SAMP8 mice by regulating AMPK/mTOR/ULK1 signaling pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on physical strength and expression levels of hepatic AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), unc-51 like autophagy activating kinase 1 (ULK1) proteins and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNAs in aging (senescence accelerated mouse/prone 8, SAMP8)mice, so as to exp lore its mechanism underlying delaying aging by activating AMPK/mTOR/ULK1 signaling pathway. METHODS: Twenty-four male SAMP8 mice were randomly divided into model group, rapamycin (autophagy inducer) group, EA group and EA+autophagy inhibitor (EA+inhibitor) group, with 6 mice in each group, and 6 homologous anti-rapid aging male (SAMR1) mice in the same age were used as the control group. Mice of the rapamycin group received intraperitoneal injection of rapamycin solution (2 mg·kg-1·d-1). EA (2 Hz, 1 mA) was applied to bilateral "Taichong"(LR3)and "Shenshu"(BL23) for 15 min each time. Mice of the EA+inhibitor group received intraperitoneal injection of mTOR inhibitor 3-methyladenine (1.5 mg·kg-1·d-1) before the EA intervention each time. The above-mentioned interventions were conducted 6 times a week for 2 consecutive weeks. Physical conditions of mice were assessed by exhaustive swimming tests. Histopathological changes of the liver were observed by H.E. staining. Western blot was used to detect the expression of AMPK, phosphorylated AMPK (p-AMPK), mTOR, phosphorylated mTOR (p-mTOR), ULK1 and phosphorylated ULK1 (p-ULk1) in the liver tissues. The expression levels of Atg5, Atg7, Atg13, Beclin1 and ULK1 (cellular autophagy-related genes) mRNAs in the liver were detected by quantitative real-time PCR. The immunoactivity (IA) of heme oxygenase 1 (HO-1) in the liver was detected by immunohistochemistry, and the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) of the liver were measured by hydroxylamine method for assessing the level of oxidative stress. RESULTS: Compared with the control group, the duration of exhaustive swimming, the expression levels of AMPK, p-AMPK, ULK1 and p-ULK1 proteins, and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNA, HO-1 IA and SOD activity were considerably down-regulated (P<0.01), while the expression levels of mTOR and p-mTOR and MDA content were significantly up-regulated (P<0.01) in the model group. In comparison with the model group, the duration of the exhausted swimming, the expression levels of AMPK, p-AMPK, ULK1 and p-ULK1 proteins, and Atg5, Atg7, Atg13, Beclin1 and ULK1 mRNAs, HO-1 IA and SOD activity were significantly up-regulated (P<0.01, P<0.05), whereas the expression levels of mTOR and p-mTOR proteins and MDA content were notably down-regulated (P<0.01, P<0.05) in the rapamycin, EA and EA+inhibitor groups. The improvement of the abovementioned indexes of EA+inhibitor group was not as good as rapamycin and EA groups (P<0.01), suggesting an elimination of the therapeutic effects after administration of 3-methyladenine. No significant differences were found between the rapamycin and EA groups in the abovementioned indexes (P>0.05) except p-mTOR and mTOR which were higher in the EA group (P<0.01). H.E. staining showed ambiguous boundary of the liver lobule, disordered arrangement of hepatocytes with a large amount of fat vacuoles at different size and deviation of nucleus, and lysis of some hepatocytes. These situations were relatively milder in the rapamycin and EA groups. CONCLUSION: EA may enhance physical strength and promote cellular autophagy in the liver of aging mice by regulating AMPK/mTOR/ULK1 signaling, thereby inhibiting excessive oxidative stress, and delaying aging process to some extent.

[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.