RESUMEN

OBJECTIVE: To investigate the mechanism of electroacupuncture （EA） in promoting the browning of white adipose tissue in middle-aged and aged obese rats induced by high fat by regulating AMP-activated protein kinase （AMPK） /silence-information regulatory factor 1 （Sirt1） pathway and neuregulin 4 （Nrg4）. METHODS: Twenty-four male SD rats were randomized into blank control, model and EA groups （n=8 per group）. The obesity model was established by feeding the rats with high-fat diet for 6 weeks. For the EA group, EA （2 Hz/15 Hz, 1.5 mA） was applied to "Guanyuan" （CV4） and bilateral "Shenshu" （BL23）, "Fenglong" （ST40） and "Tianshu" （ST25） for 20 min, once a day, 5 days a week for 6 weeks. Rats of the blank control and model groups were also restrained for 20 min. The body mass and food intake were measured every week, and the Lee's index, epididymal fat, perirenal fat and brown adipose tissue were weighed. The contents of serum total cholesterol （TC）, triglyceride （TG）, high density lipoprotein cholesterol （HDL-C）, low density lipoprotein cholesterol （LDL-C） and norepinephrine （NE） were determined by ELISA. H.E. staining was used to observe the morphological changes of white and brown adipose tissue. The mRNA expression levels of mitochondrial uncoupling protein 1 （UCP1）, peroxisome proliferator activated receptor γ co-activator 1α （PGC-1α）, PR-domain protein 16 （PRDM16）, peroxisome proliferator activated receptor γ （PPARγ） and Nrg4 in the adipose tissue were detected by quantitative real time PCR, and the protein expression levels of Nrg4, AMPKα, Sirt1 and interleukin-6 （IL-6） in the white and brown adipose tissue were detected by Western blot. RESULTS: Compared with the blank control group, the body mass, food intake, the Lee's index, epididymal fat and perirenal fat mass, and serum TG, TC and LDL-C contents and the expression level of IL-6 protein were significantly increased （P<0.01, P<0.05, P<0.001）, and the brown adipose mass, serum HDL-C and NE contents, the expression levels of UCP1, PGC-1α, PRDM16, PPARγ and Nrg4 mRNAs, and the protein expression levels of AMPKα, Sirt1 and Nrg4 proteins in both white and brown adipose tissues were significantly decreased in the model group （P<0.01, P<0.05）. After EA intervention, the increased levels of body mass, food intake, Lee's index, epididymal fat and perirenal fat mass, serum TG, TC and LDL-C contents, and the expression of IL-6 protein, and the decreased levels of brown adipose mass, serum HDL-C and NE contents, expression levels of UCP1, PGC-1α, PRDM16, PPARγ and Nrg4 mRNAs, and those of AMPKα, Sirt1 and Nrg4 proteins in both white and brown adipose tissues were apparently reversed（P<0.05, P<0.01, P<0.001）. H.E. staining showed an increase of the volume and content of intracellular vacuoles of both white and brown adipose tissues, disordered arrangement of cells with vague boundary in the model group, which was relatively milder including a decrease of volume and content of vacuoles of both white and brown adipose, neat arrangement of cells with clear boundary. CONCLUSION: EA intervention can improve lipid metabolism and promote white adipose tissue browning in middle-aged and aged obese rats, which is possibly associated with its functions in activating AMPK/Sirt1 signaling pathway and up-regulating the level of Nrg4.

Asunto(s)

Electroacupuntura , Metabolismo de los Lípidos , Animales , Masculino , Ratas , Tejido Adiposo Pardo , Tejido Adiposo Blanco , Proteínas Quinasas Activadas por AMP/genética , LDL-Colesterol , Interleucina-6 , Metabolismo de los Lípidos/genética , Obesidad/genética , Obesidad/terapia , PPAR gamma , Ratas Sprague-Dawley , Sirtuina 1/genética

RESUMEN

This study aims to investigate the neuroprotective effect of tetramethylpyrazine on mice after spinal cord injury and its mechanism. Seventy-five female C57BL/6 mice were randomly divided into 5 groups, namely, a sham operation group, a model group, a tetramethylpyrazine low-dose group(25 mg·kg~(-1)), a tetramethylpyrazine medium-dose group(50 mg·kg~(-1)), and a tetramethylpyrazine high-dose group(100 mg·kg~(-1)), with 15 mice in each group. Modified Rivlin method was used to establish the mouse model of acute spinal cord injury. After 14 d of tetramethylpyrazine intervention, the motor function of hind limbs of mice was evaluated by basso mouse scale(BMS) and inclined plate test. The levels of inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1ß(IL-1ß) in the spinal cord homogenate were determined by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe the histology of the spinal cord, and Nissl's staining was used to observe the changes in the number of neurons. Western blot and immunofluorescence method were used to detect the expression of glial fibrillary acidic protein(GFAP) and C3 protein. Tetramethylpyrazine significantly improved the motor function of the hind limbs of mice after spinal cord injury, and the BMS score and inclined plate test score of the tetramethylpyrazine high-dose group were significantly higher than those of the model group(P<0.01). The levels of TNF-α, IL-6, and IL-1ß in spinal cord homogenate of the tetramethylpyrazine high-dose group were significantly decreased(P<0.01). After tetramethylpyrazine treatment, the spinal cord morphology recovered, the number of Nissl bodies increased obviously with regular shape, and the loss of neurons decreased. As compared with the model group, the expression of GFAP and C3 protein was significantly decreased(P<0.05,P<0.01) in tetramethylpyrazine high-dose group. In conclusion, tetramethylpyrazine can promote the improvement of motor function and play a neuroprotective role in mice after spinal cord injury, and its mechanism may be related to inhibiting inflammatory response and improving the hyperplasia of glial scar.

Asunto(s)

Fármacos Neuroprotectores , Traumatismos de la Médula Espinal , Ratas , Ratones , Femenino , Animales , Ratas Sprague-Dawley , Fármacos Neuroprotectores/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6 , Ratones Endogámicos C57BL , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/genética , Médula Espinal/metabolismo

RESUMEN

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.

RESUMEN

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.

Asunto(s)

Electroacupuntura , Remielinización , Traumatismos de la Médula Espinal , Traumatismos Vertebrales , Animales , Axones , Trasplante de Células , Femenino , Ratas , Ratas Sprague-Dawley , Células de Schwann , Médula Espinal , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/terapia