| Objectives:1.Evaluate the regulatory effect of electro-acupuncture on oxidative stress and energy metabolism in type 2 diabetic model rats,and investigate whether electro-acupuncture can achieve the goal of alleviating insulin resistance and intervening in type 2 diabetes by increasing the proportion of slow muscle fibers in skeletal muscles.2.Study whether electro-acupuncture can upregulate the expression of glucose transporter 4(GLUT4)protein in skeletal muscles by activating the adenosine 5’-monophosphate-activated protein kinase(AMPK)/peroxisome proliferator-activated receptor-Gamma coactivator 1 Alpha(PGC-1α)signaling pathway,and thus verify the feasibility of the proposed pathway of electro-acupuncture.Methods:96 four-week-old male SPF-grade Wistar rats were selected as experimental subjects.After one week of adaptive feeding,12 rats were randomly selected as the blank group and given regular feed and free drinking water.The remaining 80 rats were induced with high sugar and high fat diet for four weeks,and rats with insulin resistance characteristics and body weight exceeding the normal group were selected for intraperitoneal injection of streptozotocin(STZ)solution at a concentration of 35mg/kg,with 2 consecutive fasting blood glucose levels≥11.1mmol/L or(and)random blood glucose levels>16.7mmol/L taken as the criteria for diabetes model rats.The final 60 model rats were randomly divided into model group,electro-acupuncture group,metformin group,electro-acupuncture+metformin group,and electro-acupuncture+inhibitor group,and fed with high sugar and high fat diet.Electro-acupuncture group was treated with BL-20,EX-B3,ST36,and SP6 connected to an electro-acupuncture instrument,with needles retained for 20 minutes;metformin group was treated with synchronous oral administration of 5ml/kg of metformin solution and the same acupuncture intervention as the electro-acupuncture group;electro-acupuncture+metformin group was treated with the same intervention as the electro-acupuncture group and the metformin group;electro-acupuncture+inhibitor group was treated with the same intervention as the electro-acupuncture group and intraperitoneal injection of 10mg/kg of Compound C.The blank group and model group were treated with the same gripping and fixing methods as the intervention groups.The above interventions were performed once a day,and there was no intervention on the 7th,14th,21st,28th,35th,and 42nd days,for a total of 6 weeks of intervention.Each group of rats’ fasting blood glucose level was measured before and after intervention every week.After 6 weeks,serum was collected to detect fasting serum insulin levels using a radioimmunoassay.Insulin resistance index and pancreatic β-cell function index were calculated.Skeletal muscle paraffin sections were prepared for HE staining.Serum MDA,T-AOC,GSH,and SOD levels were measured using a colorimetric method,and serum ROS levels were detected using a fluorescence immunoassay.Detection of AMP/ATP ratio using high-performance liquid chromatography.The expression levels of IRS-1,Mfn1,Mfn2,Drp1,Fis1,Ampk-α1,Ampk-α2,p-Ampk,PGC-1α,and Glut4 in each group of rats were detected using Western Blot.The expression of Mhy7,Myh1,Mhy2,Myh4,ATP synthase,Ampk-α1,Ampk-α2,and PGC-1α genes in skeletal muscles of each group of rats was detected using qRT-PCR.The distribution of Ampkα1+α2,PGC-1α and Glut4 in skeletal muscles of each group of rats was detected using immunohistochemistry.Results:1.Glucose tolerance-related indicators:starting from the end of the third week of intervention,the fasting blood glucose levels of the electro-acupuncture group,metformin group,and electro-acupuncture combined with metformin group showed a significant decrease,which was lower than that of the model group(P<0.05);although there was no significant difference among the three groups,starting from the fourth week of intervention,the electro-acupuncture combined with metformin group showed a significant decrease in fasting blood glucose levels compared to before intervention(P<0.05),indicating that electro-acupuncture,metformin,and acupuncture-medication can effectively reduce blood sugar,but the combination of acupuncture and medication has a better hypoglycemic effect.The insulin resistance index and pancreatic β-cell function index of the electro-acupuncture group,metformin group,and electro-acupuncture combined with metformin group were lower than those of the model group(P<0.05),and there was no significant difference among the three groups,all of which could alleviate insulin resistance;there was no significant difference between the electro-acupuncture inhibitor group and the model group(P>0.05).Furthermore,in the study of skeletal muscle insulin resistance,it was found that the expression levels of IRS-1 protein in the skeletal muscle of rats in the electro-acupuncture group,metformin group,and electro-acupuncture combined with metformin group were higher than those of the model group and the electro-acupuncture inhibitor group(P<0.05).There was no significant difference in the expression level of IRS-1 protein in the skeletal muscle of rats between the electro-acupuncture combined with metformin group and the blank group(P>0.05),which also proves at the molecular mechanism level that the combination of acupuncture and medication has significant advantages in improving insulin receptor substrate expression and increasing glucose uptake.2.Oxidative stress-related indicators:The serum MDA and ROS levels of the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were lower than those of the model group and the electro-acupuncture inhibitor group(P<0.05).The mean score comparison among the three groups showed that the metformin group>electro-acupuncture group>electro-acupuncture plus metformin group,indicating that the accumulation of oxidative by-products was the least in the electro-acupuncture plus metformin group.The serum T-AOC,GSH,and SOD levels of the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were higher than those of the model group and the electro-acupuncture inhibitor group(P<0.05),suggesting that the three intervention methods could improve antioxidant capacity and alleviate oxidative stress.The advantage of the electro-acupuncture plus metformin group mainly lies in reducing the accumulation of oxidative by-products.In addition,the detection of skeletal muscle mitochondrial fusion and fission proteins showed that the expression levels of fusion proteins Mfnl and Mfn2 in the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were higher than those of the model group and the electro-acupuncture inhibitor group(P<0.05),and the expression levels of fusion proteins in the electro-acupuncture plus metformin group were higher than those in the electro-acupuncture group and the metformin group(P<0.05).The expression levels of the skeletal muscle fission proteins Drpl and Fisl in the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were lower than those of the model group and the electro-acupuncture inhibitor group(P<0.05).The expression level of Drpl protein in the electro-acupuncture plus metformin group was lower than that in the electro-acupuncture group and the metformin group(P<0.05).Both mitochondrial fusion proteins in the electro-acupuncture plus metformin group showed overexpression,and the expression level of fission protein was also lower than that in the single intervention group,indicating a better effect in inhibiting cell apoptosis and improving mitochondrial function.3.Skeletal muscle fiber energy metabolism-related indicators:The expression levels of I-type slow muscle fiber genes in the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were higher than those in the model group and the electro-acupuncture inhibitor group(P<0.05),and there was no significant difference compared with the blank group(P>0.05).There was no significant difference in the expression levels of slow muscle fiber genes among the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group(P>0.05),indicating that all three interventions could improve slow muscle fiber gene expression in rats.Comparing the expression levels of fast muscle fiber genes in each group of rats,it was found that the expression levels of Ⅱx fiber genes in the model group and the electro-acupuncture inhibitor group were higher than those in the blank group(P<0.05),and there was no significant difference between the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group and the blank group(P>0.05).The mean score comparison showed that the model group>electro-acupuncture inhibitor group>metformin group>electro-acupuncture plus metformin group>electro-acupuncture group>blank group.The three interventions could to some extent lower the Ⅱx fiber gene expression level.There was no significant difference in the expression levels of Ⅱa fast muscle fiber genes among the groups of rats(P>0.05),and the mean score comparison showed that the electro-acupuncture inhibitor group>model group>electro-acupuncture group>metformin group>blank group>electro-acupuncture plus metformin group.The expression levels of Ⅱb fast muscle fiber genes in the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were lower than those in the model group and the electro-acupuncture inhibitor group(P<0.05).There was no significant difference in the expression levels of Ⅱb fast muscle fibers in the electro-acupuncture plus metformin group compared with the blank group(P>0.05),and there was no significant difference in the expression levels of Ⅱb fast muscle fibers in the electro-acupuncture inhibitor group compared with the model group(P>0.05).Overall,the results suggest that the effect of the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group on Ⅱa fiber type is minimal,while the combination of acupuncture and medication has the best effect on lowering Ⅱb fiber type.When comparing ATPase gene expression levels,there was no significant difference between the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group compared with the blank group(P>0.05),but the expression levels were higher in the model group and the electro-acupuncture inhibitor group(P<0.05).There was no significant difference between the electro-acupuncture inhibitor group and the model group(P>0.05).This suggests that all three interventions,including electro-acupuncture,metformin,and acupuncture and medication,could improve ATPase gene expression levels,effectively improving skeletal muscle energy metabolism.4.AMPK/PGC-1α pathway-related indicators:The electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group had a higher AMP/ATP ratio in skeletal muscle compared to the model group(P<0.05),and there was no significant difference compared to the blank group(P>0.05).Protein-level tests showed that the expression levels of Ampkal,Ampka2,p-Ampk,and PGC-1α proteins in the skeletal muscle of the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were higher than those in the blank group,model group,and electro-acupuncture inhibitor group(P<0.05).The expression levels of Glut4 protein in skeletal muscle of the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group were higher than those in the model group and the electro-acupuncture inhibitor group,and there was no significant difference compared to the blank group(P>0.05).There was no significant difference among the three groups(P>0.05),and the expression level of Glut4 protein in the electro-acupuncture inhibitor group was lower than that in the model group(P<0.05).At the gene level,there was no significant difference in the expression levels of Ampkal and Ampka2 genes in skeletal muscle between the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group compared with the blank group(P>0.05),but these levels were higher than those in the model and electro-acupuncture inhibitor groups(P<0.05).There was no significant difference in the expression level of Ampkα1 gene between the electro-acupuncture inhibitor group and the model group(P>0.05).The expression levels of PGC-la gene in skeletal muscles showed no significant difference between the electro-acupuncture group,metformin group,and blank group(P>0.05).The electro-acupuncture plus metformin group had a higher expression level of PGC-1α gene compared to the blank group(P<0.05),and all three groups had a higher expression level of PGC-lα gene compared to the model group and electro-acupuncture inhibitor group(P<0.05).The electro-acupuncture plus metformin group had a higher expression level of PGC-1α gene compared to the electro-acupuncture group and metformin group(P<0.05).This suggests that the electro-acupuncture group,metformin group,and electro-acupuncture plus metformin group can all enhance the ability of skeletal muscles to transport glucose by activating the AMPK/PGC-1α signaling pathway through the increase in the AMP/ATP ratio.The result of the electro-acupuncture inhibitor group indicates that the mechanism of electro-acupuncture is closely related to this pathway.Moreover,the combination of acupuncture and medication is more effective in increasing the expression level of PGC-1α compared to single intervention.Conclusions:1.The combination of electro-acupuncture at the spleen Yu,stomach Wanxia Yu,Zusanli,and Sanyinjiao points can alleviate the symptoms of T2DM in rats,effectively reduce blood sugar,improve insulin resistance in T2DM rats,and protect pancreatic β-cells.The combination of electro-acupuncture and metformin is more advantageous in reducing skeletal muscle insulin resistance.2.Electro-acupuncture can protect mitochondrial function by increasing the expression of skeletal muscle cell mitochondrial fusion protein,decreasing the expression of fission protein,relieving oxidative stress,mediating skeletal muscle fiber type transition,up-regulating slow muscle fiber gene expression,promoting aerobic metabolism,improving energy metabolism,and thereby alleviating T2DM skeletal muscle insulin resistance.The combination of acupuncture and medication is more effective than electro-acupuncture or metformin alone in reducing the accumulation of oxidative byproducts,inhibiting myocyte apoptosis,improving mitochondrial function,and increasing the proportion of slow muscle fibers.3.The mechanism of electro-acupuncture improving skeletal muscle insulin resistance is closely related to the activation of AMPK phosphorylation,the increase in skeletal muscle AMPKα1 and α2 protein and gene expression levels,and the downstream opening of the PGC-1α pathway,which improves the ability of muscle cells to transport glucose.The abnormality of various indicators in electro-acupuncture rats after AMPK inhibitor intervention further indicates the importance of the AMPK/PGC-1α signaling pathway in the mechanism of electro-acupuncture.Moreover,the combination of acupuncture and medication is more effective in increasing the expression level of PGC-1α compared to single intervention. |
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