Based On MST1, The Mechanism Of Intervention Of T2DM By Electroacupuncture "Yupu", "Spleen" And "Shenshu" Was Explored

BACKGROUND:Type 2 diabetes mellitus(T2DM)has a high morbidity,mortality and economic consumption,brought about a heavy burden to the community and the family.Previous studies on T2DM paid more attention to insulin resistance,and oral drugs are mainly insulin sensitizer and insulin secretion agent.While beta cell dysfunction and reduced beta cell mass play an important role in the onset and development of T2DM,there are no drugs can inhibit the failure and apoptosis of beta cell.To restore functional beta cell mass through suppressing MST1 may be an promising target to inhibit the onset or development of T2DM.Clinical and experimental studies have proved that acupuncture is effective for treatment of T2DM.Yishu(Weiwanxiashu),Pishu(BL20)and Shenshu(BL23)are commonly used points for treating T2DM,and these points are all located in the nerve segments associated with pancreas.This study was designed based on above theories,to observe whether there were differences in the therapeutic effects on T2DM model rats among electroacupuncture at the Yishu,BL20 and BL23 which are located in the nerve segments associated with pancreas,as well as to explore whether electroacupuncture at the three back-shu points affect the beta cell function and apoptosis through the MST1-related pathway.This experiment is to attract people to pay more attention to the impotence of beta cell function and beta cell apoptosisin in the development of T2DM;as well as to reflect the advantage of acupuncture to intervene T2DM through many ways compared to existing drugs.MST1 is the upstream indicators of many pathway that regulates beta cell function and apoptosis,the study focused on the MST1,so as to put forward a new focus for the study of acupuncture treatment for T2DM,and to explore the mechanism from a more macro perspective.In addition,if it has been verified that acupuncture has a benign effect on regulating MST1,it can promote the research of efficacy and intervention mechanism of acupuncture on T1DM,so as to expand the application range of acupuncture.OBJECTIVE:1.By investigating the effects of electroacupuncture at the Yishu,BL20 and BL23,on the body weight,fasting blood glucose(FBG),oral glucose tolerance test(OGTT),glycated serum protein(GSP),serum lipid,fasting insulin(INS),insulin resistance index(HOMA-IR),and islet beta cell morphology of the T2DM model rats,to observe the effects of electroacupuncture at the Yishu,BL20 and BL23 on T2DM,and to compare the advantages and disadvantages.2.By investigating the effects of electroacupuncture at the Yishu,BL20 and BL23,on the expression of MST1 gene and protein,MST1 protein cleavage and phosphorylation,activation of BIM and caspase-3 protein,expression of PDX1 protein,expression of insulin gene and protein of the T2DM model rats,to observe the effects of electroacupuncture at the Yishu,BL20 and BL23 on the islet beta cell apoptosis and function of T2DM model rats,so as to explore the mechanism of electroacupuncture at the Yishu,BL20 and BL23 on T2DM,and to compare the advantages and disadvantages.METHODS:Forty-eight male SD rats were randomly divided into 2 groups,normal group(n=8,fed with ordinary rodent chow)and type 2 diabetes mellitus model building group("model building group" for short,n=40).Rats in the model building group were fed with high-fat and high-sugar fodder(HFSD)for 50 days,and then injected intraperitoneally with a freshly prepared streptozotocin(STZ)solution at 35mg/kg.BW.Blood samples were collected from the tail vein after 72 hours of injection and random blood glucose was measured by Roche blood glucose meter.OGTT was performed on rats after 2 weeks of STZ injection.Rats with random blood glucose>16.7mmol/L and OGTT 2h time-point blood glucose?11.1mmol/L were selected as successful T2DM model rats.Then T2DM model rats were divided into 4 groups according to the blood glucose level:model group(n=8),electroacupuncture(EA)Yishu group(n=8),EA BL20 group(n=8),EA BL23 group(n=8).The EA groups were intervened once a day,20min for each time,6 times a week,continuous intervention for 4 weeks.Observe and record the general situation of rats daily,test weight and fast blood glucose(FBG)of rats at each weekend,OGTT was performed before and after the intervention,and the area under the curve(AUC)was calculated.After 4-weeks intervention,rats were sacrificed,collected the blood from abdominal aorta,then collected blood serum by centrifuging.The level of serum triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL),high density lipoprotein(HDL)and glycated serum protein(GSP)were measured by automatic biochemical analyzer.And measured the level of insulin with radioimmunoassay.Take a part of the pancreatic tissue,and then fixed it with paraformaldehyde,which would be used to observe the islet morphology with HE staining and used to detected the expression of MST1,clMST1,pMST1,BIM,caspase-3,PDX1 and insulin protein in pancreatic beta cells by immunohistochemical method.Take another part of the pancreatic tissue to be fixed with liquid nitrogen,to be used to detected the expression levels of MST1 and insulin genes by Real-time PCR.RESULTS:1.Blood glucose metabolism related indicators:During the 4 week intervention,the body weight of rats in the model group was basically decreased,and it was decreased significantly after 4 weeks compared with that before the intervention.While FBG of rats in the model group has been maintained at a high level,after 4 weeks it were significantly higher than that before intervention.GSP also showed that the average blood glucose level in the past 2-3 weeks of rats in the model group was significantly higher than the normal group.The OGTT results showed that the glucose tolerance state of rats in the model group were significantly impaired before and after intervention compared with those of rats in the normal group;after 4 weeks intervention,the area under the curve of OGTT was significantly increased compared with that before intervention,consistent with the change trend of FBG.After 4 weeks intervention,HOMA-IR of rats in the model group was also significantly higher than that of rats in the normal group.Overall,it showed that after 4 weeks,the blood glucose levels,glucose tolerance,and insulin resistance worsened in the model group,and T2DM progressed.The body weight of the rats in the EA Yishu group changed consistent with the trend of that of the model group during 4 weeks of intervention,and there were no significant differences in comparison with that of the model group at any time.Compared with itself,FBG was significantly lower in the first week of the intervention than that before intervention,but remained at a high level since the second week of the intervention.Compared with the model group,the level of FBG in EA Yishu group was significantly decreased at the end of the second and fourth week after intervention.GSP levels also showed a significant decrease in blood glucose levels over the past 2-3 weeks compared with the model group.OGTT results changed consistent with the change trend of FBG,there was no significant change before and after intervention,but the area under the curve after 4 weeks of intervention was significantly lower than that of the model group.After 4 weeks of intervention,HOMA-IR in the EA Yishu group was significantly lower than that in the model group.In general,the intervention of EA at Yishu acupoint on the T2DM model rats for 4 weeks,could significantly inhibit the increase of blood glucose,improve the impaired glucose tolerance and the deterioration of insulin resistance,and inhibit the development of T2DM.The body weight of the rats in the EA BL20 group changed consistent with the trend of that of the model group and the EA Yishu group during 4 weeks of intervention,and there were no significant differences in comparison with that of the model group and the EA Yishu group at any time.Compared with itself,FBG was significantly lower in the first and second week of the intervention than that before intervention,but rebound significantly in the third and fourth weeks of the intervention.Compared with that of the model group at the same time,the FBG level declined at every weekend,but the difference was not significant.GSP levels also showed a significant decrease in blood glucose levels over the past 2-3 weeks compared with the model group.OGTT results changed consistent with the change trend of FBG,there was no significant change before and after intervention,and the area under the curve after 4 weeks of intervention was decreased than that of the model group,but the difference was not significant.After 4 weeks of intervention,HOMA-IR in the EA BL20 group was also significantly lower than that in the model group.Overall,the intervention of EA at BL20 acupoint on the T2DM model rats for 4 weeks,could inhibit the increase of blood glucose,improve the impaired glucose tolerance and the deterioration of insulin resistance,but not as significant as EA at the Yishu.The body weight of the rats in the EA BL23 group changed consistent with the trend of that of the model group,the EA Yishu group and theEA BL20 group during 4 weeks of intervention,and there were no significant differences in comparison with that of the model group the EA Yishu group and theEA BL20 group at any time.Compared with itself,FBG was significantly lower in the first week of the intervention than that before intervention,but remained at a high level since the second week of the intervention.Compared with the model group,the FBG level of the rats in the EA BL23 group decreased at the third and fourth weekend,but the difference was not significant;while compared with that of the rats in the EA BL20 group,the FBG level was higher than that of the EA BL20 group at the second weekend of the intervention,that is to say the hypoglycemic effect of the EA at BL20 was better than EA at BL23 at the second weekend of the intervention.GSP levels also showed a significant decrease in blood glucose levels over the past 2-3 weeks compared with the model group.OGTT results changed consistent with the change trend of FBG,there was no significant change before and after intervention,and the area under the curve after 4 weeks of intervention was decreased than that of the model group,but the difference was not significant.After 4 weeks of intervention,HOMA-IR in the EA BL23 group was also significantly lower than that in the model group.Overall,the intervention of EA at BL23 acupoint on the T2DM model rats for 4 weeks,could inhibit the increase of blood glucose,improve the impaired glucose tolerance and the deterioration of insulin resistance,but not as significant as EA at the Yishu.2.Blood lipid-related indicators:The TC,TG and LDL levels of rats in the T2DM model group were significantly higher than those in the normal group,while the HDL level of rats in the T2DM model group was not significantly changed compared with that of the normal group.This is consistent with the dyslipidemia characteristics HFD/STZ model rats.EA at the Yishu on T2DM model rats for 4 weeks reduced the levels of TC,TG and LDL and improved lipid metabolism.EA at BL20 on T2DM model rats for 4 weeks could reduce LDL level,but had no significant effect on TC,TG and HDL levels.EA at BL23 acupuncture on T2DM model rats for 4 weeks reduced the levels of TC and LDL,but had no significant effect on TG and HDL levels.EA at Yishu had better effect on regulating TG level than EA at BL20.Overall,the ability of EA at Yishu to regulate dyslipidemia was more significant than that of EA at BL20 and BL23 acupoints.This may be one of the reasons for the much more significant effect of EA at BL20 on regulating blood sugar than that EA at BL20 and BL23 acupoints.3.Insulin level:There was no significant difference between the insulin levels in the model group and the normal group.While EA at Yishu can significantly reduce the insulin levels compared with the model group,EA at BL20 and BL23 points have a tendency to lower insulin levels,but the effect is not significant.4.MST1 related mechanism indicators:The expression of MST1 gene and protein of rats in the model group increased significantly,and MST1 protein cleavage and phosphorylation level also increased,that was,MST1 protein activation increased.The expression of pro-apoptotic factors BIM protein and cleavaged caspase-3 protein of the mitochondrial apoptotic pathway was increased,that was,the activation of BIM and caspase-3 was increased and cell apoptosis was increased.The expression of PDX1 protein was decreased,and the expression of INS gene and protein was also decreased,that was to say the function of beta cell to synthesize insulin was reduced.EA at Yishu and EA at BL20 could significantly reduce the expression of MST1 gene and protein,and inhibite he cleavage and phosphorylation of MST1 protein,that was to say,they could inhibit the activation of MST1 protein.So as to inhibite the activation of BIM and caspase-3,resulting in inhibition of apoptosis.At the same time,EA at Yishu and EA at BL20 could promote the expression of PDX1 protein and promote the expression of INS gene and protein,that was to say,they could promote the synthesis of insulin in pancreatic beta cells..EA at BL23 can also inhibit the expression of MST1 gene and protein,and inhibit the cleavage of MST1 protein,but its effect on phosphorylation was not obvious.However,it still could further inhibit the activation of BIM and caspase-3,so as to inhibit beta cell apoptosis.At the same time,EA at BL23 can significantly promote the expression of PDX1 protein,but it had no obvious effect on INS gene transcription,but it could significantly promote the expression of INS protein,which also reflect that EA at BL23 could promote the synthesis of insulin in pancreatic beta cells.It can be seen that,EA at Yishu,BL20 and BL23 acupoints could regulate the apoptosis and the function of beta cell by regulating MST1-related factors.CONCLUSION:1.The intervention of EA at Yishu,BL20 and BL23 acupoint on the T2DM model rats for 4 weeks,could significantly inhibit the increase of blood glucose,improve the impaired glucose tolerance and the deterioration of insulin resistance,and improve lipid metabolism in some extend.While EA at Yishu had a much obvious effect on regulating blood glucose and lipid metabolism than EA at BL20 and BL23.Therefore,Yishu has certain advantages and specificity to interfere with the treatment of T2DM.Since Yishu,BL20 and BL23 are all located at the nerve segments that associated with pancrease,but they overlap with the different nerve segments of the pancreas,we could not determine whether the differences among Yishu,BL20 and BL23 in effects and mechnanisms are related to the nerve segment,and the but the specific relationship remains to be further explored.2.EA at Yishu,BL20 and BL23 could regulate the apoptosis and the function of beta cell by regulating MST1-related factors,so as to regulating blood glucose and lipid metabolism.And there were no significant differences among EA at Yishu,BL20 and BL23 on regulating MST1-related factors.The specific mechanism of Yishu in intervention of T2DM still need futher exploration.