| The first part Literature ResearchInsulin resistance itself is not a disease.After consulting relevant literature,we found that "insulin resistance" is not mentioned in the name of the disease in Internal Medicine of Traditional Chinese Medicine,but this does not affect the effective intervention of traditional Chinese medicine.Under the guidance of the theory of Qi and blood,Yin and Yang in traditional Chinese medicine,syndrome differentiation and treatment can still be guided so as to block the evolution of insulin resistance and break the vicious cycle of syndrome[1].When belong to the category of "spleen" of traditional Chinese medicine,pancreas insulin resistance from the perspective of zang-fu syndrome differentiation,belong to despair an illusion of spleen,so the treatment when the spleen to reverse insulin resistance "gripper".In addition,we always pay attention to the five elements of traditional Chinese medicine-based on "see liver disease,liver and spleen","virtual wood soil by"closely related to hepatic insulin resistance.From the perspective of meridian treatment in traditional Chinese medicine,spleen and hate belong to the category of Yang Ming meridian diseases.In the Eastern Han Dynasty,Zhang Zhongjing put forward the treatment method of "needle foot Yang Ming,so that the meridian will be cured without transmission".Modern medicine regards insulin resistance as one of the core pathologies of metabolic syndrome,and it has also been widely regarded as the common pathogenesis of obesity,hypertension,non-alcoholic fatty liver disease,type 2 diabetes and other diseases[2].The dysfunction of insulin receptor and its target organs as well as post-receptor dysfunction are recognized as the main pathogenesis of insulin resistance,but there are relatively few studies on insulin resistance caused by chronic inflammation.The target organs involved in chronic inflammation are systemic,which has something in common with the holistic understanding of diseases in traditional Chinese medicine.Therefore,it is feasible to explore the mechanism of the action of traditional Chinese medicine by taking inflammation as the starting point.On the basis of previous studies,this study took ZDF(fa/fa)as the insulin resistance model and pioglitazone with effective effect as the reference object to explore the mechanism of acupuncture on the inflammatory damage of liver,kidney and islet,the important target organs of insulin resistance.The second part Experimental StudyObjectiveZDF(fa/fa)rats fed with special induction feed Purina#5C08 for 4 weeks were used as research subjects.The ZDF(fa/+)rats were used as blank control to observe the improvement effect of electricacupuncture(EA)on chronic inflammation of insulin resistance and to explore its molecular signaling mechanism.To provide beneficial evidence-based basis for EA treatment of insulin resistance and make it widely used in the clinical prevention and treatment of various metabolic diseases.MethodsBefore the experiment,30 male ZDF(fa/fa)rats were randomly divided into three groups according to random number table method:ZDF group(n=10),ZDF+EA group(n=10),ZDF+Pi group(n=10),and ZDF(fa/+)rats were divided into control group(n=10).ZDF(fa/fa)were fed a special induced diet for 4 weeks with Purina#5C08(Labdiet,Inc.,USA),and ZDF ZDF(fa/+)rats were fed a normal diet.Orbital venous sinus blood of rats in each group was collected,FINS,C-P,and HOMA-IR index were determined.In addition,IL-6 and INF-? were determined.Oral glucose tolerance test(OGTT)was used to evaluate insulin resistance of ZDF(fa/fa)rats.When the above indexes of ZDF(fa/+)rats fed Purina#5C08 diet were significantly decreased compared with ZDF(fa/+)rats fed ordinary diet(P<0.05).This indicates that the model of insulin resistance has been successfully constructed.After modeling,30 male ZDF(FA/FA)rats were randomly divided into three groups according to random number table method:ZDF group(n=10),ZDF+EA group(n=10),ZDF+Pi group(n=10),and ZDF(fa/+)rats were divided into control group(n=10).Rats in the ZDF+EA group were treated with electroacupuncture;rats in the ZDF+Pi group were given pioglitazone hydrochloride suspension(10mg/kg/d)by gavage;rats in the control group and the ZDF group were given constant volume distilled water by gavage.Rats in each group were gavaged for 8 weeks,once a day.When rats in ZDF group were stimulated,they were bound with special rat clothing.Rats in the other 3 groups were restrained for the same time every day without EA stimulation.Acupoints on both sides of Fenglong(ST40)and Sanyinjiao(SP6)were electrified.Density wave was used for 20min/time,once a day,5 times a week for 8 consecutive weeks.The weight of the rats were measured and recorded weekly during the intervention.After 8 weeks of intervention,all rats were anesthetized with intraperitoneal injection of 10%pentobarbital solution,and 6ml blood was extracted directly through abdominal aorta by opening the abdominal cavity.The serum was collected by centrifugation and the above indexes were measured again.In addition,TC,TG,HDL-C and LDL of each group were measured to evaluate the situation of blood lipid.In addition,the levels of C-P,TC,TG,HDL-C and LDL in each group were detected to evaluate the situation of blood lipid,and the levels of CRP,MCP-1,glutamic oxalacetic transaminase(ALT),glutamic pyruvic transaminase(AST),creatinine(CREA)and UREA nitrogen(UREA)in plasma of all rats were detected to evaluate the liver and kidney function.The morphological changes of liver,kidney,and islet were observed by HE staining and the ultrastructural changes were observed by electron microscopy.The expression of IKK?-I?B?/NF-?B P65 protein and gene in liver,kidney and pancreatic islet of ZDF rats were observed by RT-PCR and Western blot.IKK? and IRS-1 protein locations and their relationship in liver and pancreas were observed by double immunofluorescence assay.The apoptosis of islet ? cells was detected by TUNEL to evaluate the apoptosis of ?cells in insulin resistant rats and the ameliorate effect of EA treatment.Results1 Evaluation of model formation in ZDF(fa/fa)ratsAfter feeding Purina#5C08 for 4 weeks,compared with ZDF(fa/+)group,the FPG,FINS,HOMA-IR index,IL-6 and TNF-? of rats in ZDF,ZDF+ Pi and ZDF+EA groups were significantly increased(all P<0.05).The blood glucose values of ZDF(fa/fa)rats in different time periods were significantly higher than those of the control group(all P<0.05),and there was an interaction between the test time and the group(all P<0.05).The blood glucose value at OGTT 2h in the ZDF group was greater than 11.1.2 Effect of EA and pioglitazone on the weight of ZDF ratsThe body weight of ZDF rats in control group was significantly lower than that in the other three groups.Compared with the ZDF group,the body weight of rats in the ZDF+Pi group increased significantly from before to after intervention(P<0.05).After 2 weeks of intervention,the weight of rats in the ZDF+EA group was significantly decresed compared with that in the ZDF+Pi group(P<0.05).By the 8 weeks of intervention,the ZDF+EA group was compared with the ZDF group and the ZDF+Pi group,respectively.The trend was the same as that of the 5th week(P<0.05).3 Effects of EA on insulin resistance and inflammatory factors in ZDF(fa/fa)ratsAt the 8th weekend of intervention,compared with control group,the levels of FPG,FINS,HOMA-IR index and C-P of rats in ZDF group and ZDF+EA group were significantly decreased(P<0.05),and there was no significant difference between ZDF+Pi group and control group(P>0.05).Compared with ZDF group,the above indexes were also significantly decreased in ZDF+Pi group and ZDF+EA group(P<0.05),and ZDF+Pi group was significantly decreased than ZDF+EA group(P<0.05).Compared with control group,the levels of CRP,IL-6,MCP-1 and TNF-? in ZDF,ZDF+Pi and ZDF+EA groups were significantly decreased(P<0.05),and the expression levels of the above indexes in ZDF+Pi group and ZDF+EA group were significantly decreased compared with ZDF group(P<0.05),and ZDF+PI group was lower than ZDF+EA group(P<0.05).4 Effect of EA on blood lipid in ZDF(fa/fa)ratsAfter 8 weeks of intervention,compared with control group,the levels of TC,TG and LDL in ZDF group were significantly increased(P<0.05),while the level of HDL was significantly decreased(P<0.05).Compared with ZDF group,TC and TG levels in ZDF+Pi group and ZDF+EA group were significantly decreased(P<0.05),HDL level was significantly increased(P<0.05),and ZDF+Pi group was significantly decreased than ZDF+EA group(P<0.05).5 Effect of EA on liver and kidney function in ZDF(fa/fa)ratsCompared with control group,the levels of AST,ALT,CREA and UREA in ZDF group were significantly increased(P<0.05).Compared with ZDF group,TC and TG levels in ZDF+Pi group and ZDF+EA group were significantly decreased(P<0.05)and ZDF+Pi group was better than ZDF+EA group(P<0.05).6 Effect of EA on liver histomorphology in ZDF(fa/fa)ratsThe liver tissue was observed under a high power(400 times)light microscope.By comparing the difference of liver tissue between ZDF+EA group and ZDF group,it was suggested that electroacupuncture therapy could improve the vacuolar degeneration and cytoplasmic thinning of liver cells,which was second only to pioglitazone.At the same time,transmission electron microscopy showed that liver cell injury in ZDF+EA group was significantly improved compared with that in ZDF group.Mitochondria are round or dumbbell shaped,sparse distribution,clear structure,mild edema,part of the cleft crest.Rough endoplasmic reticulum was layered around the nucleus and mitochondria,and ribosomes were attached on the surface.No obvious damage was observed.It was second only to pioglitazone in protecting mitochondria and rough endoplasmic reticulum from inflammatory damage.7 Effect of EA on renal histomorphology in ZDF(fa/fa)ratsThe renal tissue was observed under a high power(400x)light microscope.The comparison of liver tissue pathology between ZDF+EA group and ZDF group suggested that electroacupuncture therapy could improve the inflammatory response of nephron,which was second only to pioglitazone.At the same time,the ultrastructural changes of each group were observed by transmission electron microscope.Compared with the ZDF group,renal damage was significantly improved in the ZDF+EA group.The number of mitochondria is small,oval or long,moderate swelling,local vacuolation,crest expansion,fracture and disappearance;Rough endoplasmic reticulum was slightly dilated with ribosome attachment on its surface.The Golgi apparatus is slightly dilated and hyperplastic.It is suggested that electroacupuncture is second only to pioglitazone in protecting mitochondria and rough endoplasmic reticulum from inflammatory injury.8 Effect of EA on islet morphology in ZDF(fa/fa)ratsThe morphology of islet tissue after HE staining was observed under high power(400x)light microscope.There was low concentration of inflammatory reaction in the islet tissue of ZDF(fa/fa)rats.By comparing the pathological changes of islet cells in the ZDF+EA group and the ZDF group,the morphology and structure of islet cells in the ZDF+EA group were improved compared with the ZDF group.Although there was a small amount of lymphocyte infiltration in the surrounding tissue,the boundary of islet cells was clear,and no obvious necrosis of islet cells was observed.At the same time,the ultrastructural changes of islet ? cells were observed by transmission electron microscopy.Mitochondrial structure of pancreatic islet 3 cells in both ZDF+EA and ZDF+Pi groups showed severe swelling,enlargement,absence of cristae,and unclear flocculent in the lumen.ZDF+EA group rat islet beta cells rough endoplasmic reticulum were neatly arranged,the number of rich,only mild expansion,including tips and pioglitazone may both by reducing the inflammatory cell infiltration in the pancreas tissue,and cupping mainly by inhibition of endoplasmic reticulum stress,improve function of endoplasmic reticulum steady state,and not dependent on the mitochondrial pathway.9 Effects of 10 EA on the protein expression levels of NF-?B P65,I?B?,IKK?,p-I?B?,p-NF-?B P65,p-IKK?,and IRS-1 in liver and kidney of ZDF(fa/fa)ratsCompared with control group,the expressions of NF-?B P65,I?B?,IKK?,p-I?B?,p-NF-?B P65 and p-IKK(3 in liver and kidney tissues of rats in ZDF group,ZDF+Pi group and ZDF+EA group were significantly increased(P<0.05),and the protein expression levels of IRS-1 were significantly decreased(P<0.05).Compared with the ZDF group,the protein expressions of NF-?B P65?I?B?,IKK?,p-I?B?,p-NF-?B P65 and p-IKK? in ZDF+EA group and ZDF+Pi group were significantly decreased(P<0.05),and the protein expression levels of IRS-1 were significantly increased(P<0.05),the ZDF+Pi group was also statistically significant compared with the ZDF+EA group(P<0.05).10 The effect of EA on the protein expression of p-NF-?B P65,p-I?B? and p-IKK? in islet of ZDF(fa/fa)ratsCompared with the control group,the expressions of p-NF-?B P65,p-I?B? and p-IKK? in the pancreatic islets of rats in the ZDF,ZDF+Pi and ZDF+EA groups were significantly increased(P<0.05).Compared with ZDF group,the protein expression levels of p-NF-?B P65,p-I?B? and p-IKK? in ZDF+EA group and ZDF+PI group were significantly decreased(P<0.05),and ZDF+Pi group was lower than ZDF+EA group(P<0.05).11 Expression of NF-?B P65,IKK? and I?B? in liver,kidney and pancreas of ZDF(fa/fa)rats by electrotherapyCompared with the control group,the mRNA expressions of NF-?B P65,IKK? and I?B? in ZDF group,ZDF+Pi group and ZDF+EA group were significantly increased(P<0.05).Compared with the ZDF group,the mRNA expression levels of NF-?B P65,IKK? and IKBa in ZDF+EA group and ZDF+Pi group were significantly decreased(P<0.05),and ZDF+Pi group was lower than ZDF+EA group(P<0.05).12 IKK(3 and IRS-1 protein locations and their relationship in liver and pancreas of ZDF rats were observed by double immunofluorescence assayAt high magnification(400X),IKK? and IRS-1 proteins were expressed in both liver and pancreas,and most of them were nuclear.In some cells,IKK? and IRS-1 were co-localized in the nucleus.The number of IRS-1 cells in the control group and the ZDF+EA group was higher than that in the IKK? group,while the number of IRS-1 cells in the ZDF group was significantly lower than that in the IKK? group.The ratio of IRS-1/IKK? in the control group and the ZDF+EA group was greater than 1,and the expression difference was more obvious in the control group,while the ratio of the ZDF group and the ZDF+Pi group was less than 1,and the expression difference in the ZDF group was smaller.13 Effect of EA on apoptosis of islet ? cells in ZDF ratsIn the control group,there was no obvious apoptotic islet ? cells,and the chromatin of the nuclei was normal and the nuclei were uniformly blue.However,no matter in the model group or the treatment group,only sporadic and a small amount of islet ?-cell apoptosis,chromatin concentration and green nuclei were observed under the fluorescence microscope.There was no significant difference in the apoptosis rate of islet ?-cell between ZDF group,ZDF+EA group and ZDF+Pi group(P>0.05).ConclusionIn this study,we found that EA can inhibit the activation of IKK?-I?B?/NF-?B P65 signaling pathway in liver,kidney and pancreatic islet of ZDF(fa/fa)rats,reduce the inflammatory response and increase the activity of insulin substrate IRS-1,thereby improving insulin resistance and protecting the liver and kidney function of ZDF(fa/fa)rats.Among them,IKK? may be a potential target for electroacupuncture intervention of insulin resistance.This provides favorable evidence for the widespread use of EA in the treatment of insulin resistance such as diabetes,obesity and cardiovascular disease. |
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