Bio-analysis Of Caffeic Acid Para-nitrophenylethyl Ester Against Visceral Tissue Damage And Insulin Resistance Related Proteins In Type 2 Diabetic Mice

The typical pathological feature of type 2 diabetes mellitus is insulin resistance,which accompanied with a relative decrease in insulin secretion caused by dysfunction of beta cells.Insulin insufficiency results in elevated blood glucose and lipid levels,resulting in pathological damage to pancreas,liver and heart,and eventually leading to complications of type 2 diabetes mellitus.In type 2 diabetes mellitus,the changes of blood-related indicators and insulin resistance-related proteins reflect the development of complications of type 2 diabetes mellitus,and many of these indicators and factors have become targets for the treatment of type 2diabetes mellitus.It is an important method to detect the therapeutic effect of drugs by analyzing the changes of expression of these factors in vivo after drug treatment.Previous studies have found that para-nitrocaffeic acid phenylethyl ester?CAPE-pNO₂?had better anti-inflammatory,anti-oxidation,anti-tumor and anti-diabetic complications than natural caffeic acid phenylethyl ester?CAPE?.In this paper,the effects of CAPE-pNO₂ on blood parameters,morphological changes and insulin resistance regulatory proteins in type 2 diabetic mice were analyzed.The research was divided into three parts:1)the changed of blood parameters were detected in model mice after treated with CAPE-pNO₂;2)the morphological changes of pancreas,liver and heart tissues were analyzed in model mice after treated with CAPE-pNO₂ by histological method.3)the expression of insulin resistance-related protein were analyzed in model mice after treated with CAPE-pNO₂ by Western blotting.The model of type 2 diabetic mice:six-week-old male Kunming mice were fed with high-fat diet for one month,and then injected with the dosage of 40 mg/kg/day streptozotocin?STZ?for five days to induce type 2 diabetic model.Fifty mice were randomly divided into five groups:1)the model control group was intraperitoneally injected with the same volume of saline,2)the CAPE group was intraperitoneally injected with the 10 mg/kg/day dosage of CAPE,3)the Low-pNO₂ group was intraperitoneally injected with the 5 mg/kg/day dosage of CAPE-pNO₂,4)the Medium-pNO₂ group was intraperitoneally injected with the 10 mg/kg/day dosage of CAPE-pNO₂,5)the High-pNO₂ group was intraperitoneally injected with the 15mg/kg/day dosage of CAPE-pNO₂.6)Another blank control group was set,and fed with normal diet and intraperitoneally injected with the same volume of saline.The weight of mice was monitored every day and fasting blood glucose of mice was noted every three days during administration.After one month of intraperitoneal injection,the mice were executed for i.p.glucose tolerance assay.The mice were euthanized,then the pancreas,liver,heart tissues and blood samples were collected.The changed of blood biochemical indexes:the results of blood glucose test showed that CAPE-pNO₂ could control the level of fasting blood glucose in type 2diabetic mice,the results of automatic biochemical analyzer showed that CAPE-pNO₂could reduce the levels of total cholesterol?TC?,triglyceride?TG?,low density lipoprotein total cholesterol?LDL-C?,creatine kinase?CK?,lactate dehydrogenase?LDH?,alanine aminotransferase?ALT?and aspartate aminotransferase?AST?.The results of ELISA showed that CAPE-pNO₂ could decrease the serum insulin level of model mice.In addition,the results of glucose tolerance test showed that CAPE-pNO₂could increase the glucose tolerance of mice;the results of HOMA-IR showed that CAPE-pNO₂ decreased the insulin resistance level of type 2 diabetic mice.The changed of morphological in tissue damage in type 2 diabetic mice:the histomorphological changes of pancreas,liver and heart were analyzed by HE staining,the contents of hepatic glycogen were analyzed by PAS staining,and the changes of islet and islet beta cells were analyzed by immunofluorescence staining.HE staining of pancreas,liver and heart tissues showed that CAPE-pNO₂ improved the pathological damage of pancreas,liver and heart tissues in type 2 diabetic mice;PAS staining showed that CAPE-pNO₂ increased hepatic glycogen synthesis in liver tissues;immunofluorescence staining showed that CAPE-pNO₂ increased the size of islet and the number of islet?cell.The expression of insulin resistance-related proteins in type 2 diabetic mice:the expression levels of p-AMPK,p-Akt,p-JNK,GLUT4,PPAR?and GSK3?in liver tissues were analyzed by Western blotting.The results showed that CAPE-pNO₂ could increase the expression of p-AMPK and GLUT4,it indicated that CAPE-pNO₂ could increase the utilization of glucose;CAPE-pNO₂ could increase the expression of p-Akt and decrease the expression of GSK3?,it indicated that CAPE-pNO₂ could increase the synthesis of hepatic glycogen and decrease the output of hepatic glucose;CAPE-pNO₂ could increase the expression of PPAR?and decrease the expression of p-JNK,it indicated that CAPE-pNO₂ could enhance fatty acid oxidation.The above analysis results of physiological and biochemical indexes and morphological changes of organs and protein expression levels in type 2 diabetic mice indicated that CAPE-pNO₂ had better effects on controlling blood glucose and decreasing blood lipid and alleviating tissue damage and improving the islet morphology and protecting the islet?cell and improving liver insulin resistance,especially on improving insulin resistance level of type 2 diabetic mice.This might be concerned with the intake of glucose in liver tissues,promoting fatty acid oxidation,reducing liver glucose output and increasing liver glycogen synthesis.