The Study On The Effect And Mechanism Of Lizhong Decoction On Inhibiting Renal Fibrosis In Type 2 Diabetes Mellitus Rats

Part 1 Experimental study on Type 2 Diabetes Mellitus in Food-induced Obesity SD Rats.BackgroundHigh-fat diet combined with low-dose streptozotocin(STZ)is the most commonly used modeling method in experimental type 2 diabetic models.This method has been widely used in the study of type 2 diabetes mellitus and type 2 diabetic nephropathy.High-fat diet can induce food-borne obesity in animals.And the animal model of food-borne obesity has significant insulin resistance.Therefore,the injection of low-dose STZ into the model can greatly improve the modeling rate of type 2 diabetes mellitus model.However,the method of high-fat diet can only induce food-borne obesity in about 50%of animals.And there is no standard of STZ injection doses.It is still necessary to explore and improve the modeling methods of type 2 diabetes mellitus model.ObjectiveFirstly,the model of foodborne obesity in rats was observed after given different high-fat diet formulations,so as to find a better way to establish the rat model of food-borne obesity.Then,the effects of different doses of STZ on the establishment of type 2 diabetic rat model were investigated on the basis of food-induced obesity rat model.The above steps are intended to provide a reference for the establishment of type 2 diabetic rat model.MethodThis experiment is divided into two steps.The first step is to establish the rat model of food-borne obesity.And the second step is to establish the rat model of type 2 diabetes mellitus by injecting low dose STZ on the basis of the rat model of food-borne obesity.Step 1:male SD rats were randomly divided into control group,high fat and high sugar diet group and triple method group.The control group was fed with common diet,the high fat and high sugar diet group was fed with high fat and high sugar diet,and the triple method group was given high fat and high sugar diet+high fat milk gavage + free drinking of sucrose water.The rats fed with high fat and high sugar were weighed at the end of the 6th week.If the body weight?mean+2 standard deviation of the body weight in the control group,the food-induced obese rats were included respectively.After the food-induced obese rats were included,the daily food intake,drinking water and urine volume of the rats in each group were collected and calculated by metabolic cage,and the body weight of the rats was measured.Step 2:the food-borne obese rats were randomly divided into 7 groups according to different injection doses of STZ after fasted for 12 hours.These groups were:group A(original high fat and high sugar diet group,STZ 26mg/kg),group B(original high fat and high sugar diet group,STZ 28mg/kg),group C(original high-fat and high-sugar diet group,STZ 30mg/kg),group D(original triple method group,STZ 26mg/kg),Group E(original triple method group,STZ 28mg/kg),Group F(original triple method group,STZ 30mg/kg)and control group(intraperitoneal injection of the same dose of sodium citrate buffer).Fasting blood glucose was measured through caudal vein on 7 days,14 days,21 days and 28 days after STZ injection in different groups.Four consecutive fasting blood glucose greater than 11.lmmol/L was defined as type 2 diabetic rats.After type 2 diabetic rats were included in the study,the daily food intake,drinking water and urine volume of each group were collected and calculated by metabolic cage immediately,and the body weight of the rats was measured.On the 31st day after STZ injection,fasting blood glucose,total cholesterol,triglyceride and insulin were measured,and some pancreatic tissues were stained with HE.Result(1)After 6 weeks of high fat and high sugar diet,the modeling rate of food-borne obesity in the triple diet group(81.25%)was significantly higher(P<0.05)than that in the high fat and high sugar diet group(47.92%).(2)The food intake,drinking water and urine volume of group A to F after 4 weeks of STZ injection were significantly higher than those before STZ injection(P<0.05),and the body weight of group A to F after 4 weeks of STZ injection were significantly decreased than those before STZ injection(P<0.05).There was an obvious phenomenon of "three more and one less".(3)The fasting blood glucose,total cholesterol,triglyceride and insulin resistance index in group A to F were significantly higher than those in the control group(P<0.05)on the 31st day after injection of STZ.(4)Compared with the control group,on the 31st day after STZ injection,the islet tissue in group A to F showed obvious atrophy,the number and number of islets were relatively small,lipid deposition and uneven nuclear size appeared in some islet cells.(5)Although both groups C and F(STZ 30 mg/kg dose group)had animal death,there was no significant difference(P>0.05)in survival rate between group A to F after injection of STZ.The modeling rate of type 2 diabetes mellitus in group E(28mg/kg dose group)was significantly higher than that in group D STZ(STZ26mg/kg dose group)(P<0.05).Conclusion(1)Triple method feeding for 6 weeks is a better method to establish the rat model of food-borne obesity.(2)Triple method feeding for 6 weeks combined with one-off intraperitoneal injection of STZ 28mg/kg is a better method to establish the model of type 2 diabetes mellitus.Part 2 The Study on the effect and Mechanism of Lizhong decoction on inhibiting Renal Fibrosis in Type 2 Diabetes Mellitus.BackgroundDiabetes mellitus is the most common metabolic disease,and the main type of diabetes is type 2 diabetes mellitus.Diabetic nephropathy is one of the main chronic complications of diabetes mellitus,and it is also the main cause of end-stage renal disease.Studies have reported that 20%to 30%of patients with type 2 diabetes will develop diabetic nephropathy.As the incidence of type 2 diabetes mellitus increases,the incidence of diabetic nephropathy increases.Traditional Chinese medicine(TCM)has a long history in the treatment of diabetes mellitus so that it has accumulated a lot of experience.Summing up the experience of traditional and modern medicine in the treatment of diabetes mellitus,we can find that "qi xu" is the basic pathogenesis throughout the whole process of diabetes mellitus.At the same time,from the point of view of“treating diseases that have not yet occurred”,the treatment of diabetic nephropathy should Wenyang Jianpi,which nourished the kidneys by replenishing the spleen.Therefore,we think that the treatment of early diabetic nephropathy should adopt the therapy of“Yiqi Wenyang Jianpi".Lizhong decoction is a common prescription for the clinical treatment of diabetes mellitus and its complications.It has the function of“Wenzhong Quhan Yiqi Jianpi ".Therefore,Lizhong decoction can be used for the treatment of early diabetic nephropathy.However,little research about the effect of Lizhong decoction on type 2 diabetic nephropathy was investigated,which needs to be further studied.ObjectiveIn order to observe the effects of Lizhong decoction on renal function,renal morphology,protein expression of VEGFA,TGF-?1 and COL-1 and gene expression of MiR-200b in type 2 diabetic rats,so as to confirm that Lizhong Decoction has anti-fibrotic effect on the kidney of type 2 diabetic rats and to discuss its preliminary mechanism.MethodMale SD rats were randomly divided into control group and module group.The control group was fed with common diet and drinking water in animal house,and the model group was fed with triple diet of high fat and high sugar diet+high fat milk+sucrose water.The body weight was weighed after 6 weeks of continuous feeding.If the body weight of the module group?the body weight of mean+2 SD in the control group,the rats in the module group was defined as food-borne obese rats,otherwise the rest would be eliminated.After the food-induced obese rats were included,the rats in the module group were fed with high-fat and high-sugar feed and drinking water instead of the triple diet of high fat and high sugar diet+high fat milk+sucrose water in animal house until the end of the experiment.Then the food-induced obese rats were injected intraperitoneally with 1%STZ 28 mg/kg,while the rats in the control group were injected intraperitoneally with the same dose of sodium citrate buffer.Fasting blood glucose was measured through caudal vein on 7 days,14 days,21 days and 28 days after STZ injection in different groups.Four consecutive fasting blood glucose greater than 11.lmmol/L and accompanied by polydipsia,polyphagia,polyuria were defined as type 2 diabetic rats.Then the type 2 diabetic rats were randomly divided into three groups:model group,losartan potassium group and Lizhong decoction group.The rats in losartan potassium group were given losartan potassium intragastrically.The rats in Lizhong decoction group were given Lizhong decoction intragastrically.The rats in the control group and the model group were given the same volume of distilled water intragastrically for 12 weeks.At the end of 12 weeks after intervention,the 24-hour free diet urine of rats was collected with metabolic cage,the urine volume and the 24-hour urinary microalbumin were recorded.After 12 weeks of intervention,fasting blood glucose,total cholesterol,triglyceride,urea nitrogen,serum creatinine,glycosylated hemoglobin and fasting insulin were measured,and insulin resistance index was calculated.The body weight and kidney weight of rats were weighed and the renal weight index(renal weight/body weight)was calculated.Some renal cortical tissues were used for HE,PAS,PASM,MASSON staining.Pathological changes in kidney tissue were observed and photographed by light microscopy and transmission electron microscopy.The percentage of positive staining area of PAS,PASM and MASSON staining was calculated by software.Some renal cortex tissues were used to detect the expression of VEGFA,TGF-?1,COL-I protein and miR-200b gene by Western blot method and Real-time PCR respectively.Result(1)At 0 week,4 weeks,8 weeks and 12 weeks after intervention,the body weight of each group decreased compared with the control group(P<0.05).However,there was no significant change in the body weight of Lizhong decoction group and losartan potassium group compared with the model group(P>0.05).(2)After 12 weeks of intervention,the renal mass index of each group was significantly higher than that of the control group(P<0.05).Compared with the model group,the renal mass index of Lizhong decoction group and losartan potassium group decreased significantly(P<0.05).However,there was no significant difference between Lizhong decoction group and losartan potassium group(P>0.05).(3)After 12 weeks of intervention,fasting blood glucose,total cholesterol,triglyceride,creatinine,urea nitrogen,insulin resistance index,glycosylated hemoglobin and urinary Microalbumin in each group were significantly higher than those in the control group(P<0.05).Compared with the model group,blood glucose,total cholesterol,triglyceride,insulin resistance index and glycosylated hemoglobin in Lizhong decoction group were decreased(P<0.05),while there was no significant change in losartan potassium group(P>0.05).After 12 weeks of intervention,creatinine,urea nitrogen and urinary microalbumin were significantly decreased in Lizhong decoction group and losartan potassium group compared with the model group(P<0.05),but there was no significant difference between the two groups(P>0.05).(4)After 12 weeks of intervention,the pathological changes in Lizhong decoction group and losartan potassium group were significantly less than those in model group,and the positive rates of PAS,PASM and MASSON staining in Lizhong decoction group and losartan potassium group were lower than those in model group.There were significant difference(P<0.05).(5)After 12 weeks of intervention,the expression of VEGFA,TGF-?1 and COL-I protein in each group was higher than that in the control group(P<0.05).The expression of VEGFA,TGF-?1 and COL-I protein in Lizhong decoction group and losartan potassium group were significantly lower than that in model group(P<0.05),but there was no significant difference between Lizhong decoction group and losartan potassium group(P>0.05).(6)After 12 weeks of intervention,the expression of miR-200b gene in each group was significantly lower than that in the control group(P<0.05).The expression of miR-200b gene increased in Lizhong decoction group and losartan potassium group compared with the model group(P<0.05).However,there was no significant difference in miR-200b gene expression between Lizhong decoction group and losartan potassium group(P>0.05).Conclusion(1)Lizhong decoction could improve renal function in type 2 diabetic rats;(2)Lizhong decoction can improve the pathological changes of kidney and inhibit the progression of fibrosis in type 2 diabetic rats;(3)The anti-fibrosis effect of Lizhong decoction on kidney of type 2 diabetic rats may be related to lower blood sugar,and regulation of lipid metabolism and regulation of TGF-?1/miR-200b/VEGFA pathway.