| With the rapid development of economic and the improvement of people's living standard,the incidence rate of Diabetes is increasing year by year.One of the most common complications of Diabetes is Diabetic Nephropathy(DN).The incidence rate of diabetic nephropathy is about 20%-30%of diabetes mellitus.DN is one of the main reasons leading to End Stage Renal Disease(ESRD).There are many reasons for DN.Generally speaking,it can be divided into two parts:congenital factors and postnatal habits.It's pathological mechanism includes oxidative stress reaction.insufficient insulin secretion,decreased sensitivity of tissue cells to insulin effect.microinflammatory reaction.etc.It's pathological changes including glomerulosclerosis and glomerular filtration membrane destruction,vacuole like changes of renal tubular epithelial cells and renal interstitial fibrosis.At present,it is clinically believed that in the development of diabetes,immune system,oxidative stress,endocrine disorders,abnormal fat metabolism and other factors interact with inflammatory factors,which can eventually lead to the occurrence of DN.At present,it is clinically believed that Pyroptosis will occur during the development of diabetes.Through the Pyroptosis signaling pathway NLRP3/Caspase-1/GSDMD,the integrity of the cell membrane is destroyed,and the cell membrane loses the ability to regulate the entry and exit of substances.The cell swell because a large number of small inorganic molecules enter the cell,causing the cell membrane to dissolve and the cell to disintegrate.As a results,a large amount of IL-1? and IL-18 are released into the environment around the cell,and released the pro-inflammatory signal to affect the surrounding cells at the same time.The surrounding cells are activated,recruit inflammatory cells,and eventually pyrolysis will occur.Purpose:The animal model of DN rats with type 2 DM was established by using highfat feed+low-dose STZ to observe the effect of Tangshenping on the pharmacodynamics and oxidative stress level.Observing the pathological morphology of kidney tissue in DN rats through HE and Mallory staining and transmission electron microscopy.Testing the expression of GSDMD,NLRP3,IL1?,Caspase-1,IL-18 protein and mRNA in kidney tissue through RT-PCR,immunohistochemistry,in-situ-hybridization,and Western blotting.Studing the protective effect of Tangshenping on the kidneys of DN rats and its effects on the NLRP3/Caspase-1/GSDMD signal pathway to provide the basis for the theory of"Kidney Atrophy".Method:1.42 male SD rats(weight of 200±20g)were used in the current experiments.SD rats were fed in clean animal cupboard,dieting freely.After 7 days of adaptive feeding,rats were divided into 10 controls and 60 models.The models was fed with high fat and sugar diet,and the controls was fed with ordinary diet.The feeding environment was room temperature(23±2)?,relative humidity(55±10)%.alternating cycle of day and night.2.After 8 weeks of high-fat diet,the rats in the models were injected with lowdose streptozotocin(35mg/kg)once,and the blood glucose in the tail vein was measured 48 hours later.When the random blood glucose was ?16.7mmol/l,and the urine glucose testing was positive(++++),it was considered that the establishment of DN models was successful.The models were fed with high-fat diet continuously,and the rats in the controls were fed with ordinary diet.3.The models were randomly divided into model group,irbesartan group.Tangshenping low dose group,middle dose group and high dose group according to the weight range.Each dose group of Tangshenping was given according to the clinical dosage,which was 3.5 times(0.525mg/kg·D-1)of the clinical dosage for the small dose group,7 times(1.05mg/kg·D-1)for the medium dose group,14 times(2.1mg/kg·D-1)for the large dose group,and 10 times(25mg/kg·D-1)for the Irbesartan group.The treatment groups were administrated by gavage.Controls and models were administrated by the same volume of deionized water.According to the weight coefficient of 0.1ml/100g/day,the gavage dose was calculated.The models and the drugs were fed with high-fat diet and free water.Replacing the litter every day and taking samples after 15 weeks.4.Observing the performence of rats,including weight,mental state,activity,appearance,fur,weight change,etc.Monitoring blood lipid,oxidative stress index,inflammation index,24-hour urine protein quantitative.The changes of Caspase-1,NLRP3,GSDMD,IL-1?,IL-18 in renal tissue were observed by immunohistochemistry,in situ hybridization,RT-PCR and Western blottingting.Result:1.General status:Compared with the models,the controls and Tangshenping in small,medium and large dose were significantly improved in body weight.After 15 weeks of administration in drugs,the body mass of rats in middle and large dose Tangshenping group was significantly increased(P<0.01),the ratio of kidney mass to body mass was significantly reduced(P<0.01),the 24-hour urine protein quantity was significantly reduced(P<0.01),and the serum BUN content was significantly reduced.The level of TG and SCR decreased(P<0.01),and the pathological changes of kidney decreased.Compared with the models,the thickness of glomerular basement membrane in Tangshenping high dose group was significantly thinner(P<0.01).The oxidative stress indexes in Tangshenping groups were improved,the level of NO,SOD was significantly increased(P<0.01),and the level of MDA was significantly reduced(P<0.01).2.The effect of Tangshenping on the signal transduction pathway of NLRP3/Caspase-1/GSDMD in renal tissue of DN rats induced by high-fat diet and low-dose STZ:compared with the models,the levels of Caspase-1,NLRP3,GSDMD,IL1-?,IL-18 protein and mRNA in Tangshenping groups were significantly reduced,and Tangshenping high-dose group was better.Conclusion:1.Tangshenping can improve the symptoms of diabetic nephropathy in DN rats,improve the oxidative stress state in DN rats,reduce the damage of DN,reduce the urine protein in DN rats,protect the renal function,delay the micro inflammatory state and the process of cell death in DN rats,and its effect is dosedependent.2.Tangshenping can reduce the expression of IL-1? and IL-18 of DN rats,so as to inhibit the micro inflammatory state in DN rats and delay the process of proptosis.3.Tangshenping can inhibit the development of proptosis and inflammation by interfering with the expression of protein and mRNA in NLRP3/Caspase-1/GSDMD signal transduction pathway. |
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