Study On The Mechanism Of Tongluo Digui Decoction In Improving Diabetic Kidney Disease By Inducing Autophagy To Regulate Pyrolysis Of Renal Tubular Epithelial Cells

Objectives : This study intends to replicate the DKD mouse model to observe the effects of Tongluo Digui decoction on kidney cell autophagy,NLRP3 inflammasome and cell pyrolysis in diabetic mice.To explore the potential mechanism of Tongluo Digui decoction’s intervention in diabetic kidney disease.Research methods:1.With reference to the literature and previous research methods on this disease,C57BL/6J mice were injected intraperitoneally with streptozotocin to replicate type 1diabetes animal models.After 8 weeks,according to the quantitative results of urine protein,the successfully modeled mice were randomly divided into model groups,Tongluo Digui decoction high-dose group,Tongluo Digui decoction medium-dose group,Tongluo Digui decoction low-dose group and Rapamycin group,another normal control group was set up,and the related indexes were tested after 12 weeks of continuous gastric administration.2.After gavage,collect blood and urine specimens of mice for biochemical index detection.3.HE staining detects the pathological changes of mouse kidney tissue,Masson staining detects the level of collagen deposition in kidney tissue,and PAS staining detects the level of glycogen deposition in mouse kidney tissue.Electron microscopy observed the occurrence of renal tubular autophagy in mice.4.Immunohistochemistry,Western blotting and Real-time PCR were used to detect the expression levels of autophagy indicators Beclin-1 and p62 in kidney tissues,inflammatory body-related proteins NLRP3,IL-1 β,and pyrolysis-related proteins GSDMD and caspase-1 expression.Results:1.After 12 weeks of drug intervention,blood biochemical indicators were tested.Compared with the normal group,the blood glucose,urea nitrogen level,blood creatinine level and 24-hour urine protein quantitatively increased in the model group(P<0.01).Compared with the model group,the high and medium doses of Tongluo Digui decoction group can reduce blood sugar,urea nitrogen level,blood creatinine level and 24-hour urine protein level(P<0.05),while the effect of low dose group is not significant(P> 0.05);Compared with the normal group of mice,the C peptide and insulin levels of the model group were lower(P<0.01),while the high and medium doses of Tongluo Digui decoction intervention groups were significantly higher than the model group(P<0.05).2.The results of pathological staining of kidney tissue showed that compared with the normal group,the model group had increased glomerular volume,proliferation of mesangial matrix,enlarged mesangial area of the glomerulus,swelling,fusion,necrosis,and collagen deposition of renal tubular epithelial cells in the model group.Obviously,the intervention of Tongluo Digui decoction on the pathological damage of the kidney tissue in mice was significantly improved,but the low-dose group did not improve significantly.Electron microscope observation revealed that the number of autophagosomes in the renal tubules of mice increased after the intervention of Tongluo Digui decoction,and the high dose group of Tongluo Digui decoction was significant.3.The expression levels of autophagy-related protein,NLRP3 inflammasome and pyrolysis-related protein in mouse kidney tissues were detected.Compared with the normal group of mice,the autophagy-related protein p62 protein,NLRP3 protein,IL-1β protein and the pyrolysis-related protein GSDMD and caspase-1 levels in the kidney tissue of the model group were significantly increased(P<0.01);The high and medium doses of Tongluo Digui decoction decreased to varying degrees after intervention(P<0.01,P<0.05),but the low dose group did not improve significantly(P>0.05);The level of autophagy-related protein Beclin-1 in the model group was lower than that in the normal group.The high and medium doses of Tongluo Digui decoction had different degrees of increase(P<0.01,P<0.05),while the low-dose group did not improve significantly(P>0.05).4.By detecting the expression of p62,Beclin-1,NLRP3,GSDMD,and caspase-1m RNA in the kidney tissue,compared with the normal group of mice,the expression level of p62,NLRP3,GSDMD,and caspase-1 m RNA in the kidney tissue of the model group increased significantly(P<0.01);the high and medium doses of Tongluo Digui decoction had different degrees of reducing effects(P<0.01,P<0.05),while the low dose group did not improve significantly(P>0.05);while the model group Beclin-1 m RNA levels were lower than those in the normal group.The high and medium doses of Tongluo Digui decoction had different degrees of increasing effects(P<0.01,P<0.05),while the low-dose group did not improve significantly(P>0.05).Conclusions:1.Tongluo Digui decoction can improve the kidney function of DKD mice and delay the progression of the disease.2.Tongluo Digui decoction may play a therapeutic effect by enhancing the autophagy level of DKD mice,inhibiting the activation of NLRP3 inflammatory bodies,and inhibiting the pyrolysis of kidney tissue cells in DKD mice.