The Effect Of Tangtongyin Delaying Diabetic Kidney Disease-induced Renal Fibrosis By Regulating The TGF-β₁/Smad3 Signaling Pathway Through MiRNA-21

Objective:（1）To observe the effects of Tangtongyin on the TGF-β₁/Smad3 signaling pathway and miRNA-21 in diabetic kidney disease（DKD）rats through in vivo animal experiments,so as to investigate the molecular mechanism by which Tangtongyin inhibits DKD-induced renal EMT and ECM accumulation,protects renal function,and delays renal fibrosis.（2）Through in vitro cell experiments,the protein expression of TGF-β₁,Smad3,and other proteins in HBZY-1 of DKD rats under high glucose conditions was observed after intervention with Tangtongyin serum and miRNA-21 inhibitor to further explore whether Tangtongyin targets miRNA-21 to regulate the TGF-β₁/Smad3 signaling pathway,inhibit the proliferation of HBZY-1 cells under high glucose conditions,and delay the progression of renal fibrosis in DKD.Methods:Part I（In vivo experiments）:Sixty male SPF-grade SD rats were randomly selected,with 10 assigned to the normal group and the remaining 50 rats induced with DKD using multiple small-dose intraperitoneal injections of high-sugar and high-fat diets combined with streptozotocin（STZ）.After successful modeling,the rats were randomly divided into five groups:model group,high,medium,and low dose(24.84,12.42,6.21 g·kg^-1)of Tangtongyin group,and irbesartan group（13.5 mg·kg-¹）,with 8rats in each group.The medication groups were administered by gavage,and the model and normal groups were given equal volumes of physiological saline by gavage once a day for 8 weeks.After 8 weeks of intervention,all rats were fasted for 12 hours（without water deprivation）,and blood samples were taken from the tail vein to measure fasting blood glucose（FBG）.Metabolic cages were used to collect 24-hour urine samples.After taking the whole blood from the femoral artery,the rats were sacrificed,and their kidney tissues were quickly removed and weighed to calculate the kidney hypertrophy index.Some kidney tissues were packaged and frozen at-80℃,while the rest were fixed in 4%polyformaldehyde.The levels of serum creatinine（Scr）,blood urea nitrogen（BUN）,and 24-hour urine microalbumin（m Alb）were measured using an automatic biochemical analyzer.The changes in the kidney morphology of each group of rats were observed using Masson and HE staining.The m RNA expression of TGF-β₁,miRNA-21,Smad3,FN,and Vimentin in the rat kidney tissues of each group was detected using RT-PCR.The protein expression of TGF-β₁,P-Smad3,Smad3,FN,and Vimentin in the rat kidney tissues of each group was detected using Western blot.Finally,the protein expression of TGF-β₁,Smad3,FN,and Vimentin in the rat kidney tissues of each group was detected using immunohistochemical staining.Part II（in vitro experiments）:Thirty healthy male SPF SD rats were used after a one-week adaptation feeding period.Fifteen rats were randomly assigned to the blank group,while the remaining fifteen were assigned to the Tangtongyin group.The Tangtongyin group received administration of Tangtongyin granules at a dose of12.42 g·（kg.d）^-1by gavage,while the blank group received an equal volume of saline by gavage.After 7 days of treatment,rats were anesthetized with ether inhalation one hour after the last gavage administration,and femoral artery blood was collected to prepare serum containing the drug.HBZY-1 cells were cultured and used to establish a normal group,high glucose group,and Tangtongyin high,medium,and low concentration serum groups to detect cell proliferation using the CCK-8 method.Additionally,HBZY-1 cells were cultured and used to establish a high glucose group,miRNA-21 inhibitor group,Tangtongyin group,and Tangtongyin+miRNA-21inhibitor group.After cell transfection,Western blot was used to detect the protein expression of TGF-β₁,P-Smad3,Smad3,and FN in HBZY-1 cells under high glucose conditions with intervention of Tangtongyin and miRNA-21 inhibitor.Results:Part 1（in vivo experiments）:（1）Comparison of kidney hypertrophy index,FBG,Scr,BUN,and 24 h m Alb levels in each group of rats:Compared to the normal group,rats in the model group showed an increase in kidney hypertrophy index,FBG,Scr,BUN,and 24 h m Alb levels（P<0.05）.In contrast,rats in the Tangtongyin intervention groups exhibited a decrease in kidney hypertrophy index,FBG,Scr,BUN,and 24 h m Alb levels to varying degrees（P<0.05）,with the high and medium dose groups showing a greater reduction than the low dose group（P<0.05）,but there was no significant difference between the high and medium dose groups（P>0.05）.The irbesartan group had lower kidney hypertrophy index,Scr,BUN,and 24 h m Alb levels（P<0.05）.（2）Renal histopathology comparison among rat groups:HE staining exhibited that:Renal tissue structure in the normal group was normal and well-arranged,while the model group displayed thickening of the glomerular basement membrane,mesangial proliferation,volume enlargement,and renal tubule atrophy.In the irbesartan and various doses of Tangtongyin groups,pathological changes were reduced.Masson staining revealed disordered structure of renal tissue cells,as well as a significant increase in collagen fibers between glomeruli and renal tubular interstitium in rats from the model group compared to the normal group（P<0.05）.Compared to the model group,collagen fibers decreased in the irbesartan and high/medium doses of Tangtongyin groups（P<0.05）.No significant difference was observed among the three groups（P>0.05）.（3）Comparison of TGF-β₁,Smad3,miRNA-21,FN,and Vimentin m RNA expression in rat renal tissues among groups:In the model group,the expression of TGF-β₁,Smad3,miRNA-21,FN,and Vimentin m RNA in rat renal tissues significantly increased compared to the normal group（P<0.05）.However,there was no significant difference in the low-dose group of Tangtongyin（P>0.05）.In the Irbesartan group and high and medium-dose groups of Tangtongyin,the expression of TGF-β₁,Smad3,miRNA-21,FN,and Vimentin m RNA in rat renal tissues decreased（P<0.05）,and there was no significant difference among the three groups（P>0.05）.（4）Comparison of TGF-β₁,P-Smad3,Smad3,FN,and Vimentin protein expression in rat renal tissues of each group:The expression of TGF-β₁,P-Smad3,Smad3,FN,and Vimentin proteins in rat renal tissues of the model group increased compared to the normal group（P<0.05）.However,in the low-dose group of Tangtongyin,there was no significant difference in the expression of these proteins compared to the model group（P>0.05）.In contrast,the Irbesartan group and the high and medium-dose groups of Tangtongyin showed a significant decrease in the expression of TGF-β₁,P-Smad3,Smad3,FN,and Vimentin proteins（P<0.05）,with no significant difference observed among the three groups（P>0.05）.（5）Comparison of TGF-β₁,Smad3,FN,and Vimentin expression in rat kidney tissues among the groups observed using immunohistochemistry:In the normal group,TGF-β₁and Smad3 were expressed in small amounts mainly in the mesangial area of the renal tissues,while FN and Vimentin were mainly expressed in the renal tubules and interstitium with a small amount of expression in the renal glomerulus.The expression showed brown-yellow clusters or granular scattered distribution.In the model group,the positive expression of TGF-β₁,Smad3,FN,and Vimentin increased significantly（P<0.05）compared with the normal group.However,in the Irbesartan group and the high and medium-dose groups of Tangtongyin,the expression of TGF-β₁,Smad3,FN,and Vimentin decreased significantly（P<0.05）compared with the model group,and there was no significant difference among the three groups（P>0.05）.Part II（in vitro experiments）:（1）The effect of Tangtongyin on HBZY-1 proliferation under high glucose conditions:Cell proliferation rate of the high glucose group was significantly higher than the normal group（P<0.05）.Tangtongyin serum groups at various concentrations showed a significant decrease in cell proliferation rate compared to the model group（P<0.05）,with a more pronounced effect observed in the high and medium concentration serum groups compared to the low concentration serum group（P<0.05）.No significant difference was observed between the high and medium concentration serum groups（P>0.05）.（2）Expression of miRNA-21 in each group after transfection:In comparison to the normal group,the high glucose group showed a significant rise in the expression of miRNA-21（P<0.05）;In comparison to the high glucose group,the miRNA-21inhibitor group、the Tangtongyin group、the Tangtongyin+miRNA-21 inhibitor group showed a decrease in the expression of the miRNA-21（P<0.05）,and there was no significant difference between the three groups（P>0.05）.（3）The effect of Tangtongyin on the protein expression of TGF-β₁,P-Smad3,Smad3,and FN in HBZY-1under high glucose conditions via miRNA-21 regulation:In comparison to the normal group,the high glucose group showed a significant decrease in the protein expression of TGF-β₁,P-Smad3,Smad3,FN（P<0.05）.In comparison to the high glucose group,the miRNA-21 inhibitor group showed a significant decrease in the protein expression of TGF-β₁,P-Smad3,Smad3,FN（P<0.05）.Both the Tangtongyin group and the Tangtongyin+miRNA-21 inhibitor group showed a decrease in the expression of the aforementioned indicators in comparison to the miRNA-21 inhibitor group（P<0.05）,and there was no significant difference between the two groups（P>0.05）.Conclusion:（1）Tangtongyin is effective in controlling the FBG of DKD rats,reducing m ALB,BUN,and Scr,protecting kidney function,alleviating renal pathological changes,and inhibiting the proliferation of HBZY-1under high glucose conditions,thereby delaying the progression of DKD-associated renal fibrosis.（2）Tangtongyin can reduce the expression of miRNA-21,TGF-β₁,P-Smad3,Smad3,FN,and Vimentin in the kidneys of DKD rats,thereby inhibiting the progressive accumulation of EMT and ECM in DKD rat kidneys and delaying renal fibrosis.This mechanism is achieved by regulating miRNA-21 to target the TGF-β₁/Smad3 signaling pathway.（3）Tangtongyin’s regulation of the TGF-β₁/Smad3 signaling pathway is multi-targeted and may involve additional targets beyond miRNA-21.