Study On The Effect And Molecular Mechanism Of Colquhounia Root Tablet In Diabetic Nephropathy Rats

Objective: Diabetic nephropathy(DN)is a severe microvascular complication of diabetes and a major cause of death in end-stage renal disease(ESRD).However,the exact pathogenesis of DN is yet to be clarified,and effective treatments are lacking.Growing clinical evidences show the potentials of Colquhounia root tablet(CRT)in alleviating diabetic nephropathy.However,its pharmacological properties and underlying mechanisms remain unclear.Here,we observed the effects of CRT on apoptosis and autophagy in DN rats,and used transcriptome sequencing(RNA-sequencing,RNA-seq)technology to initially explore the underlying mechanisms.Methods: Thirty SPF grade male SD rats were randomly and equally divided into normal control group(Con group),diabetic nephropathy group(DN group),and CRT treatment group(CRT group).In the DN and CRT groups,rats were given streptozotocin(60 mg/kg)intraperitoneally to induce the DN model.After successful modeling,the rats in the CRT group were treated with 600mg/(kg·d)CRT by gavage,and the changes of blood glucose(BG)and urinary microalbumin(m ALB)in each group were monitored every 2 weeks during the dosing period.All rats were dissected and sampled at the end of the 8th week of administration.We evaluated BG,m ALB,serum creatinine(Scr),body weight and kidney index(KI)in the different groups.The renal histopathological changes in each group of rats were observed by HE,PAS and Masson staining,and TUNEL assay was used to evaluate apoptosis in kidney tissues.Western blot was used to detect the expression of autophagy-related proteins(LC3,Beclin-1,p62)and apoptosis-related proteins(Bax,Bcl-2)in rat kidney tissues.Transcriptome analysis of kidney tissues from each group of rats using RNA-seq technology to screen for differentially expressed genes and enrich them for KEGG pathway analysis.We searched the relevant literature to further identify the molecules and signaling pathways related to autophagy downstream of the drug,and verified the expression of related molecules and signaling pathways using q RT-PCR and Western blot.Results:1.Changes of main metabolic parameters: Compared with Con group,BG,m ALB,Scr and KI levels in DN group were significantly increased(P<0.05),while body weight was decreased(P<0.05).Compared with DN group,m ALB,Scr and KI levels of rats in CRT group were significantly decreased(P<0.05),while body weight was significantly increased(P<0.05).However,in our study,CRT treatment did not alleviate the significantly elevated BG levels in the DN group,suggesting that CRT could alleviate DN without affecting BG levels.2.Pathological changes of kidney tissue: In HE staining,compared with the Con group,the DN group rats the lobulated glomeruli,severely atrophied glomerular vessels,and vacuolar degeneration cytoplasm,inflammatory infiltration.PAS staining showed increased glycogen deposition within the thylakoid membranes of the DN group rats.In addition,a slight increase in renal fibrosis was observed in DN rats by MASSON staining.Compared with the DN group,CRT treatment effectively improved the renal histopathological changes.3.TUNEL staining results: Compared with the Con group,apoptosis was significantly increased in the kidney tissue of rats in the DN group;apoptosis was reduced in the CRT group compared with the DN group.4.RNA-Seq results: RNA-Seq found that CRT significantly downregulated the expression of 1367 m RNAs and upregulated the expression of 447 m RNAs in the kidney of DN rats.Among them,CD36 was one of the most significantly down-regulated m RNAs.KEGG pathway enrichment analysis revealed that CD36 was enriched in the autophagy-associated AMPK signaling pathway.5.q RT-PCR results: Compared with the Con group,CD36 expression was significantly increased(P<0.05)in DN group.Compared with the DN group,CD36 expression was significantly decreased(P<0.05)in CRT group.6.Western blot results: Compared with Con group,the expression of autophagy-related proteins Beclin-1 and LC3II/LC3 I was significantly decreased(P<0.05)and the expression of p62 protein level was significantly increased(P<0.05)in DN group;the expression of pro-apoptotic protein Bax was significantly increased(P<0.05)and the expression of anti-apoptotic protein Bcl-2 was decreased(P<0.05);the expression of CD36 was significantly increased(P<0.05);expression of pathway protein p-AMPK was significantly decreased(P<0.05).Compared with DN group,the expressions of Beclin-1,LC3II/LC3 I,Bcl-2 and p-AMPK in CRT group were significantly increased(P<0.05),and the protein levels of p62,Bax and CD36 were significantly decreased(P<0.05).Conclusion:1.CRT can improve the Scr and urinary m ALB levels in DN rats and reduce the pathological kidney damage,confirming the protective effect of CRT on the kidneys of DN rats;2.CRT can promote protective autophagy in the kidney of DN rats;3.CRT can inhibit the apoptosis in the kidney of DN rats;4.CRT may enhance autophagy and inhibit apoptosis in the kidney of DN rats through the CD36/AMPK signaling pathway to achieve renoprotective effects in DN rats.