基于cAMP信号通路探讨补肾开玄通络方改善糖尿病肾病的作用机制OA

Objective:To investigate the molecular mechanism by which the Bushen Kaixuan Tongluo prescription exerts a renal protective effect in mice with diabetic kidney disease(DKD)by regulating the cyclic adenosine monophosphate(cAMP)signaling pathway.Methods:Thirty specific pathogen-free(SPF)male db/db mice were adaptively fed for three weeks.Mice with a random tail vein blood glucose level ≥11.1 mmol·L-1 and urinary albumin-creatinine ratio(ACR)≥ 30 mg·g1 were considered successfully modeled.The successfully modeled mice were randomly divided into five groups with six mice in each group:the model group,the low-,medium-,and high-dose Bushen Kaixuan Tongluo prescription groups(administered at doses of 7,14,28 g·kg-1·d-1 respectively),and the positive drug irbesartan group(administered at a dose of 20 mg·kg-1·d-1).Additionally,six db/m mice were selected as the blank group.Mice in each group were given intragastric administration of the Bushen Kaixuan Tongluo prescription at the corresponding concentrations,irbesartan,or an equal volume of pure water,and the intervention lasted for 12 weeks.During the experiment,the general conditions,body weight changes,and renal function indicators of the mice were dynamically monitored.After the intervention,a blood glucose meter was used to measure the fasting blood glucose(FBG)of the mice.An automatic biochemical analyzer was employed to detect the levels of serum creatinine(SCr),blood urea nitrogen(BUN),urinary microalbumin(uALB),ACR,aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC),triglycerides(TG),leptin(LEP),glycosylated serum protein(GSP),and insulin(INS)in the mice.Renal tissues were collected for hematoxylin-eosin(HE)staining,periodic acid-Schiff(PAS)staining,and Masson's trichrome staining to observe the histopathological changes.Immunohistochemistry(IHC)was used to detect the expressions of protein kinase A(PKA)and cAMP response element-binding protein(CREB)in the mice.Western blot analysis was performed to determine the expression levels of PKA,phosphorylated protein kinase A(p-PKA),CREB,phosphorylated cAMP response element-binding protein(p-CREB),and B-cell lymphoma-2(Bcl-2)proteins in the renal tissues of the mice.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression levels of PKA,CREB,and Bcl-2 in the renal tissues of the mice.Results:Compared with the blank group,the mice in the model group showed listlessness,decreased activity,and a significant increase in body weight(P＜0.01).Biochemical indicators revealed that the levels of BUN,uALB,ACR,AST,ALT,TC,TG,FBG,LEP,GSP,and INS were significantly increased(P＜0.01),while SCr showed an increasing trend with no statistically significant difference.Compared with the model group,the mice in the Bushen Kaixuan Tongluo prescription intervention groups had improved general conditions and a decreasing trend in body weight.Biochemical indicators showed that the levels of BUN,uALB,ACR,TC,GSP,and INS were significantly decreased(P＜0.05),while SCr,AST,ALT,TG,and LEP showed a decreasing trend with no statistically significant difference.Renal histopathological analysis showed that the model group exhibited typical DKD pathological features such as thickening of the glomerular basement membrane,expansion of the mesangial matrix,and deposition of collagen fibers in the renal tubulointerstitium,and all treatment groups could alleviate the above pathological damages.The IHC results showed that compared with the blank group,the expression levels of p-PKA and p-CREB in the renal tissues of the model group were significantly decreased(P＜0.01).Compared with the model group,the expression level of p-PKA in the medium-dose Bushen Kaixuan Tongluo prescription group was significantly increased(P＜0.01),while the expression level of p-CREB showed an increasing trend with no statistically significant difference.Western blot results showed that compared with the blank group,the expression levels of p-PKA/PKA,p-CREB/CREB,and Bcl-2 in the model group were significantly decreased(P＜0.05).Compared with the model group,the expression levels of these proteins in the medium-dose Bushen Kaixuan Tongluo prescription group were significantly increased(P＜0.01).Real-time PCR results showed that compared with the blank group,the mRNA expressions of PKA,CREB,and Bcl-2 in the model group were significantly down-regulated(P＜0.05).Compared with the model group,the mRNA expressions of these genes in the medium-dose Bushen Kaixuan Tongluo prescription group were significantly up-regulated(P＜0.05).Conclusion:The Bushen Kaixuan Tongluo prescription can improve the liver and kidney functions of db/db mice,correct lipid metabolism disorders and glucose metabolism imbalance.Its renal protective effect is associated with up-regulating the cAMP signaling pathway to improve renal fibrosis and reduce the level of oxidative stress,thereby protecting renal function.