Formononetin Activates The Nrf2/ARE Signaling Pathway Via Sirt1 To Improve Diabetic Renal Fibrosis

ObjectiveDiabetic nephropathy is one of the most common microvascular complications of diabetes and the main cause of death and disability of diabetes mellitus.At present,due to the lack of comprehensive research on diabetic nephropathy in modern medicine,the conventional treatment methods such as glucose control,lipid control and pressure control can only delay the occurrence and development of diabetes.Therefore,it is of great clinical significance to further reveal the pathogenesis of diabetic nephropathy and search for new effective drugs.At present,a large number of studies have shown that one of the main pathological changes of diabetic nephropathy is renal fibrosis,and oxidative stress is the main cause of diabetic renal fibrosis,and improving the oxidative stress state of the body is an effective way to prevent diabetic renal fibrosis and improve diabetic nephropathy.The up-regulation of Sirtl then activates the Nrf2/ARE signaling pathway and regulates the expression of corresponding antioxidant enzymes to resist oxidative stress-induced fibrosis injury,which is of great significance for alleviating diabetic renal fibrosis.Traditional Chinese medicine has a long history in the prevention and treatment of diabetic nephrotic disease.As a traditional Lingnan drug,Niudali has a good clinical effect.Related studies also show that the main chemical component of Niudali--the flavonoid formononetin has significant antioxidant activity and Sirtl agitative effect.However,the specific mechanism of its improvement in diabetic nephropathy is not clear.So,is the improvement of diabetic renal fibrosis by formononetin related to the activation of Sirtl/Nrf2/ARE signaling pathway?This remains to be clarified.The aim of this study was to further investigate the ameliorating effect of formononetin on diabetic renal fibrosis.At the same time,the effect of formononetin on Sirtl/Nrf2/ARE signaling pathway was explored to clarify its mechanism of action and provide a scientific basis for clinical research.Methods1.Forononetin improves renal function and renal fibrosis in diabetic micedb/db diabetic nephropathy mouse model was selected,low and high doses of formonetin were given,and metformin hydrochloride was used as the positive drug group.After continuous intragastric administration for 8 weeks,serum and renal tissue of db/db mice were collected for the next experiment.To observe the effects of formononetin on blood glucose,body weight,total cholesterol(TC),serum triglyceride(TG),blood urea nitrogen(BUN)and other related indexes of DB/DB model mice.Immunofluorescence,Western blotting and immunohistochemistry were used to detect the histopathological changes of kidney and the protein expressions of type Ⅰ collagen(COLⅢ),α-smooth muscle actin(α-SMA),fibronectin(FN),intercellular adhesion molecule(Icam-1),SIRT 1,NRF2,KEAP1,HO-1 and SOD-1 in kidney.2.Forononetin activates the NRF2/ARE pathway by up-regulating Sirtl,and regulates the expression of fibrosis components in GMCs induced by high glucose.The rat mesangial cells stimulated by high glucose were selected as the model in vitro.Firstly,the effect of formononetin on the activity of glomerular mesangial cells(GMCs)was detected by MTT assay.The effect of oxidation index ROS in GMCs was detected by superoxide anion fluorescent probe.Western blotting was used to detect the effects of formononetin on proteins related to Sirtl/Nrf2/ARE signaling pathway in GMCs cultured with high glucose.The mediating role of Sirtl in formononetin activation/Nrf2/ARE signaling pathway was demonstrated by small molecule interfering RNA technology.The effect of formononetin on nuclear translocation of Nrf2 protein was observed by immunofluorescence assay.Results1.Animal experiment results showed that,compared with db/db model group,formononetin significantly improved the blood glucose level of model group mice;It significantly improved the blood lipid and renal function such as TG,TC and BUN in model group.It can significantly inhibit the expression of fibrosis index proteins such as COLⅠ,COLⅢ,α-SMA,FN and Icam-1 in kidney tissue of model group mice.At the same time,formononetin also significantly improved the pathological changes of kidney in model group.Forononetin can also increase the expression of Nrf2,HO-1,SOD-1,Sirtl and other proteins in kidney tissue,and inhibit the expression of Keap1 protein.2.The cell experiment results showed that formononetin could significantly reverse the upregulation of fibrosis indexes such as FN and Icam-1 in high glucose exposed GMCs.Meanwhile,formononetin can promote the expression of Nrf2 protein and increase its distribution in the nucleus.In addition,formononetin inhibits the overproduction of ROS induced by high glucose by activating Nrf2/ARE.Further studies showed that formononetin can up-regulate the expression of Sirtl,and the absence of Sirtl can block the activation of the Nrf2/ARE signaling pathway in high-glucose-induced GMCs,and reverse the down-regulation of FN and Icam-1.ConclusionFormononetin has a significant effect on diabetic nephropathy,and has a good effect on improving glucose and lipid metabolism and renal dysfunction in db/db diabetic nephropathy model mice.The specific mechanism of action may be through the up-regulation of Sirtl expression and then activation of Nrf2/ARE signal Pathway to improve the oxidative stress of diabetic nephropathy to prevent the process of renal fibrosis,and play a therapeutic effect on diabetic nephropathy.