The Mechanism Of Formononetin On Inhibiting Ferroptosis-associated Fibrosis And Improving Chronic Kidney Diseae Through Smad3/ATF3/SLC7A11 Signaling Pathway

Objective: Chronic kidney disease(CKD)is a disease of kidney damage caused by various reasons.Renal fibrosis(RF)is the key pathological process in its occurrence and development.Ferroptosis of renal tubular epithelial cells can promote the occurrence and development of RF.Formononetin(FN)is a widely used and valuable monomer component in the traditional Chinese medicine Astragalus membranaceus.Studies have shown that FN can inhibit the expression of Smad3,while ATF3 can be directly combinded with Smad3 to inhibit the expression of SLC7A11.Therefore,targeting Smad3 to treat ferroptosis related RF has become a new treatment strategy.This study intends to explore the role of small molecular monomer FN from traditional Chinese medicine in improving CKD and its regulatory mechanism through Smad3/ATF3/SLC7A11 signaling pathway from the perspective of ferroptosisRF process.Methods: 1)In vivo experiments: the CKD models of mice with folic acid(FA-CKD)and unilateral ureteral obstruction(UUO-CKD)are constructed and mice were treated with FN(40 mg/kg)and positive control drug Ang Ⅱreceptor antagonist VST(20 mg/kg)by gavage.The mice were divided into the Vehicle group,FA group,FA+FN group,FA+VST group,Sham group,UUO group,UUO+FN group,and UUO+VST group.To evaluate the effects of FN on renal function and pathology(serum creatinine,serum urea nitrogen,HE staining),ferroptosis level(GSH,MDA,tissue iron content,ferroptosis markers SLC7A11 and GPX4),oxidative stress indicators(Keap1,Nrf2 and 4-HNE),fibrosis degree(Masson staining,ɑ-SMA,fibronectin and Col1a1).Immunohistochemical staining and Western blot were used to evaluate the effects of FN on Smad3 phosphorylation and ATF3 activation.2)In vitro experiments: CCK8 experiment was conducted to determine the appropriate concentration of FN in treating p TECs.The p TECs were treated with ferroptosis agonists RSL3 and erastin to simulate the occurrence of renal ferroptosis.And different doses of FN(20-80 μM)were incubated with p TECs.Ferroptosis level(GSH,MDA,tissue iron content,ferroptosis markers SLC7A11 and GPX4,degree of lipid peroxidation,intracellular iron content)and fibrotic genes(ɑ-SMA,fibronectin and Col1a1)were detected.Cytoplasmic and nuclear isolation was used to detect the effects of FN on the activation of Smad3,ATF3 and the entry of Nrf2 into the nucleus.We also verified the effects of FN on impeding the binding of exogenous Smad3 and ATF3 in 293 FT cells by Co-Immunoprecipitation(Co-IP)test and evaluated the effects of FN on the downstream of ATF3 SLC7A11.Results: The in vivo experiments showed that: 1.The serum creatinine and urea nitrogen levels of mice in the FA model group were significantly higher than those detected in the Vehicle/Sham group,while FN treatment significantly reduced this level.2.HE staining showed that the renal tubular lumen damage in FA and UUO models was significantly improved after FN intervention.3.Masson staining,the detection of ɑ-SMA,fibronectin and Col1a1 by IHC staining,PCR detection and Western blot showed that the expression of collagen fibers and fibrin deposited in the renal interstitial tissue were significantly reduced in the FN group,and the effect of FN was better than that of VST.4.After FN treatment,the expression of 4-HNE was reduced and the entry of Nrf2 into the nucleus was promoted.5.FN reversed the increased tissue iron level,MDA level and decreased GSH level in FA and UUO models,as well as the expression of ferroptosis-negative related proteins GPX4 and SLC7A11.6.The levels of activated p-Smad3 and ATF3 were increased in FA and UUO models and were significantly decreased in the FN treatment group.The in vitro experiments showed that: 1.The appropriate concentration of p TECs for FN treatment is 0-80 μM.Over 80 μM FN treatment showed an obvious inhibitory effect on cells.2.Western blot showed that the expression of fibroin increased under the stimulation of RSL3/erastin,and its content decreased significantly after FN intervention.3.The expression of oxidative stress biomarker 4-HNE and the negatively related proteins GPX4 and SLC7A11 of ferroptosis were consistent with those in vivo.The increased MDA level and the increased degree of lipid peroxidation,as well as the decreased GSH level and decreased intracellular iron content in the ferroptosis were reversed after FN treatment.4.In the ferroptosis microenvironment,FN promotes the entry of Nrf2 into the nucleus.5.Smad3 and ATF3 translocated from the cytoplasm to the nucleus under the stimulation of ferroptosis agonists RSL3/erastin,and the translocation was significantly inhibited after FN intervention.6.FN intervention blocked the combination of Smad3 and ATF3.Conclusion: 1.FN improved the renal function damage of FA mice and repaired the renal tubular damage.2.FN inhibited renal fibrosis and ferroptosis in vivo and in vitro.3.FN alleviated CKD by blocking the binding of Smad3 and ATF3 and promoting the expression of SLC7A11,further blocking the ferroptosis of renal tubular epithelial cells and inhibiting fibrosis.