Nanoformulation Of Carbon Monoxide Releasing Molecule Protects Against Cyclosporin A-induced Nephrotoxicity And Renal Fibrosis Via Suppressing NLRP3 Inflammasome Mediated TGF-β/Smad Pathway

Background:Cyclosporin A（Cs A）is a clinic typical immunosuppressor,which has been widely used in the treatment of heart,liver and kidney transplantation.However,long-term use of Cs A can cause renal toxicity,induce renal fibrosis and lead to chronic renal failure.Carbon monoxide（CO）is an important gaseous molecule in vivo,which has multiple cellular protective functions such as anti-inflammatory and antioxidant.CO donor or CO induction have been proved to be an effective therapeutic method in a series of inflammation models.However,the effect and the molecular mechanism of CO donor,SMA/CORM2,on Cs A-induced renal fibrosis have not been elucidated.Objective:This study aims to explore the potential effect and the molecular mechanism of CO donor,SMA/CORM2,on Cs A-induced renal fibrosis.Methods: Animal experiments: to establish the Cs A-induced renal fibrosis model,adult male CD-1 mice are injected subcutaneously with Cs A（30 mg/kg）every day for 4weeks.Based on the Cs A-induced renal fibrosis model,adult male CD-1 mice are injected intravenously with SMA/CORM2（1 mg/kg）3 times per week for 4 weeks to establish the SMA/CORM2 intervention model.In order to explore the effect of NLRP3 inflammasome on Cs A-induced renal fibrosis,adult male CD-1mice are intraperitoneal injected with NLRP3 inhibitor-MCC950（10 mg/kg）every other day for 4 weeks.After4 weeks,the mouse serum and kidney have been collected to detect the inflammation and renal fibrosis related indicators.Cell experiment: HK-2 cells（human kidney proximal tubular cells）are pretreated with SMA/CORM2,CORM2,SMA,and inactivated SMA/CORM2,and then treated with Cs A.After 24 h treatment,cytotoxicity assay has been assessed by CCK8.Result:（1）The mouse kidney wight and kidney coefficient have been significantly reduced by Cs A.Meanwhile,HE staining shows that Cs A remarkably disrupts the renal tubular structure.Chem-Bio detection reveals that Cs A significantly damages the renal function.However,this phenomenon has been reversed by SMA/CORM2.The above results show that SMA/CORM2 reverses Cs A-changed mouse renal histomorphology and renal function.（2）Masson staining displays that Cs A significantly induces the renal fibrosis,and immunoblot shows the protein levels of α-SMA,col,TGF-β and p-Smad2/3 have been elevated upon Cs A treatment.Inversely,SMA/CORM2 remarkably inhibits Cs A-caused renal fibrosis and fibrosis-related molecular expression.The above results suggest that SMA/CORM2 alleviates Cs A-induced renal fibrosis through inhibiting the TGF-β/Smad pathway.（3）Immunoblot shows that the protein levels of NLRP3,cleaved caspase1,mature IL-1β,IL-6 and TNF-α have been increased by Cs A treatment.Nonetheless,SMA/CORM2 remarkably reduced the protein levels of NLRP3,cleaved caspase1,mature IL-1β,IL-6 and TNF-α.The above results suggest that SMA/COM2 ameliorates Cs A-caused renal inflammation.（4）Masson staining reveals that NLRP3 inhibitor-MCC950 remarkably improves CSA-induced renal fibrosis,and immunoblot displays that MCC950 reduces the protein levels of α-SMA,col,TGF-β,NLRP3,cleaved caspase1 and mature IL-1β compared to Cs A group.To sum up,SMA/CORM2 alleviates Cs A-caused renal fibrosis via inhibiting NLRP3 inflammasome mediated TGF-β/Smad pathway.（5）HK-2 cytotoxicity assessment suggests that SMA/CORM2 markedly alleviates Cs A-caused cytotoxicity.Conclusion: SMA/CORM2 alleviates Cs A-caused renal fibrosis via inhibiting NLRP3 inflammasome mediated TGF-β/Smad pathway.