Nodakenin Attenuates Renal Interstitial Fibrosis By Inhibiting Snail Protein Expression

Objective:Fibrosis is not only a characteristic disease of chronic kidney disease,but also a key factor leading to acute renal failure.There is no effective method for treating chronic kidney disease.A number of studies had shown that the recombinant human TGF-?1 could stimulate the increase of the index of renal interstitial fibrosis?RIF?.In the previous research,our group found that nodakenin could down-regulate TGF-?1/Smad signaling pathway,which couldattenuate renal fibrosis,but the mechanism was unclear.Therefore,in this study,we demonstrated whether nodakenin can inhibitrenal fibrosis by inhibiting the expression of snail protein in vivo and in vitro experiments.Methods:In vivo experiment:Twenty eight Balb/c mice?male?aged 6-8 weeks,weighing 17-20 g were randomly divided into sham operation group?Sham group?,model group?Unilateral ureteral obstraction,UUO group?,low dose group?1mg/kg Noda,L group?and High dose group?10mg/kg Noda,H group?.After 1 week of routine feeding,the treatment group was given the nodakenin,while the sham operation group and the model group were given the same volume of solvent?10%Tween 80?.The mice were sacrificed 7 days after gavage,and the changes of Serum creatinine?SCr?and Blood urea nitrogen?BUN?in each group were observed.The structural changes of the kidney tissues and the infiltration of inflammatory cells were observed by HE staining.The glycogen staining?PAS staining?,masson staining and sirius red?Sirius Red?was used to observe the severity of renal interstitial matrix deposition and fibrosis,and the protein of the fibrosis index was detectedby western blot,including?-SMA,vimentin,fironectin.ThendetectedSnailand other protein associated with TGF-?1/Smad signal pathway by western blot.In vitro experiments:Rat kidney tubular epithelial cells?NRK-52E cells?were placed in an incubator of 5%CO ₂,37°C with sufficient humidity,and cultured in a DMEM high glucose medium containing 10%FBS.When the cell growth reached 80%-90%convergence,it was digested with 0.25 g/L trypsin and passaged for later use.The cells were divided into control group?CTL group?,model group?TGF-?1 group?,low-dose group,middle-dose group and high-dose group?TGF-?1+10?m Noda group,TGF-?1+20?m Noda group,TGF-?1+40?m Noda group?.We selected 6 ng/?L of human recombinant TGF-?1 stimulator cells and different concentrations of Nodakenin?Noda?for intervention.After 48 hours of treatment,cell morphology changes were observed by using an inverted microscope and immunofluorescence staining.Western blot and real-time fluorescent quantitative PCR were performed to detect the expression of fibrosis-related proteins,and detectedSnailand other protein associated with TGF-?1/Smad signal pathway.The snail protein of NRK-52E cells was overexpressed?snail-oe?by lentiviral transfection,and then Western blot was used to determine whether the transfection was successful.The cells were divided into snail-oe group,snail-oe TGF-?1 noda group and snail-oe noda group.The expression of fibrosis protein,snailand other protein associated with TGF-?1/Smad signal was detected by Western blot after 48 hours of intervention.Results Invivo experiment:1.Compared with UUO group,the serum creatinine?SCr?and urea nitrogen?BUN?inhigh dose were significantly decreased.2.Pathological staining showed that the renal tubule dilatation and tubular epithelial cell shedding can observed in the UUO group.At the same time,the expanded renal tubular region showed a large amount of matrix deposition,serious interstitial fibrosis region occurs,the renal tubular expansion of the H group is reduced,the deposition of the matrix is reduced,and the fibrosis region is obviously reduced.3.Western blot was used to detect the overexpression of the protein?-SMA,Vimentin and fibronectin in the UUO group,and the fibrosis-related protein in thegroup H was decreased compared with the UUO group.More interesting,the expression of Snail protein ingroup H was significantly lower and the expression of the protein in TGF-?1/Smad signal pathway was increased in the model group?P<0.01?.The effect of the low dose group?L group?was not significant as compared to the UUO group.In vitro experiment:1.Compared with the CTL group,after the TGF-?1 group was stimulated for 48hours,the volume of the cells was large,the polarity was lost,and the expression of E-cadherin was relatively weak.2.Compared with the CTL group,the expression of the fibrosis-related protein as?-SMA,vimentin and fibronectinof the TGF-?1 group was significantly higher than that of the CTL group.In contrast,especially 20?m nodakenin was added in the cells.The expression of these proteins can be significantly reduced.3.The results showed that the expression of the protein in the TGF-?1/Smad signal pathway and Snail protein of the treatment group was significantly lower than that of the TGF-?1 group.4.After the overexpression of snail,the expression of fibrosis protein could not be inhibited effectively after the intervention of nodakenin.Conclusion:1.Nodakenin can partial restore the damaged renal function of UUO mice by delaying renal interstitial fibrosis.2.Nodakenin can reduce the expression of related fibrotic proteins produced by TGF-?1 stimulation in NRK-52E.3.Nodakeninrelay on Snail protein expression to attenuating renal interstitial fibrosis.