Effect And Mechanism Of Nodakenin On Transdifferentiation Of Human Renal Tubular Epithelial Cells Induced By Serum Of Uremia Patients

Objective: To investigate the effect and possible mechanism of nodakenin on serum-induced epithelial-mesenchymal transition syndrome(EMT)induced by serum of uremic patients,and to provide a new possible and effective way for prevention and treatment of renal interstitial fibrosis and to provide cell biology evidence for the clinical application of nodakenin as a effective way.Methods: To collected 10 uremic patients and 10 normal serum from the Department of Nephrology,Affiliated Hospital of Southwest Medical University from June 2016 to October 2016 on Aseptic conditions.were taken in equal quantities mixed standby.HK-2 cells were divided into normal group(10% normal human serum),model group(10% uremic serum),nodakenin control group(10%normal serum +nodakenin 80 ng / ml),Low dose of nodakenin intervention group(10% uremia serum + nodakenin 20 ng / ml),mediate dose of nodakenin intervention group(10% uremia serum + nodakenin 40 ng / ml),high dose of nodakenin intervention group(10% uremia serum +nodakenin 80 ng / ml).The morphological changes of cells in each group were observed by inverted phase contrast microscope.The cell proliferation activity of each group was determined by CCK-8 assay.Transforming growth(TGF-?)was measured by enzyme-linked immunosorbent assay(ELISA)The expression of ?-SMA,ZO-1,E-cadherin,smad3,P-smad3,?-sma,E-cadherin and epithelial tight junction Zonula occludens 1(ZO-1),Fibronectin(FN),interleukin-6(IL-6),Toll-like receptor 4(TLR4),IKK? The expression of P-IKK?,NF-?B P65 and P-NF-KB P65 protein were detected by Western blot(WB).Immunofluorescence(IF)was used to detect the expression of P-smad3 and ZO-1.Immunohistochemistry(IHC)was used to detect the expression of interleukin-6(IL-6)The effects of sera from patients with uremia on HK-2 cells: Compared with the normal group,the proliferative activity of 20% and 40% uremia serum cells decreased significantly(P<0.05),while the cells in 5% and 10% urease sera Serum TGF-?content was significantly increased,with significant statistical difference(P <0.01).After 48 hours of stimulation with 10% urease serum,the cells started to thin,became longer and fusiform,the expression of ?-SMA protein was enhanced,and the expression of E-cadherin and ZO-1 protein was weakened(P <0.01).The proliferative activity of HK-2 cells had no significant difference(P> 0.05)at the concentration of 10?g / ml,20?g/ ml,40?g / ml,80?g / ml and 160?g / ml of nodakenin.Compared with the normal group,the cell bodies of the model group were significantly thinner,longer,long fusiform shape,after the intervention of nodakenin,the cells gradually transformed from the long fusiform back tothe normal polygons or triangles.Compared with the normal group,the expression of TGF-?,P-smad3,?-SMA and Fibronectin in the model group was significantly increased,Glycosides were more obvious and statistically significant(P <0.01).There was no significant difference in smad3 protein expression in all groups.Compared with normal group,the expression of E-cadherin and ZO-1 in the model group was weakened,and the expression of E-cadherin and ZO-1 in the model group decreased.The effect of middle-dose and high-dose of nodakenin was more obvious.Compared with the normal group,the expression of TLR4,IL-6,P-NF-KB P65 and P-IKK? protein increased significantly after uremia serum was stimulated for 24 hours.The middle and high doses of nodakenin reduce their expression,and the difference was statistically significant(P <0.01).The levels of NF-KB P65,IKK? each group show no significant change.Conclusion: In vitro,nodakenin can inhibit serum-induced transdifferentiation of HK-2 cells induced by serum of uremic patients to some extent,and its mechanism may be through the inhibition of TLR4/NF-?B pathway to reduce the secretion of inflammatory cytokines.