Effect Of Erythropoietin And Astragalus On Expression Of TLR₄/MyD88 In Human Renal Tubular Cells Induced By Postasphyxial-Serum Of Neonates

Objective To investigate the effect of Erythropoietin(EPO) and Astragalus on expression of TLR4/MyD88 (Toll-like receptors 4/myeloid differentiation factor 88) in human renal tubular cells(HK-2) induced by postasphyxial-serum of neonates.Methods(1)Human renal proximal tubular cell(HK-2) was used as target cell. (2)The serum of neonates in 24 hours after asphyxia(Apgar Scoring less than 4),whose concentration were 20%(volum fraction),were applied as attacking factor. (3) The experiment was divided as control group,asphyxia group, group of pretreatment with erythropoietin (EPO), group of pretreatment with Astragalus. (4) The following parameters were detected:the changes of morphology were observed under inverted microscope; the expression of the positive binding rate of special fluorescent antibody in TLR4 on HK-2 cell were detected by Flow Cytometry (FCM).The expression of MyD88 on cytoplast were detected by the use of immunohistochemical method. (5) All data were expressed as mean±SD, one-way ANOVA were used throughout, statistical analysis was performed using the statistical programs SPSS 14.0, differences were considered significant when p< 0.05.Results (1) Under inverted microscopy:HK-2 cells in control group were significantly increased,sticked to each other tightly and grew very quickly.Their adhesion were better,multy-ang applan,and refraction raised.The quantity and structure of HK-2 cell was normal.Compared with control group, the change in morphology of HK-2 was most obvious and serious in asphyxia group,the cells grew slowly,the mount decreased,structure of the cells changed from typical multy-ang applan to off-normal round or ellipse.Refraction rate was decreased,and contour enhanced, The vacuolus,lipid droplet and granulation appeared in the kytoplasm. There was much cell debris in accrescent intercellular space. But compared with asphyxia group,the changes in morphology of HK-2 were obviously improved in the groups of pretreatment with EPO. Compared with asphyxia group,the changes in morphology of HK-2 were obviously improved in the groups of pretreatment with Astragalus. (2) The expression of the positive binding rate of special fluorescent antibody in TLR4 on HK-2 cell:Compared with controls group (8.84±1.58),the expression of the positive binding rate of special fluorescent antibody in TLR4 of asphyxia group (28.68±7.84) is obviously inceased; Compared with asphyxia group (28.68±7.84), the expression of the positive binding rate of special fluorescent antibody in TLR4 of EPO group (14.76±3.52) is significantly decreased; Compared with asphyxia group (28.68±7.84), the expression of the positive binding rate of special fluorescent antibody in TLR4 of Astragalus group (13.45±2.08) is the same decreased(P<0.05). (3) The expression of MyD88 on cytoplast were detected by the use of immunohistochemical method: Compared with controls group (1.23±0.56),the expression of MyD88 on HK-2 were significantly increased in asphyxia group (7.35±2.08); Compared with asphyxia group, the expression of MyD88 on HK-2 were significantly decreased in EPO group (2.75±1.69);Compared with asphyxia group, the expression of MyD88 on HK-2 were the same decreased in Astragalus group (1.93±1.64), statistical significance was accepted for p values of<0.05.Conclusions These results suggest that EPO and Astragalus could relieve the expression of TLR4/MyD88 signal pathway in renal tubular cells induced by postasphyxial-serum in neonates,the protection function of postasphyxial-renal injury by EPO and Astragalus is exerted.