Effect Of Human Amniotic Mesenchymal Stem Cells On Autophagy Of Human Renal Tubular Epithelial Cells HK2 In Hyperglycemia Environment

Objective:Human tubular epithelial cells(HK2)were cultured in high and low glucose medium,which were co-cultured by Transwell with Human amniotic mesenchymal stem cells(hAMSCs).To investigate the effect of hAMSCs on autophagy of HK2 in high glucose environment by observing the expression changes of autophagy markersMethods:Cell lines HK2 were divided into a Rapamycin group in hAMSCs co-culture at Low glucose(LG group,5.5mm)and High glucose(HG group,30mM)groups.The cell viability of HK2 at 24h,48h and 72h in RAPA co-culture group(3-Methyladenine group,3-MA group)and 3-MA co-culture group(CCK8)was detected in RAPA co-culture group(5.5mm 10mM 20mM 30mM).Western Bolt detected the changes of HK2 autophagy markers(LC3II/LC3I?Beclinl?P62).The apoptosis rate of HK2 was detected by flow cytometry and Hoechst/PI fluorescence staining.The expression of LC3B was detected by immunofluorescence.The changes of autophagy markers(LC3II/LC3I?Beclin1?P62)mRNA were detected by RT-PCR.Transmission electron microscopy was used to detect the number of autophagosomes.The cell activity of HK2 was detected by CCK8.The glucose content of the medium at 24h,48h and 72h was measured by a hypoglycemic meter.Results:?CCK8 results showed that the culture activity of HK2 in 30mM sugar medium was significantly lower than that in 5.5mm medium at 24h(P<0.05).?Western Bolt results:Beclinl expression of HK2 autophagy marker LC3II/LC3I in HG group was inhibited and P62 expression was increased,while hAMSCs co-culture reversed they expression.?The results of flow cytometry were consistent with Hoechst/PI fluorescence staining results.The apoptosis rate of HK2 in HG group was significantly increased(P<0.05),and hAMSCs treatment could reduce the apoptosis rate.?HK2 cells of HG group autophagy activation and inhibition of processing,again after cocultivation,WB test results showed that HG group of autophagy inhibition(accelerate LC3II/LC3I Beclinlexpression and reduce P62 expression),the rise in autophagy activity after co-culture(LC3?/LC3?Beclinl expression raise P62 express lower)and HK2 autophagy activity up from HG after dealing with RAPA,hAMSCs treatment could further activate autophagy,After 3-MA treatment,autophagy activity to achieve the minimum,a certain degree of recovery after co-culture HK2 autophagy activity.? The expression trend of LC3B in each group was consistent with that of WB.?mRNA expression of autophagy markers(LC3?/LC3?Beclinl P62)in each group was detected by RT-PCR,and the overall trend was consistent with that of WB.? Autophagosomes were observed in LG group and significantly decreased in HG group.After co-culture,the number of autophagosomes increased compared with HG group.After RAPA activated autophagosomes,the number increased.Autophagosomes were hardly observed in the 3-MA group,and a small number of autophagosomes were observed after co-culture.?CCK8 detected the activity of HK2 in each group and found that the cell activity was positively correlated with autophagy activity.?The glucose concentration in the medium of each group was measured by the stable glucose meter,which showed that the co-culture of hAMSCs could significantly reduce the sugar concentration in the medium(P<0.05),and the reduction rate increased with the increase of time.Conclusion:The autophagy of HK2 was inhibited in the high-glucose environment,while co-culture of hAMSCs could activate the autophagy activity of HK2 impaired in the high-glucose environment.