ABSTRACT
Objective To observe the effects of myriocin(ISP-1) on improvement of glomerular messangial cell(GMC) apoptosis and on gene expression profiles of cell cycle regulatory proteins in GMC.Methods Rat GMC was cultured with 100 ?mol/L ISP-1 for 6,12,24,and 48 h,apoptosis was evaluated through flow cytometry,Hoechst33258/PI fluorescence stainig and DNA fragmentation analysis was carried out under Sepharose electrophoresis to observe the changes of apoptosis and DNA fragmentation.Gene expression profiles of cell cycle regulatory proteins were detected by SuperArray Real-Time PCR microarray analysis.The expression of Bax and Bcl-2 proteins was investigated by Western blotting.Results ISP-1 could significantly induce GMC apoptosis in a time-dependent manner,which was the most significant after 48 h.Apoptosis bodys of GMC were observed by Hoechst/PI fluorescence staining,and a typical ladder pattern was identified in DNA electrophoresis.SuperArray Real-Time PCR microarray analysis revealed that ISP-1 could up-regulate the expression of genes involved in DNA damage,apoptosis and cell cycle regulation,such as Rad51,Atm,Brcal,caspases,cyclinA2,cyclinC,chek1,cyclinB1,cyclinB2,cyclinD2,cyclinF,Cdc25a,and P27.Western blotting showed that ISP-1 could significantly up-regulate Bax protein expression and down-regulate Bcl-2 protein expression,respectively.ConclusionISP-1 could induce GMC apoptosis in a time-dependent manner,propabably through influencing gene expression of cell cycle regulatory proteins and apoptosis proteins.
ABSTRACT
Objective To observe the changes of extracellular matrix(ECM) production and hypertrophy of glomerular mesangial cells(GMCs) induced by high glucose(HG),and the inhibitory role of myriocin(ISP-1).Methods GMC cultured with normal glucose(5.6 mmol/L D-Glucose,NG),HG(25.2 mmol/L D-Glucose) and HG plus ISP-1(100 mg/L) for different durations(0,24,48,(72 h)).The sizes of GMC were indicated by forward scatter intensity,measured by flow cytometery,and the levels of fibronection(FN),collagen Ⅳ(Col Ⅳ),laminin(LN),precollagen Ⅲ(Pcol Ⅲ) and hyaluronic acid(HA) in the supernatant of cultured GMC were detec-(ted) by ELISA.Results Compared with NG,HG could induce GMC hypertrophy(P