Heat shock pretreatment improves mesenchymal stem cell viability by heat shock proteins and autophagy to prevent cisplatin-induced granulosa cell apoptosis

摘要

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) can partially repair chemotherapy-induced ovarian damage. However, low survival rate after transplantation hampers the therapeutic efficiency of BMSCs. Heat shock pretreatment (HSP) effectively improves the cell survival. This study attempted to investigate the mechanisms of HSP on BMSCs survival and the effects of heat shock-pretreated BMSCs (HS-MSCs) on cisplatin-induced granulosa cell (GC) apoptosis.METHODS:BMSCs were isolated, cultured, and identified. After receiving HSP for different duration times in a 42?°C water bath, the apoptotic rates of BMSCs were detected by Annexin V-FITC/PI to determine the optimal condition of HSP. Cisplatin was added to the medium of HS-MSCs to simulate chemotherapy environment. The proliferative curve, apoptotic rate, and viability of HS-MSCs were determined by CCK-8, Annexin V-FITC/PI, and Hoechst33342/PI respectively to explore the alteration of biological characteristics. The levels of heat shock protein 70 and 90 (HSP70 and HSP90) and the expressions of autophagy-related markers (Beclin1 and LC3B) were detected by Western blot. In addition, the autophagosomes were observed by transmission electronic microscopy to discuss the possible mechanisms. The GCs were isolated, cultured, and identified. The HS-MSCs were co-cultured with GCs before and after the addition of cisplatin. Then, the apoptotic rate and viability of GCs were detected to investigate the therapeutic and preventive effects of HS-MSCs on GC apoptosis.RESULTS:After receiving HSP at 42?°C for 1?h, BMSCs represented the lowest apoptotic rate. After the addition of cisplatin, the apoptotic rate of HS-MSCs (11.94%?±?0.63%) was lower than that of BMSCs (14.30%?±?0.80%) and the percentage of HS-MSCs expressing bright blue/dull red fluorescence was lower than that of BMSCs. The expression of HSP70 and HSP90 increased, while the number of autophagosomes, the expression of Beclin1, and the LC3BII/LC3BI ratio decreased in HS-MSCs. The apoptotic rates of GCs co-cultured with HS-MSCs before and after the addition of cisplatin were 39.88%?±?1.65% and 36.72%?±?0.96%, both lower than those of cisplatin-induced GCs (53.81%?±?1.89%).CONCLUSION:HSP can alleviate the apoptosis and improve the survival of BMSCs under chemotherapy environment. The mechanism may be associated with the elevated expression of HSP70 and HSP90 and the attenuation of autophagy. Moreover, HS-MSCs have both therapeutic and preventive effects on cisplatin-induced GC apoptosis.