Glutamine regulates the effect of hormones on the apoptosis of bone microvascular endothelial cells

Zan Yongfeng; Song Keguan; Liu Yuda

doi:10.12307/2026.660

Abstract：BACKGROUND:Glucocorticoids can significantly inhibit the expression of proteins related to the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in bone microvascular endothelial cells,thereby triggering programmed cell death and necrosis.Glutamine has been shown to activate the PI3K/Akt/mTOR signaling pathway. OBJECTIVE:To explore how glutamine regulates the effect of glucocorticoids on the apoptosis of bone microvascular endothelial cells. METHODS:Bone microvascular endothelial cells were extracted from the femoral head tissues of Sprague-Dawley rats under aseptic conditions.Passage 3 bone microvascular endothelial cells were cultured for 12 hours and then divided into four groups:the control group was routinely cultured for 24 hours,the glucocorticoid group was cultured with methylprednisolone sodium succinate for 24 hours,the glucocorticoid+glutamine group was cultured with methylprednisolone sodium succinate for 12 hours and then with glutamine for 12 hours,and the glucocorticoid+glutamine+LY294002 group was cultured with methylprednisolone sodium succinate for 12 hours followed by addition of glutamine treatment for 6 hours and the PI3K/Akt signaling pathway inhibitor LY294002 for 6 hours.After culture,cell apoptosis was detected by AO-PI double fluorescence staining and Annexin V/PI double staining by flow cytometry;BCL-2,Bax,PI3K,Akt,mTOR mRNA expression was detected by RT-qPCR;and western blot was used to detect BCL-2,Bax,PI3K,Akt,mTOR,p-PI3K,p-Akt,and p-mTOR protein expression. RESULTS AND CONCLUSION:(1)The apoptosis rate was higher in the glucocorticoid group than in the control group and the glucocorticoid+glutamine group(P<0.05),and the apoptosis rate was higher in the glucocorticoid+glutamine+LY294002 group than in the glucocorticoid+glutamine group(P<0.05).(2)BCL-2,PI3K,Akt,mTOR mRNA expression in the glucocorticoid group was lower than that in the control group and glucocorticoid+glutamine group(P<0.05),and Bax mRNA expression was higher than that in the control group and glucocorticoid+glutamine group(P<0.05);BCL-2,PI3K,and mTOR expression in the glucocorticoid+glutamine+LY294002 group was lower than that in the glucocorticoid+glutamine group(P<0.05),and Bax mRNA expression was higher than that in the glucocorticoid+glutamine group(P<0.05).(3)BCL-2,PI3K,Akt,mTOR,p-PI3K,p-Akt,p-mTOR protein expression in the glucocorticoid group was lower than that in the control group and the glucocorticoid+glutamine group(P<0.05),and Bax proteins were higher than that in the control group and the glucocorticoid+glutamine group(P<0.05);BCL-2,PI3K,Akt,mTOR,p-PI3K,p-Akt,p-mTOR protein expression in the glucocorticoid+glutamine+LY294002 group was lower than that in the glucocorticoid+glutamine group(P<0.05)and Bax protein expression was higher than that in the glucocorticoid+glutamine group(P<0.05).To conclude,glutamine reduces glucocorticoid-induced apoptosis in bone microvascular endothelial cells through activation of the PI3K/Akt/mTOR signaling pathway.