The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II / 药学学报

ABSTRACT

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.