Effects Of Bone Marrow Stromal Stem Cell Transplantation On The Expression Of Cyclins In Neuronal Cells After Cerebral Infarction

Transplantation of bone marrow stromal cells (BMSCs) improves animal neurological functional recovery after cerebral ischemia. But the mechanism remains unclear. As cell cycle machinery plays an important role in the pathogenesis of stroke, we investigated cell cycle elements expression in the injured brain of middle cerebral artery occlusion (MCAO) of rat models with or without BMSCs transplantation by RT-PCR, immunohistochemistry, and western blotting.The cell cycle markers, cdk4 and its cyclin partner cyclin D1 increased from 24 h after ischemia–reperfusion in the MCAO models(n=28) and decreased gradually at 7day. Similarly, phosphorylation of the retinoblastoma protein (pRb), the cyclin D/cdk4 complex mutual target, was upregulated at 12h, peaked at 72h and dereased at 7 day. Proliferating cell nuclear antigen (PCNA), an S phase marker, also increased. However, intravenously administrated BMSCs (2×106)at 6h after ischemia- reperfusion facilitated cyclin D1 and cdk4 decrease at early time and phosphorylated pRb was downregulated accordingly in rats(n=28). Meanwhile PCNA decreased at 24 h after BMSCs transplantation. Interestingly, p27 did not show any change at molecular expression and dramatically decreased at protein level in the absence but enhanced in the presence of BMSCs.These findings showed that BMSCs might lead to cell cycle arrest in the injured brain via the downregulation of cyclin D/cdk4/pRb pathway as well as upregulation of p27 level. It can also explain the treatment effect of BMSCs for stroke. Transplantation of bone marrow stromal cells (BMSCs) decreased astrocytic apoptosis, reduced astrogliosis and the thickness of scar wall after brain damage. However, the interactions between BMSCs and astrocytes are unclear. Thus, whether BMSCs could modulate cell cycle in injured astrocytes was investigated in this study. There are four experimental groups used to explore this question: 1) normal cultured astrocytes, 2) glutamate (Glu, 100mM )-treated astrocyte group, 3) Glu-treated astrocyte cocultured wtih BMSCs group, 4) Glu-treated astrocyte +BMSCs + L-Canavanine( iNOS inhibitor ) group. Cell cycle proteins, cyclin D1, cdk4 and proliferating cell nuclear antigen (PCNA) were induced in astrocytes damaged by glutamate in vitro. However, BMSCs coculture significantly inhibited the expression of cyclin D1, cdk4 and PCNA whereas increased the expression of p27. Flow cytometry showed that cell cycle progression in damaged astrocytes was blocked in the G1 phase by BMSCs. An inhibitor of inducible nitric oxide synthase largely reversed this process in vitro.These data indicated that BMSCs might inhibit cell cycle control system by targeting a signaling pathway involved in not only downregulating the cyclin D1-cdk4 complex but also upregulating p27 expression. NO might play an important role in the interaction between these two cells. The phenomenon that BMSCs conferred decline of astrogliosis post-ischemia may derive from inhibiting cell cycle progression in astrocytes. This experiment may provide additional evidence that BMSCs have an effect of modulating cell cycle outside the immune system.