Inhibitory effect on the apoptosis of disc nucleus pulposus cells in rats by silencing COL9A3 gene to mediate MAPK signaling pathway: a study on the function and mechanism.

Abstract

To explore the effect of collagen IX alpha 3 chain (COL9A3) gene silencing on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration (IVDD) and its regulatory mechanism, so as to provide potential reference for the treatment of IVDD. The model of IVDD in rats were constructed to isolate, culture and identify nucleus pulposus cells for subsequent experiment. With the construction of lentivirus vectors, cells were divided into Blank group, negative control (NC) group, COL9A3 shRNA group, COL9A3 overexpression group, mitogen-activated protein kinase (MAPK) pathway inhibitor (Theaflavin 3,3'-digallate, TF3) group and COL9A3 shRNA+TF3 group according to different transfection treatments. After cell transfection, the expression of COL9A3, extracellular regulated protein kinase 1/2 (ERK1/2) and phosphorylated-ERK1/2 (p-ERK1/2), MEK1/2 and p-MEK1/2, as well apoptosis related indexes were detected by using quantitative real-time PCR (qRT-PCR) and Western Blot. Furthermore, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of transfected cells, and flow cytometry to detect changes of cell cycle and apoptosis. The model of IVDD in rats was established successfully, and rat nucleus pulposus cells were cultured subsequently. After transfection, compared with NC group, there was significant downregulation in COL9A3 expression, significant upregulation in ERK1/ERK2 and MEK1/MEK2 expressions, evident increase in Bax and Caspase3 expressions, and a decrease Bcl-2 expression in transfected cells of COL9A3 shRNA group (all p<0.05). The opposite trends were detected in the above indexes in cells of COL9A3 overexpression group (all p<0.05). Furthermore, in TF3 group, there was significant decrease in ERK1/ERK2 and MEK1/MEK2 expressions, reduction in Bax and Caspase3 expressions, increase in Bcl-2 expression (all p<0.05), yet without evident change in the expression of COL9A3. Meanwhile, COL9A3 expression was decreased in COL9A3 shRNA+TF3 group (p<0.05); however, there was no significant statistical difference in the remaining indexes when compared with those in the NC group (all p>0.05). Subsequently, compared with NC group, COL9A3 overexpression group and TF3 group revealed evident increased cell proliferation and significant decreased cell apoptosis (all p<0.05); whereas it was remarkably the opposite in COL9A3 shRNA group (all p<0.05). However, no evident difference was detected in the comparison of cell proliferation and apoptosis among Blank group, NC group and COL9A3 shRNA+TF3 group (all p>0.05). Overexpression of COL9A3 gene and inhibition of MAPK signaling pathway can induce proliferation and inhibit apoptosis of nucleus pulposus cells. Significantly, silence of COL9A3 gene expression can activate MAPK signaling pathway and affect the expression of apoptosis related factors, so as to inhibit the proliferation of nucleus pulposus cells and promote cell apoptosis in rats with IVDD.