The effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells

Abstract

Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt), X-linkedinhibitor of apoptosis protein (XIAP), cysteinyl aspartate specific proteinase-3 (caspase-3), with the treatment of thymic stromal lymphopoietin. Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope. Second or third passage cells maintained in monolayers were used for the following experiments. The groups were divided randomly into normal group, TNF-α treated group, TSLP treated group, TSLP+LY94002 treated group and TSLP+Embelin treated group. As a control, normal group was treated with PBS. TNF-α treated group was treated with 500 ng/ml TNF-α as a positive control. TSLP treated group was treated with 10 ng/ml rhTSLP. TSLP+LY94002 treated group and TSLP+Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 min in different corresponding experiments, for which 10 μmol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt, XIAP, caspase-3 were investigated by Western blot analysis. Results As the culture cell type II collagen staining was positive observed by fluorescence microscopy, we confirmed that the cultured cells were nucleus pulposus cells. In comparison with negative control, the levels of p-Akt, XIAP in TSLP treated group were elevated (t=9.510, P=0.001; t=8.851, P=0.001). Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance. Compared with TSLP treated group, downregulated level of p-Akt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798, P=0.001; t=7.032, P=0.002; t=5.908, P=0.004). Upregulated caspase-3 activity were also observed in TSLP+ Embelin treated group (t=7.990, P=0.001). Furthermore, significant increased apoptotic cell rate was observed in TSLP+LY294002 or TSLP+Embelin treated groups (t=21.268, P=0.001; t=21.279, P=0.001). Conclusion TSLP may have a potential anti-apoptotic effect on mouse NP cells via upregulating XIAP in PI3K/Akt signaling pathway to restrain the activation of caspase-3. Key words: Cell culture techniques; Aopotosis; Apoptosis regulatory proteins