香菇多糖抑制STZ诱导的INS-1细胞凋亡的作用及机制

Effects and Mechanisms of Lentinan on Streptozotocin-induced Apoptosis in INS-1 Cells

目的探讨香菇多糖（lentinan，LNT）对链脲佐菌素（streptozoein，STZ）诱导的大鼠胰岛素瘤细胞系（INS-1）细胞凋亡的影响，并探索其可能的作用机制。方法四甲基偶氮唑盐微量酶反应比色（MTT）法观察STZ单独处理、STZ和不同浓度LNT共同处理INS-1细胞24h后细胞活力的改变：AnnexinV-PI双染法检测细胞凋亡；Hoechst33258染色和末端转移酶介导的dUTP切口末端标记（TUNEL）法验证STZ和LNT的处理对INS-1细胞凋亡的影响；放免法检测经药物处理后INS-1细胞贮存、分泌胰岛素的功能；Western印迹分析经STZ、LNT处理后INS-1细胞相关信号通路蛋白PDX-1、Bcl-2（抗凋亡基因）、Bax和Cleavedeaspase-3（促凋亡基因）的表达差异。结果与空白组相比，LNT能浓度依赖性地抑制STZ引起的INS-1细胞活力下降，浓度为200μg／ml时开始有统计学差异（P〈0．05）；LNT能抑制STZ诱导的INS-1细胞凋亡；LNT可抑制STZ诱导的INS-1细胞贮存、分泌胰岛素功能受损和PDX-1蛋白表达量下降；LNT能影响细胞凋亡信号通路相关蛋白Bcl-2、Bax、Cleavedcaspase．3表达水平。结论香菇多糖能抑制STZ诱导的INS-1细胞凋亡，可能是通过影响凋亡信号通路中相关蛋白分子的水平实现的。

Objective To investigate the effect of lentinan （LNT） on the apoptosis of rat in- sulinoma cell line （INS-1） induced by streptozotocin （STZ） and explore its possible mecha- nism. Methods MTT assay was used to observe the changes of cell viability of INS-1 treated with STZ alone, STZ and different concentrations of LNT for 24 h. Annexin V-PI staining was used to de- tect the cell apoptosis, Hoechst33258 staining and terminal transferase-mediated dUTP nick end la- beling （TUNEL） to verify the effect of STZ and LNT treatment on the apoptosis of INS-1 cells, and radioimmunoassay to detect the insulin storage and secretion function of INS-1 cells after treat- ment. The expression levels of PDX-1, Bel-2, Bax, and cleaved caspase-3 were measured by Western blotting in INS-1 cells treated with STZ and LNT. Results LNT could inhibit the STZ-in- duced decrease of INS-1 cell viability in a concentration-dependent manner, which showed signifi- cant difference from the blank control group when the concentration of LNT was 200μg/mL （P 〈 0.05 ） . LNT could inhibit STZ-induced cell apoptosis and improve the impaired storage and secre- tion of insulin by INS-1 cells. It could decrease the expression of PDX-1 protein and greatly affect the expression levels 3. Conclusion of apoptosis signaling pathway-related proteins Bcl-2, Bax, and cleaved caspase- LNT can inhibit the apoptosis of INS-1 cells induced by STZ by affecting the levelsof apoptotic signaling pathway-related proteins.