铁皮石斛多糖对神经细胞凋亡抑制作用的研究

Study on the inhibitory effect of dendrobium polysaccharide on neuron apoptosis

目的探究铁皮石斛多糖(DOP)能否抑制由肿瘤坏死因子-α(TNF-α)引起的神经细胞凋亡,以及其抑制凋亡的最佳浓度。方法分别采用不同浓度的TNF-α对小鼠海马神经元细胞系(HT22)进行培养,确定TNF-α诱导HT22细胞凋亡的最佳浓度;再将不同浓度的DOP与最佳浓度的TNF-α共同培养HT22细胞24 h后,采用PCR、Western blot检测凋亡相关基因和蛋白的表达,查找DOP抑制HT22细胞凋亡的最佳作用浓度;之后,同时通过吉姆萨染色、赫斯特(Hoechst)染色、原位末端转移酶标记技术(Tunel)染色、流式细胞术观察HT22细胞凋亡数量及比例的变化。结果与正常培养组比较,TNF-α可增加HT22细胞中凋亡相关基因的表达,且最佳浓度为5 ng/ml(P<0.0001)。与5 ng/ml TNF-α组比较,各浓度DOP均可抑制HT22细胞凋亡基因的表达,其中20μg/ml和25μg/ml DOP组的抑制效果最为明显(P<0.001);且20μg/ml和25μg/ml DOP组相关蛋白的表达、HT22细胞的凋亡数量和比例均显著低于5 ng/ml TNF-α组(P<0.001)。结论DOP可以抑制由TNF-α引起的HT22细胞凋亡,且其浓度在20μg/ml和25μg/ml时的抑制效果最佳。

Objective To explore whether dendrobium polysaccharide(DOP)can inhibit the apoptosis of nerve cells caused by tumor necrosis factor-α(TNF-α)and the optimal concentration of DOP.Methods Mouse hippocampal neurons(HT22)were cultured with different concentrations of TNF-αto determine the optimal concentration of TNF-αinduced HT22 cell apoptosis.After co-cultured with different concentrations of DOP and the optimal concentration of TNF-αfor 24 hours,the expression of apoptosis related genes and proteins was detected by PCR and Western blot to find out the inhibition of DOP on HT22 cells.At the same time,the number and proportion of HT22 cells apoptosis were observed by Giemsa staining,Hoechst staining,Tunel staining and flow cytometry.Results Compared with the normal culture group,TNF-αincreased the expression of apoptosis related genes in HT22 cells,and the optimal concentration was 5 ng/ml(P<0.0001).Compared with the 5 ng/ml TNF-αgroup,the expression of apoptosis gene in HT22 cells was inhibited by different concentrations of DOP,especially in the 20μg/ml and 25μg/ml groups(P<0.001),and the expression of related proteins in the 20μg/ml and 25μg/ml groups,the number and proportion of apoptosis in HT22 cells were significantly lower than those in the 5 ng/ml TNF-αgroup(P<0.001).Conclusion DOP can inhibit the apoptosis of HT22 cells induced by TNF-α,and the best inhibition effect is at the concentration of 20μg/ml and 25μg/ml.