辣椒素对高盐致大鼠血管平滑肌细胞增殖的抑制作用

Inhibitory effects of capsaicin on the proliferation of rat vascular smooth muscle cells induced by high sodium

目的研究辣椒素对高盐诱导的大鼠血管平滑肌细胞(VSMC)增殖的抑制作用。方法组织贴壁法培养原代大鼠血管平滑肌细胞,根据MTT结果绘制大鼠血管平滑肌细胞的生长曲线,大鼠血管平滑肌细胞传至第4代去血清同步化24 h,不同浓度辣椒素(0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L、100μmol/L)进行高盐培养72 h,MTT比色法检测大鼠血管平滑肌细胞增殖情况,选取抑制血管平滑肌细胞增殖的辣椒素浓度进行后续实验。CCK-8检测渗透压对大鼠血管平滑肌细胞增殖的影响,流式细胞仪分析细胞增殖周期,免疫荧光染色法和Western blot检测增殖细胞核抗原(PCNA)的表达,实时荧光定量PCR检测瞬时受体电位家族香草醛1型受体(TRPV1)mRNA的表达,Western blot检测瞬时受体电位家族香草醛1型受体的蛋白表达。结果 MTT结果显示,辣椒素浓度为10μmol/L时大鼠血管平滑肌细胞增殖开始受抑制,100μmol/L时抑制作用增强(P<0.05),选取10μmol/L辣椒素进行后续实验。CCK-8结果显示,高盐组细胞明显增殖,而甘露醇组、正常组和高盐+辣椒素组无差异。流式细胞仪测得高盐组G0/G1、G2/M期细胞比例减少(P<0.05),S期细胞比例增多(P<0.05);与高盐组比较,高盐+辣椒素组G0/G1、G2/M期细胞比例增加(P<0.05),S期细胞比例下降(P<0.05)。免疫荧光及Western blot结果显示,高盐组增殖细胞核抗原阳性细胞核增多,蛋白表达增加(P<0.05),而高盐+辣椒素组增殖细胞核抗原阳性细胞核减少,蛋白表达减少(P<0.05)。实时荧光定量PCR和Western blot结果显示高盐组瞬时受体电位家族香草醛1型受体mRNA及蛋白表达减少(P<0.05),辣椒素组则增加(P<0.05)。结论辣椒素可抑制高盐所致的大鼠血管平滑肌细胞增殖,其作用机制可能与激活瞬时受体电位家族香草醛1型受体表达有关。

Aim To investigate the effect of capsaicin on the inhibition of rat vascular smooth muscle cells （VSMC） induced by high sodium.Methods Vascular smooth muscle cells were isolated from thoracic aorta of male Sprague-Dawley rats by tissue adherent method,and the growth curve of vascular smooth muscle cells was made according to MTT assay.The experimental procedures were performed with the fourth generation of vascular smooth muscle cells.Vascular smooth muscle cells were synchronized by serum starvation for 24 h before capsaicin and high sodium treatment.Vascular smooth muscle cells were treated with different concentration of capsaicin （0.01,0.1,1,10 and 100 μmol/L） for 72 h in high sodium.The effects of different concentration capsaicin and osmotic pressure on the proliferation induced by high sodium medium in vascular smooth muscle cells were evaluated by MTT assay and CCK-8,respectively.The cell cycle of vascular smooth muscle cells was analyzed by flow cytometry technology.The localization and protein expression of PCNA were employed by immunofluorecence staining and Western blot.The mRNA and protein expressions of TRPV1 were detemined by real-time PCR and Western blot.Results MTT results showed that when the concentration of capsaicin was 10μmol/L,the proliferation of rat vascular smooth muscle cells began to be inhibited,and the inhibitory effect was increased at 100 μmol/L （P〈0.05）,so 10 μmol/L capsaicin was selected for subsequent experiments.CCK-8 results showed that the cells in high sodium group proliferated obviously,but there was no difference between mannitol group,normal group and capsaicin group.Capsaicin prevented notably the decrease of cell number in G0/G1 and G2/M stage and increase of cell number of S stage in vascular smooth muscle cells treated with Na＋（159 mmol/L）.PCNA protein level was also decreased by capsaicin.The decrease of mRNA and protein level of TRPV1 stimulated Na＋（159 mmol/L） was alleviated by capsaicin.Conclusion Capsaicin can inhibit the proliferation of vascular smooth muscle cells induced by Na＋（159 mmol/L）,the mechanism may be related to the up-regulation of TRPV1 expression.