豆甾醇通过抑制钙离子内流抑制大鼠前列腺间质细胞收缩

Stigmasterol inhibits the contraction of rat prostatic stromal cells by inhibiting calcium influx

[目的]检测豆甾醇对前列腺间质细胞收缩的抑制作用。[方法]体外培养大鼠原代前列腺间质细胞,通过鬼笔环肽染色法对前列腺间质细胞进行表型鉴定;用细胞增殖及乳酸脱氢酶(LDH)试剂盒检测豆甾醇对前列腺间质细胞活力和毒性的影响;用胶原凝胶收缩实验检测对前列腺间质细胞收缩功能的影响;运用逆转录-聚合酶链反应(RT-PCR)法研究豆甾醇对收缩相关的α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(CollagenⅠ)的mRNA水平表达的影响;用蛋白免疫印迹(Western Blot)法检测豆甾醇对转化生长因子-β1(TGF-β1)诱导的α-SMA表达的影响;流式细胞术检测豆甾醇对乙酰胆碱(ACh)诱导的细胞内钙离子浓度的影响。[结果]大鼠原代前列腺细胞培养获得的细胞以间质平滑肌细胞为主;浓度低于10μmol/L的豆甾醇对前列腺间质细胞的活力无明显影响,且无明显毒性;胶原凝胶实验结果显示,血清可以使前列腺间质细胞收缩,差异具有统计学意义(P<0.01),10μmol/L的豆甾醇可显著抑制该收缩作用,抑制率可达到77.1%,差异具有统计学意义(P<0.05);RT-PCR结果显示,豆甾醇显著降低大鼠前列腺间质细胞中α-SMA和CollagenⅠmRNA水平的表达,差异具有统计学意义(P<0.01);Western Blot结果显示,豆甾醇可以抑制TGF-β1诱导的α-SMA蛋白的表达;流式细胞实验发现ACh刺激导致的钙离子浓度明显增加可以被豆甾醇显著抑制。[结论]豆甾醇可抑制前列腺间质收缩,其机制与抑制其钙离子内流有关。

[Objective]To detect the inhibitory effect of stigmasterol on the contraction of prostate stromal cells.[Method]Rat primary prostatic stromal cells were cultured in vitro and the phenotypes were identified by phalloidin staining.The effects of stigmasterol on cell viability and toxicity were investigated by CCK8 and LDH assays.The contractile response of prostate stromal cells was analyzed by collagen gel assay.RT-PCR and Western Blot were performed to detect the basal and transforming growth factor-β1(TGF-β1)induced expressions ofα-smooth muscle actin(α-SMA)and CollagenⅠ.Acetylcholine(ACh)induced intracellular calcium levels was established and the effect of stigmasterol on ACh-induced Ca2+concentration was further detected by flow cytometry.[Results]Cells obtained from the rat prostate were mainly identified as the interstitial smooth muscle cells;stigmasterol had no significant effects on the viability and toxicity of prostatic stromal cellswith concentrations lower than 10μM;collagen gel assay showed that seruminducedthe contraction of the prostatic stromal cells(P<0.01),which was significantly inhibited bystigmasterol,andthe inhibition rate reached 77.1%at 10μmol/L(P<0.05).RT-PCR data showed that stigmasterol significantly down-regulated the mRNA expressions ofα-SMA and CollagenⅠin rat prostatic stromal cells(P<0.01).Western Blot also showed that stigmasterol inhibited TGF-β1 inducedα-SMA expression.The increased calcium levelin response to ACh stimulation was also significantly inhibited by stigmasterol.[Conclusion]Stigmasterol inhibits the contraction of prostate stroma through inhibition of calcium influx.