缺氧诱导SD大鼠阴茎海绵体平滑肌细胞内炎症反应及番茄红素的干预作用

Hypoxia-induced inflammatory response in penile corpora cavernosal smooth muscle cells of sd rats and the interventional roleof lycopen

目的:探索缺氧对SD大鼠海绵体平滑肌细胞(CCSMCs)内炎症反应的诱导作用及番茄红素调节效应。方法:分离SD大鼠原代CCSMCs并连续观察第4代海绵体平滑肌细胞内的形态稳定性;为了验证所培养的CCSMCs的纯度,进行了α-平滑肌肌动蛋白(α-SMA)和结蛋白(Desmin)的免疫荧光染色;不同浓度的CoCl_(2)和番茄红素单独或联合干预海绵体平滑肌细胞24 h后,使用细胞计数试剂盒-8(CCK8)检测细胞活力;设立3个细胞处理组别:对照组、缺氧组(CoCl_(2)200μmol/L)和番茄红素治疗组(40μmol/L);培养24 h后,应用蛋白质印迹法(Western blotting)和反转录定量聚合酶链式反应(RT-qPCR)检测缺氧诱导因子1α(HIF-1α)、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、IL-18、IL-1β水平。结果:提取的SD大鼠海绵体平滑肌细胞在培养至第4代时依旧保持了形态上的均一性,经α-SMA和Desmin染色均显示出纯度较高的阳性反应。200μmol/LCoCl_(2)处理后CCSMCs的活力下降到50%,且Western blotting和RT-qPCR检测细胞内HIF-1α、TNF-α、IL-6、IL-18的水平均呈上升趋势(P<0.05),IL-1β的mRNA表达亦显著升高(P<0.05),但蛋白表达量仅有升高趋势(P>0.05);40μmol/L番茄红素能将CCSMCs的活力得以提升至正常状态的80%,同时显著降低HIF-1α、TNF-α、IL-18的蛋白表达量和TNF-α、IL-18、IL-1β的mRNA表达量(P<0.05),IL-6的蛋白、mRNA及IL-1β的蛋白表达量仅有下降趋势(P>0.05)。结论:缺氧条件下,CCSMCs中炎症因子表达上调,番茄红素能有效抑制炎症因子表达。

Objective To explore the hypoxia-induced inflammatory response in corpus cavernosum smooth muscle cells(CCSMCs)of SD rats and the regulatory effect of lycopene.Methods Primary CCSMCs were isolated from SD rats and observed for morphological stability up to the fourth passage.Immunofluorescence staining forα-smooth muscle actin(α-SMA)and desmin was performed to verify the purity of cultured CCSMCs.After 24 hours of exposure to different concentrations of CoCl_(2)and lycopene,either alone or in combination,cell viability was assessed using the Cell Counting Kit-8(CCK8).The experiment included three treatment groups:control,hypoxia(200μmol/LCoCl_(2)),and lycopene treatment(40μmol/L).Western Blotting and reverse transcription quantitative polymerase chain reaction(RT-qPCR)were used to detect the levels of hypoxia-inducible factor 1-alpha(HIF-1α),tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),interleukin-18(IL-18),and interleukin-1 beta(IL-1β)after 24 hours of culture.Results The CCSMCs maintained morphological uniformity up to the fourth passage.High purity was confirmed through positive staining forα-SMA and desmin.Following 200μmol/L CoCl_(2)treatment,CCSMCs viability decreased to 50%,with increases in intracellular levels of HIF-1α,TNF-α,IL-6,IL-18(P<0.05),and a significant rise in IL-1βmRNA expression(P<0.05),though protein levels showed only an increasing trend(P>0.05).Lycopene at 40μmol/L elevated CCSMC viability to 80%of normal levels and significantly reduced HIF-1α,TNF-α,IL-18 protein expression,and TNF-α,IL-18,IL-1βmRNA levels(P<0.05).IL-6 protein,IL-6 mRNA,and IL-1βprotein levels showed a decreasing trend(P>0.05).Conclusion Under hypoxic conditions,the expression of inflammatory factors in CCSMCs is upregulated,and lycopene can effectively inhibit the expression of these inflammatory factors.