木姜子和忍冬藤配伍乙醇提取物对气管平滑肌细胞增殖及凋亡的影响

Effect of ethanolic extract from Litsea pungens combined with Caulis Lonicerae on proliferation and apoptosis of airway smooth muscle cell

目的 观察木姜子和忍冬藤配伍乙醇提取物对气管平滑肌细胞(ASMC)增殖、凋亡的影响,并探讨其可能机制。方法 配制5.65μg/mL、11.3μg/mL、22.6μg/mL浓度的木姜子和忍冬藤配伍乙醇提取物。体外培养ASMC,将其分为空白组、模型组、低浓度组、中浓度组、高浓度组。各组均加入1%胎牛血清+杜氏改良伊格尔培养基/汉氏F-12营养培养基,除空白组外,其余各组均加入20μg/mL血小板源性生长因子,此外各浓度组加入相应浓度的木姜子和忍冬藤乙醇提取物。培养6h、12h、18h、24h、36h后检测各组细胞增殖情况,并计算增殖抑制率。培养24h后,观察各组细胞凋亡情况,检测各组转化生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)、白细胞介素(IL)-4、IL-13、IL-17水平,以及B细胞淋巴瘤2(Bcl-2)、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达水平。结果 各浓度组的抑制率均高于模型组,低浓度组、中浓度组、高浓度组的抑制率依次升高,且抑制率均随时间延长而升高(均P<0.05)。低浓度组可以观察到有部分细胞凋亡和核固缩现象,中浓度组和高浓度组该现象增多。与空白组比较,模型组TGF-β1、VEGF、IL-4、IL-13、IL-17水平及Bcl-2蛋白表达水平升高,Caspase-3蛋白表达水平降低(均P<0.05);与模型组比较,各浓度组TGF-β1、VEGF、IL-4、IL-13、IL-17水平及Bcl-2蛋白表达水平均降低,Caspase-3蛋白表达水平升高(均P<0.05)。结论 木姜子和忍冬藤配伍乙醇提取物可抑制ASMC增殖,并可诱导ASMC凋亡,其机制可能与促进凋亡蛋白表达、抑制抗凋亡蛋白表达以及降低相关细胞因子水平有关。

Objective To observe the effect of ethanolic extract from Litsea pungens combined with Caulis Lonicerae on proliferation and apoptosis of airway smooth muscle cell(ASMC),and to explore the possible mechanisms.Methods The ethanolic extract from Litsea pungens combined with Caulis Lonicerae with the concentration of 5.65 μg/mL,11.3 μg/mL,or 22.6 μg/mL was prepared.ASMCs were cultured in vitro,and then were divided into blank group,model group,low concentration group,medium concentration group and high concentration group.All groups were added with 1% fetal calf serum+Dulbecco′s modified Eagle medium/Ham′s nutrient mixture F12,except the blank group,the remaining groups were added with 20 μg/mL platelet-derived growth factor(PDGF),besides,each concentration group was added with ethanolic extract from Litsea pungens and Caulis Lonicerae of corresponding concentration.After 6,12,18,24 and 36 hours of culture,cell proliferation was detected in each group,and the proliferation inhibitory rate was calculated.After 24 hours of culture,cell apoptosis was observed in each group,the levels of transforming growth factor β1(TGF-β1),vascular endothelial growth factor(VEGF),interleukin(IL)-4,IL-13 and IL-17 were measured,as well as the expression levels of B-cell lymphoma 2(Bcl-2) and cysteinyl aspartate specific proteinase 3(Caspase-3).Results Each concentration group had a higher inhibitory rate than the model group,the inhibitory rates of the low concentration group,the medium concentration group and the high concentration group increased successively,and also ascended with time(all P<0.05).Apoptosis and pyknosis were observed in partial cells of the low concentration group,and that phenomenon was more common in the medium concentration and high concentration groups.Compared with the blank group,the model group obtained higher TGF-β1,VEGF,IL-4,IL-13,IL-17 levels and Bcl-2 protein expression level,but lower Caspase-3 protein expression level(all P<0.05);compared with the model group,each concentration group obtained lower TGF-β1,VEGF,IL-4,IL-13,IL-17 levels and Bcl-2 protein expression level,but higher Caspase-3 protein expression level(all P<0.05).Conclusion The ethanolic extract from Litsea pungens combined with Caulis Lonicerae not only can inhibit proliferation of ASMC,but also can induce apoptosis of ASMC.The mechanisms may be related to promoting apoptosis protein expression,inhibiting anti-apoptotic protein expression,and reducing relevant cytokines levels.