香烟烟雾提取物对SZ95人皮脂腺细胞增殖、凋亡及脂质合成的影响

Effects of cigarette smoke extract on proliferation, apoptosis, and lipid synthesis of human SZ95 sebocytes

[背景]吸烟与痤疮及反向痤疮关系密切,香烟是否通过影响皮脂腺细胞生物学功能参与相关疾病的发生,目前仍不清楚。[目的]研究香烟烟雾提取物(CSE)体外对SZ95人皮脂腺细胞增殖、凋亡、脂质合成的影响及机制。[方法]体外培养永生化SZ95人皮脂腺细胞,设置不同浓度CSE刺激组及相应的PBS对照组进行研究。采用MTT法检测体积百分比(余同)为5%、2.5%、1%、0.5%、0.25%、0.1%的CSE染毒24、48、72 h后细胞活力的变化;FITC Annexin/PI双染法检测5%、2.5%、1%CSE染毒24、48、72 h后细胞凋亡的变化;尼罗河红及油红染色法检测1%CSE对细胞脂质合成的影响;实时荧光定量PCR法及蛋白印迹法检测1%CSE作用前后细胞内芳香烃受体(AhR)及其下游经典靶位基因细胞色素P4501A1(CYP1A1)的基因及蛋白表达情况。[结果]MTT实验结果显示,与对照组相比,2.5%和5%CSE诱导细胞不同时间(24、48、72 h)的细胞活力下降(P<0.01):2.5%CSE组细胞活力分别为81%、60%、66%,5%CSE组细胞活力分别为54%、16%、12%。2.5%和5%CSE剂量依赖性诱导细胞晚期凋亡:24、48、72 h后2.5%CES组细胞晚期凋亡比例(Annexin+/PI+)分别为3.74%、6.71%、56.6%,5%CSE组分别为22.9%、27.1%、64.5%。1%CSE作用48 h后细胞脂质合成减少(P<0.01),CSE组的脂质合成量是对照组的85%。1%CSE作用2 h时细胞内AhR及CYP1A1 mRNA的相对表达水平分别是对照组的1.68倍和4.53倍(P<0.01),48 h时蛋白的相对表达水平是对照组的2.10倍和3.48倍(P<0.01)。[结论]CSE呈剂量依赖性抑制体外细胞增殖,诱导细胞晚期凋亡,一定浓度时抑制细胞脂质合成,上调AhR及CYP1A1蛋白及mRNA的表达。本研究结果提示香烟烟雾可能通过干扰人皮脂腺细胞的正常生物学功能而参与皮脂腺相关疾病的发生。

[Background] Cigarette smoking is closely related to acne and acne inversa(AI). Whether cigarettes affect the biological functions of sebocytes and then participate in the occurrence of related diseases is still unclear.[Objective] This study aims to determine the effects of cigarette smoke extract(CSE) on the proliferation, apoptosis, and lipid synthesis of human SZ95 sebocytes and its mechanisms. [Methods] Immortalized human SZ95 sebocytes were cultured in vitro. CSE stimulation groups and corresponding PBS control groups were set up. Cell viabilities of SZ95 sebocytes were examined after exposure to 5%, 2.5%, 1%, 0.5%, 0.25%, and 0.1% CSE(volume fraction, thereafter) for 24, 48 and 72 h of incubation by MTT method. Cell apoptosis rates were measured by FITC Annexin/PI double staining after being incubated with 5%, 2.5%, and 1% CSE for 24, 48, and 72 h. Lipid contents in SZ95 sebocytes were analyzed after Nile red and Oil red staining. The expressions of aryl hydrocarbon receptor(Ah R) and its downstream classic target gene cytochrome P4501 A1(CYP1A1) before and after 1% CSE exposure were detected by real-time PCR and Western blotting. [Results] The MTT method results showed that, compared with the control groups, 2.5% and 5% CSE induced significant decreases in cell viabilities at multiple time points(24, 48, and 72 h)(P < 0.01): the viabilities of the 2.5% CSE group were 81%, 60%, 66%, respectively;and those of the 5% CSE group were 54%, 16%, and 12%, respectively. Cell late apoptosis was induced by 2.5% and 5% CSE in a dose-dependent manner: after incubation for 24, 48, and 72 h, the ratios of late apoptosis cells(Annexin+/PI+) were 3.74%, 6.71%, and 56.6% in the 2.5% CSE group, and 22.9%, 27.1%, and 64.5% in the 5% CES group, respectively. Cell lipid synthesis decreased after incubation with 1% CSE for 48 h(P < 0.01), and the amount of lipid synthesis in the CSE group was 85% of that of the control group. After 1% CSE treatment, the relative m RNA expression levels of Ah R and CYP1A1 in the cells at 2 h were 1.68 times and 4.53 times of the control group, respectively(P < 0.01), and the relative protein expression levels at 48 h were 2.10 times and 3.48 times of the control group, respectively(P < 0.01). [Conclusion] CSE dose-dependently inhibits cell proliferation and induces cell apoptosis in vitro, as well as inhibits lipogenesis and upregulates Ah R and CYP1 A1 protein and m RNA expressions in SZ95 seboctyes at a certain concentration, which indicates that cigarette smoke may participate in the development of sebaceous gland related diseases by interfering with the normal biological functions of sebocytes.