基于TRPV1/AMPK通路探讨石榴皮总多酚对亚油酸诱导的人皮脂腺细胞脂质合成的影响

Effects of pomegranate peel polyphenols on linoleic acid-induced lipid synthesis in human sebocytes based on TRPV1/AMPK pathway

目的 探讨石榴皮总多酚(pomegranate peel polyphenols,PPPs)对亚油酸(linoleic acid,LA)诱导的永生化人皮脂腺SZ95细胞脂质合成的影响及其作用机制。方法 体外培养SZ95细胞,运用LA诱导构建脂质过度合成模型,采用CCK-8法检测不同质量浓度PPPs对SZ95细胞活力的影响;尼罗红染色检测细胞脂质合成情况;qRT-PCR检测瞬时受体电位香草酸亚型1(transient receptor potential vanilloid 1,TRPV1)、腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)mRNA表达;Western blotting检测TRPV1、AMPK、磷酸化AMPK(phosphorylated AMPK,p-AMPK)蛋白表达。采用siRNA进行细胞转染,观察沉默TRPV1后PPPs对LA诱导的SZ95细胞脂质合成及TRPV1/AMPK通路的影响。结果 1.25、2.5、5μg/mL的PPPs对SZ95细胞活性无明显影响,且均可显著抑制LA诱导的脂质过度合成(P<0.01),其中5μg/mL PPPs的抑制效应最为明显。与对照组比较,模型组细胞脂质合成明显增加(P<0.01),TRPV1、AMPK mRNA表达及TRPV1、p-AMPK/AMPK蛋白表达水平均明显降低(P<0.01);PPPs对无LA诱导的SZ95细胞基础脂质合成无明显影响,TRPV1、AMPK mRNA表达水平升高(P<0.01),但TRPV1、p-AMPK/AMPK蛋白表达水平差异无统计学意义。与模型组比较,PPPs明显减少LA诱导下的SZ95细胞脂质合成(P<0.01),明显提高TRPV1、AMPK mRNA表达及TRPV1、p-AMPK/AMPK蛋白表达水平(P<0.05、0.01)。沉默TRPV1显著增加了LA诱导下的SZ95细胞脂质合成(P<0.01),并显著减弱了PPPs对SZ95细胞脂质过度合成的抑制(P<0.01),显著降低其TRPV1、AMPK的mRNA及TRPV1、pAMPK/AMPK蛋白表达水平(P<0.05、0.01)。结论 PPPs可有效抑制LA诱导的SZ95细胞脂质合成,其作用机制可能与TRPV1/AMPK通路有关。

Objective To investigate the effect and mechanism of pomegranate peel polyphenols(PPPs) on lipid synthesis induced by linoleic acid(LA) in human immortalized SZ95 sebocytes.Methods SZ95 sebocytes were cultured in vitro and lipid over-synthesis model were induced by LA.The effect of different concentrations of PPPs on viability of SZ95 sebocytes was measured by CCK-8assay.Lipid content in cells was measured by nile red staining.qRT-PCR was used to detect the mRNA expressions of transient receptor potential vanilloid 1(TRPV1) and adenosine monophosphate-activated protein kinase(AMPK).Western blotting was used to detect the protein expressions of TRPV1,AMPK and phosphorylated AMPK(p-AMPK).Cells were transfected with siRNA to observe the effect of PPPs on LA induced lipid synthesis and TRPV1/AMPK pathway in SZ95 sebocytes after TRPV1 silencing.Results PPPs(1.25,2.5 and 5 μg/mL) had no significant effect on the viability of SZ95 sebocytes but significantly inhibited LA-induced lipid over-synthesis(P<0.01),and 5 μg/mL of PPPs had the most obvious inhibitory effect.Compared with control group,the lipid synthesis of cells was significantly increased in model group(P<0.01),mRNA expressions of TRPV1 and AMPK,and protein expressions of TRPV1 and p-AMPK/AMPK were significantly decreased(P<0.01).PPPs had no significant effect on the basal lipid synthesis of SZ95 sebocytes without LA induction,and the mRNA expression levels of TRPV1 and AMPK were significantly increased(P<0.01),but there was no significant difference in the protein expression levels of TRPV1 and p-AMPK/AMPK.Compared with model group,PPPs significantly reduced the LA induced lipid synthesis in SZ95 sebocytes(P<0.01),and significantly increased mRNA expressions of TRPV1 and AMPK and protein expressions of TRPV1 and p-AMPK/AMPK(P<0.05,0.01).Silencing TRPV1 significantly increased the LA induced lipid synthesis in SZ95 sebocytes(P<0.01),significantly weakened the inhibition of PPPs on lipid over-synthesis in SZ95 sebocytes(P<0.01),significantly decreased the mRNA expression levels of TRPV1,AMPK and protein expression levels of TRPV1 and p-AMPK/AMPK(P<0.05,0.01).Conclusion PPPs can effectively inhibit LA-induced lipid synthesis in SZ95sebocytes,and its mechanism may be related to TRPV1/AMPK pathway.