杨梅素联合围脂滴蛋白基因沉默促进前脂肪细胞系3T3-L1的降脂

Myricetin combined with PLIN1 silencing promotes lipolysis of 3T3-L1 adipocytes

目的探讨杨梅素(Myric)与围脂滴蛋白(PLIN1)基因沉默对3T3-L1脂肪细胞脂解通路的影响。方法采用经典"鸡尾酒"方法诱导分化3T3-L1前脂肪细胞,筛选出杨梅素最佳干预浓度和时间。杨梅素联合sh-PLIN1干扰载体对成熟的脂肪细胞进行干预,实验共分为4组:对照组(control group)、转染组(sh-RNA group)、杨梅素组(Myric group)和联合干预组(Myric+sh-RNA group)。油红O染色脂滴,观察脂滴形态;Western blot检测细胞中ERK、p-ERK、MEK、p-MEK和激素敏感性脂肪酶磷酸化蛋白(p-HSL)的表达;ELISA测定细胞中环磷酸腺苷(cAMP)、蛋白激酶A(PKA)和磷酸化的蛋白激酶C(p-PKC)的含量。结果与Myric组和sh-RNA组相比,Myric+sh-RNA组脂滴形态变小,数量明显减少。与sh-RNA组和对照组相比,Myric+sh-RNA组和Myric组细胞内p-PKC含量、p-ERK/ERK和p-MEK/MEK的比值以及p-HSL表达显著升高(P<0.05)。同时,Myric+sh-RNA组和Myric组细胞内cAMP和PKA含量低于sh-RNA组和对照组(P<0.05)。结论杨梅素的促脂解作用可能是通过激活PKCMEK/ERK信号通路,增加该通路中p-HSL的表达量来实现的;杨梅素同时可能对cAMP/PKA信号通路中相关因子的活性有一定的抑制作用。

Objective To investigate the mechanism of myricetin（Myric） combined with PLIN1 silencing on lipolysis of 3T3-L1 adipocytes. Methods 3T3-L1 adipocytes were routinely induced to differentiation by the hormonal cocktail. According to the optimal concentration of myricetin combined with the high efficient transfection vector of sh-PLIN1, the intervention experiment was divided into four groups： control group, transfection group （sh-RNA）, myricetin group （Myric） and combination group （Myric＋sh-RNA）. The Oil Red O staining was used to observe the morphology of lipid droplets. The protein expression of ERK, p-ERK, MEK, p-MEK and p-HSL was detected by Western blot. Enzyme linked immunosorbent assay （ELISA） was used to measure the content of the cyclic adenosine monophosphate （cAMP）, protein kinase A （PKA） and p-PKC. Results Compared to Myric group and sh-RNA group, size of lipid droplets decreased obviously in Myric＋sh-RNA group. Compared to sh-RNA group and control group,the relative value of p-PKC, p-ERK/ERK, p-MEK/MEK and p-HSL increased significantly in Myric＋ sh-RNA group and Myric group （ P 〈0.05）, meanwhile, the content of cAMP and PKA decreased significantly （ P 〈 0.05）. Conclusions Mmyricetin may promote lipolysis through the activation of PKC-MEK/ERK signaling pathway, and further increase expression level of p-HSL in this pathway. Meanwhile, myricetin may also inhibit the activity of related factors in cAMP/PKA signaling pathway.