基于PI3K/AKT/mTORC信号通路探讨骨痹通消颗粒对人骨髓间充质干细胞成脂分化负性调节的机制研究

The mechanism of Gubi Tongxiao granules on the negative regulation of lipid differentiation of human bone marrow mesenchymal stem cells based on PI3K/AKT/mTORC signalling pathway

目的:观察骨痹通消颗粒对人骨髓间充质干细胞(hBMSCs)成脂分化的影响,探讨其防治激素性股骨头坏死(SANFH)的作用机制。方法:收集接受髋关节置换术的SANFH患者的骨髓组织,采用全骨髓贴壁法分离培养原代细胞并通过流式细胞术鉴定其表型。CCK-8法检测骨痹通消颗粒含药血清对hBMSCs增殖的影响,油红O、茜素红染色、RT-qPCR和Western blot检测骨痹通消颗粒含药血清作用后,hBMSCs骨脂分化、过氧化物酶体增殖物激活受体γ(PPARγ)、胆固醇调节原件结合蛋白(SREBP)、人骨形态发生蛋白(BMP2)、生长转化因子β1(TGF-β1)mRNA和通路蛋白的表达情况。结果:不同体积分数的骨痹通消颗粒含药血清均可促进hBMSCs增殖,且存在着一定的量效和时效关系,尤以5%组作用48 h时促进细胞增殖最为明显;与模型组比,5%含药血清作用后可明显减少胞质中的脂滴着色进而增加钙盐沉积处的橙红色矿化结节(P<0.05),同时下调细胞中PPARγ、SREBP mRNA和上调BMP2、TGF-β1 mRNA表达水平(P<0.05);此外,骨痹通消颗粒含药血清还可明显降低细胞中PI3K、p-AKT和mTORC等蛋白表达水平,上调Maf1蛋白表达(P<0.05)。结论:骨痹通消颗粒可以负性调节hBMSCs的成脂分化,促进成骨分化,进而发挥治疗SANFH的作用,其机制可能与调控PI3K/AKT/mTORC信号通路有关。

Objective:To observe the effect of Gubi Tongxiao granules on the lipid differentiation of human bone marrow mesenchymal stem cells(hBMSCs),and to explore its mechanism of preventing and treating steroid-induced necrosis of femoral head(SANFH).Methods:The bone marrow tissue of SANFH patients undergoing hip replacement was collected,and the original cells were isolated and cultured by the whole bone marrow wall method and their phenotype was detected by flow cytometry.CCK-8 method detected the effect of Gubi Tongxiao granules on the proliferation of hBMSCs.Oil red O,lucin red staining,RT-qPCR and Western blot detected the drug-containing serum effect of Gubi Tongxiao granules,the expression of hBMSCs bone lipid differentiation,expression of peroxisome proliferator-activated receptorγ(PPARγ),sterol-regulatory element binding proteins(SREBP),bone Morphogenetic Protein 2(BMP2),transforming growth factor-β1(TGF-β1)mRNA and pathway proteins.Results:Different volume fractions of Gubi Tongxiao granules could promote the proliferation of hBMSCs,especially in 5%group after 48 h.Compared with the model group,5%drug-containing serum could significantly reduce the lipid droplet coloring in the cytoplasm and increase the orange-red mineralized nodules in the calcium salt deposit(P<0.05),down-regulate the mRNA expression levels of PPARγand SREBP,and up-regulate the mRNA expression levels of BMP2 and TGF-β1(P<0.05).In addition,after the intervention of drug-containing serum,the expression of PI3K、p-AKT and mTORC protein was significantly decreased,and the expression of Maf1 protein in cells was increased(P<0.05).Conclusion:Gubi Tongxiao granules can negatively regulate the fat differentiation of hBMSCs,promote osteogenesis differentiation,and play a role in the treatment of SANFH.Its mechanism may be related to the regulation of PI3K/AKT/mTORC signalling pathway.