人参皂苷Rg_3调节免疫检查点PD-L1抑制肺癌Lewis细胞增殖的作用及机制研究

Effect and mechanism of ginsenoside Rg_3 on inhibition of LLC proliferation in non-small cell lung cancer cells by immune checkpoint PD-L1

目的研究人参皂苷Rg_3对小鼠非小细胞肺癌Lewis细胞(LLC)中免疫检查点程序性死亡分子1配体(PD-L1)的调节作用及其作用机制。方法通过MTT法及细胞长时程动态监测法观察人参皂苷Rg_3对LLC增殖的影响;20 ng/mLγ干扰素(IFN-γ)处理LLC制备PD-L1高表达体外模型,采用人参皂苷Rg_3进行干预,流式细胞术及免疫荧光检测人参皂苷Rg_3对PD-L1表达的影响;采用Western blotting法验证人参皂苷Rg_3对PI3K/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)通路相关蛋白表达的影响。结果人参皂苷Rg_3 16、32、64、128μmol/L能够显著抑制LLC增殖(P<0.01)及减少IFN-γ诱导的PD-L1表达(P<0.05);人参皂苷Rg_3 32、64μmol/L能够降低PI3K、m TOR蛋白的表达水平(P<0.01);人参皂苷Rg_3 16、32、64μmol/L能够抑制Akt蛋白的磷酸化(P<0.05)。结论人参皂苷Rg_3能够显著抑制LLC中PD-L1表达,通过抑制PI3K/Akt/m TOR通路,阻断PD-L1介导的肿瘤细胞免疫逃逸,增强T细胞的免疫应答作用,抑制LLC生长。

Objective To investigate the effect of ginsenoside Rg3 on immune checkpoint PD-L1 in Lewis cells(LLC) and to explore the related mechanism. Methods The effects of ginsenoside Rg3 on the proliferation of LLC were observed by MTT assay and cell long-term dynamic monitoring. LLC cells treated with 20 ng/m L IFN-γ were used to construct experimental model with high expression of PD-L1 in vitro. The effect of ginsenoside Rg3 on expression of PD-L1 was detected by flow cytometry and immunofluorescence. The effect of ginsenoside Rg3 on the protein expression of PI3 K/Akt/mTOR pathway was verified by Western blotting. Results Ginsenoside Rg3 at 16, 32, 64, and 128 μmol/L significantly inhibited the proliferation of LLC(P < 0.01) and reduced the expression of PD-L1 induced by IFN-γ(P < 0.05). Ginsenoside Rg3 at 32 and 64 μmol/L significantly decreased the protein expression of PI3 K and mTOR(P < 0.01), and ginsenoside Rg3 at 16, 32, and 64 μmol/L significantly inhibited the phosphorylation of Akt proteins(P < 0.05). Conclusion Ginsenoside Rg3 significantly inhibited the expression of PD-L1 in LLC cells by inhibiting PI3 K/Akt/mTOR pathway. Ginsenoside Rg3 blocked the tumor cells escaping immune response by PD-L1 over-expression, enhanced the immune response of T cells, and inhibited the growth of non-small cell lung cancer cells.