【摘要】 目的 探究清肺合剂减少Lewis肺癌细胞犬尿氨酸产生并降低T细胞芳烃受体（AhR）表达的作用及其机制。方法 在外源性补充色氨酸的基础上，用清肺合剂、程序性死亡受体-1(PD-1)抑制剂等干预CD8~+T、Lewis肺癌细胞共培养体系，通过细胞计数试剂盒-8(CCK-8)检测细胞活力，酶联免疫吸附试验（ELISA）检测细胞上清液中犬尿氨酸表达水平，蛋白免疫印迹（WB）检测CD8~+T细胞AhR、PD-1蛋白表达。结果 清肺合剂显著抑制Lewis肺癌细胞生长（P<0.05）。与CD8~+共培养对照组比较，清肺合剂含药血清组和PD-1抑制剂组细胞上清液中犬尿氨酸含量均显著下降[（86.05±8.70）pmol/mL、（113.83±11.64）pmol/mL比（138.07±15.71）pmol/mL,P<0.05],CD8~+T细胞AhR[(0.48±0.05)、（0.59±0.08）比（0.82±0.12）,P<0.05]、PD-1蛋白[（0.67±0.06）、（0.51±0.04）比（0.91±0.07）,P<0.05]表达水平均显著降低。与清肺合剂含药血清组和PD-1抑制剂组比较，PD-1抑制剂+清肺合剂含药血清组细胞活力以及细胞上清液中犬尿氨酸含量显著下降[（59.11±7.63）pmol/mL比（86.05±8.70）pmol/mL、（113.83±11.64）pmol/mL,P<0.05],CD8~+T细胞AhR[(0.26±0.07)比（0.48±0.05）、（0.59±0.08）,P<0.05]、PD-1蛋白[（0.28±0.03）比（0.67±0.06）、（0.51±0.04）,P<0.05]表达水平显著降低。与PD-1抑制剂+清肺合剂含药血清组比较，PD-1抑制剂+清肺合剂含药血清+信号传导及转录激活因子1(STAT1)激动剂组和PD-1抑制剂+清肺合剂含药血清+AhR受体激动剂组细胞上清液中犬尿氨酸含量均显著增加[（98.26±11.35）pmol/mL、（92.03±10.16）pmol/mL比（59.11±7.63）pmol/mL,P<0.05],CD8~+T细胞AhR[(0.54±0.07)、（0.48±0.04）比（0.26±0.07）,P<0.05]、PD-1蛋白[（0.49±0.12）、（0.62±0.10）比（0.28±0.03）,P<0.05]表达水平均显著升高。结论 清肺合剂能降低CD8~+T细胞AhR和PD-1表达水平，其可能是通过抑制Lewis肺癌细胞犬尿氨酸产生实现，且与PD-1抑制剂合用有协同增效作用。更多还原

【Abstract】 Objective To investigate the effects and mechanisms of Qingfei Mixture in reducing the production of kynurenine in Lewis lung cancer cells and decreasing the expression of aryl hydrocarbon receptor(AhR) in T cells. Methods In a co-culture system of CD8~+T cells and Lewis lung cancer cells, exogenous supplementation of tryptophan was applied, followed by intervention with Qingfei Mixture, programmed cell death receptor-1(PD-1)inhibitors. Cell viability was detected using the cell counting kit-8 assay. The expression levels of kynurenine in the cell supernatants were measured by enzyme-linked immunosorbent assay. Protein expression levels of AhR and PD-1 in CD8~+T cells were detected by Western blotting. Results Qingfei Mixture significantly inhibited the growth of Lewis lung cancer cells(P<0.05). Compared with the CD8~+co-culture control group, the levels of kynu-renine in the cell supernatant were significantly decreased [(86.05±8.70),(113.83±11.64) vs.(138.07±15.71)pmol/mL, P<0.05] in the Qingfei Mixture-containing serum group and the PD-1 inhibitor group. The expression levels of AhR[(0.48 ±0.05),(0.59 ±0.08) vs.(0.82 ±0.12), P <0.05] and PD-1 [(0.67 ±0.06),(0.51 ±0.04) vs.(0.91 ±0.07), P <0.05] in CD8~+T cells were significantly decreased. Compared with the Qingfei Mixture-containing serum group and the PD-1 inhibitor group, the combination treatment group(PD-1 inhibitor + Qingfei Mixture-containing serum)showed significantly reduced cell viability and a lower level of kynurenine [(59.11±7.63) vs.(86.05±8.70),(113.83±11.64)pmol/mL, P<0.05]. The expression levels of AhR [(0.26±0.07) vs.(0.48±0.05),(0.59±0.08), P<0.05] and PD-1 protein [(0.28±0.03) vs.(0.67±0.06),(0.51±0.04), P<0.05] in CD8~+T cells were significantly decreased. Compared with the com-bination treatment group(PD-1 inhibitor + Qingfei Mixture-containing serum), the levels of kynurenine in the cell supernatant were significantly increased [(98.26±11.35),(92.03±10.16) vs.(59.11±7.63)pmol/mL,P <0.05] in the STAT1 agonist group and the AhR agonist group(PD-1 inhibitor + Qingfei Mixture-containing serum + STAT1 agonist or AhR agonist). The expression levels of AhR [(0.54±0.07),(0.48±0.04) vs.(0.26±0.07),P<0.05] and PD-1 protein [(0.49±0.12),(0.62±0.10) vs.(0.28±0.03), P<0.05] in CD8~+T cells were significantly increased. Conclusion Qingfei Mixture reduces expression levels of the AhR and PD-1 in CD8~+T cells. This effect may be mediated by inhibiting kynurenine production in Lewis lung cancer cells. Moreover, Qingfei Mixture exhibits synergistic effects when combined with PD-1 inhibitors.更多还原