健脾除痰解毒方通过PD-1/PD-L1通路平衡Th1/Th2漂移而增强Lewis肺癌小鼠免疫功能

Jianpi-Chutan-Jiedu Formula enhances immune function in mice with Lewis lung carcinoma by modulating PD-1/PD-L1 pathway and shifting Th1/Th2 balance

目的:探讨健脾除痰解毒方(JCJF)增强Lewis肺癌小鼠免疫功能是否与其通过程序性死亡受体1(PD-1)/程序性死亡受体配体1(PD-L1)通路平衡Th1/Th2细胞相关。方法:取C57BL/6J小鼠60只并建立Lewis肺癌模型,按随机数字表法分为模型(model)组,顺铂(DDP)组,低、中、高剂量JCJF(L-JCJF、M-JCJF和H-JCJF)组,以及H-JCJF+DDP组,每组10只;另取10只健康C57BL/6J小鼠作为空白组。各组小鼠均给予相应处理,连续治疗14d。处死小鼠后取小鼠瘤体和脾脏,计算抑瘤率和脾脏指数;HE染色观察小鼠肿瘤组织和肺组织的形态学变化;TUNEL法检测小鼠肿瘤细胞凋亡;免疫组化染色检测肿瘤组织PD-L1蛋白表达;ELISA法检测小鼠血清干扰素γ(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-13含量;RT-qPCR检测肿瘤组织PD-1、PD-L1、Bax和Bcl-2的mRNA表达;Western blot检测肿瘤组织PD-1、PD-L1、Bax、Bcl-2、IL-10和IL-13蛋白表达。结果:与model组相比,H-JCJF+DDP明显抑制移植瘤生长,能在最大程度上减小肿瘤体积和质量(P<0.05),抑瘤率达59.72%,亦显著降低小鼠脾脏指数(P<0.05);H-JCJF+DDP组小鼠肿瘤组织凋亡率显著升高(P<0.05),PD-L1蛋白表达显著减少(P<0.05);H-JCJF+DDP能显著升高小鼠血清IFN-γ和IL-2水平(P<0.05),降低IL-10和IL-13水平(P<0.05);H-JCJF+DDP亦降低肿瘤组织PD-1、PD-L1和Bcl-2的mRNA和蛋白表达水平(P<0.05),增加Bax的mRNA和蛋白表达(P<0.05),且显著减少IL-10和IL-13蛋白表达(P<0.05)。结论:JCJF可诱导肺癌细胞凋亡,明显抑制肺癌小鼠瘤体生长,还可上调小鼠血清IFN-γ和IL-2水平,下调IL-10和IL-13水平,从而平衡Th1/Th2漂移,改善小鼠免疫功能,抑制肿瘤细胞免疫逃逸,其机制可能与下调PD-1/PD-L1通路有关。

AIM:To investigate the potential link between the immune enhancement induced by Jianpi-Chu‐tan-Jiedu Formula(JCJF)and the balance of Th1/Th2 cells through programmed death-1(PD-1)/programmed death-ligand 1(PD-L1)pathway in mice with Lewis lung carcinoma.METHODS:Sixty C57BL/6J mice were randomly divided into six groups(10 mice each):model group,cisplatin(DDP)group,low-,middle-and high-dose JCJF(L-JCJF,M-JCJF and H-JCJF,respectively)groups,and H-JCJF+DDP group.Another 10 healthy C57BL/6J mice were taken as blank group.The treatment was administered for 14 d.The mice were euthanized,and the tumor and spleen were collected to calculate tumor inhibitory rate and spleen index.Morphological changes in tumor tissue and healthy lung tissue were observed by HE staining.Tumor cell apoptosis was detected by TUNEL assay.The expression of PD-L1 protein was detected by immu‐nohistochemistry.The serum levels of interferon-γ(IFN-γ),interleukin-2(IL-2),IL-10 and IL-13 were determined by ELISA.The mRNA expression of PD-1,PD-L1,Bax and Bcl-2 was detected by RT-qPCR,and the protein expression of PD-1,PD-L1,Bax,Bcl-2,IL-10 and IL-13 was detected by Western blot.RESULTS:Compared with model group,the mice in H-JCJF+DDP group exhibited significant tumor growth inhibition(P<0.05),leading to the greatest reduction in tumor volume and weight,with an inhibitory rate of 59.72%.The mice in H-JCJF+DDP group also exhibited a significantly lower spleen index,a higher apoptotic rate of tumor tissue,and lower expression of PD-L1 protein than those in model group(P<0.05).Furthermore,the serum levels of IFN-γand IL-2 were significantly increased and the levels of IL-10 and IL-13 were significantly decreased in H-JCJF+DDP group compared with model group(P<0.05).The mRNA and protein ex‐pression of PD-1,PD-L1 and Bcl-2 in tumor tissues was lower in H-JCJF+DDP group than that in model group(P<0.05),while Bax was higher(P<0.05).Furthermore,the protein levels of IL-10 and IL-13 in H-JCJF+DDP group were significantly decreased compared with model group(P<0.05).CONCLUSION:Treatment with JCJF induces lung cancer cell apoptosis and significantly inhibited tumor growth in mice.This effect may be related to the down-regulation of PD-1/PD-L1 pathway.