升降理肺消瘤汤对Lewis肺癌小鼠免疫炎性反应和JAK2/STAT3信号通路的影响

Effect of Shengjiang Lifei Xiaoliu Decoction(升降理肺消瘤汤)on Immune Inflammatory Response and JAK2/STAT3 Signal Pathway in Lewis Lung Cancer Mice

目的探讨升降理肺消瘤汤对Lewis肺癌小鼠免疫和炎性反应的调控作用,以及对JAK2/STAT3信号通路的影响。方法适应性喂养C57BL/6J雄性小鼠1周,其中空白组8只,荷瘤组60只。荷瘤组小鼠注射Lewis肺癌细胞造模成功后,将瘤体肉眼可见的荷瘤小鼠分为模型组、PD-1抑制剂组、升降理肺消瘤汤(XLT)低剂量组、XLT中剂量组、XLT高剂量组、XLT中剂量联合PD-1抑制剂组(联合用药组),每组8只。空白组和模型组每日0.4 mL/20 g生理盐水灌胃;PD-1抑制剂组每3 d腹腔注射100μg信迪利单抗;XLT低、中、高剂量组每日0.4 mL/20 g相应浓度中药灌胃;联合用药组每日0.4 mL/20 g中浓度中药灌胃,每3 d腹腔注射100μg信迪利单抗,各组连续给药14 d。最后一次给药的24 h后,称量记录小鼠体质量,摘眼球取血,颈椎脱臼法处死小鼠,比较各组小鼠抑瘤率、去瘤体质量、胸腺指数和脾指数;ELISA法检测血清中白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、IL-6浓度水平;Western Blot检测Janus激酶2(JAK2)、p-JAK2、信号转导和转录激活因子3(STAT3)、p-STAT3蛋白表达水平。结果与模型组比较,各用药组小鼠平均瘤重均较少,PD-1抑制剂组、XLT中、高剂量组瘤重明显减少(P<0.01);PD-1抑制剂组和XLT中剂量组去瘤体质量增加(P<0.01);各用药组小鼠胸腺指数和脾指数均显著增加(P<0.01);PD-1抑制剂组、XLT中剂量组及联合用药组IL-2水平显著升高(P<0.05),各用药组小鼠IFN-γ水平均明显升高(P<0.05);除XLT低剂量组,各用药组TNF-α和IL-6水平均明显降低,差异有统计学意义(P<0.05);各用药组小鼠JAK2蛋白相对表达量均降低(P<0.05),XLT高剂量组和联合用药组p-JAK2降低差异有统计学意义(P<0.05);除PD-1抑制剂对p-STAT3蛋白的表达抑制作用不明显外,各用药组小鼠STAT3、p-STAT3蛋白相对表达量均较模型组显著降低(P<0.05)。结论升降理肺消瘤汤能够抑制Lewis肺癌小鼠瘤体生长,可能与其增强瘤鼠抗肿瘤免疫反应,减轻炎性反应,抑制JAK2/STAT3信号通路的激活有关。

Objective To explore the regulatory effects of Shengjiang Lifei Xiaoliu Decoction(升降理肺消瘤汤,SJLFXLD)on immune and inflammatory responses in Lewis lung cancer mice,as well as its impact on the JAK2/STAT3 signaling pathway.Methods C57BL/6J male mice were fed adaptively for one week,with 8 in the blank group and 60 in the tumor bearing group.After successfully injecting Lewis lung cancer cells into the tumor bearing group mice for modeling,the mice whose tumor was visible to the naked eye were divided into a model group,a PD-1 inhibitor group,a low dose group of SJLFXLD,a medium dose group of SJLFXLD,a high dose group of SJLFXLD,and a combination of medium dose of SJLFXLD and PD-1 inhibitor group(combined medication group),with 8 mice in each group.The blank group and model group were given 0.4 mL/20 g normal saline by gavage every day.In the PD-1 inhibitor group,100μg of Sinilimab was injected intraperitoneally every 3 days.The low,medium and high dose groups of SJLFXLD were given 0.4 mL/20 g of corresponding concentration of Chinese herbal medicine by gavage every day.In the combined treatment group,0.4 mL/20 g of Chinese herbal medicine with medium concentration was given by gavage every day,and 100μg of Sinilimab was injected intraperitoneally every 3 days.Each group was continuously administered for 14 days.24 hours after the last administration,the weight of the mice was measured and recorded.Eyeballs were removed for blood collection,and the mice were euthanized using cervical dislocation method.The tumor inhibition rate,tumor removal body mass,thymus index,and spleen index of each group of mice were compared.ELISA method for detecting interleukin-2(IL-2)and interferon in serum-γ(IFN-γ),tumor necrosis factor-α(TNF-α),IL-6 concentration level.Western blot was used to detect the expression levels of Janus kinase 2(JAK2),p-JAK2,signal transducer and activator of transcription 3(STAT3),and p-STAT3 proteins.Results Compared with the model group,the average tumor weight of mice in each medication group was lower,while the tumor weight of PD-1 inhibitor group,medium and high dose groups of SJLFXLD was significantly reduced(P<0.01).The tumor mass of PD-1 inhibitor group and medium dose group of SJLFXLD increased(P<0.01).The thymus index and spleen index of mice in each medication group significantly increased(P<0.01).The levels of IL-2 in the PD-1 inhibitor group,medium dose group of SJLFXLD,and combination therapy group were significantly increased(P<0.05),and the levels of IFN-γof mice in each medication group were significantly increased.Except for the low dose group of SJLFXLD,the levels of TNF-αand IL-6 of mice in each medication group were significantly increased,with a statistically significant difference(P<0.05).The relative expression level of JAK2 protein in mice of each medication group decreased(P<0.05),and there was a statistically significant difference in p-JAK2 reduction in the high-dose SJLFXLD group and the combined medication group(P<0.05).Except for the unclear inhibitory effect of PD-1 inhibitor on the expression of p-STAT3 protein,the relative expression levels of STAT3 and p-STAT3 protein in each medication group of mice were significantly reduced compared to the model group(P<0.05).Conclusion Shengjiang Lifei Xiaoliu Decoction can inhibit the growth of tumor of Lewis lung cancer mice,which may be related to its enhancement of anti-tumor immune response,reduction of inflammatory response,and inhibition of the activation of JAK2/STAT3 signaling pathway.