通关藤总皂苷通过IL-6/STAT3/NF-κB信号通路改善肝癌H22小鼠肿瘤炎症的研究

Study on the Improvement of Tumor Inflammation in H22 Mice with Hepatocellular Carcinoma Through IL-6/STAT3/NF-κB Signaling Pathwayin the Total Saponins from Marsdenia Tenacissima

目的探讨通关藤总皂苷对H22荷瘤小鼠抗肿瘤炎症作用及机制。方法建立H22荷瘤小鼠模型,随机分为模型组,阳性组(阿霉素),联合给药组(阿霉素^(+)通关藤总皂苷中剂量),通关藤总皂苷低(62.5 mg/kg)、中(125 mg/kg)、高(250 mg/kg)剂量组,另设空白对照组。给药14 d。观察各组小鼠的行为学变化;剥离肿瘤组织进行称重,计算抑瘤率。采用HE染色、流式细胞术、酶联免疫吸附法(ELISA)、反转录实时定量聚合酶链式反应(RTPCR)和蛋白免疫印迹法(Western blot)对H22荷瘤小鼠的肝脏组织和肿瘤组织病理形态、外周血淋巴细胞比例、血清炎症因子、STAT3和核转录因子-κB(NF-κB)通路相关基因和蛋白等指标进行检测。结果通关藤总皂苷能够明显改善H22荷瘤小鼠的精神、行为等状态;各给药组抑瘤率明显,联合给药的抑瘤率高于通关藤总皂苷和阿霉素单独给药,差异具有统计学意义(P<0.01);模型组小鼠肝脏组织肝窦可见肿瘤细胞,且有炎症细胞浸润现象,各给药组情况得到不同程度改善。模型组小鼠肿瘤组织内肿瘤细胞生长密集,可见异型性和病理性核分裂现象,炎细胞浸润,各给药组肿瘤细胞出现大片坏死现象;相较于模型组,各给药组CD3^(+)、CD4^(+)T淋巴细胞百分率升高,CD8^(+)T淋巴细胞百分率降低,CD19^(+)、CD20^(+)B淋巴细胞和CD3^(-)CD16^(+)NK细胞百分率升高,差异具有统计学意义(P<0.01);通关藤总皂苷中、高剂量组血清中白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的浓度较模型组降低(P<0.01);各给药组中STAT3、MCL-1、XIAP、BCL-2、NF-κB mRNA的表达和STAT3、p-STAT3、MCL-1、XIAP、BCL-2、NF-κB蛋白的表达均较模型组降低(P<0.01)。结论通关藤总皂苷对H22荷瘤小鼠的肿瘤炎症有抑制作用,其机制可能与通关藤总皂苷下调STAT3和NF-κB信号通路中相关基因和蛋白的表达量及提高机体免疫功能有关。

Objective The purpose of this study was to explore the anti-tumor inflammatory effect and mechanism of total saponins from marsdenia tenacissima on the H22 tumor-bearing mouse model.Methods The H22 tumor-bearing mouse model was established and randomly divided into model group,positive(adriamycin),combined administration group(medium dose of adriamycin^(+)total saponins from marsdenia tenacissima),low total saponins from marsdenia tenacissima(62.5 mg/kg),medium(125 mg/kg),high(250 mg/kg)dose group,and blank control group.Dosing for 14 days.The behavioral changes of the mice in each group were observed;the tumor tissue was peeled off and weighed to calculate the tumor inhibition rate.HE staining,flow cytometry,ELISA,RT-PCR and Western blot analysis of liver tissue and tumor tissue pathological morphology,peripheral blood lymphocyte ratio,serum inflammatory factors and STAT3 and NF-κB pathway-related genes of H22 tumor-bearing mice and protein indicators.Results Total saponins from marsdenia tenacissima could significantly improve the mental and behavioral states of H22 tumor-bearing mice;the tumor inhibition rate of each administration group was obvious,and the tumor inhibition rate of combined administration was higher than that of total saponins from marsdenia tenacissima and doxorubicin alone,with statistical difference(P<0.01);tumor cells and inflammatory cell infiltration were seen in the liver sinuses of the mice in the model group,and the conditions of each administration group were improved to varying degrees.The tumor cells in the tumor tissue of the mice in the model group grew densely,atypia and pathological mitosis were seen,and inflammatory cells were infiltrated,and the tumor cells in each administration group appeared massive necrosis;Compared with the model group,the percentages of CD3^(+)T and CD4^(+)T lymphocytes in each administration group increased,the percentages of CD8^(+)T lymphocytes decreased,and the percentages of CD19^(+),CD20^(+)B lymphocytes and CD3^(-)CD16^(+)NK cells increased.Statistical difference(P<0.01);The serum concentrations of IL-6,IL-8 and TNF-αin the total saponins from marsdenia tenacissima middle and high dose groups were lower than those in the model group(P<0.01);The expression of STAT3,MCL-1,XIAP,BCL-2,NF-κB mRNA and the expression of STAT3,p-STAT3,MCL-1,XIAP,BCL-2 and NF-κB protein were all lower than those in the model group(P<0.01).Conclusion The total saponins from marsdenia tenacissima have inhibitory activity on tumor inflammation in H22 tumor-bearing mice,and the mechanism may be related to the down-regulation of the expression of related genes and related proteins in the STAT3 and NF-κB signaling pathways,and the improvement of the body's immune function.related.