迷迭香酸抑制荷瘤小鼠H22肝癌移植瘤生长的作用机制

Mechanism of rosmarinic acid inhibiting the growth of H22 hepatocellular carcinoma transplant tumors bearing mice

目的 观察迷迭香酸抑制荷瘤小鼠H22肝癌移植瘤生长的作用机制。方法 建立肝癌H22荷瘤小鼠模型,根据随机数字表法分为模型组、迷迭香酸组、环磷酰胺(CTX)组,每组5只。迷迭香酸组灌胃给予迷迭香酸,CTX组腹腔注射CTX,模型组灌胃给予等体积0.9%氯化钠注射液,3组均连续给药14 d。比较3组肿瘤生长情况、免疫器官指数、肿瘤组织病理变化、T淋巴细胞(CD3^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+))、CD3^(+)、CD4^(+)凋亡率、肿瘤组织中免疫细胞因子[干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白介素-10(IL-10)]水平。结果 给药14 d后,CTX组和迷迭香酸组的肿瘤重量和体积低于模型组(P均<0.01);迷迭香酸组小鼠体质量、胸腺指数和脾脏指数与模型组比较差异无统计学意义(P>0.05);CTX组小鼠体质量和胸腺指数低于模型组(P均<0.01),脾脏指数与模型组比较差异无统计学意义(P>0.05)。HE切片染色结果显示,模型组小鼠肿瘤组织中肿瘤细胞分布密集且数目丰富,坏死细胞较少;CTX组肿瘤细胞数明显减少,细胞排列稀疏,肿瘤细胞坏死较多;迷迭香酸组出现肿瘤细胞凋亡和坏死,肿瘤细胞数目减少。CTX组脾脏组织中CD3^(+)、CD4^(+)水平高于模型组(P均<0.05),CD8^(+)水平、CD4^(+)/CD8^(+)与模型组比较差异无统计学意义(P>0.05);迷迭香酸组CD4^(+)水平、CD4^(+)/CD8^(+)高于模型组,CD8^(+)水平低于模型组(P<0.05或P<0.01),CD3^(+)水平与模型组比较差异无统计学意义(P>0.05);CTX组荷瘤小鼠脾脏组织中CD4^(+)、CD8^(+)凋亡率高于模型组,迷迭香酸组CD4^(+)凋亡率低于模型组,CD8^(+)凋亡率高于模型组(P<0.05或P<0.01);CTX组与迷迭香酸组肿瘤组织中IFN-γ水平高于模型组,TNF-α、IL-10水平低于模型组(P<0.05或P<0.01)。结论 迷迭香酸可抑制H22肝癌移植瘤的生长,其机制可能为通过调节免疫细胞功能和免疫细胞因子分泌,改善机体肿瘤免疫抑制微环境,减少肿瘤细胞免疫逃逸,发挥抗肿瘤作用。

Objective To observe the mechanism of rosmarinic acid inhibiting the growth of H22 hepatocellular carcinoma transplant tumors bearing mice.Methods The H22 tumor bearing mice model of liver cancer was established and divided into rosmarinic acid group,cyclophosphamide(CTX) group and model group according to the random number table method,with 5 mice in each group.The rosmarinic acid group was given rosmarinic acid by gavage,the CTX group was given CTX by intraperitoneal injection,and the model group was given equal volume of 0.9% sodium chloride injection by gavage.The three groups were given drugs continuously for 14 days.The tumor growth,immune organ index,pathological changes of tumor tissue,T lymphocyte(CD3^(+),CD4^(+),CD8^(+),CD4^(+)/CD8^(+)),CD3^(+) & CD4^(+) apoptosis rate,immune cytokines in tumor tissue(IFN-γ,TNF-α,IL-10) levels.Results After 14 days of administration,the tumor weight and volume of CTX group and rosmarinic acid group were lower than those of model group(P<0.01);There were no significant differences in body weight,thymus index and spleen index between rosmarinic acid group and model group(P>0.05);The body weight and thymus index of mice in CTX group were lower than those in model group(P<0.01),and there was no significant difference in spleen index between CTX group and model group(P>0.05).The results of slices HE staining showed that the tumor cells in the tumor tissue of the model group were densely distributed and abundant,and the necrotic cells were less;In CTX group,the number of tumor cells were significantly reduced,the cells were sparse,and the tumor cells were more necrotic;In rosmarinic acid group,apoptosis and necrosis of tumor cells occurred,and the number of tumor cells decreased.The levels of CD3^(+) and CD4^(+) in spleen tissue of CTX group were higher than those of model group(P<0.05),but there were no significant differences in CD8^(+) level and CD4^(+)/CD8^(+) between CTX group and model group(P>0.05);The levels of CD4^(+) and CD4^(+)/CD8^(+) in rosmarinic acid group were higher than those in the model group,while the levels of CD8^(+) were lower than those in the model group(P<0.05 or P<0.01).There was no significant difference in the levels of CD3^(+) between rosmarinic acid group and the model group(P>0.05);The apoptosis rates of CD4^(+) and CD8^(+) in spleen tissue of tumor bearing mice in CTX group were higher than those in model group,while the apoptosis rate of CD4^(+) in rosmarinic acid group was lower than that in model group,and the apoptosis rate of CD8^(+) was higher than that in model group(P<0.05 or P<0.01);The levels of IFN-γ in CTX group and rosmarinic acid group were higher than those in the model group,the levels of TNF-αand IL-10 were lower than those in the model group(P<0.05 or P<0.01).Conclusion Rosmarinic acid can inhibit the growth of H22 hepatocellular carcinoma transplant tumors,and its mechanism may be through regulating immune cells function and immune cytokine secretion,improving the body′s tumor immunosuppressive microenvironment,reducing tumor cells immune escape,and playing an anti-tumor role.