熊果酸对小鼠H22移植瘤的抑制作用研究

Inhibitory effects of ursolic acid on tumor growth in hepatocarcinoma 22-bearing mice

目的:观察熊果酸(ursolic acid,UA)对小鼠H22移植瘤的抑制作用并对其机制进行探讨。方法:将75只昆明种小鼠于左前腋下皮下接种H22肝癌细胞,24h后随机分为5组,每组15只,模型组、环磷酰胺(cyclophosphamide,CP)对照组[25mg/(kg·d)]和UA低、中、高剂量药物组[50、100、150mg/(kg·d)]。UA各剂量组和CP对照组分别以相应剂量UA和CP灌胃,模型组以等体积花生油灌胃,每天灌胃1次,连续给药2周。末次给药24h后,称小鼠体质量,摘除眼球取血后处死。无菌条件下完整剥离小鼠肿瘤及肝、肾、脾组织,计算抑瘤率及肝、肾、脾指数;HE染色观察肿瘤组织病理学改变;分离脾淋巴细胞,CCK8法测定小鼠脾++++淋巴细胞增殖活性;免疫组化法检测肿瘤组织中CD4、CD8T细胞亚群的分布情况,计数CD4T细胞和CD8T细胞个数。并采用酶联免疫吸附法(ELISA)检测血清中白细胞介素12(interleukin-12,IL-12)的浓度。结果:UA各剂量组小鼠H22肿瘤质量增长均较缓慢,其中UA中、高剂量组肿瘤质量明显减小,与模型组比较,差异均具有统计学意义(P<0.05);UA中、高剂量组抑瘤率分别为30.15%和39.80%。UA各剂量组肝、肾指数与模型组比较差异无统计学意义(P>0.05),而脾指数则随UA剂量增加逐渐升高,其中,UA中、高剂量组脾指数与模型组比较差异具有统计学意义(P<0.05)。HE染色病理观察结果显示,模型组肿瘤细胞生长旺盛,细胞体积较大,胞质丰富,少见坏死区域;UA干预组肿瘤组织中瘤细胞生长增殖明显受到抑制,肿瘤细胞数量减少,密度降低,排列稀疏,细胞核固缩深染或破裂,细胞间质较多,核浆比例减少,坏死区域明显增多。CCK8实验结果显示,随着UA剂量增加,脾淋巴细胞增殖活性逐渐升高,其中,UA中、高剂量组脾淋巴细胞增殖活性显著升高,与模型组间的差异具有统计学意义++(P<0.05)。免疫组化结果显示,UA中、高剂量组肿瘤组织中CD4、CD8T细胞个数与模型组比较均明显增加,差异具有统计学意义(P<0.05)。UA高、中、低剂量组血清中IL-12的含量均较模型组升高,差异均有统计学意义(P<0.05);UA高剂量组与CP对照组相比亦显著升高(P<0.05)。结论:一定剂量UA可抑制H22荷瘤小鼠肿瘤生长,其作用机制可能与UA促进机体免疫器官发育和淋巴细++胞增殖,增加CD4、CD8T细胞向肿瘤组织浸润,并提高血清中IL-12等抗肿瘤活性细胞因子的浓度诱导细胞免疫有关。

OBJECTIVE： To observe the effect of ursolic acid （UA） on tumor growth in H22 mice and explore its mechanism. METHODS： H22 cells were inoculated subcutaneously into left anteromedial axilla of KM mice. 24 h later, mice were randomly divided into five groups： the model group, the cyclophosphamide（CP） control group [（25 mg/（kg · d）] and the UA low, mid, high dose groups [50, 100, 150 mg/（kg · d）]. Animals of the UA groups and the CP control group were treated with ig UA of different dosage and CP for 2 weeks. The model group were treated with equal volume of peanut oil intragastrically. 24 h after the last administration, the mice were weighed, and sacrificed after taking blood sample from eyeball extraction. Tumor, liver, kidney and spleen were removed under aseptic condition. Tumor inhibition rate and liver, kidney, spleen indexes were calculated. Tumor histopathological change were examined by HE staining. Cell counting kit8 （CCK8） was used to determine proliferation activity of spleen lymphocyte. The CD4＋and CD8＋T cell subsets in tumor tissue were assessed by immunohistochemistry. The number of CD4＋T, CD8＋T ceils in five random microscopic fields （ × 400） of each section were counted and the average number of every microscopic field calculated. The cells with cytoplasm containing claybank granules were positive cells. Serum IL-12 concentration was measured by enzyme linked immunosorbent assay. RESULTS： The H22 tumors in UA groups grew slowly. Compared with model group, the tumor weights of mid-dose and high-dose UA groups were reduced significantly （P〈0.05）. The tumor inhibition rate of mid-dose and high-dose UA groups were 30.15% and 39.80%, respectively. The liver and kidney indexes of UA groups and model group showed no significant change, but the spleen index gradually increased with dose of UA. The spleen index of mid-dose and high-dose UA groups was significant different with model group （P〈0.05）. Tumor cells in model group grew well and had less necrotic area, while the growth of tumor ceils in UA groups were significantly inhibited. Karyopyknosis, karyolysis, intercellular substance and necrotic area were increased, whilst the karyoplasmic ratio was reduced. Proliferation activity of spleen lymphocyte increased with the dose of UA. Proliferation activities of mid-dose and high-dose UA groups were higher than model group （P〈0.05）. The number of CD4＋T, CD8＋ T cells in tumor per high power field （ ×400） of mid-dose and high-dose UA groups were significantly more than model group （P〈0.05）. Serum IL- 12 concentrations of low-dose, mid-dose and high-dose UA groups were significantly higher than model group （P〈0.05）. Serum IL-12 concentration of high-dose UA group was significantly higher than CP group （P〈0.05）. CONCLUSION： UA could inhibit the growth of H22 tumor in mice. The possible mechanism is growth promotion of immune organs and lymphocyte proliferation, inducing CD4＋, CD8＋ T cell infiltration in tumor tissue and enhancing serum IL-12 concentration to induce cellular immunity. [KEY WORDS1 ursolic acid ; mice ; neoplasms