山茱萸多糖对免疫抑制小鼠免疫功能的影响

Regulation of polysaccharide of Fructus Corni on the immune function of immunosuppressive mice

为探讨山茱萸多糖(polysaccharide of Fructus Corni, PFC)对环磷酰胺(cyclophosphamide, CTX)诱导的免疫抑制小鼠免疫功能的影响及其相关机制,将小鼠随机分为正常组、免疫抑制模型(CTX)组、PFC低剂量(CTX+LD)组、PFC中剂量组(CTX+MD)和PFC高剂量(CTX+HD)组,每组12只。腹腔注射CTX建立免疫抑制小鼠模型。各给药组小鼠灌胃对应剂量PFC。给药结束后通过碳粒廓清实验检测小鼠腹腔巨噬细胞的吞噬能力;H-E染色观察脾脏组织病理学变化;菌落计数法检测肠道内容物中乳酸杆菌、双歧杆菌和大肠埃希菌数量;FACS检测小鼠脾脏中CD4~+和CD8~+T细胞百分比;ELISA试剂盒检测小鼠血清中IL-2和IL-10表达水平;Western blotting检测小鼠脾脏组织中IL-2、IL-10、IFN-γ、IL-4、T盒子转录因子(T-box transcription factor, T-bet)和GATA结合蛋白3(GATA binding protein 3,GATA3)蛋白表达水平。结果显示,与Control组相比,CTX组小鼠廓清指数、吞噬指数、脾脏中CD4~+、CD8~+T细胞百分比及CD4~+/CD8~+T细胞比值均显著降低(均P<0.05);肠道乳酸杆菌和双歧杆菌数量均显著降低(均P<0.05),大肠埃希菌数量显著增加(P<0.05);血清中IL-2表达显著减少(P<0.05),IL-10表达显著增加(P<0.05);脾脏组织结构紊乱,其中T-bet、IFN-γ和IL-2蛋白表达显著减少(均P<0.05),GATA3、IL-4和IL-10蛋白表达显著增加(均P<0.05);与CTX组相比,中剂量及高剂量PFC给药组小鼠的廓清指数、吞噬指数、脾脏中CD4~+、CD8~+T细胞水平及CD4~+/CD8~+T细胞比值均显著升高(均P<0.05);肠道乳酸杆菌和双歧杆菌数量显著增加(均P<0.05),大肠埃希菌数量显著减少(P<0.05);血清中IL-2表达显著增加(P<0.05),IL-10表达显著减少(P<0.05);脾脏组织结构损伤减轻,其中T-bet、IFN-γ和IL-2蛋白表达显著增加(均P<0.05),GATA3、IL-10和IL-4蛋白表达显著减少(均P<0.05)。以上结果提示,PFC可提高CTX诱导的免疫低下小鼠的免疫功能,其具体机制可能与调节肠道菌群数量及Th1/Th2比值有关。

The goal of this study was to understand the effect and mechanism of polysaccharide of Fructus Corni(PFC) on the immune response of cyclophosphamide(CTX) induced immunosuppressive mice. Mice were randomly divided into the normal group(control group), immunosuppressive model group(CTX group), low-dose PFC group(CTX+LD group), medium-dose PFC group(CTX+MD group), and high-dose PFC group(CTX+HD group), with 12 mice per group. The immunosuppressive mouse model was established using intraperitoneal injection of CTX. The treatment groups were given a corresponding dose of PFC solution by gavaging. At the end of the treatments, the peritoneal macrophages’ phagocytic capacity was detected by carbon particle clearance test, the spleen pathological change by H-E staining, and the amount of Lactobacillus, Bifidobacterium and Escherichia coli in the intestine by colony counting. In addition, the percentage of CD4and CD8T cells in the spleen was detected by FACS, the expression levels of serum IL-2 and IL-10 were measured by ELISA and the protein levels of IFN-γ, IL-2, IL-4, IL-10, T-box transcription factor(T-bet) and GATA binding protein 3(GATA3) in the spleen tissue were detected by Western blotting. The results showed that, compared to the control group, the clearance index, phagocytic index, splenic CD4, CD8T cell percentages and CD4/CD8T cell ratio in the CTX group were all significantly decreased(all with P<0.05). The amount of intestinal Lactobacillus and Bifidobacterium was also significantly decreased(both with P<0.05), while the number of Escherichia coli was significantly increased(P<0.05). At the same time, the serum level of IL-2 was significantly decreased(P<0.05), whereas the expression of IL-10 was significantly increased(P<0.05). The spleen of the CTX group showed tissue damage and the expressions of T-bet, IFN-γ and IL-2 protein in the spleen significantly decreased(all with P<0.05), while those of GATA3, IL-10, and IL-4 protein significantly increased(all with P<0.05). Compared to the CTX group, the clearance index, phagocytic index, CD4, CD8T cell frequencies, and CD4/CD8T cell ratio in the spleen of mice treated with medium-or high-dose PFC were significantly increased(all with P<0.05). The amount of intestinal Lactobacillus and Bifidobacterium was also significantly increased(both with P<0.05), while the number of Escherichia coli was significantly decreased(P<0.05). The expression of IL-2 in the serum of PFC-treated mice was significantly increased whereas the expression of IL-10 was significantly decreased(both with P<0.05). In addition, the PFC groups showed alleviated spleen tissue damage, and the protein levels of T-bet, IFN-γ and IL-2 were significantly increased(all with P<0.05), while those of GATA3, IL-10, and IL-4 were significantly decreased(all with P<0.05). In summary, our results suggest that PFC may improve the immune function of CTX-induced immunosuppressive mice. The underlying mechanism may involve the regulation of intestinal flora composition and Th1/Th2 ratio.