基于TLR4信号通路探讨扶正膏增强细胞免疫功能机制

Mechanism of Fuzheng ointment enhancing cellular immune function based on TLR4 signaling pathway

目的基于Toll样受体4(TLR4)信号通路探讨扶正膏增强细胞免疫功能的机制。方法(1)将20只雄性ICR小鼠随机分为正常组、免疫功能低下组、扶正膏组、左旋咪唑组,每组5只。除正常组外,其余组小鼠均连续3 d腹腔注射环磷酰胺建立免疫功能低下模型。造模成功后,扶正膏组给予扶正膏2250 mg/kg灌胃,左旋咪唑组给予左旋咪唑20 mg/kg灌胃,空白组和免疫功能低下组给予等量生理盐水灌胃,均连续灌胃28 d。流式细胞术检测脾脏组织中CD3^(+)、CD4^(+)、CD8^(+)T细胞比例,Western blot法检测脾脏组织中TLR4、髓样分化因子88(MyD88)、白细胞介素-1受体相关激酶4(IRAK4)、肿瘤坏死因子受体相关因子6(TRAF6)蛋白表达情况。(2)取大鼠20只,给予扶正膏69.6 g/(kg·d)连续灌胃14 d制备扶正膏含药血清。体外分离、培养正常小鼠脾淋巴细胞,分为4组:正常组体外正常培养;扶正膏含药血清组加入10%扶正膏含药血清培养48 h;MyD88抑制剂组加入5μmol/L MyD88抑制剂ST2825培养8 h;MyD88抑制剂+含药血清组加入5μmol/L ST2825培养8 h后,离心去上清,然后加入10%扶正膏含药血清培养48 h。ELISA法检测细胞上清液中白细胞介素-2(IL-2)、干扰素-γ(IFN-γ)表达水平,Wsetern blot法检测脾淋巴细胞中TLR4、MyD88、IRAK4、TRAF6蛋白表达情况。结果免疫功能低下组脾脏组织中CD4^(+)T细胞比例和TLR4、MyD88、IRAK4、TRAF6蛋白相对表达量均明显低于正常组(P均<0.05),扶正膏组和左旋咪唑组CD4^(+)T细胞比例和TLR4、MyD88、IRAK4、TRAF6蛋白相对表达量均明显高于免疫功能低下组(P均<0.05)。扶正膏含药血清组脾淋巴细胞中IL-2、IFN-γ表达水平和TLR4、MyD88、IRAK4、TRAF6蛋白相对表达量均明显高于正常组、MyD88抑制剂组和MyD88抑制剂+含药血清组(P均<0.05),MyD88抑制剂组和MyD88抑制剂+含药血清组IL-2、IFN-γ表达水平和TLR4、MyD88、IRAK4、TRAF6蛋白相对表达量均明显低于正常组(P均<0.05),MyD88抑制剂组和MyD88抑制剂+含药血清组上述各指标比较差异均无统计学意义(P均>0.05)。结论扶正膏可能通过激活TLR4信号通路增强细胞免疫功能。

Objective It is to explore the mechanism of Fuzheng ointment enhancing cellular immune function based on Toll-like receptor 4(TLR4)signaling pathway.Methods①Twenty male ICR mice were randomly divided into normal group,immunocompromised group,Fuzheng ointment group and levamisole group,with 5 mice in each group.The mice of all the groups except for the normal group were continuously treated with cyclophosphamide by intraperitoneal injection for 3 days to establish the immunocompromised models.After successful modeling,the Fuzheng ointment group was given Fuzheng ointment 2250 mg/kg by gavage,the levamisole group was given levamisole 20 mg/kg by gavage,and the blank group and immunocompromised group were given equal amounts of normal saline by gavage,all continuously gavaged for 28 days.The proportions of CD3^(+),CD4^(+),and CD8^(+)T cells in splenic tissue were detected by Flow cytometry,the expressions of TLR4,myeloid differentiation factor 88(MyD88),interleukin-1 receptor-associated kinase 4(IRAK4),and tumor necrosis factor receptor-associated factor 6(TRAF6)were detected by Western blot.②Twenty rats were taken and continuously treated with Fuzheng ointment 69.6 g/(kg·d)by gavage for 14 days to establish medicated serum of Fuzheng ointment.Splenic lymphocytes were isolated and cultured in vitro in normal mice,and divided into 4 groups:the normal group was cultured in vitro,Fuzheng ointment-containing serum group was cultured for 48 h with 10%Fuzheng ointment-containing serum,MyD88 inhibitor group was cultured for 8 h with 5μmol/L MyD88 inhibitor ST2825,and MyD88 inhibitor+containing serum group was cultured for 8 h with 5μmol/L ST2825,then centrifuged to remove supernatant,and then cultured with 10%Fuzheng ointment-containing serum.The expression levels of interleukin-2(IL-2)and interferon-γ(IFN-γ)in the cell supernatant were detected by ELISA,and the protein expressions of TLR4,MyD88,IRAK4,and TRAF6 in the splenic lymphocytes were detected by Wsetern blot.Results The proportion of CD4^(+)T cells and the relative expressions of TLR4,MyD88,IRAK4,and TRAF6 proteins in spleen tissues of the immunocompromised group were significantly lower than those of the normal group(all P<0.05),and the proportion of CD4^(+)T cells and the relative expressions of TLR4,MyD88,IRAK4,and TRAF6 proteins in the Fuzheng ointment group and levamisole group were significantly higher than those of the immunocompromised group(all P<0.05).The expression levels of IL-2,IFN-γand the relative expressions of TLR4,MyD88,IRAK4 and TRAF6 proteins in splenic lymphocytes in the Fuzheng ointment-containing serum group were significantly higher than those in the normal group,MyD88-inhibitor group and MyD88 inhibitor+drug-containing serum group(all P<0.05),the expression levels of IL-2,IFN-γand the relative expression of TLR4,MyD88,IRAK4 and TRAF6 proteins in the MyD88 inhibitor group and MyD88 inhibitor+drug-containing serum group were significantly lower than those in the normal group(all P<0.05),and the differences in the above indexes were not statistically significant between the MyD88 inhibitor group and MyD88 inhibitor+drug-containing serum group(all P>0.05).Conclusion Fuzheng ointment can enhance cellular immune function maybe by activating TLR4 signaling pathway.