大黄游离蒽醌对重症急性胰腺炎大鼠肠道免疫功能的影响及其作用机制

摘要

Objective To observe the effects of free anthraquinones of rhubarb on immune function in mesenteric lymph nodes (MLN) of severe acute pancreatitis (SAP) rats.Methods Twenty-four healthy male SD rats were randomly divided into the sham operation group , model group, free rhubarb anthraquinone group and rhubarb decoction group , 6 rats in each group.SAP model was made by retrograde pancreatic duct injection of sodium taurocholate in the model group , free rhubarb anthraquinone group and rhubarb decoction group .The sham operation group was injected with equal volume of sterilized normal saline by retrograde injection of pancreatic duct .Rhubarb decoction group and free rhubarb anthraquinone group were respectively administrated by intragastric administration with 200 mg/kg free anthraquinone of rhubarb and 6.6 g/kg rhubarb decoction at 12 hours before and 2 hours after the inducement of SAP .Sham operation group and model group were not treated .MLN was collected 24 hours after molding .The histopathological changes of MLN were observed under light microscope after HE staining .The levels of TNF-αand IL-1 in MLN homogenate were detected by ELISA .The ex-pression of NLRP3 and ASC was tested in the MLN by immunoflurescence .The number of CD4 +, CD4 +CD25+Foxp3+, Th1, Th2, Th1/Th2 ratio and Treg percentage were tested by flow cytometry .Results Lymphoid nodules appeared to be enlarged, cortex was thickened , the quantity of lymphoid nodules was increased in the cortex area of the model group as compared with those of the sham operation group .The quantity and size of lymphoid nodules in the cortex area were smaller in free rhubarb anthraquinone group and rhubarb decoction group than that of the model group .No difference was found in the lymph nodule volume between the free rhubarb anthraquinone group and rhubarb decoction group .Expression level of NLRP3, IL-1 and TNF-α:the model group was significantly higher than the sham operation group (P<0.05), the free rhubarb anthraquinone group and rhubarb decoction group was significantly lower than the model group (P<0.05).CD4 +CD25+Foxp3+, CD4 +and Treg ratio:the model group was significantly higher than the sham operation group (P<0.05), except for CD4 +in the free rhubarb anthraquinone group , the free rhubarb anthraquinone group and rhubarb decoction group was significantly lower than the model group (all P<0.05).Th1, Th2 and Th1/Th2:Compared with the sham op-eration group, Th1 and Th1/Th2 was higher, and the Th2 in the model group was significantly lower (all P<0.05).Th1 and Th1/Th2 was significantly lower in the free rhubarb anthraquinone group and rhubarb decation group than in the model group, and the Th2 in free rhubarb anthraquinone group and rhubarb decoction group was significantly higher than that of the model group (P<0.05).No statistically significant difference was found in the above indicators between the free rhu -barb anthraquinone group and rhubarb decoction group (all P>0.05).Conclusions Free anthraquinone of rhubarb can significantly reduce immune inflammatory response in intestine , and its effect is similar to rhubarb decoction .The mecha-nism may be that free anthraquinone of rhubarb may regulate the body ′s immune balance through inhibiting the expression of inflammasome, regulating Th1/Th2 imbalance and intervening the immune response in early stage of acute pancreatitis .%目的：观察大黄游离蒽醌对重症急性胰腺炎（ SAP）大鼠肠道免疫功能的影响，并探讨其作用机制。方法将24只健康雄性SD大鼠随机分为假手术组、模型组、大黄游离蒽醌组及生大黄水煎液组，每组6只。模型组、大黄游离蒽醌组及生大黄水煎液组经胰胆管逆行注射牛磺胆酸钠制作SAP模型，假手术组经胰胆管逆行注射等量灭菌生理盐水。大黄游离蒽醌组、生大黄水煎液组建模前12 h及建模2 h分别给予大黄游离蒽醌200 mg／kg和生大黄水煎液6．6 g／kg灌胃，假手术组、模型组不予处理。建模24 h，取各组肠系膜淋巴结（MLN），采用HE染色法观察MLN组织病理形态变化，ELISA法检测MLN组织匀浆上清液TNF-α、IL-1，免疫荧光法检测核苷酸结合寡聚化结构域样模式识别受体（NLRP3）、凋亡相关斑点样蛋白（ASC）表达，流式细胞仪检测CD4＋、CD4＋CD25＋Foxp3＋细胞及Th1、Th2细胞数量，计算Treg比例、Th1／Th2。结果与假手术组比较，模型组淋巴小结增大，皮质区增厚，皮质区淋巴小结数量增加；与模型组比较，大黄游离蒽醌组、生大黄水煎液组皮质区淋巴小结数量少、体积小；大黄游离蒽醌组、生大黄水煎液组淋巴小结大小相当。 TNF-α、IL-1和NLRP3、ASC水平：模型组明显高于假手术组（ P＜0．05），游离蒽醌组及生大黄组明显低于模型组（P＜0．05）。 CD4＋CD25＋Foxp3＋、CD4＋、Treg比例：模型组明显高于假手术组（P均＜0．05），除大黄游黄蒽醌组CD4＋外，大黄游离蒽醌组、生大黄水煎液组均明显低于模型组（P均＜0．05）。 Th1、Th2及Th1／Th2：模型组Th1、Th1／Th2均高于假手术组（P均＜0．05），Th2低于假手术组（P＜0．05）；大黄游离蒽醌组、生大黄水煎液组Th1、Th1／Th2较模型组均明显降低（P均＜0．05），而Th2较模型组明显升高（P＜0．05）。大黄游离蒽醌组、生大黄水煎液组上述各检测指标比较差异均无统计学意义（ P均＞0．05）。结论大黄游离蒽醌能减轻SAP大鼠肠道的免疫炎性反应，其效果与生大黄水煎液相当；其机制可能是通过抑制炎性小体的表达和调节Th1／Th2失衡，干预SAP早期的免疫过激反应，继而调节机体的免疫平衡。