清化益肠方对急性放射性肠损伤模型小鼠肠组织NLRP3炎症小体的影响

Effect of Qinghua Yichang Formula(清化益肠方)on NLRP3 Inflammasome in Intestinal Tissue of Mice with Acute Radiation-Induced Intestinal Injury

目的基于NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体探讨清化益肠方对急性放射性肠损伤模型小鼠的作用及可能分子机制。方法60只C57BL/6J小鼠随机分为对照组、模型组、造模前给药组、造模后给药组、抑制剂组、中药联合抑制剂组,每组10只。除对照组外,其余5组小鼠均采用单次全剂量照射构建小鼠急性放射性肠损伤模型。造模前给药组、中药联合抑制剂组造模前7天连续给予浓度为4 g/ml清化益肠方水煎液灌胃,每次0.2 ml,每日1次。造模后给药组、造模前给药组和中药联合抑制剂组造模后连续给予4 g/ml清化益肠方水煎液灌胃14天;对照组、模型组和抑制剂组给予0.2 ml生理盐水灌胃,每日1次,连续14天。自照射后2 h起,抑制剂组和中药联合抑制剂组予NLRP3抑制剂MCC950(浓度为10 mg/kg)腹腔注射0.2 ml,隔日1次,共7次。HE染色观察肠组织病理改变,Western Blot法及RT-qPCR法检测肠组织NLRP3蛋白及mRNA表达水平,免疫组织化学法检测肠组织NLRP3、Caspase-1、GSDMD蛋白表达水平;流式细胞术检测小鼠脾脏CD4+、CD8+T细胞比例;ELISA法测定小鼠血清γ干扰素(IFN-γ)、白细胞介素18(IL-18)、白细胞介素1β(IL-1β)含量。结果HE染色结果显示,对照组小鼠肠组织无病变;模型组小鼠肠绒毛长度减短、黏膜层变薄,固有层多灶性黏膜坏死,局部伴有中性粒细胞浸润;各药物干预组小鼠肠组织病理损伤有不同程度的改善,其中中药联合抑制剂组改善程度最明显。与对照组比较,模型组小鼠肠组织中NLRP3蛋白及mRNA表达水平升高,肠组织NLRP3、Caspase-1、GSDMD蛋白表达增加,脾脏CD4+T细胞比例上升、CD8+T细胞比例下降,血清IFN-γ、IL-18、IL-1β含量升高(P<0.05)。与模型组比较,其余各给药组上述各指标均有所改善(P<0.05)。造模前给药组NLRP3、Caspase-1、GSDMD蛋白较造模后给药组降低(P<0.05);中药联合抑制剂组NLRP3 mRNA水平较抑制剂组降低(P<0.05)。结论清化益肠方可能是通过抑制NLRP3炎症小体发挥防治急性肠损伤的作用。

Objective To explore the effect and possible molecular mechanism of Qinghua Yichang Formula(清化益肠方,QYF)in treating acute radiation-induced intestinal injury mice via NOD-like receptor thermal protein domain associated protein 3(NLRP3).Methods Sixty C57BL/6J mice were randomly divided into control group,model group,pre-modeling medication group,post-modeling medication group,inhibitor group,and QYF plus inhibitor group,with 10 mice in each group.Except for the control group,the other five groups were irradiated with a single full dose to establish the acute radiation-induced intestinal injury mice model.The pre-modeling medication group and the QYF plus inhibitor group were continuously given 4 g/ml of QYF decoction by gavage before modeling,0.2 ml each time,once a day for 7 days.The post-modeling medication group,pre-modeling medication group and QYF plus inhibitor group were given 4 g/ml of QYF decoction for 14 days after modeling.The control group,model group and inhibitor group were given 0.2 ml of normal saline once a day for 14 consecutive days.Two hours after irradiation,the inhibitor group and the QYF plus inhibitor group were given an intraperitoneal injection of 0.2 ml of the NLRP3 inhibitor MCC950(concentration:10 mg/kg),once every two days.To observe the pathological changes in intestinal tissues,hematoxylin-eosin(HE)staining was used.Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of NLRP3 in intestinal tissues.Immunohistochemistry was used to detect the expression level of NLRP3,Caspase-1 and GSDMD in intestinal tissues.The proportion of CD4+and CD8+T cells in the spleens of mice were detected by flow cytometry.ELISA was used to determine the levels of IFN-γ,IL-18,and IL-1βin mice serum.Results HE staining showed no lesions in the intestinal tissue of mice in the control group.The mice in the model group had shortened intestinal villi,thinned mucosal layers,multifocal mucosal necrosis in the lamina propria,and local neutrophil infiltration.The pathological damage of intestinal tissue of mice in each medication group was improved to varied degrees,among which the QYF plus inhibitor group showed most obvious improvement.Compared to those in the control group,the protein and mRNA expression levels of NLRP3 in the intestinal tissue of mice in the model group significantly increased,with higher NLRP3,Caspase-1,and GSDMD protein expression in the intestinal tissue,increased proportion of CD4+T cells in spleen,decreased proportion of CD8+T cells,and increased levels of IFN-γ,IL-18 and IL-1βin serum(P<0.05).Compared to those in the model group,the above indicators in the other medication groups were all improved(P<0.05).The NLRP3,Caspase-1,and GSDMD proteins in the pre-modeling medication group were lower than those in the post-modeling medication group(P<0.05);and the NLRP3 mRNA level in the QYF plus inhibitor group was lower than that in the inhibitor group(P<0.05).Conclusion QYF may play a role in preventing and treating acute radiation-induced intestinal injury by inhibiting the expression of NLRP3.