文献类型：期刊文献

中文题名：寒湿瘀结证子宫内膜异位症纤维化小鼠腹腔微环境NLRP3炎性小体活化相关蛋白及Th17/Treg的表达特点

英文题名：Expression characteristics of NLRP3 inflammasome-activated protein and Th17/Treg in the peritoneal microenvironment of endometriosis-fibrosis mice with cold-dampness stasis syndrome

作者：黄灿灿[1];吉秀家[1];张作良[1];毛海燕[2];张小花[1];岳斌[1];靳灵[1];武权生[1]

第一作者：黄灿灿

机构：[1]甘肃中医药大学,甘肃兰州730000;[2]甘肃省人民医院,甘肃兰州730000

第一机构：甘肃中医药大学

年份：2023

卷号：34

期号：8

起止页码：2016

中文期刊名：时珍国医国药

外文期刊名：Lishizhen Medicine and Materia Medica Research

收录：北大核心:【北大核心2020】；CSCD:【CSCD_E2023_2024】；

基金：国家自然科学基金(82260949,81760879);甘肃中医药大学科学研究与创新基金项目(2021KCYB-2);甘肃中医药大学研究生创新创业基金(2022CX-01);甘肃省科技计划项目优秀博士生项目(23JRRA1220);西北环境与营养相关疾病中医药防控协同创新中心开放项目(ZYXT-20-06)。

语种：中文

中文关键词：子宫内膜异位症纤维化;寒湿瘀结证;NLRP3炎性小体;Th17/Treg失衡

外文关键词：Endometriosis-fibrosis;Syndrome of cold-damp stasis;NLRP3 inflammasome;Th17/Treg imbalance

摘要：目的探讨寒湿瘀结证子宫内膜异位症纤维化模型小鼠腹腔微环境中NLR家族蛋白3(NOD-like receptor protein 3,NLRP3)炎性小体活化相关蛋白及Th17/Treg的表达规律。方法将55只雌性BALB/c小鼠随机分为假手术组、供体组、模型组(分为模型7天组、模型14天组、模型21天组),模型组采用异体移植腹腔注射法和冷水浴法构建寒湿瘀结证子宫内膜异位症纤维化模型。比较各组小鼠的自主活动次数、粪便含水率及热痛潜伏期以评价证候;采用腹腔粘连评分评价粘连程度;比较各组异位组织总重量,采用苏木精-伊红(hematoxylin-eosin,HE)染色,评价病理;马松三色(Massontrichrome,Masson)染色,免疫组化法检测异位病灶中转化生长因子-β(transforming growth factor-β,TGF-β)、Ⅰ型胶原蛋白(Collagen type 1,Col-1)的表达,评价纤维化;采用蛋白免疫印迹法(Western Blot)测定异位病灶组织中肿瘤坏死因子受体相关因子6(TNF receptor associated factor 6,TRAF6)、NLRP3、含CARD的凋亡斑点样(apoptosis-associated speck-like protein containing a CARD,ASC)、含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase 1,caspase-1)蛋白的表达水平,评价NLRP3炎性小体的活化;采用流式细胞术检测辅助性T淋巴细胞17(T-helper 17,Th17)、调节性T淋巴细胞(regulatory T lymphocyte,Treg),评价Th17/Treg水平。结果与假手术组对比,模型小鼠异位病灶总重量增加、热痛潜伏期缩短、腹腔粘连评分增高(P<0.05),且随着造模时间延长,变化更为显著(P<0.05);与假手术组对比,模型小鼠腹腔可见明显异位灶且HE染色显示柱状上皮及腺体增生明显,且随着随着造模时间延长,变化更为明显;Masson染色显示:与假手术组对比,模型小鼠胶原沉积面积增大(P<0.05),且随着造模时间延长,变化更为显著(P<0.05);免疫组化显示:与假手术组对比,模型小鼠异位灶中TGF-β,Col-1表达量增加(P<0.05),且随着造模时间延长,变化更为显著(P<0.05);Western Blot检测显示:与假手术组对比,模型小鼠异位灶中TRAF6、NLRP3、ASC、Caspase-1蛋白的表达水平增高(P<0.05),且随着造模时间延长,变化更为显著(P<0.05);流式细胞学检测显示:与假手术组对比,模型小鼠腹腔液中Th17细胞占比、Treg细胞占比以及Th17/Treg比值升高(P<0.05),其中各模型组小鼠腹腔液Th17占比和Treg占比,随着造模时间的延长而逐渐升高(P<0.05),而Th17/Treg比值无统计学差异。结论腹腔微环境中NLRP3炎性小体活化及Th17/Treg失衡可能参与了子宫内膜异位症纤维化的形成。
Objective To investigate the expression of NLRP3 inflammasome activation key protein and Th17/Treg in the perito?neal microenvironment of the endometriosis-fibrosis model mice of cold-dampness stasis.Methods 55 BALB/c female mice were randomly divided into sham operation group,donor group,and model group(divided into 7 days group,14 days group and 21 days group).The model group used allogeneic transplantation intraperitoneal injection and cold water swimming to construct a cold and wet intertwined endometriosis-fibrosis model,which was divided into 7 days(model 7 days group,sham operation group),14 days(model 14 days group),and 21 days(model 21 days group).The number of spontaneous activities,fecal water content and latency of heat pain were compared in each group to evaluate the syndrome;The degree of adhesion was evaluated by abdominal adhesion score;The total weight of ectopic tissue in each group was compared,and hematoxylin-eosin(HE)staining was used to evaluate the pathology;Masson trichrome(Masson)staining and immunohistochemical method were used to detect transforming growth factor in ectopic lesions-β(transforming growth factor-β,TGF-β),Collagen type 1(Col-1)expres?sion to evaluate fibrosis;Western blot was used to detect tumor necrosis factor receptor associated factor 6(TRAF6),NLR family protein 3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC)The expression level of cysteinyl aspar?tate specific protein 1(caspase-1)protein was used to evaluate the activation of NLRP3 inflammasome;T-helper 17(Th17)and regulatory T lymphocyte(Treg)were detected by flow cytometry to evaluate the level of Th17/Treg.Results Compared with the sham-operated group,the total weight of heterotopic lesions in the model mice increased,the latency of heat pain shortened,and the score of abdominal adhesion increased(P<0.05);Compared with the sham-operated group,the abdominal cavity of the model mice showed obvious ectopic foci,and HE staining showed that the columnar epithelium and gland hyperplasia were obvious(P<0.05),and the changes were more obvious with the prolongation of modeling time(P<0.05);Masson staining showed that compared with sham operation group,the area of collagen deposition in model mice increased(P<0.05);Immunohistochemistry showed that compared with sham operation group,in endometriosis lesion of model mice,The expression of TGF-βand Col-1 increased(P<0.05);Western blot showed that the expression levels of TRAF6,NLRP3,ASC and Caspase-1 protein in the endometriosis lesion of the model mice increased compared with the sham operation group(P<0.05);Flow cytometry showed that compared with sham operation group,the proportion of Th17 cells,Treg cells and Th17/Treg ratio in the peritoneal fluid of model mice increased(P<0.05).Conclusion The activation of NLRP3 inflammasome and Th17/Treg imbalance in the peritoneal mi?croenvironment may be involved in the formation of endometriosis-fibrosis in the syndrome of cold-damp stasis.