摘要
目的探讨左归降糖舒心方对糖尿病动脉粥样硬化小鼠肠道黏膜屏障的影响及机制。方法Apoe-/-雄性小鼠适应性饲养1周后,给予小剂量链脲佐菌素(40 mg·kg^(-1))腹腔注射,每日1次,连续5 d;检测小鼠空腹血糖>11.1 mmol·L^(-1)后,继续以高脂饲料喂养12周。造模结束后,按照随机数字表法将造模小鼠分为模型组、二甲双胍组(0.065 g·kg^(-1))、左归降糖舒心方组(28.86 g·kg^(-1)),另取C57BL/6雄性小鼠作为空白对照组,每组5只。各给药组按照上述剂量灌胃给药(15 mL·kg^(-1)),空白对照组及模型组给予等量蒸馏水灌胃,每天1次,连续8周。采用ELISA法检测小鼠血清中胰高血糖素样肽1(GLP-1)、胆囊收缩素(CCK)、脂多糖(LPS)、白细胞介素18(IL-18)、白细胞介素1β(IL-1β)的含量;苏木精-伊红(HE)染色法观察主动脉、回肠组织病理变化;过碘酸雪夫(PAS)染色法观察回肠组织杯状细胞数量;RT-qPCR法检测小鼠回肠组织肠道紧密连接蛋白1(Claudin-1)、闭锁小带蛋白1(ZO-1)、咬合蛋白(Occludin)mRNA表达情况;Western Blot法检测回肠组织NOD样受体热蛋白结构域相关蛋白3(NLRP3)、IL-1β及Gasdermin D-N端片段(GSDMD-N)蛋白表达水平。结果与空白对照组比较,模型组小鼠血清GLP-1、CCK含量显著降低(P<0.01);血清LPS、IL-1β、IL-18含量均显著升高(P<0.01);主动脉血管内膜增厚且结构紊乱,内壁不平整,存在大面积粥样斑块突出管腔,高倍镜下可见斑块内胆固醇结晶,平滑肌细胞排列紊乱,内膜下可见泡沫细胞及脂质堆积,炎症细胞浸润;回肠组织结构完整性被破坏,肠黏膜上皮排列紊乱,部分发生固有层增厚、中断,肠绒毛存在疏松断裂;回肠绒毛高度及隐窝深度显著降低(P<0.01);回肠PAS阳性细胞表达量显著降低(P<0.01);回肠组织Claudin-1、ZO-1、Occludin mRNA表达显著下调(P<0.01);回肠组织NLRP3、IL-1β、GSDMD-N蛋白表达水平均显著升高(P<0.01)。与模型组比较,左归降糖舒心方组及二甲双胍组小鼠血清GLP-1、CCK含量显著升高(P<0.01);血清LPS、IL-1β、IL-18含量均显著降低(P<0.01);主动脉管腔狭窄程度及斑块病变减轻,血管内壁欠光滑,散在粥样斑块,脂质蓄积及炎症细胞浸润情况均有不同程度改善;回肠黏膜上皮损伤均有所改善,回肠结构较完整,肠绒毛密度增加,绒毛断裂状态减少;肠绒毛高度及隐窝深度明显增加(P<0.05,P<0.01);回肠PAS阳性细胞表达量明显增加(P<0.05,P<0.01);回肠组织Claudin-1、ZO-1、Occludin mRNA表达明显上调(P<0.05,P<0.01);回肠组织NLRP3、IL-1β、GSDMD-N蛋白表达水平均明显降低(P<0.05,P<0.01)。结论左归降糖舒心方可以改善糖尿病动脉粥样硬化小鼠的胃肠激素分泌,降低炎症因子水平,修复肠道黏膜屏障,其作用机制可能与调控NLRP3炎症小体信号通路,抑制肠道炎症因子表达有关。
Objective To investigate the effect and mechanism of Zuogui Jiangtang Shuxin Formula(ZGJTSXF)on the intestinal mucosal barrier in mice with diabetic atherosclerosis.Methods After one week of acclimatization,male Apoe-/-mice were intraperitoneally injected with a low dose of streptozotocin(40 mg·kg^(-1))once daily for five consecutive days.After confirming fasting blood glucose levels>11.1 mmol·L^(-1),the mice were continuously fed a high-fat diet for 12 weeks.Upon successful modeling,the mice were randomly divided into the model group,the metformin group(0.065 g·kg^(-1)),the ZGJTSXF group(28.86 g·kg^(-1)),and male C57BL/6 mice served as the blank control group with five mice per group.The treatment groups received the respective drugs by gavage(15 mL·kg^(-1)),while the blank control and model groups received an equal volume of distilled water,once daily for 8 consecutive weeks.Serum levels of glucagon-like peptide-1(GLP-1),cholecystokinin(CCK),lipopolysaccharide(LPS),interleukin-18(IL-18),and interleukin-1β(IL-1β)were measured by ELISA.Pathological changes in the aorta and ileum were observed by hematoxylin-eosin(HE)staining.The number of goblet cells in ileal tissue was assessed by periodic acid-Schiff(PAS)staining.The mRNA expression levels of intestinal tight junction proteins Claudin-1,zonula occludens-1(ZO-1),and Occludin in the ileum were detected by RT-qPCR.The protein expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),IL-1β,and Gasdermin D-N-terminal fragment(GSDMD-N)in the ileum were measured by Western Blot.Results Compared with the blank control group,the model group showed significantly decreased serum levels of GLP-1 and CCK(P<0.01),and significantly increased serum levels of LPS,IL-1β,and IL-18(P<0.01).The aortic intima was thickened and structurally disordered,with an uneven inner wall and large atherosclerotic plaques protruding into the lumen.Under high magnification,cholesterol crystals within the plaques,disordered smooth muscle cell arrangement,subintimal foam cells,lipid accumulation,and inflammatory cell infiltration were observed.The structural integrity of the ileum was disrupted,with disordered arrangement of the intestinal mucosal epithelium,partial thickening and interruption of the lamina propria,and loose and broken intestinal villi.The ileal villus height and crypt depth were significantly reduced(P<0.01).The number of PASpositive cells in the ileum was significantly decreased(P<0.01).The mRNA expression levels of Claudin-1,ZO-1,and Occludin in the ileum were significantly downregulated(P<0.01).The protein expression levels of NLRP3,IL-1β,and GSDMD-N in the ileum were significantly increased(P<0.01).Compared with the model group,the ZGJTSXF group and the metformin group showed significantly increased serum levels of GLP-1 and CCK(P<0.01),and significantly decreased serum levels of LPS,IL-1β,and IL-18(P<0.01).The degree of aortic lumen stenosis and plaque lesions was reduced,with a relatively smoother vascular wall,scattered atherosclerotic plaques,and varying degrees of improvement in lipid accumulation and inflammatory cell infiltration.Damage to the ileal mucosal epithelium was ameliorated,the ileal structure was relatively intact,intestinal villus density increased,and villus breakage was reduced.The ileal villus height and crypt depth were significantly increased(P<0.05,P<0.01).The number of PASpositive cells in the ileum was significantly increased(P<0.05,P<0.01).The mRNA expression levels of Claudin-1,ZO-1,and Occludin in the ileum were significantly upregulated(P<0.05,P<0.01).The protein expression levels of NLRP3,IL-1β,and GSDMD-N in the ileum were significantly decreased(P<0.05,P<0.01).Conclusion ZGJTSXF can improve gastrointestinal hormone secretion,reduce inflammatory factor levels,and repair the intestinal mucosal barrier in mice with diabetic atherosclerosis.Its mechanism of action may be related to the regulation of the NLRP3 inflammasome signaling pathway and the inhibition of intestinal inflammatory factor expression.
作者
杨金伟
周毅
肖凡
喻嵘
YANG Jinwei;ZHOU Yi;XIAO Fan;YU Rong(College of Integrative Medicine,Hunan University of Chinese Medicine,Changsha 410208 Hunan,China;The Second Affiliated Hospital of Integrative Medicine,Hunan University of Chinese Medicine,Liuyang 410300 Hunan,China;School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208 Hunan,China)
出处
《中药新药与临床药理》
北大核心
2025年第12期2034-2043,共10页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家自然科学基金项目(82205077)
湖南省自然科学基金项目(2022JJ40315)
湖南省教育厅资助科研项目(21B0390)
湖南省卫生健康委科研计划项目(D202303067601)
中药粉体与创新药物省部共建国家重点实验室培育基地开放基金项目(21PTKF1016)。
