凉血活血方对脓毒症急性肾损伤小鼠肠道菌群及NLRP3/caspase-1/GSDMD焦亡通路的影响  

Effect of Liangxue Huoxue decoction on intestinal flora and NLRP3/caspase-1/GSDMD signaling pathway in mice model of sepsis-induced acute kidney injury

作　　者：周旻[1] 杨磊[2] 卓玉珍[2] 李棣华[2] 张兰秋 崔凌志 李家瑞[1] Zhou Min;Yang Lei;Zhuo Yuzhen;Li Dihua;Zhang Lanqiu;Cui Lingzhi;Li Jiarui(Department of Nephrology,Nankai Hospital,Tianjin 300100,China;Institute of Acute Abdomen,Tianjin Nankai Hospital,Tianjin 300100,China;Graduate School of Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津市南开医院肾内科,天津300100 [2]天津市南开医院中西医结合急腹症研究所,天津300100 [3]天津医科大学研究生院,天津300070

出　　处：《中华危重病急救医学》2023年第3期250-255,共6页Chinese Critical Care Medicine

基　　金：天津市自然科学基金(19JCZDJC36200,18JCQNJC13400);内蒙古自治区自然科学基金(2022MS08029);天津市卫建委中西医结合课题(2019038,2021009,2021044)。

摘　　要：目的:探讨凉血活血方对脓毒症急性肾损伤(AKI)小鼠肠道菌群、肠道屏障及肾组织NOD样受体蛋白3/天冬氨酸特异性半胱氨酸蛋白酶1/焦孔素D(NLRP3/caspase-1/GSDMD)细胞焦亡信号通路的影响。方法:将30只雄性C57BL/6小鼠随机分为假手术组(Sham组)、脓毒症组(CLP组)、脓毒症+凉血活血方组(CLP+LXHX组),每组10只。通过盲肠结扎穿孔术(CLP)建立脓毒症AKI小鼠模型;Sham组仅进行开腹处理。制模前2 h,CLP+LXHX组给予凉血活血方水煎剂(6 g/kg)灌胃治疗;Sham组及CLP组给予等体积生理盐水灌胃。制模后24 h,麻醉处死小鼠,取结肠和肾组织及结肠内新鲜粪便。光镜下观察小鼠肾组织和结肠组织的病理变化;采用实时聚合酶链反应(RT-PCR)检测小鼠肾脏组织炎症因子白细胞介素(IL-1β和IL-18)的mRNA表达;采用蛋白质免疫印迹试验(Western blotting)检测小鼠肾组织中NLRP3、caspase-1和GSDMD的蛋白表达;采用16S rDNA高通量测序检测小鼠肠道菌群的变化。结果:与Sham组比较,CLP组肾脏组织炎症细胞浸润增加,肾脏空泡化,肾脏组织中IL-1β和IL-18的mRNA表达及NLRP3、caspase-1和GSDMD的蛋白表达均明显升高;结肠组织病理损伤显著,肠道菌群的物种丰富度显著降低,肠球菌和埃希菌-志贺菌的相对丰度显著增加,回肠杆菌属、拟普雷沃菌属、毛螺菌属、克雷伯菌属和副萨特菌属的相对丰度显著升高。与CLP组比较,凉血活血方可以显著减轻脓毒症小鼠肾脏组织和结肠组织病理损伤,并降低病理评分(分:肾脏组织为1.75±0.43比3.50±0.50,结肠组织为1.25±0.43比4.50±0.50,均 P<0.05),改善肠道菌群组成,降低肠球菌和埃希菌-志贺菌的相对丰度,并显著增加乳酸杆菌和阿克曼菌属的相对丰度,显著降低肾脏组织中IL-1β和IL-18的mRNA表达〔IL-1β mRNA(2 ^(-ΔΔCt)):1.59±0.05比4.61±0.88,IL-18 mRNA(2^(-ΔΔCt)):1.69±0.17比2.86±0.63,均 P<0.05〕及NLRP3、caspase-1和GSDMD的蛋白Objective To investigate the effect of Liangxue Huoxue decoction on intestinal flora,intestinal barrier and NOD-like receptor protein 3(NLRP3)/caspase-1/gasdermin D(GSDMD)pyroptosis signaling pathway in mice model of sepsis-induced acute kidney injury(AKI).Methods The model of AKI was established by cecal ligation and perforation(CLP).Thirty male C57BL/6 mice were randomly divided into sham operation group(Sham group),sepsis group(CLP group)and sepsis+Liangxue Huoxue decoction(CLP+LXHX group),with 10 mice in each group.Mice in Sham group only underwent laparotomy.Two hours before model establishment,mice in CLP+LXHX group were treated with Liangxue Huoxue decoction(6 g/kg)by gavage;mice in Sham group and CLP group were given equal volume of normal saline by gavages.After 24 hours of modeling,all mice were sacrificed under anesthesia,and the colon and kidney tissues and fresh feces in the colon were taken.The pathological changes of kidney and colon were observed by hematoxylin-eosin(HE)staining under light microscope.Real-time polymerase chain reaction(RT-PCR)was used to detect inflammatory factors(interleukins,IL-1βand IL-18)in renal tissue.The expressions of NLRP3,caspase-1 and GSDMD were detected by Western blotting.The changes of intestinal flora in mice were detected by 16S rDNA high-throughput sequencing.Results Compared with the Sham group,the inflammatory cell infiltration of the kidney tissue was increased and the kidney became vacuolated in CLP group,the mRNA expressions of IL-1β,IL-18,and the protein expressions of NLRP3,caspase-1 and GSDMD were significantly increased in CLP group,the species richness of intestinal microflora decreased significantly,the relative abundance of Enterococcus and Escherichia-Shigella increased significantly,and the relative abundance of Ileibacterium,Alloprevotella,Lachnospiraceae,Klebsiella and Parasutterella increased significantly in CLP group.Compared with CLP group,Liangxue Huoxue decoction can significantly reduce the pathological changes of kidney and colon tissue

关 键 词：脓毒症 凉血活血方 细胞焦亡 肠道菌群 急性肾损伤 

分 类 号：R285.5[医药卫生—中药学]