【作者】 李文斌；

【导师】 杨明施； 高敏；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2022， 硕士

【摘要】 目的:研究异甘草酸镁对脓毒症小鼠多器官功能的保护作用,并初步探讨异甘草酸镁对细胞焦亡信号通路的影响。方法:选取8周龄雄性Balb/C小鼠,随机分配为六组:假手术+溶剂对照组(Sham+V)、假手术+异甘草酸镁组(Sham+Mg IG)、6h脓毒症模型+溶剂对照组(6h CLP+V)、6h脓毒症模型+异甘草酸镁组(6h CLP+Mg IG)、24h脓毒症模型+溶剂对照组(24h CLP+V)、24h脓毒症模型+异甘草酸镁组(24h CLP+Mg IG)。采用盲肠结扎穿刺(CLP)建立脓毒症模型。异甘草酸镁干预组在假手术或造模后立即腹腔注射异甘草酸镁注射液(50mg/kg),3h追加一次;余组予等量0.9%Na Cl注射液处理。术后6小时、24小时处死小鼠收集小鼠血清及心、肝、肺及肾组织;采用苏木素-伊红染色处理各器官组织,光镜下观察各组小鼠肺组织、肝组织、心肌组织、肾组织病理变化,评估器官病理损伤情况;全自动分析仪检测谷丙转氨酶(ALT)、谷草转氨酶(AST)、肌酐(SCR)、尿素氮(BUN)、乳酸脱氢酶(LDH)、肌酸激酶同工酶(CKMB)水平;采用酶联免疫吸附法(ELISA)检测小鼠血清肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)水平;以比色法检测血清丙二醛(MDA)水平及超氧化物歧化酶(SOD)活性。采用Western blot检测肝脏、肾脏及肺组织中Caspase-11、GSDMD、IL-1β的表达,观察异甘草酸镁对各器官细胞焦亡信号通路的影响。结果:与Sham+V组相比,6h及24h CLP+V组小鼠肝、肾、肺组织病理损伤明显,损伤评分上升(P<0.01),血清ALT、AST、BUN、SCR、LDH、CK-MB指标明显上升(P<0.05),血清SOD活性下降(P<0.01),MDA水平上升(P<0.05),炎症因子TNF-α、IL-6、IL1-β水平上升(P<0.01,P<0.05);经异甘草酸镁干预后,6h及24h CLP+Mg IG组小鼠肝、肾、肺组织病理学评分下降(P<0.01),组织形态学损伤减轻,血清ALT、AST、LDH、CK-MB、BUN、SCR指标下降(P<0.01),血清SOD活性相对升高(P<0.01),MDA水平下降(P<0.01),24h CLP+Mg IG组小鼠炎症因子TNF-α、IL-1β、IL-6水平下降(P<0.01);在Western blot中,24h CLP+V组小鼠肝脏、肺组织及肾组织内Caspase-11、GSDMD、IL-1β蛋白表达较Sham+V组小鼠明显上升(P<0.05,P<0.01),与24h CLP+V组小鼠比,24h CLP+Mg IG组小鼠上述蛋白表达显著下降(P<0.05,P<0.01)。结论:异甘草酸镁注射液对脓毒症小鼠多器官功能具有保护作用,能明显减轻脓毒症小鼠多器官组织病理损伤并改善了多器官功能,其机制可能与异甘草酸镁抗炎抗氧化作用有关;异甘草酸镁干预后能使脓毒症小鼠肝脏、肺组织及肾组织中焦亡相关蛋白Caspase-11、GSDMD表达下降并减少炎症介质IL-1β的释放,提示异甘草酸镁可能在脓毒症小鼠细胞焦亡中发挥保护作用。更多还原

【Abstract】 Objective:Investigate the protective effect of magnesium isoglyclycinate on multi-organ function in mice with sepsis,and to investigate the effect of magnesium isoglyrrhizinate on pyroptosis signaling pathway.Methods:Eight-week-old male Balb/C mice were randomly assigned to six groups: sham surgery + solvent control group(Sham+V),sham surgery + magnesium isoglyrrhizinate group(Sham+Mg IG),6h sepsis model + solvent control group(6h CLP+V),6-hour sepsis model +magnesium isoglyrrhizinate group(6h CLP + Mg IG),24 h sepsis model +solvent control group(24h CLP + V),24 h sepsis model + magnesium isoglyrrhizinate group(24h CLP + Mg IG).A sepsis model was established using cecum ligation and puncture(CLP).Magnesium isoglyrrhizinate intervened with intraperitoneal injection of magnesium isoglyrrhizinate injection(50 mg/kg)immediately after sham surgery or molding,and was supplemented once 3 hours,and an equal amount of 0.9% Na Cl injection was given.The serum and heart,liver,lung and kidney tissues of mice were collected at 6 hours and 24 hours after surgery,and the tissues of various organs were treated with hematoxylin-eosin staining,and the lung tissue,liver tissue,myocardial tissue and kidney tissue of each group of mice were observed under light microscopy Pathological changes to assess organ pathological damage;fully automated analyzer to detect alanine transaminases(ALT),aspartate aminotransferase(AST),creatinine(SCR),Urea nitrogen(BUN),lactate dehydrogenase(LDH),creatine kinase isoenzyme(CK-MB)levels,enzyme-linked immunosorbent assay(ELISA)The serum tumor necrosis factor α(TNF-α),interleukin-1 β(IL-1 β),and interleukin-6(IL-6)levels in mice were measured Serum malondialdehyde(MDA)levels and superoxide dismutase(SOD)activity were detected by colorimetric method.Western blot was used to detect the expression of Caspase-11,GSDMD,IL-1 β in liver,kidney and lung tissues,and the effect of magnesium isoglyrrhizinate on the cytojectrosis signaling pathway of various organs was observed.outcome :Sham+V Compared to 6h and 24 h CLP+V Mice had an increased liver,kidney,and lung tissue injury score(P<0.01),serum ALT、AST、BUN、LDH、CK-MB indicator rise(P<0.05),serum SOD activity descent(P<0.01),MDS level rise(P<0.05),TNF-α、IL-6、IL-1β level rise(P<0.01,P < 0.05);After intervention with magnesium isoglyrrhizinate,6h and 24 h CLP+Mg IG group liver,kidney,lung tissue in mice pathology rating drop(P<0.01),serum ALT、AST、LDH、CK-MB、BUN、SCR indicator drop(P<0.01),Serum SOD activity descent(P<0.01),MDA decreased level(P<0.01),TNF-α、IL-1β、IL-6 decreased level(P<0.01);In Western blot,24 h CLP+V in mouse liver,lung tissue and kidney tissue Caspase-11、GSDMD、IL-1β protein expression increased significantly compared with Sham+V mice(P<0.05,P<0.01),compared with mice in the 24 h CLP+V group,24 h CLP+Mg IG group Mice The expression of the above protein decreased significantly(P<0.05,P<0.01)。Conclusion:Magnesium isoglyrrhizinate injection has a protective effect on the multi-organ function of mice with sepsis,and its mechanism may be related to the anti-inflammatory and antioxidant effect of magnesium isoglyrrhizinate,and the expression of pyroptosis-related protein Caspase-11 and GSDMD in the liver,lung tissue and kidney tissue of mice with sepsis can be reduced and the expression of inflammatory mediator IL-1β can be reduced.更多还原