出 处:《生物医学转化》2023年第1期78-83,100,共7页Biomedical transformation
基 金:国家自然科学基金项目(82172122);天津市医学重点学科(专科)建设项目(TJSYXZDXK010)。
摘 要:目的评估栀子苷元(Genipin)对盲肠结扎穿孔术(Cecal Ligation and Puncture,CLP)诱导的脓毒症小鼠模型的治疗效果,并探讨其潜在的作用机制。方法将雄性C57BL/6小鼠分为假手术组(Sham组)、脓毒症模型组(CLP组)、栀子苷元治疗脓毒症组(Genipin+CLP组)。栀子苷元治疗组在建模后0 h和20 h经尾静脉注射2.5 mg/kg栀子苷元,而Sham组和CLP组注射等体积生理盐水。实验1,观察栀子苷元对脓毒症小鼠120 h生存率的影响;实验2,基于生存率的结果选取建模后36 h为采样时间点,并行以下方法检测:利用酶联免疫吸附法(Enzym-linked Immunosorbent Assay,ELISA)检测肿瘤坏死因子α(Tumor Necrosis Factorα,TNF-α)和白细胞介素6(Interleukin 6,IL-6)含量;常规苏木精-伊红(Hematoxylin eosin,HE)染色法检测肺和肝组织病理损害;Western Blot法检测脾脏内质网应激相关蛋白的表达变化,包括葡萄糖调节蛋白78(Glucose Regulated Protein 78,GRP78)、蛋白激酶R样内质网激酶(Protein Kinase R-like Endoplasmic Reticulum Ki‐nase,PERK)、磷酸化真核翻译起始因子2α(p-eukaryotic Initation Factor 2α,p-eIF2α)和C/EBP同源蛋白(C/EBP Homolo‐gous Protein,CHOP);脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(Terminal-deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling,TUNEL)检测脾细胞凋亡变化。结果与CLP组相比,栀子苷元治疗提高了脓毒症小鼠120 h生存率(P<0.05),降低了血清TNF-α和IL-6含量(P<0.05),并减轻肺和肝脏组织病理学损伤。另外,栀子苷元显著下调了脾脏GRP78、PERK、p-eIF2α和CHOP蛋白的表达水平(P<0.05),并进而减少内质网应激诱导的脾细胞凋亡数量(P<0.05)。结论栀子苷元能够明显减轻脓毒症所导致的脏器损害和过度炎症反应,改善脓毒症预后。该效应的作用机制可能与栀子苷元减轻脾脏内质网应激导致其凋亡机制的启动被阻断,进而减轻脾细胞凋亡有关。Objective This study is undertaken to elucidate the protective effect and molecular mechanism of genipin against cecal ligation and puncture(CLP)-induced sepsis.Methods Male C57BL/6 mice were assigned to three groups:vehicle-treated sham group,vehicle-treated CLP group,and genipin-treated CLP group.Genipin(2.5 mg/kg)and vehicle(0.9%saline)were respectively administered to corresponding group via the tail vein at 0 h and 20 h after the CLP.First,for the survival experiment,mortality was monitored up to 120 h after the CLP procedure.Second,based on the survival test,36 h time point after CLP was selected for further tests.The serum concentrations of tumor necrosis factorα(TNFα)and interleukin 6(IL-6)were detected by enzym-linked immunosorbent assay(ELISA).The pathological damages to lung and liver tissue were detected by hematoxylin eosin staining(HE staining).The expression levels of endoplasmic reticulum(ER)stress-related proteins such as glucose regulated protein78(GRP78),protein kinase r-like endoplasmic reticulum kinase(PERK),p-eukaryotic initation factor 2α(p-eIF2α),and C/EBP homologous protein(CHOP)were determined by Western Blot.Detection of splenocyte apoptosis was performed by using the terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL)method.Results Compared with the vehicle-treated CLP group,genipin significantly improved the survival of septic mice,decreased the serum levels of TNF-αand IL-6,and alleviated histopathological damages to lung and liver such as edema and inflammatory cell infiltration.Moreover,genipin significantly downregulated the expression levels of splenic GRP78,PERK,p-eIF2α,and CHOP,and lessened ER induced splenocyte apoptosis.Conclusion Genipin significantly attenuates sepsis-induced organ injury and the excessive inflammatory response and improves the outcome of sepsis.The effect mechanisms of genipin against sepsis are probably associated with decreasing splenocyte apoptosis by attenuating ER stress to further block the ER stress-induced apoptosis.
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