肠道菌群介导生脉方对脓毒症小鼠的治疗作用

Gut microbiota-mediated therapeutic effect of Shengmai formula(SMF)on sepsis in mice

探究生脉方(Shengmai formula,SMF)对脓毒症小鼠组织损伤、血清炎症因子和外周血固有免疫细胞比例的作用;考察肠道菌群在SMF治疗脓毒症中的作用。灌胃0.3,0.6,1.2 g/kg或腹腔注射0.6 g/kg SMF 4 d后腹腔注射20 mg/kg脂多糖(LPS)建立脓毒症模型,考察小鼠生存率,通过H&E染色观察小鼠肝、肺、肾组织病理改变,检测血清IL-6、TNF-α、谷丙转氨酶(ALT)、谷草转氨酶(AST)、尿素氮(BUN)和肌酐(Cr)水平;建立LPS和盲肠结扎穿刺(CLP)脓毒症模型,通过流式细胞术检测SMF灌胃或腹腔注射对外周血单核细胞、巨噬细胞和中性粒细胞比例的影响;考察抗生素(ABX)处理对SMF灌胃治疗脓毒症的影响;考察SMF灌胃小鼠的粪菌对脓毒症的治疗作用。结果表明,SMF灌胃显著提高LPS模型小鼠生存率,减轻肝、肺、肾损伤和炎性浸润,降低血清IL-6、ALT、AST、BUN、Cr水平;显著降低LPS模型24 h外周血巨噬细胞比例,下调CLP模型24 h外周血单核、巨噬细胞和中性粒细胞比例。SMF腹腔注射对脓毒症模型上述指标均无显著作用。ABX处理逆转了SMF灌胃的脓毒症治疗作用;SMF灌胃小鼠的粪便移植到伪无菌鼠,显著减轻其脓毒症症状。结果表明,SMF通过调节肠道菌群,降低血清炎症因子,缓解组织损伤,发挥对脓毒症小鼠的治疗作用。本研究为SMF治疗临床脓毒症提供理论依据。

To investigate the effects of Shengmai formula(SMF)on tissue damages,serum inflammatory factors and the proportion of innate immunocytes in peripheral blood,sepsis models using either intraperitoneal injection of 20 mg/kg lipopolysaccharide(LPS)or cecal ligation and puncture(CLP)were established.The role of gut microbiota in septic mice during SMF treatment was further investigated.LPS-induced sepsis model was carried out 4 days after daily gavage administration with 0.3 g/kg,0.6 g/kg,1.2 g/kg SMF or intraperitoneal injection with 0.6 g/kg SMF.Survival rates of septic mice were determined.Histological evaluations of liver,lung and kidney were analyzed by H&E staining.Serum IL-6,TNF-α,Alanine transaminase(ALT),Aspartate aminotransferase(AST),Blood urea nitrogen(BUN)and Creatinine(Cr)levels were determined.LPS and CLP-induced sepsis models were established,and the proportion of monocytes,macrophages and neutrophils in peripheral blood were analyzed by flow cytometry after gavage administration or intraperitoneal injection of SMF.The therapeutic effects of SMF after antibiotics treatment were further determined,and the therapeutic effects of fecal microbiota from SMF-treated mice were investigated.The results show that LPS-induced sepsis caused death of mice,damages in liver,lung and kidney with increased infiltration of leukocytes and elevated levels of serum IL-6,ALT,AST,BUN and Cr,which were all reversed by gavage administration of SMF.Gavage administration of SMF could significantly reduce the proportion of peripheral macrophages in LPS model and monocytes,macrophages,neutrophils in CLP model.Intraperitoneal injection of SMF showed no therapeutic benefits in septic mice.Depletion of gut microbiota using antibiotics cocktail reversed the therapeutic effects of SMF on sepsis,indicating the involvement of gut microbiota.Fecal microbiota from SMF-treated donors was transplanted into pseudo-sterile recipients,and we found FMT could significantly ameliorate sepsis of recipients.These results showed that gavage administration of SMF reduced serum inflammatory factors and alleviated tissue damages in septic mice by regulating gut microbiota.This study provides a theoretical basis for the treatment of clinical sepsis with SMF.