| Objective: Based on the theory of "gut-muscle axis",this study investigated the influence of gut microbiota and its metabolites in the development of sepsis-induced muscle weakness,and explored the role and potential mechanism of the differential metabolite vitamin K1 in sepsis-induced muscle weakness.Methods:Part ⅠIn the sepsis susceptibility experiment,eighty healthy adult male Specific Pathogen Free(SPF)C57BL/6J mice,6-8 weeks old,weighing 20-22 g,were selected.Forty mice were injected intraperitoneally with Lipopolysaccharide(LPS)10 mg/kg to establish sepsis-induced muscle weakness,and every mouse was numbered before modeling and collected feces to make fecal fluid.According to the survival time of mice after modeling,the mice were divided into sepsis sensitive mice group(S-sen group)and sepsis resistant mice group(S-res group).The mice that were moribund or even died within 1 day were defined as sepsis sensitive mice(S-sen),and the mice that survived to 7 days and recovered active were defined as sepsis resistant mice(S-res).The Compound muscle action potential(CMAP)of the sciatic nerve-gastrocnemius muscle complex was detected before moribund or after 7 days in both groups,and tissue apoptosis level was detected by TUNEL staining.In the fecal microbiota transplantation experiment,forty SPF adult male C57BL/6J mice were randomly divided into four groups by random number table:(1)control group(Ctrl group,n=10): intraperitoneal injection of equal amount of saline;(2)antibiotic group(ABX group,n=10): gavage with compound antibiotics for 5 days,followed by intraperitoneal injection of equal amount of saline;(3)resistant group(Res group,n=10): gavage with compound antibiotics for 5 days,gavaged with fecal fluid of S-sen for 3 days,followed by intraperitoneal injection of LPS 15 mg/kg;(4)sensitive group(Sen group,n=10): gavage with compound antibiotics for 3 days,followed by intraperitoneal injection of LPS 15 mg/kg.The severity of sepsis was evaluated immediately 24 hours after intraperitoneal injection of saline or LPS and CMAP was detected.The diameter and cross-sectional area of gastrocnemius muscle fibers were determined by H&E staining.The relative expressions of E3 ubiquitin ligase proteins Mu RF-1 and MAFbx,and tight junction proteins ZO-1and Occludin in colon were determined by western blotting.The composition of fecal gut microbiota of mice in both groups was determined by 16 Sr DNA sequencing.Nontargeted metabolomics analysis was performed to determine fecal metabolite composition in both groups.Ultra Performance Liquid Chromatography Tandem Mass Spectrometry(UPLC-MS/MS)detected the content of vitamin K1 in serum of the two groups.Part ⅡThirty healthy adult male SPF-grade C57BL/6J mice,6-8 weeks old,weighing 20-22 g were selected and randomly divided into three groups by random number table :(1)control group(Ctrl group,n=10): intraperitoneal injection of equal amount of saline;(2)lipopolysaccharide group(LPS group,n=10): intraperitoneal injection of lipopolysaccharide 15 mg/kg;(3)vitamin K1 group(VK1 group,n=10)group: Intraperitoneal injection of lipopolysaccharide 15 mg/kg followed by gavage of vitamin K1 1 mg/kg.The severity of sepsis was evaluated immediately 24 hours after modeling,and CMAP was detected.The diameter and cross-sectional area of gastrocnemius muscle fibers were determined by H&E staining.The relative expressions of E3 ubiquitin ligase proteins Mu RF-1 and MAFbx in tibial anterior muscle and tight junction proteins ZO-1 and Occludin in colon were determined by western blotting.Serum levels of IL-1,IL-6 and TNF-α were determined by ELISA.The levels of MDA,GR and SOD were determined by colorimetry.Results:Part Ⅰ(1)Compared with the S-res group,the score of sepsis severity and the degree of tissue damage in the S-sen group were significantly increased(P <0.05),the incubation period of CMAP was significantly prolonged(P < 0.05),and the amplitude was significantly decreased(P < 0.05).(2)Compared with Ctrl group and ABX group,sepsis severity score in Sen group and Res group was significantly increased after 24h(P < 0.05).Compared with Ctrl group,there was no significant difference in the severity of sepsis in ABX group(P > 0.05).Compared with Res group,sepsis severity score of Sen group was significantly increased(P < 0.05).(3)Compared with the Res group,the forelimb grip of mice in Sen group was significantly decreased(P < 0.05),the latency of CMAP was significantly prolonged(P < 0.05),and the amplitude was significantly decreased(P < 0.05).(4)Compared with Ctrl group,the muscle fiber diameter and cross-sectional area in ABX group had no statistical significance(P > 0.05),while the muscle fiber diameter and cross-sectional area in Res and Sen groups were significantly decreased(P < 0.05).Compared with Res group,the diameter and cross-sectional area of gastrocnemius muscle fiber in Sen group were significantly decreased(P < 0.05).(5)Compared with Ctrl group,the protein expression levels of Mu RF1 and MAFbx in anterior tibial muscle of ABX group and Res group had no statistical difference(P > 0.05),while the protein expression levels of Mu RF1 and MAFbx in anterior tibial muscle of Sen group were significantly increased(P <0.05).There was no significant difference in protein expression levels of Mu RF1 and MAFbx in anterior tibial muscle of Ctrl group,ABX group and Res group(P > 0.05).(6)Compared with Ctrl group,the expression of ZO-1 and Occludin in colon of ABX group and Res group had no statistical difference(P > 0.05),while the expression of ZO-1 and Occludin in colon of Sen group significantly decreased(P < 0.05).The protein expression levels of ZO-1 and Occludin in Ctrl group,ABX group and Res group had no statistical significance(P > 0.05).(7)Compared with Sen group,there was no significant difference in Chao1 index,Good-coverage index and Simpson index of intestinal flora in Res group(P > 0.05),while Shannon index was increased(P < 0.05),and there was no significant difference in β diversity in Res group(P > 0.05).Nontargeted metabolomics analysis suggested that vitamin K1 content in feces of mice in Res group was significantly increased compared with Sen group(P < 0.05),and UPLC-MS/MS suggested that serum vitamin K1 content of mice in Res group was significantly increased compared with Sen group(P < 0.05).Part Ⅱ(1)Compared with Ctrl group,sepsis severity scores in LPS group and VK1 group were significantly increased 24 h later(P < 0.05).Compared with VK1 group,the severity of sepsis in LPS group was significantly increased(P <0.05).(2)Compared with VK1 group,CMAP latency of mice in LPS group was significantly prolonged(P < 0.05),and amplitude was significantly decreased(P < 0.05).(3)Compared with Ctrl group and VK1 group,the protein expressions of Mu RF-1 and MAFbx in the anterior tibial muscle of mice in LPS group were significantly increased(P < 0.05).There was no significant difference in the protein expressions of Mu RF-1 and MAFbx in anterior tibial muscle between Ctrl group and VK1 group(P > 0.05).(4)Compared with the Ctrl and VK1 groups,the colonic ZO-1 and Occludin protein expressions were increased in the LPS group significantly(P< 0.05);the differences between the colonic ZO-1 and Occludin protein expressions in the Ctrl and VK1 groups were not statistically significant(P >0.05).(5)Compared with Ctrl group and VK1 group,the expression of p NF-κB/NF-κB protein in the anterior tibial muscle of mice in LPS group was significantly increased(P < 0.05).There was no significant difference in the expression of p NF-κB/NF-κB protein in colon between Ctrl group and VK1group(P > 0.05).Compared with VK1 group,SIRT1 in LPS group was significantly decreased(P < 0.05).(6)Compared with Ctrl group and VK1 group,serum concentrations of IL-1,IL-6 and TNF-α in LPS group were significantly increased after 24 h(P <0.05).There was no significant difference in serum IL-1,IL-6 and TNF-αconcentrations between Ctrl group and VK1 group(P > 0.05).(7)Compared with Ctrl and VK1 groups,the GR and SOD levels of the anterior tibial muscle of mice in LPS group were significantly decreased(P <0.05),while the MDA level was significantly increased(P < 0.05).Conclusion: Mice with the same genetic background showed sepsis heterogeneity when injected with lipopolysaccharide intrapitoneally to construct sepsis models,which was mediated by fecal bacteria transplantation.Intestinal differential metabolite vitamin K1 can antagonize the phosphorylation of NF-κB by up-regulating the expression of SIRT1,reduce the level of oxidative stress and inflammation in skeletal muscle,and improve the acquired myasthenia in sepsis mice. |
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