| Objective: In this study,we constructed an animal model of SIDD,applied rapamycin to block the mTOR signaling pathway,and examined its effects on mTOR/LDLR,diaphragm lipid metabolism,diaphragm morphology and function to provide new directional targets for the mechanism of SIDD occurrence and prevention measures.Methods: 18 adult male SD rats were randomly divided into three groups(n=6 each):Control group,rats were intraperitoneally injected with the same volume of normal saline;Sepsis(LPS)group,rats were intraperitoneally injected with LPS(10mg/kg);In the sepsis +mTOR inhibitor(LPS+RAPA)group,the septic rats were intraperitoneally injected with rapamycin(2mg/kg).Changes in body weight and general conditions of the rats were recorded before and after modelling.(1)RM6240E biological information acquisition system was used to detect the mechanical contraction performance of the isolated diaphragm after electrical stimulation;(2)the expression of inflammatory factors in each group were detected;(3)The pathological changes of the diaphragm tissue were observed by HE staining.Lipid deposition in the diaphragm was observed by oil red staining and immunofluorescence,and the muscle fiber types of the diaphragm were observed by ATPase staining.(4)Western Blot assay was used to detect the protein expressions of mTOR,LDLR,Anx A2,and PCSK9.(5)Lipidomic analysis of rat diaphragm tissue by applying LC-MS based high-throughput analysis technique.Results:(1)Morphological examination of diaphragm: HE staining results showed that the diaphragm fibers of Control group rats were basically normal in shape;In the LPS group,the diaphragm was obviously damaged,and the interstitial edema and congestion were accompanied by obvious inflammatory cell infiltration.Compared with the LPS group,the diaphragm damage was significantly reduced in the LPS+RAPA group.(2)Diaphragmatic inflammation index detection: qRT-PCR showed that the relative expression of TNF-α and IL-6 mRNAs were significantly up-regulated in the LPS group compared with the Control group(P<0.0001)(3)Diaphragmatic fiber typing: compared with the Control group,the ratio of slow muscle fibers to fast muscle fibers in the LPS group did not change significantly.(4)Lipid deposition in the diaphragm: Oil red staining showed that compared with the Control group,fibrolipid deposition in the diaphragm was significantly increased in the LPS group(P<0.05).Compared with the LPS group,the lipid deposition in the diaphragm was significantly reduced in the LPS+RAPA group(P<0.05).(5)Mechanical contraction performance of electrically stimulated isolated diaphragm:Compared with the Control group,the single contraction tension(Pt)and the maximum tetanic contraction force(Po)of the isolated diaphragm in the LPS group and the LPS+RAPA group decreased significantly(P<0.05),and the contraction-stimulation frequency curve shifted downward.The muscle strength indexes in LPS group were lower than those in Control group and LPS+RAPA group.However,there was no significant difference in muscle strength between Control group and LPS+RAPA group.(6)Lipidomic analysis: In total,we detected 1935 lipid molecules in rat diaphragm tissues,and its 776 and 1159 lipid molecules were detected in positive and negative ion mode,respectively.Further analysis revealed that there were significant differences in the lipid metabolites of rat diaphragm between Control and LPS groups.(7)Changes in lipid metabolism-related proteins: Western Blot results showed that the expression of mTOR,LDLR and Anx A2 in diaphragm fibres was significantly increased in the LPS group compared to the Control group(P<0.05),while the expression of PCSK9 was significantly decreased.Compared with the LPS group,the expression of mTOR,LDLR,and Anx A2 in diaphragm fibers was significantly decreased in the LPS+RAPA group(P<0.05),and the expression of PCSK9 was significantly increased.Conclusion: Sepsis can activate the mTOR signaling pathway,up-regulate LDLR expression,leading to lipid deposition in the diaphragm,decreased diaphragmatic contractility,and diaphragmatic dysfunction.Rapamycin,a targeted inhibitor of mTOR,can inhibit the activity of mTOR,down-regulate the expression of LDLR,improve the abnormal lipid metabolism of diaphragm,and alleviate Sepsis-induced diaphragm dysfunction.Abnormal lipid metabolism due to mTOR activation may be a potential early initiating mechanism of sepsis-related diaphragm dysfunction... |
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