Study On Inhibition Of Rictor And Down-regulation Of MTOR Signaling Pathway Enhance Autophagy In Sepsis Myocardial Cell

Objective:To investigate the key signaling pathways and key genes of that involved in the change in the autophagy regulation of sepsis cardiac dysfunction（SIMD）,and to explore the effects of key genes on the autophagy level of SIMD myocardial cells.Methods:1.Data sets of gene expression levels in genomic sepsis（GSE65682,GSE131761,GSE134364）and myocardial tissue of SIMD mice（GSE69345,GSE171546）were used as research objects.Autophagy related genes were extracted from sepsis’data sets,and the expression levels of autophagy related genes in each data set were standardized,and gene set enrichment analysis（GSEA）was performed to screen significantly enriched autophagy related signaling pathways.GSEA analysis was performed on the data set of gene expression levels in myocardium of SIMD mice,and the same significantly enriched signaling pathway as that of sepsis patients was screened,and the key genes in this signaling pathway were extracted.The gene expression levels of lipopolysaccharide（LPS）in human sepsis model and mouse SIMD animal model were collected by GSE134364,and to study the variation trend of key genes and autophagy effect markers at each time node.2.The patients with SIMD diagnosed in the Internal Medicine Intensive Care Unit of the Affiliated Hospital of Guizhou Medical University were selected as the research objects,and healthy people were selected as the control group.The peripheral blood of SIMD patients was collected.The expression levels of Rictor and autophagy effect markers rapamycin-insensitive companion of m TOR（LC3B）,Beclin-1 were detected by real-time quantitative reverse transcription PCR（q RT-PCR）.The intra-group correlation analysis was performed on the expression levels of each gene,and the inter-group comparison analysis was performed with the control group.At the same time,the case information and related test results of SIMD patients were collected,and the correlation analysis with each gene expression was carried out.3.Thirty-six Specific Pathogen Free（SPF）male Kunming mice were divided into sham operation group,model group and experimental group by random number method,with 12 mice in each group.The SIMD mouse model was established by Cecum Ligation and Puncture（CLP）.The experimental group was given Rictor specific inhibitor JR-AB2-011 10mg/kg intraperitoneal injection,sham operation group and model group were given the same amount of solvent intraperitoneal injection.The levels of Serum Cardiac Troponin I（c Tn I）and Creatine kinase MB,CK-MB were detected at 12 and 24 hours after operation.The change of protein expressions of Rictor,protein kinase B（PKB/AKT）and LC3B in mouse myocardium were detected.TEM and Hematoxylin-Eosin（H-E）staining were performed to observe the pathological changes of myocardial tissue.Results:1.Among all data sets of sepsis patients,422 common autophagy related genes were significantly enriched in the"BIOCARTA MTOR PATHWAY"signaling pathway.SIMD mouse cardiac tissue genes are mainly enriched in"BIOCARTA MTOR PATHWAY"and"KEGG MTOR SIGNALING PATHWAY"signaling way,m TOR signaling pathway is the key pathway for SIMD autophagy regulation.Rictor is one of the key genes in it.The expression of Rictor was significantly upregulated at 6 h after LPS injection in human model of sepsis established by LPS in GSE134364 data set（P<0.05）.The expression of Rictor gene in the SIMD animal model was significantly higher than that in the sham operation group（P<0.05）.2.A total of 37 subjects were included in the study,including 25 in the SIMD group,aged 55.16±20.95 years,including 14（56.0%）males and 11（44.0%）females;A total of 12 healthy subjects,aged 43.33±8.78 years,were included in the control group,including 6（50.0%）males and 6（50.0%）females.The expression of Rictor in the SIMD group was significantly increased compared with the control group（P<0.05）,while AKT,LC3B and BEClin-1 were significantly decreased compared with control group（P<0.05）.The relative expression of Rictor in SIMD group was negatively correlated with AKT2（P<0.05,r=-0.76）,Beclin-1（P<0.05,r=-0.65）and LC3B（P<0.05,r=-0.67）,and positively correlated with PCT（P<0.05,r=0.67）;The relative expression of LC3B was negatively correlated with APACHE II score in the SIMD group（P<0.05,r=-0.77）.3.The expression levels of cTnI,CK-MB,Rictor,Phospho-Ak T^Ser473 and LC3B in SIMD model group were higher than those in sham group at each time node（P<0.05）.The protein expression levels of c Tn I,CK-MB,Rictor and Phospho-Aktser473 in the experimental group were lower than those in the model group,while the protein expression level of LC3B was higher than that in the model group at each time node（P<0.05）.H-E staining of mouse myocardium:myocardium tissue of sham operation group was normal;Moderate hydropic degeneration of myocardial fibers was observed in the model group.Mild hydropic degeneration of myocardial fibers was observed in the experimental group.Transmission electron microscopy of mouse myocardium:in the sham operation group,no myofibers were broken and were closely arranged;In the model group,the myofibers were fragmented,sparsely arranged and narrowed,with a large number of fractures,severe cytoplasm edema,and 1 autophagic lysosome was observed;In the experimental group,muscle fibers were arranged relatively closely,with many muscle fibers broken and mild edema.One autophagosome and four autophagolysis could be seen in the visual field.Conclusions:1.The level of autophagy in SIMD patients and SIMD mice decreased significantly,and markers of myocardial injury increased.Low level of autophagy in SIMD patients was significantly correlated with myocardial cell injury.2.MTOR signaling pathway is the main signaling pathway that regulates the autophagy level of SIMD cardiomyocytes,and it is activated at the early stage to inhibit the autophagy level of myocardium,leading to myocardial cell injury.3.Rictor is a key target for the m TOR signaling pathway to regulate the autophagy of cardiomyocytes.By targeting inhibition the expression level of Rictor,the activation of m TOR signaling pathway can be reduced,the autophagy of cardiomyocytes can be increased,the damage of cardiomyocytes can be alleviated,and cardiac dysfunction caused by sepsis can be improved.