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Real-time quantitative PCR analysis of cytokines and chemokines in C57BL/6 mouse liver ischemic-reperfusion injury (IRI) model.

ABSTRACT: We assessed mouse cytokines and chemokines gene expression in fatty liver ischemic-reperfusion injury (IRI). C57BL/6 mice were fed with a normal chow diet (ND, 4.09 kcal/gram,13.4% kJ/fat) or a high-fat diet (HFD, 5.10 kcal/gram, 60% kJ/fat) and further grouped to treat with orally administered N-acetylcysteine (NAC, 10g/L). To induce liver ischemic-reperfusion injury in mice, an atraumatic micro clip was placed across the hepatic hilus, which interrupted the blood supply to the left and median lobes of the liver for 45-minutes partial warm ischemia time. After 24 hours of liver IRI, the affected left lobe of the liver was harvested and stored in Allprotect Tissue Reagent (Qiagen). We used Qiagen RT² Profiler™ PCR Array for mouse cytokines & chemokines to distinguish immunologically related gene signatures specific to the liver IRI in mice and characterize the effect of NAC treatment in HFD mice.

ORGANISM(S): Mus musculus

PROVIDER: GSE209733 | GEO | 2022/07/30

REPOSITORIES: GEO

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Project description:INTRODUCTION Ischemia and reperfusion injury (IRI)-elicited tissue injury contributes to morbidity and mortality in a wide range of pathologies, including myocardial infarction, ischemic stroke, acute kidney injury, trauma, circulatory arrest 1. Ischemia-reperfusion injury is also a major challenge during organ transplantation and cardiothoracic, vascular and general surgery 1. IRI, one of the biggest challenges for organ transplantation, continues to be a vital source of morbidity among recipients, especially in liver transplantation 2. With the enlarging shortage of available donor livers, the increased use of extended criteria donor grafts further increases IRI, adversely affecting both short-term and long-term outcomes of graft and patient survival 3. Numerous studies have investigated the benefits of pharmacological, heat shock, and ischemic preconditioning interventions aimed at decreasing liver IRI 4. However, the benefit was limited. Our center has been making great effort in conquering IRI and have developed a novel surgical technique called ischemia-free liver transplantation 5. It is an ultimate method to overcome IRI in liver transplantation, but there is still a long way to popularize it. As a result, it is still of great significance to study IRI and identify the core genes in the process and the underlying mechanism. Comprehensive bioinformatics analysis has been increasingly important as a method to study various pathological and physiological condition 6. By enrolling multiple omics or combining different types of omics, comprehensive bioinformatics analysis was able to recognize key factors that could have potentially pathogenic impact such as gene expression, protein function, and downstream pathways. With the rapid development of high-throughput sequencing technologies, several transcriptomic datasets on IRI of liver transplantation have become available in the Gene Expression Omnibus (GEO) database. Herein, we recruited 3 GEO datasets to conduct comprehensive analysis with the GEO dataset from our center. Moreover, we performed the first proteome of liver tissues to study liver IRI. Then the transcriptome and proteome were used for combined analysis to reveal key factors in liver IRI.

Project description:Background: N-acetylcysteine (NAC), a scavenger of free radicals, possesses strong capability to improve high fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in mice. However, the underlying mechanism(s) of NAC-ameliorated NAFLD are still not fully illustrated. This study was designed to clarify the involvement of long chain non-coding RNAs (lncRNAs) in the beneficial effects of NAC on NAFLD in HFD-induced obese mice. Methods: C57BL/6J mice were arranged into normal fat diet (10% energy from fat), HFD (45% energy from fat), and HFD plus NAC (2 g/L in drinking water) groups, respectively. After 14-week intervention, the role of NAC was determined by immunohistochemistry and biochemical indexes in both plasma and liver. The profiles of hepatic lncRNAs were analyzed by whole transcriptome sequencing, and were further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Meanwhile, the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were carried out to identify the functions of candidate lncRNAs. Furthermore, lncRNA-related ceRNA network was constructed in combination with miRNA and mRNA profiles by Cytoscape software. Results: NAC treatment obviously rescued the deleterious alterations induced by HFD, including body and liver weight, hepatic triglyceride (TG), plasma alanine aminotransferase (ALT), plasma aspartate transaminase (AST), and liver histomorphology (H&E and Oil red O staining). Through whole transcriptome sequencing technology, 52167 (50758 known and 1409 novel) lncRNAs were detectable in liver samples. Our data revealed that the expressions of 175 lncRNAs were significantly changed by HFD but reversed by NAC treatment through cross-comparison. Among those lncRNAs, 5 lncRNAs, including lncRNA-NONMMUT148902.1 (NO_902.1), lncRNA-XR_001781798.1 (XR_798.1), lncRNA-NONMMUT141720.1 (NO_720.1), lncRNA-XR_869907.1 (XR_907.1), and lncRNA-ENSMUST00000132181 (EN_181), were selected based on the absolute value of log2 fold change (FC) being greater than 4, P-value ＜ 0.01, and P-adjust ＜ 0.01. Further qRT-PCR identified that lncRNA-NO_902.1, lncRNA-XR_798.1, and lncRNA-EN_181 were dramatically decreased by HFD while rescued by NAC treatment, consistent with the RNA-sequencing. Finally, we further screened and constructed a ceRNA network, including lncRNA-EN_181, 3 miRNAs, and 13 mRNAs, which was associated with the beneficial role of NAC in NAFLD. Conclusions: This study identified for the first time that lncRNA-EN_181 might be a potential target in NAC-ameliorated NAFLD. The finding enhanced our understanding of biological mechanisms in the beneficial role of NAC.

Project description:Metabolic syndrome is a growing global health problem and is strongly associated with the development of chronic kidney disease (CKD). Recently, the prevalence of CKD has been increasing worldwide, particularly among the older adults. We previously demonstrated that aged kidneys are prone to developing tertiary lymphoid tissues (TLTs) and sustain chronic inflammation after injury; however, the relationship between renal TLT formation and metabolic syndrome is unknown. In the present study, we demonstrated that high-fat diet (HFD) feeding partly promoted TLT formation and chronic inflammation in the kidneys via sterol-O acyltransferase (SOAT) 1-dependent lipid deposition. Wild-type mice fed a HFD prior to ischemic reperfusion injury (IRI) exhibited pronounced TLT formation with respect to size, number and maturity in the kidneys, and sustained chronic inflammation compared to the controls. Untargeted lipidomics revealed that the levels of a selective group of lipids, including cholesteryl esters (CEs), were significantly increased in aged kidneys associated with TLT formation after renal IRI. Consistently, the gene expression level of Soat1 was significantly increased in aged kidneys associated with TLT formation after renal IRI. Notably, treatment with avasimibe, a SOAT inhibitor, significantly attenuated TLT maturation and improved renal inflammation and fibrosis in HFD-fed mice subjected to IRI. Together, our findings suggest the importance of dysregulated lipid metabolism, particularly SOAT1-dependent CE accumulation, in the pathophysiology of CKDs associated with TLT formation. Mice+diet analysis (dataset 2) is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS4994' rel='noopener noreferrer' target='_blank'>MTBLS4994</a>.Mice+injury analysis (dataset 1) is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS4988' rel='noopener noreferrer' target='_blank'>MTBLS4988</a>.

Project description:Metabolic syndrome is a growing global health problem and is strongly associated with the development of chronic kidney disease (CKD). Recently, the prevalence of CKD has been increasing worldwide, particularly among the older adults. We previously demonstrated that aged kidneys are prone to developing tertiary lymphoid tissues (TLTs) and sustain chronic inflammation after injury; however, the relationship between renal TLT formation and metabolic syndrome is unknown. In the present study, we demonstrated that high-fat diet (HFD) feeding partly promoted TLT formation and chronic inflammation in the kidneys via sterol-O acyltransferase (SOAT) 1-dependent lipid deposition. Wild-type mice fed a HFD prior to ischemic reperfusion injury (IRI) exhibited pronounced TLT formation with respect to size, number and maturity in the kidneys, and sustained chronic inflammation compared to the controls. Untargeted lipidomics revealed that the levels of a selective group of lipids, including cholesteryl esters (CEs), were significantly increased in aged kidneys associated with TLT formation after renal IRI. Consistently, the gene expression level of Soat1 was significantly increased in aged kidneys associated with TLT formation after renal IRI. Notably, treatment with avasimibe, a SOAT inhibitor, significantly attenuated TLT maturation and improved renal inflammation and fibrosis in HFD-fed mice subjected to IRI. Together, our findings suggest the importance of dysregulated lipid metabolism, particularly SOAT1-dependent CE accumulation, in the pathophysiology of CKDs associated with TLT formation. Mice+injury analysis (dataset 1) is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS4988' rel='noopener noreferrer' target='_blank'>MTBLS4988</a>.Mice+diet analysis (dataset 2) is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS4994' rel='noopener noreferrer' target='_blank'>MTBLS4994</a>.

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Real-time quantitative PCR analysis of cytokines and chemokines in C57BL/6 mouse liver ischemic-reperfusion injury (IRI) model.

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