Effect Of MCPIP1/SIRT1/NF-?B Signaling Pathway On Sepsis-mediated Organ Damage And Inflammatory Responses

Backgroud Sepsis is a systemic inflammatory disease caused by infectious factors and is a common complication of clinical burn wounds and infectious diseases.Sepsis can lead to multiple organ dysfunction syndromes and septic shock which ultimately leads to death.Epidemiological surveys show that there are more than 19 million severe sepsis patients worldwide each year,which exceeds the incidence of common diseases such as stroke,lung cancer,breast cancer and heart disease.Although the is higher than these common diseases,the research investment in sepsis is the least.Not only the incidence of sepsis is high,but its mortality rate is also high.The survey showed that the mortality rate of severe sepsis is nearly 20%,and the mortality rate of septic shock is as high as 45%.However,unfortunately,the fundamental mechanism of the pathogenesis of sepsis has not yet been elucidated,so the treatment has not yet made a fundamental breakthrough.Therefore,it is urgent to study the pathogenesis of sepsis.The essence of sepsis is due to the uncontrolled inflammatory response of the body,the excessive activation of inflammatory cells and the imbalance of proinflammatory and anti-inflammatory factors,resulting in an uncontrolled inflammatory response.The damage and activation of macrophages is the core of the uncontrolled inflammatory response in the body.The secretion of a large number of inflammatory factors and other inflammatory mediators can cause a cascade of inflammation,leading to uncontrolled inflammatory response and multiple organ dysfunction.The sustained activation of the NF-?B signaling pathway in macrophages is an important molecular basis for the secretion of excessive inflammatory factors and inflammatory mediators by macrophages after injury.Under normal conditions,NF-?B is located in the cytoplasm and binds to its natural inhibitor I?B and cannot enter the nucleus.However,when the cells are stimulated by endogenous or exogenous risk factors,I?B can be degraded,NF-?B can be nucleated,and the target gene can be activated to produce a large amount of inflammatory mediators,and the gene product will further activate NF-?B.Therefore,inhibition of the activation of NF-?B signaling pathway is the key to alleviate the imbalance of inflammation.For sepsis,regulation of NF-?B signaling in macrophages is an important target for reducing uncontrolled inflammatory responses,increasing sepsis survival,and improving multiple organ function.MCPIP1 is a zinc finger protein family molecule with immunoregulatory function.Since its first discovery in 2006,its role in inflammatory response has received more and more attention.MCPIP1 is a RNase enzyme and deubiquitinating enzyme.Previous studies have focused on MCPIP1 as an RNase enzyme to degrade mRNA to inhibit inflammatory response and MCPIP1 as a deubiquitinating enzyme to deubiquitinate NF-?B signaling pathway.However,there are few studies on its degradation of miRNA as an RNase enzyme.Through literature review,we found that direct target miRNAs of MCPIP1 include miR-9,miR-20 b,miR-21,miR-32-5p,miR-34 a,miR-135 b,miR-143,miR-145,miR-146 a,miR-155,miR-200 and so on.miRNAs are a class of single-stranded RNA molecules encoded by endogenous genes.miRNAs are often highly conserved and tissue-specific,and can regulate target genes through transcription or post-transcriptional levels,thereby affecting target gene expression.The most common regulation of miRNAs is to form a complete complement by binding to the 3'UTR region of the targeted gene,thereby inducing degradation of the target gene mRNA,or incompletely binding to the 3'UTR region of the targeted gene,thereby inhibiting the protein translation process of the targeted gene.Then,in the regulation of macrophage inflammation,how does MCPIP1 play a role in the development of sepsis? Are there other possible mechanisms by which MCPIP1 regulates the inflammatory response?Purpose(1)To explore the effects of MCPIP1 on LPS stimulated inflammatory responses and signaling pathways in in vivo and in vitro models.(2)To analyze the role of SIRT1 in the regulation of LPS stimulated inflammatory response and signaling pathway by MCPIP1.(3)Explore the potential mechanism of MCPIP1 in regulation of SIRT1.Methods and Results(1)In the septic mouse model of intraperitoneal injection of LPS,using RT-PCR,enzyme-linked immunosorbent assay,pathological section and other tests,we found that intraperitoneal injection of LPS for 6 hours can cause obvious pathological damage of liver,lung,kidney in mice,while the release of inflammatory factors in the serum of mice,liver and kidney function indicators significantly increased,confirmed that our mouse model of sepsis was successfully established;In the LPS stimulated mouse bone marrow-derived macrophages and mice RAW 264.7 cells,using RT-PCR and other experiments,we found that inflammatory factors IL-1?,IL-6,TNF? and MCP-1 in macrophages are significantly increased with LPS stimulation,and peaked at 2-6 hours after LPS stimulation,confirming the successful establishment of our sepsis cell model.(2)In the septic cell model,the expression of MCPIP1 was detected by RT-PCR and Western blotting,and it was found that MCPIP1 was significantly increased after LPS stimulation,and peaked at 2 hours after LPS stimulation.The trend was basically the same with inflammatory factors,confirming that MCPIP1 is involved in the process of LPS induced inflammatory response.(3)In the cell model,MCPIP1 overexpression plasmid and small interfering RNA were constructed to detect the effect of MCPIP1 on LPS induced inflammatory response and signaling pathway.It was found that overexpression of MCPIP1 significantly inhibited inflammatory response,and knockdown of MCPIP1 significantly promoted inflammatory response.Overexpression of MCPIP1 significantly inhibited the nuclear translocation of NF-?B p65,while knockdown of MCPIP1 significantly promoted the nuclear translocation of NF-?B p65;in the mouse model of sepsis,the MCPIP1 overexpression plasmid was injected into the tail vein.Using RT-PCR,enzyme-linked immunosorbent assay,pathological section and other tests,it can be seen that overexpression of MCPIP1 can significantly improve the survival rate of septic mice,reduce the release of inflammatory factors in the serum of septic mice,reduce the expression of inflammatory factors in mice liver and improves the liver pathological damage and liver damage in septic mice.(4)In the cell model,the mechanism of MCPIP1 regulation of inflammatory response was explored by nuclear pulp separation assay,RT-PCR and Western blotting.It was found that overexpression of MCPIP1 significantly inhibited the acetylation level of NF-?B p65,while knockdown of MCPIP1 significantly promoted the acetylation level of NF-?B p65.At the same time,overexpression of MCPIP1 significantly promoted the expression of SIRT1,whereas knockdown of MCPIP1 significantly inhibited the expression of SIRT1.While overexpression of SIRT1 could alleviate the MCPIP1-mediated inflammatory response and the change of acetylation of NF-?B p65.(5)In the cell model,luciferase reporter gene assay,RT-PCR and Western blotting were used to explore the mechanism of MCPIP1 in regulation of SIRT1.We found that microRNA-9(miR-9)can directly bind to the 3'UTR region of SIRT1 promoter directly to regulate the expression of SIRT1,and MCPIP1 can negatively regulate the synthesis of miR-9.While overexpression of miR-9 alleviates the decrease of inflammatory response and the increase of SIRT1 due to overexpression of MCPIP1.(6)In the cell model,the mechanism of MCPIP1 in regulation of SIRT1 was explored by RT-PCR and Western blotting.It was found that microRNA-155(miR-155)can negatively regulate the expression of SIRT1,while MCPIP1 can negatively regulate the synthesis of miR-155.At the same time,overexpressing miR-155 alleviated the decrease in inflammatory response and the increase in SIRT1 due to overexpression of MCPIP1.Conclusion MCPIP1 might play an important role in LPS-induced inflammatory response of sepsis.MCPIP1 could promote the expression of SIRT1,and then inhibit the acetylation level of NF-?B p65 to block NF-?B p65 entry into the nucleus,thereby negatively regulating LPS-induced inflammatory response of sepsis;miR-9 could directly bind to the 3'UTR region of SIRT1 promoter,and then participate in the regulation of SIRT1 by MCPIP1;miR-155 could negatively regulate SIRT1,and then participate in the regulation of SIRT1 by MCPIP1.