Regulation Of LncRNA,miRNA And Related Signal Axis In Sepsis And Trauma-Related Inflammation

BackgroundSepsis mainly refers to a common clinical syndrome caused by the host's unbalanced response to infection,which can be secondary to clinical trauma such as major trauma,surgery,burns,and severe infections,with high morbidity and mortality.Even with modern new antibiotics and new treatments,the fatality rate of septic shock and multiple organ dysfunction syndrome(MODS)is still higher than 50%.The pathogenesis of sepsis is complex and involves multiple organs in the body.At present,the theory of uncontrolled inflammatory response is considered to be an important basis for the pathogenesis of sepsis,so the imbalance of inflammatory response has been the focus of research on the mechanism of sepsis development.Trauma is one of the most serious global public health problems,accounting for 10% of the global burden of disease.Imbalance of anti-inflammatory and pro-inflammatory balance caused by trauma can cause substantial damage to organs and organs on the one hand,and increase the incidence of sepsis and related complications on the other.Therefore,how to effectively prevent related complications such as sepsis and MODS is the core of improving the level of trauma treatment.However,no effective drugs or clear treatments have been found for sepsis.Therefore,early warning and early diagnosis of sepsis are particularly important,especially after trauma.This has made the exploration of related potential biomarkers a hotspot of research.So far,common C-reactive protein(CRP),procalcitonin(PCT),and interleukin(IL)have been used in the diagnosis of sepsis,but each has its own advantages and disadvantages in specific judgments.In recent years,more and more researchers have paid attention to non-coding RNAs that play an important role in major biological processes such as innate immunity and apoptosis.Studies have found that lnc RNA is closely related to the development of certain complex diseases(sepsis,cancer,metabolic disorders and cardiovascular diseases,etc.).In terms of sepsis,the expression level of lnc RNA NEAT1(hereinafter referred to as: NEAT1)in patients with sepsis has been confirmed to be significantly higher than that in healthy people,suggesting that NEAT1 may be a potential biomarker for sepsis and may be used in sepsis.It plays a regulatory role in the degree of inflammatory response during the occurrence and development.In addition,as a current research hotspot of gene expression regulation,lnc RNA can be used as a competitive endogenous RNA,combined with mi RNA,and acts as a mi RNA sponge in cells,thereby inhibiting mi RNA expression and indirectly up-regulating the expression of mi RNA-related target genes.At the same time,mi RNAs are considered to be potential biomarkers for the diagnosis and prognosis of certain diseases due to their disease specificity,relative stability,and easy accessibility.In this study,ENCORI online software was used to provide many mi RNAs that may be played by NEAT1,some of which are related to inflammation: mi R495-3p and mi R-211.Therefore,it is proposed that mi R495-3p and mi R-211 may play a role in the regulation of inflammatory response caused by sepsis,but whether NEAT1,mi R495-3p and mi R-211 are the main participants in sepsis and even trauma-related inflammation,still needs Detailed inquiry and verification.PurposesThis study aims to explain the association of lnc RNA NEAT1,mi R-495-3p,and mi R-211 with sepsis / trauma from the perspective of inflammation.By constructing a mouse model of trauma and sepsis,the survival and expression of pro-inflammatory factors were observed and their correlation with NEAT1,mi R-495-3p and mi R-211 was analyzed.Further explore the regulatory mechanism of NEAT1,mi R-495-3p and mi R-211 in the inflammatory response through an inflammatory cell model,and elucidate the mechanism of action of NEAT1,mi R-495-3p,mi R-211 and related signal axes,for sepsis and The diagnosis and treatment of trauma offers new possibilities.MethodsPart ? Effects of NEAT1,mi R-495-3p and mi R-211 gene expression levels on survival and expression of pro-inflammatory factors in a sepsis / trauma mouse modelBy constructing mice models in the sham operation group(SHAM),sepsis group(LPS),trauma group(TH),and trauma + sepsis group(TH+LPS),the survival of the mice was tracked and recorded,and the survival curve was drawn.At the same time,the serum,liver,spleen and lung tissues of mice were collected,and the liver function of mice was quantified using an automatic biochemical analyzer.The inflammatory cytokines in serum and liver tissue of mice were measured by enzyme-linked immunosorbent assay(ELISA).Evaluating the proliferation of spleen cells by MTT assay and extracted RNA from mouse lung tissues,RT-PCR was used to detect the expression of NEAT1,mi R-495-3p and mi R-211 and to analyze its correlation with mice survival rate.Finally,the method of bioinformatics analysis was used to input the above experimental results into R software to generate a correlation matrix.Part ? Role of NEAT1,mi R-495-3p and mi R-211 in inflammatory cell modelsThe inflammatory response model of monocyte macrophages was constructed by LPS,and the inflammation-related factors(p-PI3 K,PI3K,p-AKT,AKT,TNF-?,IL-8,IL-,IL-10 and MCP-1).RT-PCR was used to detect the expression of NEAT1,mi R-495-3p and mi R-211.In order to further verify the related relationship of genes,the target gene was transfected with plasmids based on cell transient technology and the targeted regulatory relationship between NEAT1 and mi R-495-3p / mi R-211 was analyzed by the luciferase reporter gene.Part ? Regulation of NEAT1 on mi R-495-3p and mi R-211 Related Signal AxisIn RAW264.7 monocyte macrophages,Western blotting was used to detect the expression of STAT3 protein in the case of NEAT1,mi R-495-3p,and STAT3 overexpression,and RT-PCR was used to detect the expression of STAT3 in NEAT1 and mi R-495-3p gene expression,and the targeting relationship between mi R-495-3p and STAT3 was detected using luciferase reporter gene technology.In the verification experiments of the signal pathway,Western blotting technology was also used to detect the expression of PI3K/AKT,and RT-PCR technology was used to detect the expression of mi R-211.ResultsPart ? Effects of NEAT1,mi R-495-3p and mi R-211 gene expression levels on survival and expression of pro-inflammatory factors in a sepsis / trauma mouse model1.Survival rate of mouse model caused by trauma and sepsis: Statistics showed that no death occurred in mice in the SHAM group,2(6.67%)deaths in the TH group,and 4(13.33%)in the LPS group.)Death,22(73.33%)of the TH + LPS group died,suggesting that sepsis caused increased mortality in mice,and trauma would further increase mortality in sepsis mouse models.2.Trauma and LPS promoted inflammatory factor expression in related mice models: Compared with the SHAM group,the serum levels of TNF-?,IL-6 and IL-1? were increased in TH and LPS mice,while the level of the mixed intervention group was the highest in TH+LPS mice(P <0.05).The same trends also appeared in the detection of inflammatory factors in liver tissues and indicators of liver function damage.Further analysis,compared with the SHAM group,the spleen cell activity of the TH group and the LPS group was significantly reduced compared with the SHAM group.The TH group and the LPS group released less IFN-? and IL-2 than the SHAM group,While the TH + LPS group released less spleen cells than the TH or LPS group(P <0.05).Based on these results,this study speculated that both trauma and sepsis promoted the production of inflammatory factors,and that post-traumatic sepsis may cause more pro-inflammatory factors in mice models than single intervention with trauma or sepsis.In thus,the inflammation was more severe.3.Correlation between NEAT1,mi R-495-3p and mi R-211 gene expression levels and survival of trauma and sepsis mice models: NEAT1 content in TH and LPS groups was higher than that in SHAM group,and TH + LPS treated mice showed higher NEAT1 levels(P <0.05).Meanwhile,in terms of mi R-495-3p and mi R-211 content,the NEAT1 content of TH and LPS mice was lower than that of SHAM group,and TH + LPS treated mice showed lower mi R-495-3p and mi R-211 levels(P <0.05).In terms of survival relationship analysis,the survival rate of mice carrying high levels of NEAT1 was worse than that of mice carrying low levels of NEAT1,while the survival rates of low-level mi R-495-3p and mi R-211 mice were relatively low.The results showed that detecting the gene levels of NEAT1,mi R-495-3p and mi R-211 may be meaningful for predicting the survival status of mice models.4.Correlation between NEAT1,mi R-495-3p and mi R-211 gene expression levels and levels of pro-inflammatory factors produced in mouse models of trauma and sepsis: NEAT1,mi R-495-3p,mi R-211,sepsis There is a correlation between the inflammatory cytokines(ie,TNF-?,IL-6,IL-10 and MCP-1)characteristic of the disease.p-PI3 K,p-AKT and STAT3 were not obvious in the mice model of the SHAM group.In the TH group and the LPS group,the correlation became stronger,and the correlation between the above molecules was most obvious in the LPS+TH group.In the mixed group,NEAT1 was positively correlated with inflammatory cytokines,while,mi R-495-3p,mi R-211 and inflammatory cytokines were negatively correlated.It was further confirmed that the levels of NEAT1,mi R-495-3p and mi R-211 can reflect the feasibility of inflammatory response in mouse models.Part ? Role of NEAT1,mi R-495-3p and mi R-211 in inflammatory cell models1.Expression of NEAT1,mi R-495-3p and mi R-211 and related inflammatory factors in cell models: up-regulated NEAT1 levels and down-regulated mi R-495-3p and mi R-211 levels were detected in inflammatory cell models(P <0.05),high levels of pro-inflammatory factors such as TNF-?,IL-6,IL-10 and inflammatory chemokines such as p-PI3 K and p-AKT were also found.2.Exploration of the correlation between LPS and NEAT1: Both the LPS group and the NEAT1(+)group can stimulate the increase of NEAT1 expression,and the expression level is higher after co-stimulation.3.Effect of NEAT1 on mi R-495-3p and mi R-211 expression in inflammatory cell models: The expression of mi R-495-3p in LPS group and NEAT1(+)group was lower than that in control group,but the expression of LPS + NEAT1(-)group The amount increased,and the same trend was also expressed in mi R-211 expression.It was shown that both LPS and NEAT1 can cause the expression of mi R-495-3p and mi R-211 to decrease,but NEAT1 may affect the expression of mi R-495-3p and mi R-211 more than LPS.4.Effect of mi R-495-3p and mi R-211 on NEAT1 expression in inflammation models: There was no difference in the expression of NEAT1 between mi R-495-3p group,mi R-211 group and control group,suggesting mi R-495-3p and mi R-211 had no effect on NEAT1 expression.5.Effect of NEAT1 on cytokines in RAW264.7 cells: The overall trend was similar.It was found that increasing NEAT1 levels led to cytokines(p-PI3 K,p-AKT,STAT3).TNF-?,IL-6,IL-10,and MCP-1)increased,and the cytokine expression produced by the dual intervention of pc DNA3.1-NEAT1 and LPS was the most significant(P <0.05).In contrast,silencing NEAT1 seemed to reverse the cytokine production promotion effect of LPS(P <0.05).6.Effects of mi R-495-3p and mi R-211 on cytokines in RAW264.7 cells: Increasing the levels of mi R-495-3p and mi R-211 leads to p-PI3 K,p-AKT,STAT3,TNF-? Low expression of IL-6,IL-10,and MCP-1,and high p-PI3 K,p-AKT,STAT3,TNF-? found in mi R-495-3p(-)and mi R-211(-),IL-6 and IL-10 were highly expressed(P <0.05).Combined with the above results,NEAT1,mi R-495-3p and mi R-211 can effectively affect the related responses in inflammatory cell models treated by LPS.7.Effect of NEAT1 as a sponge on mi R-495-3p / mi R-211 expression The transfection of pc DNA3.1-NEAT1 significantly reduced the levels of mi R-211 and mi R-495-3p,while silencing NEAT1 significantly increased mi R-211 and mi R-495-3p levels(P<0.05).However,when the levels of mi R-211 and mi R-495-3p increased or decreased,the levels of NEAT1 rarely changed(P <0.05).Therefore,there is a targeted regulatory relationship between NEAT1,mi R-495-3p and mi R-211 in RAW264.7 cells.Part ? Regulation of NEAT1 on mi R-495-3p and mi R-211 Related Signal Axis1.NEAT1 regulates mi R-495-3p / STAT3 signal axis: STAT3 expression was significantly increased in the LPS-induced inflammatory cell model(P <0.05).Treatment of macrophages with pc DNA3.1-NEAT1 and mi R-495-3p inhibitors also significantly improved the expression of STAT3 in vitro.Conversely,STAT3 expression decreased after intervention with si-NEAT1 and mi R-495-3p mimic.In reverse verification,the expression of STAT3 in the macrophages of the pc DNA-STAT3 group increased,but decreased under the action of si-STAT3(P <0.05).However,when transfected with pc DNA-STAT3 or si-STAT3,the gene levels of NEAT1 or mi R-495-3p were relatively stable(P> 0.05),suggesting that STAT3 cannot reversely regulate NEAT1.Through the luciferase reporting experiment,it was found that mi R-495-3p mimic3 + p GL3-STAT3-WT transfected with p GL3-STAT3-MUT + mi R-495-3p mimic3 group compared with p GL3-STAT3-WT + mi R-NC group.The luciferase activity of macrophages was weakened(P<0.05).These results suggest that NEAT1 may regulate the inflammatory response through the mi R-495-3p / STAT3 signaling axis.2.NEAT1 regulates mi R-211/PI3K/AKT signal axis: transfection of si-NEAT1 and mi R-211 mimics can reduce PI3K/AKT expression in macrophages,while the expression of p-PI3 K and p-AKT was enhanced in pc DNA3.1-NEAT1 and mi R-211mimics-treated macrophages(P<0.05).Further,although intervention with PI3K/AKT inhibitors could reduce PI3K/AKT expression,it failed to change the gene levels of NEAT1 and mi R-211(P>0.05).In addition,PI3K/AKT inhibitors could reverse the effect of pc DNA3.1-NEAT1 on the expression capacity of p-PI3 K,p-AKT,TNF-?,IL-6,IL-10 and MCP-1(P<0.05).After the PI3K/AKT activator was added,mi R-211mimic's promotion of TNF-?,IL-6,IL-10 and MCP-1 expression was also blocked(P<0.05).Therefore,this suggests that PI3K/AKT signaling mediates the effects of NEAT1 and mi R-211 on the inflammatory response in RAW264.7 cells.ConclusionIn summary,through animal models and cell models,this study explored the important role of inflammatory response in sepsis and trauma preliminarily,and verified that NEAT1 can modify the mi R-495-3p/STAT3 axis and mi R-211/PI3K/AKT axis to change the expression of characteristic cytokines in sepsis and enriches the regulatory mechanism of non-coding RNA in the inflammatory response.