Neuronal Nitric Oxide Synthase Regulates Lipopolysaccharide-Induced Sepsis Inflammatory Damage In Rat Cardiac H9C2 Cells Through The NF-κB Signaling Pathway

Objective: Sepsis is still a serious problem to be solved in the global public health field,and it is also one of the main causes of death in ICU.Sepsis 3.0 has made a new definition of sepsis: the disorder of patients’ body response to severe infection and the resulting organ dysfunction that is life-threatening.It can be seen that there are two characteristics of sepsis,one is severe infection,and the other is organ dysfunction.Myocardial injury caused by sepsis is an important cause of death.The activation of NF-κB signal pathway plays an important role in inflammatory response.The stimulation of the infection source on the body will activate the transcriptional activity of NF-κB,thereby stimulating the production and continuous amplification of the inflammatory response,and causing the inflammatory response to lose control and spread throughout the body,leading to dysfunction and failure of the entire body.But at present,the mechanism of sepsis has been explored continuously,and the specific mechanism is still not completely clear.Nitric oxide synthase(NOS)is a general term of isozymes that widely exist in cells.There are mainly three types: neural type,inducible type and endothelial type.At present,studies have shown that inducibleNOS(iNOS,NOS2)and endothelialNOS(eNOS,NOS3)play a role in inflammatory reaction,but the role of neuralNOS(nNOS,NOS1)in inflammatory reaction is still unclear.This study is to explore the expression of nNOS in the inflammatory model of rat cardiac H9C2 cells,and explore whether it is through NF-κB signal pathway is involved in the regulation of cellular inflammatory response.The above studies were carried out at the cellular level in order to clarify the correlation between nNOS and septic inflammatory reaction and sepsis-related myocardial injury,and provide new direction,theoretical support and research basis for the diagnosis and treatment of sepsis.Methods: In this study,we first used lipopolysaccharide(LPS)to stimulate human umbilical vein endothelial cells(HUVEC)to build a classic cellular inflammatory model.Compared with normal cells,we extracted their RNA and used high-flux transcriptome sequencing(RNA-seq)technology to detect the difference in the expression of nNOS and the possible pathway and pathway of action.After that,the rat cardiac H9C2 cells were stimulated by different concentrations of LPS to explore the construction of myocardial cell inflammation model,and the expression level of inflammatory factors in the supernatant,such as TNF-α、IL-6 and LDH,was detected to evaluate the intensity of inflammatory reaction and detected the expression level of nNOS and NF-κB p65 in myocardial cell inflammation model.Finally,the cells were pretreated with 7-Nitroindazole(7-NI),a selective inhibitor of nNOS,and then an inflammatory reaction model was established to observe the level of inflammatory factors.At the same time,we observed the effect of selective inhibition of nNOS on NF-κB p65 expression in the myocardial cell inflammation model to confirm our hypothesis,that is,nNOS is highly expressed in inflammation,and inhibition of its expression can reduce the inflammatory response of cells.At the same time,nNOS regulates the intensity of cellular inflammatory response through NF-κB signal pathway.Results: RNA-seq results showed that there were 103 differentially expressed genes between LPS group and Control group,of which 84 genes were up-regulated and 19 genes were down-regulated.nNOS was highly expressed in inflammatory reaction,and the gene signal pathway was enriched with NF-κB signal pathway.The inflammation model was constructed by stimulating H9C2 cells with high(40μg/ml),medium(20μg/ml),and low(10μg/ml)concentrations of LPS.The results showed that the high dose LPS group(H group)had the highest expression levels of TNF-α,IL-6,and LDH,followed by the medium dose LPS group(M group)and had statistically significant levels of inflammatory factors compared to C group.There was no significant difference in IL-6,LDH levels between the low dose LPS group(L group)and C group.The expression levels of nNOS and NF-κB p65 protein in the H group and the M group were significantly higher than those in the normal group,but the two protein levels in the H group increased more significantly.After that,by using 7-NI pretreated cells to inhibit the expression of nNOS,a cellular inflammation model was constructed again.It was found that with the decrease of the expression of nNOS,the expression level of NF-κB p65 also decreased,and the level of inflammatory factors in cells also decreased significantly compared with that in inflammatory cells.Conclusion: In this study,RNA-seq technology was used to find the genes with different expression between inflammatory cells and normal cells,nNOS was highly expressed in inflammatory reaction,and the model of myocardial cell inflammatory reaction was successfully constructed.This study also confirmed that nNOS is also highly expressed in the rat myocardial inflammation model,and inhibiting the expression level of nNOS can reduce the activation of NF-κB p65 and the release level of inflammatory factors,so as to reduce the inflammatory response of cells.These findings expand the potential mechanism of myocardial cell damage caused by sepsis by clarifying the role of nNOS in inflammation and related mechanisms,provide new ideas and directions for the treatment of sepsis,and provide experimental basis and theoretical support for this.