The Research Of MiRNA On The Regulation Of The Liver PMNs Apoptosis And Liver Injury After Trauma Hemorrhage In Rats

Background and objective:Trauma hemorrhage is a common clinical pathological process. After injury, cells proximal to the injury were activated to produce large amounts of inflammatory mediators, and tissue PMNs, EC cells were activated. Activated cells secreted more oxygen free radicals and various proteases and inflammatory mediators to intercellular substance, forming a vicious cycle. Liver participate in the inflammatory response after trauma hemorrhage which contains a series of physiological and biochemical reactions, such as macrophage/neutrophil cells/dendritic cells homing, the secretion of inflammatory cytokines and the pathological changes of liver tissue. Previous studies have indicated that miRNA is involved in many biological processes, such as embryonic development, hematopoiesis, glucose and lipid metabolism, cell apoptosis, cancer occurrence, et al. Thus, clarify the pathogenesis of trauma hemorrhage, reduce disease severity after trauma hemorrhage become an urgent issue.Our research was divided into four parts. Firstly, we took SD rats as the object of study and rat trauma hemorrhage model was established by fracture injury with blood loss. IHC study and serum ALT、AST content were used to analyze the liver injury at different time point after trauma hemorrhage. PMNs content was showed through FCM. IL-1β、TNF-α and chemotactic factors were taken as indexes to inducate the liver’s inflammation reaction. Secondly, by bioinformatics prediction and real-time quantitative PCR analysis, miRNA correlated with PMNs apoptosis were screened out and verified. Thirdly, PMNs isolated from liver in rat 16h after trauma hemorrhage was taken as the study object. The expression level of L-selectin/CD18/CD11b was determined by Western blot and PMNs apoptosis was showed by IHC studies. Finally, we researched the protective effect of miRNA on the regulation of the liver PMNs apoptosis after trauma hemorrhage in rats by animal experiment.Methods:In the first part, the methods of HE staining and serum ALT、AST detection at different time points after trauma hemorrhage was taken to compare liver injury in rats. The expression level of inflammatory cytokines in liver was quantified by ELISA kit. Then we analized the content of CD45+ cells in liver at different time points after trauma hemorrhage. In the second part, miRNAs highly correlated to PMNs apoptosis were predicted by bioinformatics and then real time quantitative PCR was taken for further screen. In the third part, the expression level of L-selectin/CD18/CD11b in PMNs isolated from 16h after trauma hemorrhage in model rats and miRNA intervention rats. And PMNs apoptosis rate between the two groups were compared by FCM. In the last part, the protective effect of miRNA on liver injury in rats after traumatic hemorrhage was investigated by IHC, ELISA and FCM methods.Results:The inflammatory cytokines (IL-1β, TNF-α, CC12, et al) levels were significantly increased at the time point of 16h after trauma hemorrhage compared to model level. And liver damaged most seriously at 16h after injury. FCM showed that the number of CD45+ cells was very high in 16,24,48 hours after injury, indicating that there was a strong immune response in the liver at this stage. In the second part, through a large number of bioinformatics prediction and subsequent experimental study, we screened a miRNA that was highly correlated with PMNs apoptosis, miR-126a-5p. The fourth part, rats in the miRNA intervention group were injected with miR-126a-5p before trauma hemorrhage, while the model group was injected by normal saline. IHC showed that liver injured situation in the miRNA intervention group rats were obviously lessened compared to the model group rats. And the expression level of inflammatory cytokines in miRNA group were significantly lower than that in model group.Conclusion:We successfully established the model of trauma hemorrhage rats and ascertain the time point of the severest liver injury. By bioinformatics prediction and Q-PCR study, we screened one miRNA for the first time, miRNA-126a-5p, which was highly correlated to PMNs apoptosis in rats after trauma hemorrhage. The following research confirmed that miR-126a-5p has a protective effect on liver injury induced by trauma in rats.