| BackgroundSepsis is a systemic inflammatory response syndrome(SIRS)caused by a variety of pathogenic microorganisms and endotoxin release and it is also one of serious complications for acute and severe patients such as trauma and shock(1).HMGB-1plays a key role in the occurrence and progression of sepsis,which is mainly produced by secretion and release of large amount of immune cells such as in vivo mononuclear cells,Dendritic cells,macrophages stimulated by endotoxin,inflammatory cytokines,etc.(2).It is one of the most important inflammatory mediators for the lethal effect of sepsis(3).The interaction of HMGB-1 with other inflammatory mediators plays an important role in mediating the signaling pathways of the inflammatory response,inducing the immune response and amplifying the inflammatory response(4).Studies have shown that HMGB-1 could promote the migration of macrophages and the release of various inflammatory cytokines,which caused aggregation of a variety of immune cells,playing a role in inducing inflammatory responses of sepsis(3).Studies have shown that the elevated expression of HMGB1 was closely associated with the occurrence of sepsis(5).micro RNAs are highly conserved endogenous non-coding small RNAs in eukaryotes,which can regulate the expression of target genes by complete or incomplete complementary pairing with the 3'-untranslated region(3'-UTR)of the m RNA,playing an important role in immune cell activation,inflammatory cytokine release and immune response(6-7).More and more studies have revealed that micro RNA was involved in the occurrence and progression of sepsis and would become a diagnostic marker of sepsis(8-9),and the mi R-25 expression in peripheral blood in patients with sepsis was abnormal,suggested that mi R-25 abnormalities may be related to the pathogenesis of sepsis(1-2).Bioinformatic analysis revealed that a target binding site existed between mi R-25 and HMGB1 3'-UTR.However,what is the specific role of mi R-25 in the regulation of HMGB1? It is unclear how the biological effects caused by abnormal expression in sepsis.ObjectiveThe expression of mi R-25 and HMGB1 in patients with sepsis was analyzed,the relationship between mi R-25,HMGB1 and sepsis was described,and the role of mi R-25 in regulating HMGB1 expression and the release of macrophage immune factors was explored,so as to find new targets for the diagnosis and treatment of sepsis in clinical practice and provide new molecular markers for the evaluation of patients' prognosis.MethodsIn this study,we first used macrophage induced by LPS for mi RNA microarrays,and the results were clustered to identify the differentially expressed mi RNA and predict the target mi RNA-25.Secondly,LPS induced macrophages were used to detect the differences in the expression of mi RNA-25,early inflammatory cytokine TNF-?,Il-6,and late inflammatory cytokine HMGB1 in macrophages,and the relationship between mi R-25,HMGB1 and sepsis was analyzed using the mouse model,as well as the effect on the survival rate of mice.Finally,by analyzing the expression of mi R-25 and HMGB1 in sepsis patients,we investigated the role of mi R-25 in regulating HMGB1 expression and the release of macrophage immune factors.ResultsIn the study of the mechanism of mi R-25 inhibiting the secretion of inflammatory cytokines in macrophages of sepsis,we found: First,mi R-25 can target and inhibit HMGB1 expression.The results of micro RNA.org online targeted gene prediction indicated that 3'-UTR of HMGB1 m RNA had a mi R-25 target binding site.The expression of mi R-25 mimic significantly decreased luciferase activity,suggesting that mi R-25 can target the 3'-UTR binding to HMGB1,thereby inhibiting the expression of HMGB1.Secondly,LPS can induce HMGB1 secretion in macrophages and inhibit mi R-25 expression.Flow pattern results showed that after 7 days of induction by m-CSF,the expression of CD68,a protein molecule marker specific to macrophages,was increased by 80%,indicating that the differentiation of macrophages was successfully induced,which can be further tested.Elisa test showed that the expression of the immune factors HMGB1,TNF-?,and Il-6 in the supernatant was significantly increased after the cells were treated with 100ng/m L LPS for 48 h.The results of q RT-PCR showed that the macrophages treated with LPS can significantly up-regulate HMGB1 m RNA expression and down-regulate mi R-25 expression.Western blot results showed that LPS treatment significantly enhanced the transcriptional activity of NF-?B and upregulated HMGB1 expression in macrophages.Finally,mi R-25 inhibits HMGB1 expression and cell migration in macrophages.After the cells were transfected into mi R-25 mimic or si-HMGB1,the m RNA and protein levels of HMGB1 in macrophages were significantly reduced,which also led to a significant decrease in the level of p-p65.In addition,the levels of HMGB1,TNF-?,and Il-6 in the supernatant solution were significantly down-regulated,and the migration ability of macrophages was significantly inhibited.This suggests that mi R-25 can inhibit the expression of cytoimmune factors induced by sepsis by inhibiting HMGB1 expression.(2)In the study of using the mouse model to show that mi R-25 mimics can regulation HMGB1,control group were injected with same volume of physiological saline,LPS group were injected with saline solution for three consecutive days,then were injected with LPS(dose of 10 mg/kg),LPS + mi R-25 mimics group were injected with mi R-25mimics(mi R-25 agonists,30 mg/kg/day)by tail vein injection,The expressions of mi R-25 and HMGB1 in different tissues and serum of mice were detected,the levels of cytokines TNF-?,Il-6 were measured in the serum of mice,and the death of sepsis mice in different groups were counted.We found that the expression of mi R-25 in serum,lung,liver,spleen,heart and kidney was detected by q RT-PCR.The results showed that the expression of mi R-25 in serum and different tissues of mice in the LPS group was significantly lower than that of the control group,and the difference between the two groups was statistically significant(p < 0.05).However,after the mice were injected with mi R-25 mimics,the expression of mi R-25 increased and returned to the normal level,and the difference was not statistically significant(p > 0.05).The expression of HMGB1 in serum,lung,liver,spleen,heart and kidney in different groups of mice was detected by q RT-PCR.The results showed that the expression of mi R-25 in serum and different tissues of mice in the LPS group was significantly higher than that of the control group,and the difference between the two groups was statistically significant(p< 0.05).After the injection of mi R-25 mimics,HMGB1 expression decreased and returned to the normal level,but the difference was not statistically significant(p >0.05).The expression of TNF-?and IL-6 in serum of mice in different groups was detected by ELISA.Results show that the LPS group mice serum levels of cytokines in serum TNF-?,the expression of IL-6,significantly higher than the control group,the differences between the two groups have statistical significance(p < 0.05).The mice after injection of mi R-25 mimics,cytokines in serum TNF-?,decreased the expression of IL-6,return to normal levels,with no statistically significant difference in the control group(p > 0.05).After injecting normal saline and mir-25 mimics48 h into mouse tail vein,mice sepsis was induced by injecting LPS into tail vein and survival rate was observed.The results showed that compared with the control group,the survival rate of mice after the injection of mi R-25 mimics was significantly increased(55.88%vs20.00%),and the improvement of the mortality rate of mice with sepsis by mir-255 overexpression was gradually obvious four days after LPS induction.This suggests that mirna-25 inhibits HMGB1 expression in LPS-induced sepsis mice,and further inhibits the expression of cytoimmune factors TNF-and il-6 induced by sepsis.(3)When we tested HMGB1 and mi R-25 in serum of patients with sepsis,we found that the level of HMGB1 in peripheral blood serum of patients with sepsis was significantly higher than that of the control group,and the difference between the two groups was statistically significant(p < 0.05).Western blot results showed that HMGB1 expression in PBMC of sepsis patients was significantly higher than that of the control group,and the difference between the two groups was statistically significant(p < 0.05).The results of qrt-pcr showed that compared with the healthy control group,the expression of mir-25 in peripheral blood serum and PBMC of patients with sepsis was significantly lower than that of the control group,and the difference between the two groups was statistically significant(p < 0.05).Pearson correlation analysis showed that the expression of mir-25 in serum of patients with sepsis was negatively correlated with the content of HMGB1(r =-0.591,P = 0.041).For patients with sepsis,the serum HMGB1 level was positively correlated with HMGB1 expression in PBMC(r = 0.537,P <0.001),and the serum mir-25 level was negatively correlated with HMGB1 level(r =-0.622,P<0.001).ConclusionsThe downregulation of mi R-25 and up-regulation of HMGB1 are associated with sepsis.mi R-25 can attenuate the LPS-induced HMGB1 expression and inhibit NF-?B and downstream immune factors TNF-? and IL-6.Therefore,mi R-25 can be used as a molecular marker for the diagnosis of sepsis. |
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