A Study On MicroRNA-23b Regulates Inflammatory Factor Expression On Vascular Endothelial Cells Of Sepsis And Peripheral Blood MicroRNA-23b As Sepsis-molecular Marker

Sepsis is a heterogeneous syndrome with variable physiological manifestations in patients. One of the biggest challenges in sepsis is to develop a more detailed understanding of the underlying pathophysiological mechanism. Numerous studies have found an association between endothelial cell activation and sepsis and it is becoming evident that the endothelium plays a key role in sepsis. The endothelial activation in sepsis is associated with changes in hemostatic balance, leukocyte trafficking, vascular permeability, inflammation and microcirculatory flow. Although these changes is a part of the adaptive host response to infection, they may become excessive resulting in the dysfunctional phenotype that a hallmark of sepsis.In a previous single-center study, we found compelling evidence that sepsis in humans is associated with activation of the endothelium as evidenced by increased levels of circulating endothelial biomarkers. We also found an association between these markers and severity of illness and organ dysfunction in sepsis. Given the central role of the endothelium in sepsis pathophysiology, such findings may not only improve our understanding of a component of the pathophysiology in sepsis but may also suggest targets for diagnostic platforms and specific therapeutic interventions.Emerging of mi RNA provides us a new way insight into the mechanism underlining endothelium’s role in sepsis. This article included two parts as follows:PartⅠ:Micro RNA-23 b regulates inflammatory factor expression on vascular endothelial cells of sepsisMi R-23 b is a multi-functional mi RNA which contributes to regulate multiple signal pathways. It was reported that mi R-23 b prevents many autoimmune diseases through regulating inflammatory cytokine pathways.However,its mechanism kept unkown.In the present study,the mir-23b inhibitor and mimics were transfected into human vascular endothelial cells(HUVECs)to change the expression of mir-23b individually.The negative controls(NC)was used,too.Results1 Transfection of mi R23 b Inhibitor inhibited mi R23 b expression and transfection of mi R23 b mimics increased mi R23 b expression by HUVECsHUVECs transfected with mi R23 b emitted green fluorescence under fluorescence microscopy. Quantitative PCR results indicated that mi R23 b expression decreased significantly in the group transfected with mi R23 b inhibitor sequence when compared to the blank control group and the group transfected with inhibitor NC sequence, demonstrating that transfection of mi R23 b inhibitor inhibited mi R23 b expression effectively. In contrast, mi R23 b expression increased significantly in the group transfected with mi R23 b mimics when compared to the blank group and the mimic NC group, demonstrating that transfection of mi R23 b mimic promoted mi R23 b expression.2 LPS downregulated mir-23 b expression by HUVECsVECs in sepsis were simulated by LPS stimulated HUVECs. The results showed that mir-23 b expression decreased significantly in cells transfected with mimic NC or inhibitor NC at 4h or 8h after LPS stimulation when compared to cells not stimulated with LPS(P<0.01). The results demonstrate that mir-23 b expression decreased in LPS stimulated HUVECs, that is, VECs activation in sepsis was accompanied by inhibition of mir-23 b expression. In contrast, mir-23 b expression decreased slightly in cells transfected with mimic sequence at 4h or 8h after LPS stimulation when compared to cells not stimulated with LPS, but remained still at high levels. mir-23 b expression remained low in cells transfected with Inhibitor sequence after LPS stimulation.3 LPS promoted inflammatory cytokine expression by HUVECsInflammatory cytokines NF-κB, TNF-α, IL-6, ICAM-1, selectin E, and VCAM-1 increased significantly at 4h and 8h after LPS stimulation in HUVECs(P<0.05), demonstrating that LPS stimulated HUVECs to express inflammatory cytokines and thus contributed to inflammatory reactions in sepsis.4 Mimic mir-23 b inhibited LPS stimulated expression of inflammatory factorsNF-κB, TNF-α, IL-6, ICAM-1, selectin E, and VCAM-1 m RNA increased significantly at 4h and 8h after LPS stimulation in HUVECs transfected with mimic NC(P<0.05), but decreased significantly in cells transfected with mimic(P<0.05)(Fig. 4a). Western-blot assay showed that the level of NF-κB, TNF-α, IL-6, ICAM-1, and selectin E proteins was significantly lower in the mimic group than the mimic NC group after 4h of LPS action(P<0.05), and the level of VCAM-1 protein was significantly lower in the mimic group than the mimic NC group after 8h of LPS action(P<0.05).5 Effect of inhibitor mir-23 b on inflammatory factors expression in LPS stimulated cellsNF-κB, TNF-α, IL-6, ICAM-1, selectin E, VCAM-1 m RNAs increased significantly in HUVECs transfected with inhibitor NC after 4h and 8h LPS stimulation, when compared to the pre-stimulation levels(P<0.05). The levels of these inflammatory factors increased significantly in cells transfected with inhibitor after LPS stimulation when compared to the pre-stimulation levels(P<0.05). Moreover, the levels of the inflammatory factors increased significantly after 4h or 8h LPS stimulation when compared to the levels in cells transfected with inhibitor NC. Western-blot assay showed that NF-κB, TNF-α, IL-6, ICAM-1, selectin E and VCAM-1 proteins increased significantly in cells transfected with inhibitor when compared to cells transfected with inhibitor NC after 4h or 8h of LPS action(P<0.05).Conclusion:The results showed that LPS downregulated mir-23 b expression by HUVECs 。The results demonstrated that LPS promoted HUVECs to express inflammatory factors such as NF-κB, TNF-α, IL-6, ICAM-1, selectin E, and VCAM-1. Upregulating mir-23 b inhibited NF-κB, TNF-α, IL-6, ICAM-1, selectin E and VCAM-1 expression, while downregulating mir-23 b promoted inflammatory factor expression.It has been demonstrated previously that NF-κB is activated in sepsis and regulates apoptosis, cell growth, stress reaction, immune reaction and septic shock. The levels of TNF-α and IL-6 reach a peak as early as 3h after the onset of sepsis, and the degree of increase may reflect the severity of sepsis. ICAM-1, VCAM-1 and selectin E are important cell adhesion factors, and regulate the activity of inflammatory and vascular endothelial cells, pro- and anti-inflammatory factors, as well as inflammatory cell migration to tissues and organs; hence, they play an important role in sepsis. In summary, mir-23 b regulates sepsis through inhibiting inflammatory factor expression by VECs.PartⅡ:The research of peripheral blood micro RNA-23 b as sepsis molecular marker.Objective:To evaluate the value of peripheral blood micro RNA-23b(mi RNA-23b)as the identificationmolecular marker of sepsis and SIRS, try to figure out the potential role of mi RNA-23 b as a new sepsismolecular marker applied for clinical treatment.Methods:From December 2012 to November 2014, 40 subjects in Guangzhou military command wuhan general hospital EICU were enrolled for our study peripheral blood specimens and clinic data of the subjects were gathered.They divided into sepsis group(n=20) 、SIRS group(n=10) and control group(n=10) according to theirdiagnosis. We used the mir Vana PARIS Kit to extract the serum mi RNA and All-in-One mi RNA q RT-PCR to detect the expression level of mi RNA-23 b in the serum of peripheral blood specimens. The levels of TNF-α and IL-10 inplasma was detected. We analyzed the correlation between the the expression level of peripheral blood mi RNA-23 b with clinical data of the sepsis patients. Sensitivity and specificity werecompared between the expressions of mi RNA with SOFA.Results:1) 40 studied cases included 20 sepsis patients, 10 SIRS patients, 24 cases of normal(healthy controls), mean age(49.9 ±15.7) years, 25 cases of male and 15 cases of female.2) detection sensitivity and linear range of Mi R-23bThe low detection limit of RNA in which Mi R-23 b and U6 could be detected was 6.97 pg/ L.In the 6.97-6.97 * 104pg/ L RNA range,there was a good linear relationship between the mi R-23 b and U6’s cycle threshold and the concentration of RNA(R =0. 999).3) The temperature stability of mi R-23 b in the peripheral bloodAfter peripheral blood preserved in 4 ℃forfive days, it’s total RNA quantitative results down from 51.32 ng/μL to 18.3 ng/μL. Thus,with the prolonged storage time, the degradation of the total RNA in samples increased significantly. But the expression level data of mi R-23 b and U6,in the peripheral blood leukocytes, had no obvious change.4)the analysis of illness in sepsis patients20 sepsis subjects included 12 cases of pulmonary infection, 4 cases of peritonitis, 2 cases of biliary tract infection and 2 cases of bacterial endocarditis. The 28 day mortality rate was 20%(4/20).The mean SOFA scores was 5.8 + 2.52(2-11).5) The average expression level of mi RNA-23 b in sepsis group(4.1427±2.0836)(Log2ΔCt) was significantly lower than that in healthy control group(1.6884 ±2.1499), and there was no significant difference between in sepsis group and SIRS group(3.5159 ±2.0554)(p>0.05).And the average expression level of mi RNA-23 b in SIRS group was no significantly lower than that in healthy control group(p>0.05).The white cell counts, tumor necrosis factor alpha, interleukin-10 and the ratio of interleukin-10 to tumor necrosis factor alpha were significantly higher in the peripheral blood leukocytes in sepsis group than those in heathly group(P <0. 01).There was no significant diffenrence in tumor necrosis factor alpha, interleukin-10, the ratio of interleukin-10 to tumor necrosis factor alpha or white blood counts between sepsis group and inflammatory response syndrome group(P >0. 05).6) The expression level of mi RNA-23 b was significantly lower(P < 0. 01, P=0.003), and serum IL-10 was significant higher(P<0. 01, P=0.037) in dead patients than those in survived patients in sepsis group. There was no significant diffenrence inwhite cell counts(P=0.584), tumor necrosis factor alpha(P=0.324), ratio of interleukin-10 to tumor necrosis factor alpha(P=0.263) in dead patients and those in survived patients in sepsis group.7) For patients in sepsis group, the expression level of peripheral blood mi RNA-23 b was correlated with sequential organ failure assessment(SOFA) scores( r =0.473), tumor necrosis factor alpha(r =0.485) and interleukin-10(r =0.470)(P < 0. 05).And the expression level of peripheral blood mi RNA-23 b was not correlated with white cell counts( r =0.301) and the ratio of interleukin-10 to tumor necrosis factor alpha( r =0.039)(P> 0.05).8) For patients in sepsis group,the sequential organ failure assessment scores was not correlated with tumor necrosis factor alpha(r =0.410), interleukin-10(r =0.369) and the ratio of interleukin-10 to tumor necrosis factor alpha( r =0.124)(P> 0.05).LimitationsThis study has a number of limitations. First, we did not use a consecutive patient population so the study may be subject to selection bias. Second, we only analyzed blood from the initial draw and we did not follow the biomarker dynamics over time. Third, There may have been unmeasured confounders or confounding that was not accounted for in the analysis. Fourth,, some patients enrolled with sepsis may have been misclassified and ultimately had other etiologies of disease, but included in our cohorts. Lastly, our sample size is not large. A larger study may have afforded the opportunity for more complete subset analysis.Conclusion:The expression level of mi RNA-23 b in sepsis group was significantly lower than that in healthy control group, and there was no significant difference between in sepsis group and SIRS group. The expression level of mi RNA-23 b reflects the situation of immune response,and it may be used as a molecular marker for judging the severity and prognosis of sepsis.But it still needs further study to expand the sample size before used for clinical screening.The mechanism of the action of mi RNA-23 b in the development of sepsis needs to be explored.