Expression Of P300/CBP In ARDS And Its Regulation On Differentiation Of Th17 Cells

Acute respiratory distress syndrome(ARDS)is the main cause of severe acute respiratory failure.It is characterized by increased alveolar osmotic permeability and continuous and severe hypoxemia.At present,in addition to organ support therapy,there is lack of effective treatment in clinic,it is related to the high mortality rate of patients,which is one of the main causes leading to death in critical patients.In recent decades,although protective mechanical ventilation strategies,restrictive intravenous infusion management methods,and rescue strategies such as prone position ventilation and extracorporeal membrane oxygenation(ECOM)have made great progress in severe hypoxemia patients,but since 1994,the overall mortality of ARDS has remained at around 40%.Previous studies focused on the inflammatory process of ARDS lack clinical efficacy and anti-inflammatory treatment is not satisfactory.Immune regulation has become a new research direction in recent years.As a new subset of CD4+T helper cells different from Th1 and Th2 cells in recent years,Th17 cells are involved in both innate and adaptive immune responses,and haveexcellent recruitment function of neutrophilegranulocyte.Its mediated immune response may be a potential regulatory mechanism of inflammation in ARDS.ROR?t as its specific nuclear transcription factor for Th17 cells,and its transcriptional activity determines the differentiation of Th17 cells.The transcriptional cofactor p300/CBP as the major histone acetyltransferase,regulate transcriptional activity through its acetylated nuclear transcription factor,such as p53,Foxp3,ROR?t.Acetylation can alter its stability,subcellular localization and DNA binding ability.Recently,it has been found that intranuclear accumulation of ?-catenin can promote ROR?t expression,which may be an important upstream molecule regulating the differentiation of Th17 cells.It was also reported that p300/CBP participates in the regulation of wnt/?-catenin and STAT3-ROR?t pathway activation through its acetylation.At present,the role of CBP/p300 in ARDS and the regulation of ROR?t pathway or th17 cells differentiation has never been reported.The clinical application of p300 and HDAC inhibitors has achieved good results in the field of cancer and chronic inflammatory diseases.The molecular mechanism of this molecule is still unclear in ARDS.Therefore,we have designed this topic.Objective:To clarify the expression characteristics of p300/CBP in ARDS.Through intervention on ALI animal model by p300 and HDAC inhibitor to explore their regulation on Th17 cell differentiation during ARDSinflammation and its possible mechanism.Methods:RT-PCR was used to detect the expression of p300,CBP,ROR?t,Foxp3 mRNA in peripheral blood mononuclear cells(PBMC)samples from patients with ARDS and healthy subjects.The concentration of inflammatory cytokines IL-17,IL-6 and IL-10 in plasma were measured by ELISA.Meanwhile,the baseline data of demographic and clinical testing were collected,the acute physiological and chronic health(APACHE?)score and sequential organ failure(SOFA)score were recorded.The main outcome was defined as hospital mortality within 28 days follow-up.The ARDS mice model was established by intratracheal injection of LPS.The wet to dry weight ratio of lung tissue and HE staining were measured,and the lung injury score was carried out.After successfully establishing model,p300 specific inhibitor C646 and HADC inhibitor TSA was respectively intraperitoneal injected in two intervention groups.All mice were killed 24 hours after the drug action,and the mice lung tissue and BALF were obtained.The concentrations of IL-17,IL-6 and TGF-? in lung tissue and BALF were measured by ELISA.IHC was used to observe the expression of p300,CBP and ROR?t in lung tissue.RT-PCR detected the mRNA expression of p300 and ROR?t and western-blot was used to detect the level of p300 and ROR?t protein in mice lung tissues.Results:(1)Compared with healthy controls,the mRNA expression of p300,CBP,ROR?t and IL-17,IL-6 in acute ARDS patients were significantly increased(P < 0.05),there was no significant difference between Foxp3 mRNA and IL-10 concentration in two groups.(2)Among the non-survivors showed significantly higher levels of p300,CBP and IL-6.In addition,higher mRNA expression of ROR?t in infection-caused ARDS patients than those non-infected patients.(3)Parametric and non-parametric correlation analysis showed that the mRNA levels of p300 was linearly correlated with levels of IL-6 and IL-17,correlation coefficients were respectively 0.4522 and 0.3576(P <0.05),and was rank correlated with the mRNA expression of ROR?t and CBP,correlation coefficients were 0.3382 and 0.3042(P < 0.05).However,CBP mRNA was not associated with other detection factors except for p300.(4)Logistic regression analysis revealed that p300,IL-6 and CBP might be prognostic indicators.Including baseline data and survival time,Cox regression analysis revealed the level of p300,CBP mRNA and age may be predictors of prognosis in ARDS patients.(5)IHC was used to detect the expression of p300/CBP and ROR?t in lung tissues.The results showed that p300/CBP and ROR?t were mainly expressed in interstitial leukocytes and a few alveolar epithelial cells and rarely found in bronchial epithelial cells of lung tissue.Stain mainlylocated in the nucleus and less in the cytoplasm.The staining intensity and percentage in all three model groups treated with lipopolysaccharide were higher than those in controls,lower p300 and ROR?t staining was found in the C646 intervention group.CBP staining was weaker than p300 and ROR?t in three model groups and no significant staining difference among the model groups.These results suggest that inhibition of p300 may affect the expression of ROR?t.(6)Further ELISA,WB,RT-PCR results showed that p300 inhibitor(C646)significantly inhibited p300 protein levels and caused IL-17,ROR?t mRNA and protein levels to decrease,and improved the acute lung injury pathological score,displaying its lung protective effect.HDAC inhibitor TSA showed the opposite effect,but the effect was not as strong as C646.Conclusion:p300/CBP is overexpressed in acute phase of ARDS.In particular,the expression of p300 was positively correlated with the level of ROR?t,IL-17 and may be one of the prognostic factors for patients with ARDS.Further mice models revealed that p300 promoted the differentiation of th17 cells through positive regulation of nuclear transcription factor ROR?t and p300 specific inhibitor C646 showed protective effect on ALI.