The Mechanism Of HDAC6 Promoting Sepsis By Regulating Nitric Oxide Production In Macrophages

Macrophages,as the most effective pathogen scavenger and the main source of inflammatory mediators,are the key effector cells in the face of infection.In sepsis,the severity of the disease is closely related to macrophage dysfunction.Under physiological conditions,the low level of nitric oxide(NO)is synthesized by three different isoforms of nitric oxide synthase(NOS): neuronal NOS(n NOS),endothelial NOS(e NOS),and inducible NOS(i NOS).However,the over-expressed i NOS in macrophages and neutrophils is the major source of NO in sepsis status.A large amount of NO is produced in sepsis.The excessive NO not only affects the synthesis and release of other inflammatory factors,promotes the body's inflammatory response,but also leads to vascular dysfunction during septic shock,including vasodilation,low vascular reactivity and increased vascular permeability,leading to a high mortality.Therefore,it is urgent to develop effective drugs to control NO overdose and provide new treatment strategies for sepsis.HDAC6,as a unique histone deacetylase,is mainly confined in the cytoplasm.It regulates various non-histone substrates and signaling pathways,such as ?-tubulin and HSP90 through deacetylation and ubiquitination,and exerts anti-apoptotic,anti-inflammatory and immunomodulatory effects as well.In some studies it showed that both the expression of i NOS and the level of NO increased under the influence of TNF-? and IL-1? in sepsis,while the inhibition of HDAC6 could inhibit the production of inflammatory factors,such as TNF-??IL-1? and IL-6,which are produced in macrophage.To further clarify the effects of HDAC6 on i NOS expression and NO production,the peritoneal macrophages from wild type mice were challenged by LPS after the HDAC6 inhibitor CAY10603 pretreatment,to observe i NOS expression and NO production on the basis that LPS could induce i NOS over-expression in peritoneal macrophages from wild-type mice.The experiments above were also taken in the peritoneal macrophages from HDAC6 knockout mice.Meanwhile,the mechanisms were also explored.Then in vivo experiments were carried out in mice to verify the effects of HDAC6 knockout on the production of NO and its metabolite in sepsis.The first part of this study: in vitro experiment,to explore the effects of HDAC6 on the expression of i NOS and NO production in murine peritoneal macrophages,as well as the molecular mechanisms.Establish the in vitro model by LPS challenge in murine peritoneal macrophages.It was indicated that LPS could induce the over-expression of i NOS,and elevate NOS activity as well as the nitrite concentration in the supernatant of wild type macrophages;the phosphorylation level of STAT1 at Tyr701 site was significantly increased and IRF-1 synthesis was also enhanced.In the peritoneal macrophages of HDAC6 knockout mice,or when the specific HDAC6 inhibitors CAY10603 were applied in peritoneal macrophages of wild type mice,the increased acetylated ?-tubulin were detected.Under the condition of CAY10603 pretreatment or HDAC6 gene knockout,the expression of i NOS and NO production after LPS challenge,as well as the expression of p-STAT1(Try701)and IRF-1 were significantly decreased.Considering the results of previous studies,it was indicated that the application of specific HDAC6 inhibitors CAY10603 or HDAC6 gene knockout indirectly affected the activation of STAT1 by changing the acetylation state of ?-tubulin,and afterwards affected the synthesis of IRF-1,which leading to the reduced binding of p-STAT1(Try701)and IRF-1 in i NOS promoter region,and finally decreasing the transcription level and activity of the i NOS.The second part of this study: in vivo experiment,to explore the effect of HDAC6 on NO production in septic animals.Sepsis models were established by intraperitoneal injection of endotoxin(LPS)and cecal ligation and puncture(CLP),respectively.LPS model group: the wild type C57BL/6J mice and HDAC6 knockout mice with the same gene background were challenged by LPS intraperitoneal injection,respectively;the control groups received the same amount of PBS intraperitoneal injection.6 hours later,the plasma,peritoneal lavage fluid and lung tissue samples of each group were collected.CLP model groups: the wild type C57BL/6J mice and HDAC6 knockout mice of the same gene background underwent cecal ligation and puncture,while the control groups only underwent sham surgery.16 hours after the surgery,the plasma and lung tissue samples of each group were collected.The detection indexes included(1)Ace-?-tubulin and i NOS protein expression in peritoneal macrophages of wild type LPS model mice;(2)Ace-?-tubulin,i NOS,p-STAT1 and IRF-1 protein in lung tissue of both two kinds of sepsis models;(3)the concentration of nitric oxide and its metabolites nitrotyrosine in plasma of two kinds of sepsis models.The results showed that the acetylated ?-tubulin in both peritoneal macrophages and lung tissues of HDAC6 knockout septic mice was significantly higher than that in wild type septic mice,while the level of i NOS was significantly decreased.The expression of the upstream signal molecules p-STAT1(Try701)and IRF-1 which regulates i NOS expression was significantly inhibited.The results were consistent with those of in vitro experiments.Meanwhile,the concentration of NO and nitrotyrosine in HDAC6 knockout septic mice was significantly lower than that in wild type septic mice.In addition,the survival analysis of septic mice was also carried out in this study,and it was found that the survival of HDAC6 knockout septic mice was improved significantly compared to the wild type septic mice.To sum up,the in vitro experiments have confirmed that LPS could induce the overexpression of i NOS and the excessive production of NO in mouse peritoneal macrophages.After the application of HDAC6 inhibitor CAY10603,the expression of i NOS and the production of NO can be inhibited by inhibiting the activation of STAT1(Try701)and the synthesis of IRF-1 in murine peritoneal macrophages.Further experiments were carried out on the peritoneal macrophages of HDAC6-/-mice,and the results were consistent with the results above.The in vivo experiments confirmed that both the intraperitoneal injection of LPS and CLP modeling could promote the overexpression of i NOS,and the excessive production of NO as well as its metabolites in the plasma of HDAC6 wild-type mice.HDAC6 gene knockout could inhibit the overexpression of i NOS in the peritoneal macrophages and lung tissues of septic mice by inhibiting the activation of STAT1(Try701)and the synthesis of IRF-1,thus reducing the concentration of NO and nitro tyrosine in the plasma and improving the survival condition of septic mice.The in vivo experiments and in vitro experiments were conducted with HDAC6 gene knockout mice,the results of which were consistent with that obtained from the in vitro experiments carried out with the HDAC6 inhibitor CAY10603,confirming the accuracy and specificity of the inhibitor experiment results.This study indicates that the applicationof HDAC6 inhibitors or HDAC6 gene knockout may improve the vasoconstrictive and diastolic dysfunction in septic mice by reducing the level of NO in plasma,thus reducing the hemodynamic abnormalities and improving the prognosis,which may provide a new direction for the research about drugs for sepsis treatment.