JQ1 Alleviates Liver Damage In Schistosoma Japonicum-infected Mice By Inhibiting Acetyltransferase Activity Of P300

Background:Schistosomiasis japonica is still a major public health problem,seriously impacts on human health and the social economic development,mainly distributed in tropical and subtropical regions,our country is one of the most epidemic countries.The main pathogenesis of Schistosomiasis japonica is egg granuloma lesions and secondary liver fibrosis,however,there is no ideal treatment.So it is critical to investigate new therapeutic targets for liver fibrosis on the basis of further studying the mechanism of granulomatous inflammation and fibrosis.Th17 belongs to the CD4~+T cells lineage,characterized by high Il-17 secretion.ROR?t is a key transcription factor in Th17 differentiation,which promotes the Th17differentiation of by directly activating the of Il-17 transcription.p300 is aacetyltransferase which is composed of Bromodomain,PHD,RING and HAT.HAT is acetyltransferase domain,and Bromodomain helps to recognize acetylated lysine and combine catalytic substrate.JQ1 is inhibitor of BET(Bromodomain and extra terminal domain)family.It inhibit the Bd function by combining competitively with acetylated substrates,which limit recruitment of more acetyltransferase p300,thereby the acetylation of p300 signal fail to be maintained and the promotion on the transcription of related genes was inhibited.Previous studies in our lab showed that Th17 cells not only promoted the egg induced granuloma inflammation in the acute stage,but also contributed liver fibrosis.P300upregulates ROR?t-mediated transcriptional activation of IL-17,thereby promoting Th17 differentiation by acetylating K81 of the ROR?t.Our hypothesis is:Does JQ1 down-regulate ROR?t-mediated transcription of IL-17 by inhibiting p300 acetylation,thereby affecting the differentiation of Th17,and then reducing the granuloma inflammation and fibrosis of the liver in the Schistosomiasis infection model?And does JQ1 work by competing with p300 for the bromodomain(Bd)?Methods:1.Schistosoma japonicum infected mouse model and JQ1 treatmentIn order to exclude the effect of JQ1 on adult oviposition,S.japonicum infected mice,were treated with the praziquantel by gastric gavage on 6 weeks post infetion,and then JQ1 treatment was performed.SPF C57/BL6 mice(6-8 weeks)were randomly divided into 3 groups:normal control group,DMSO solvent control group and JQ1 treated group.C57/BL6 mice were infected by 20±2 schistosome cercariae percutaneously.Praziquantel gavage(300mg/kg/d)for 2 days on 42 days post infection.JQ1(50mg/kg/d)/DMSO was intraperitoneal injection on 45 days after infection for a total of 15 days.Mice were killed for the following experiments on 60 day post infection:(1)Mouse serum were collected by enucleation sored in-80?for detection of transaminase activity.(2)Count adult and eggs;(3)The mouse weight,liver and spleen weight were recorded for calculating liver and spleen coefficients.(4)The livers tissue were fixed in formalin for the HE staining and the Sirius red staining.(5)Frozen liver tissues in-80?for measuring the content of hydroxyproline and RT-qPCR experiments.(6)Collect granulomatous cells from the liver for the FACS to test Th17 cells.2.Study on the mechanism of inhibition of p300 acetylase activity by JQ1(1)Expression and identification of recombinant p300 containing different domainsTarget genes were amplified by PCR first.Expression plasmid were constructed for transient transfection of ExpiCHO cells by reforming pcDNA3.1(+)vector.p300containing both Bd and HAT or only HAT domain were expressd in eukaryotic cell respectively.(2)Effect of JQ1 on acetyltransferases activity of recombinant p300.Recombinant p300 proteins containing different domains treated with JQ1(250 nM)for4h were assayed for acetylase activity using the Histone Acetyltransferase Activity Assay Kit.Results:1.JQ1 treatment alleviates liver granulomatous inflammation and liver fibrosis in S.japonicuminfected mice.2.The mRNA of the IL-17 and ROR?t in the livers were down-regulated by JQ1.The proportion of Th17 in granuloma cells were decreased in JQ1 treated mice.3.JQ1 inhibited the acetyltransferase activity of the p300 containing the Bd domain,but no effect on the p300 protein without Bd.Conclusions:1.JQ1 treatment alleviates liver granulomatous inflammation and liver fibrosis in S.japonicum infected mice.2.We speculated that it is reasonable inhibitting the transcription of IL-17 mediated by ROR?t through competitively binding to the Bd by JQ1,and resulting in impairment of Th17 differentiation;which in turn reduces the egg-induced liver lesions.