漆树酸通过调控KAT8介导的组蛋白H4K16ac高乙酰化改善TAC小鼠心肌纤维化

Anacardic acid attenuates myocardial fibrosis through inhibiting histone H4K16ac hyperacetylation mediated by KAT8 in TAC mice

摘要:

目的 探讨组蛋白乙酰化酶（histone acetylases，HATs）抑制剂漆树酸（anacardic acid，AA）改善胸主动脉缩窄术（thoracic aortic constriction，TAC）小鼠心肌纤维化的组蛋白乙酰化调控机制，为心肌纤维化的防治提供新靶点。方法 选择6~8周SPF级昆明小鼠为研究对象，按照随机数字表法将小鼠分为5组：正常（Normal）组、假手术（Sham）组、胸主动脉缩窄（TAC）组、胸主动脉缩窄+溶剂对照（TAC+Veh）组、胸主动脉缩窄+漆树酸（TAC+AA）组。12周后采用超声心动图检测各组小鼠模型构建情况；收集各组小鼠心脏组织进行以下检测：马松染色观察心肌组织纤维化情况；Western blot检测KAT8、H4K16ac、TGF-β1、SMAD3、Collagen Ⅰ、 Collagen Ⅲ蛋白表达情况；免疫共沉淀检测KAT8与H4K16ac的相互作用关系。结果 超声心动图表明TAC模型小鼠构建成功。马松染色结果表明，TAC组小鼠心脏较Sham组增大，心肌组织中胶原沉积增多，纤维化明显，而TAC+AA组较TAC组小鼠心脏缩小，心肌组织中胶原沉积减少，纤维化程度减轻。Western blot结果发现，TAC组小鼠心肌组织中KAT8及组蛋白H4K16ac的蛋白表达水平较Sham组明显升高（均P<0.05），且TGF-β1、SMAD3、Collagen Ⅰ、 Collagen Ⅲ蛋白表达水平较Sham组明显升高（均P<0.05）。与TAC组比较，AA明显降低TAC小鼠心肌组织中KAT8过表达及H4K16ac高乙酰化（均P<0.05）。同时，TAC+AA组TGF-β1、SMAD3、CollagenⅠ、 Collagen Ⅲ蛋白表达较TAC组明显下降（均P<0.05）。免疫共沉淀结果显示，KAT8可以与H4K16ac相互结合。此外，AA能够明显改善TAC小鼠的生存率。结论 KAT8介导的组蛋白H4K16ac高乙酰化可能通过调控TGF-β1/SMAD3信号通路参与TAC小鼠心肌纤维化，而AA能够抑制KAT8介导的组蛋白H4K16ac高乙酰化进而改善TAC小鼠心肌纤维化。

Abstract:

Objective To explore the histone acetylation mechanism of histone acetylases（HATs） to inhibit anacardic acid（AA） attenuating myocardial fibrosis in thoracic aortic constriction（TAC） mice， and provide a new target for the prevention and treatment of myocardial fibrosis.Methods SPF grade Kunming mice of 6-8 weeks were selected as the research animals and randomly divided into five groups：Normal group，Sham group，TAC group，TAC+Vehicle（TAC+Veh） group，and TAC+AA group. TAC mice models were detected by echocardiography in 12 weeks after TAC，and myocardial tissues of mice were collected for analysis. The cardiac morphology and myocardial fibrosis were observed by Masson staining. The expression of KAT8，H4K16ac，TGF-β1，SMAD3，Collagen Ⅰ， and Collagen Ⅲ were assayed by Western blot. The interaction between KAT8 and H4K16ac was detected by co-immunoprecipitation（Co-IP）.Results Echocardiography showed that TAC model mice were successfully constructed. The results of Masson staining showed that the hearts of mice in TAC group were enlarged，collagen deposition in myocardial tissues increased and fibrosis was obvious compared with that in Sham group，while the hearts of mice in TAC+AA group were smaller，collagen deposition in myocardial tissues decreased and fibrosis degree was lighter than that in TAC group. Western blot results showed that the expression of KAT8 and H4K16ac in myocardial tissues of mice in TAC group were significantly higher than those in Sham group（all P<0.05）. And the protein expression levels of TGF-β1，SMAD3，Collagen Ⅰ and Collagen Ⅲ were significantly higher than those of Sham group（all P<0.05）. Compared with TAC group，AA significantly inhibited the overexpression of KAT8 and the hyperacetylation of H4K16ac in TAC mice（all P<0.05）. Meanwhile，the protein expression levels of TGF-β1，SMAD3，CollagenⅠand Collagen Ⅲ in TAC+AA group were significantly decreased compared with TAC group（all P<0.05）. The Co-IP results showed that KAT8 could bind to H4K16ac. In addition，AA significantly improved the survival rate of TAC mice.Conclusion KAT8-mediated hyperacetylation of H4K16ac may be involved in myocardial fibrosis in TAC mice by regulating TGF-β1/SMAD3 signaling pathway，while AA could attenuate myocardial fibrosis in TAC mice by inhibiting KAT8-mediated hyperacetylation of histone H4K16ac.