【作者】 刘伟；

【导师】 辛颖；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2025， 硕士

【摘要】 研究背景肾素-血管紧张素系统(Renin-angiotensin system,RAS)的病理性激活是心血管疾病(Cardiovascular diseases,CVDs)发生发展的关键诱因。作为RAS的主要效应肽,血管紧张素Ⅱ(Angiotensin Ⅱ,AngⅡ)存在于体循环及心脏组织内。心脏局部的AngⅡ在各种CVDs的发病过程起关键促进作用,如糖尿病性心肌病、酒精性心肌病和心肌梗死等。其主要作用机制是通过诱导氧化应激、炎症、凋亡和纤维化等多种信号通路,引起心肌凋亡、肥厚和心室重构,最终导致心肌病。众所周知,含胞膜催化亚基gp91 phox的NADPH氧化酶是心血管系统活性氧(Reactive oxygen species,ROS)的主要来源。研究提示AngⅡ输注促进小鼠主动脉中gp91 phox的表达,从而激活NADPH氧化酶并诱导ROS的生成。然而,Ang Ⅱ介导的核心gp91 phox转录激活的具体分子机制尚不清楚。因此,深入探索Ang Ⅱ诱导氧化应激及心肌损伤的病理机制对于临床心肌病的预防和治疗具有重要意义。在缺血性脑卒中相关研究中发现,gp91 phox随吲哚胺2,3-双加氧酶1(Indoleamine 2,3-dioxygenase 1,IDO1)的表达增加而上调,可能是由IDO1诱导的非酶促信号介导的。我们前期实验也证实,IDO1和gp91 phox在AngⅡ组小鼠分离的心脏组织中高表达,且IDO1的高表达与Ang Ⅱ诱导其转录因子的磷酸化激活有关,即信号转导与转录激活因子1(Signal transducers and activators of transcription 1,STAT1)。因此推测,Ang Ⅱ可能通过STAT1激活IDO1的非酶促作用进而介导gp91 phox的激活。有趣的是,IDO1还可能通过非酶促过程抑制Notch1信号通路。Notch1信号在成人心脏功能的维持中起关键作用。内皮细胞Notch1信号抑制血管生成素样蛋白4(Angiopoietin-like protein 4,ANGPTL4)的表达,影响心肌脂肪酸代谢,改善心肌肥大。近期研究发现,在高糖条件下,内源性蛋白ANGPTL4通过激活整合素/局灶黏附激酶(Focal adhesion kinase,FAK)信号通路,诱导AC16心肌细胞gp91 phox表达和ROS增加,促进心肌凋亡和损伤,而抑制HUVEC的Notch1信号可导致ANGPTL4 m RNA表达增加。然而,IDO1是否亦通过调控ANGPTL4诱导gp91 phox激活?Notch1信号是否介导这一过程?目前未见报道。目前越来越多的证据表明,肠道微生物群是Ang Ⅱ相关各种病理状态的有效调节因子。肠道菌群不仅从摄入的食物中向宿主提供营养和能量,其代谢产生的生物活性物质还参与机体功能的维持或促进疾病的发生,如短链脂肪酸(Short chain fatty acid,SCFAs)和氧化三甲胺(Trimethylamine N-oxide,TMAO)。有研究发现,慢性Ang Ⅱ输注诱导高血压和肠道屏障功能障碍,并伴随产丁酸菌的减少(如Eubacterium和Butyricicoccus);而IDO1的表达依赖于肠道菌群,尤其是与丁酸产生相关的菌群。丁酸除了能够抑制IDO1的转录因子STAT1的磷酸化激活与核易位,还能诱导IDO1的泛素化降解,而这一过程可能与芳香烃受体(Aryl hydrocarbon receptor,AHR)/细胞因子信号传导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的信号激活相关。因此,Ang Ⅱ是否通过诱导肠道菌群失衡致丁酸产生减少,进而对STAT1/IDO1的抑制作用减弱?以及适当补充丁酸是否可以逆转Ang Ⅱ引起的心肌损伤,也是本课题亟待解决的问题。因此,本研究提出,Ang Ⅱ可能通过磷酸化激活STAT1诱导IDO1的表达,IDO1通过非酶促作用抑制Notch信号转导促进ANGPTL4的表达,进而转录激活gp91 phox并加重心肌氧化损伤;另一方面,Ang Ⅱ诱导肠道菌群失衡,丁酸及丁酸产生相关菌群减少,导致丁酸对STAT1/IDO1的抑制作用减弱,从而促进Ang Ⅱ诱导的心肌氧化损伤。研究目的利用Ang Ⅱ诱导的小鼠心肌病模型及人AC16心肌细胞氧化损伤模型,探讨IDO1通过Notch1/ANGPTL4的非酶促信号激活gp91 phox促心肌病的作用,解析Ang Ⅱ经肠道微生物群促IDO1非酶促反应加重心肌损伤的机制,评价补充丁酸靶向抑制STAT1及AHR/SOCS3介导的IDO1泛素化降解对Ang Ⅱ诱导的心肌损伤的防治效果,为Ang Ⅱ相关心肌损伤及氧化应激相关心肌病的防治提供新思路和新靶点。研究内容与方法1.选取8周龄健康雄性C57BL/6J小鼠,适应性饲养1周后,隔日皮下注射小剂量Ang Ⅱ(0.5 mg/kg),持续2个月,停药后观察至6个月,对照组注射同等剂量的生理盐水,建立心肌氧化损伤模型。6个月后,通过心脏超声检测心功能;收取心肌组织,提取蛋白和RNA检测氧化应激指标(4-HNE和MDA)和凋亡指标(cleaved Caspase3),以及IDO1相关通路分子(STAT1、IDO1、Notch1、HES1、ANGPTL4、FAK和gp91 phox);分别收集肠道内容物和血清进行16S r RNA测序和短链脂肪酸含量测定,明确Ang Ⅱ诱导心肌氧化损伤模型的建立,以及观察STAT1/IDO1/Notch1/ANGPTL4通路、肠道菌群及丁酸含量的改变。2.采用100 nM AngⅡ处理人AC16心肌细胞48 h,建立心肌细胞氧化损伤模型,并给予IDO1抑制剂(Epacadostat)及STAT1磷酸化抑制剂(Fludarabine),通过检测4-HNE、cleaved Caspase3、MDA和ROS等氧化损伤指标,探究IDO1对心肌细胞氧化损伤的促进作用,以及Ang Ⅱ通过激活STAT1诱导IDO1表达的机制。3.在心肌细胞氧化损伤模型中,应用IDO1抑制剂(Epacadostat)后观察ANGPTL4的表达变化;构建ANGPTL4低表达的AC16心肌细胞株,检测上述氧化损伤指标,探究IDO1是否通过激活ANGPTL4诱导心肌细胞氧化损伤。4.在心肌细胞氧化损伤模型中,应用IDO1抑制剂(Epacadostat)和IDO1酶功能激活剂(Kynurenine),检测IDO1的磷酸化水平、Notch1激活形式cleaved Notch1蛋白及其靶基因HES1 m RNA表达的变化;应用Notch1/HES1激动剂(Valproic acid),检测ANGPTL4的表达;利用JASPAR数据库预测HES1与ANGPTL4基因启动子区域的结合位点;通过染色质免疫共沉淀(Ch IP)实验验证HES1与ANGPTL4的靶向关系,探讨IDO1通过非酶促作用抑制Notch1/HES1通路上调ANGPTL4表达,从而激活gp91 phox促心肌细胞氧化损伤的机制。5.在心肌细胞氧化损伤模型中,使用STAT1磷酸化激动剂(2-NP)并给予1m M丁酸处理,探究丁酸是否通过作用于STAT1抑制IDO1/ANGPTL4通路缓解Ang Ⅱ诱导的心肌细胞氧化损伤。6.在心肌细胞氧化损伤模型中,采用小分子药物CH-223191抑制AHR/SOCS3信号并给予1 m M丁酸处理,Western blot及免疫共沉淀(IP)实验验证丁酸是否通过激活AHR/SOCS3信号诱导IDO1泛素化降解进而发挥抗心肌细胞氧化损伤的作用。研究结果1.AngⅡ低剂量、长期作用引起小鼠左心室扩张、心肌肥大和心功能不全,以及心肌氧化损伤;STAT1和FAK磷酸化增加,IDO1、ANGPTL4和gp91 phox表达上调,Notch1/HES1通路活性下调;肠道菌群失衡,群落结构和组成改变,部分丁酸产生相关菌群的丰度及血清丁酸的含量降低。2.体外心肌细胞氧化损伤模型中,给予IDO1抑制剂Epacadostat显著抑制了gp91 phox的表达,心肌细胞氧化损伤减轻;给予Fludarabine抑制STAT1磷酸化后,IDO1 m RNA及蛋白表达显著降低。3.体外心肌细胞氧化损伤模型中,ANGPTL4表达增加,且抑制IDO1可显著降低ANGPTL4的表达;而敲低ANGPTL4对AC16心肌细胞中IDO1的表达无影响,但FAK磷酸化和gp91 phox的表达显著降低,且心肌细胞氧化损伤亦减轻。4.体外心肌细胞氧化损伤模型中,cleaved Notch1蛋白及HES1 m RNA表达降低,即Notch1/HES1通路被抑制;而给予Epacadostat抑制AngⅡ诱导的IDO1激活后,恢复Notch1/HES1通路活性,但补充Kynurenine提高IDO1的酶促功能却不能恢复其对Notch1/HES1通路的抑制作用;利用Valproic acid激活Notch1/HES1通路后,ANGPTL4的表达降低,Ch IP实验进一步证实,HES1与ANGPTL4启动子区域可直接结合,且AngⅡ处理组中HES1在ANGPTL4基因启动子区域的富集显著低于对照组,提示HES1转录抑制ANGPTL4的表达。5.体外补充丁酸抑制STAT1和FAK的磷酸化,降低IDO1、ANGPTL4及gp91 phox的表达,增加Notch1/HES1通路活性,且有效抑制了AngⅡ引起的心肌细胞氧化损伤;而使用2-NP激活STAT1磷酸化后,逆转了丁酸的保护作用。6.体外补充丁酸诱导AC16心肌细胞中AHR/SOCS3蛋白表达和IDO1泛素化水平升高,且IDO1蛋白表达降低;而使用CH-223191抑制AHR/SOCS3信号后,IDO1泛素化水平降低,其蛋白表达升高。研究结论1.长期、低剂量AngⅡ刺激引起心肌氧化损伤和心功能不全,且小鼠肠道菌群失调,丁酸及其产生相关菌群减少。2.Ang Ⅱ通过激活STAT1促进IDO1的表达,IDO1则通过非酶促作用抑制Notch1/HES1通路,上调ANGPTL4表达,从而激活gp91 phox使ROS产生增加,促进AngⅡ诱导的心肌氧化损伤。3.体外补充丁酸可通过抑制STAT1减少IDO1的表达及激活AHR/SOCS3信号诱导IDO1泛素化降解而缓解Ang Ⅱ诱导的心肌细胞氧化损伤。更多还原

【Abstract】 BackgroundThe pathologic activation of the renin-angiotensin system(RAS)is a key factor in the development of cardiovascular diseases(CVDs).As,the main effective peptide of RAS,angiotensin Ⅱ(AngⅡ)exists in systemic circulation and heart tissue.Local cardiac AngⅡplays a key role in the pathogenesis of various CVDs,such as diabetic cardiomyopathy,alcoholic cardiomyopathy and myocardial infarction.Its mechanism of action is mainly through inducing oxidative stress to activate inflammation,apoptosis,fibrosis and other signaling pathways,thus causing myocardial inflammation,apoptosis,hypertrophy and ventricular remodeling,and eventually leading to the occurrence of cardiomyopathy.It is well known that NADPH oxidase containing the membrane catalytic subunit gp91 phox is the main source of reactive oxygen species(ROS)in the cardiovascular system.The study suggests that AngⅡinfusion promotes gp91 phox expression in mouse aorta,thereby activating NADPH oxidase and inducing ROS production.However,the specific molecular mechanism of Ang Ⅱ-mediated transcriptional activation of gp91 phox remains unclear.Therefore,it is of great significance to explore the pathological mechanism of Ang Ⅱ induced oxidative stress and myocardial injury for the prevention and treatment of clinical cardiomyopathy.It has been found in studies related to cerebral ischemic injury that gp91 phox increases with the expression of indoleamine 2,3-dioxygenase 1(IDO1),which may be mediated by IDO1-induced non-enzymatic signals.Our previous preliminary experiments also confirmed that IDO1 and gp91 phox are highly expressed in the heart tissues isolated from group Ang Ⅱ mice,and the high expression of IDO1 is related to the phosphorylation activation of its transcription factors,signal transducers and activators of transcription 1(STAT1),induced by Ang Ⅱ.Therefore,it is speculated that Ang Ⅱ may mediate the activation of gp91 phox through the non-enzymatic action of STAT1 activation IDO1.Interestingly,IDO1 inhibition of Notch1 signaling pathways may also be related to non-enzymatic processes.Notch1 signaling plays a key role in the maintenance of adult heart function.For example,endothelial Notch inhibits the expression of angiopoietin-like protein 4(ANGPTL4),affects myocardial fatty acid metabolism,and improves myocardial hypertrophy.Recent studies have shown that endogenous protein ANGPTL4 induces gp91 phox expression and ROS increase in AC16 cardiomyocytes by activating focal adhesion kinase(FAK)signaling pathway under high glucose conditions,promoting myocardial apoptosis and injury.However,does IDO1 also induce gp91 phox activation by regulating ANGPTL4?Does Notch1signal mediate this process?None have been reported so far.Therefore,in view of the prevention and treatment of the pathological effects of Ang Ⅱ,more and more evidence shown that intestinal microbiota is an effective regulator factor of various pathological states related to Ang Ⅱ.Gut microbiota not only provides nutrients and energy to the host from ingested food,but also produces metabolic bioactive signaling molecules to maintain health or promote the development of disease,for instance short chain fatty acid(SCFAs)and trimethylamine N-oxide(TMAO).Some studies have found that chronic Ang Ⅱ infusion induces intestinal hypertension and intestinal barrier dysfunction,accompanied by a decrease in butyric acid-producing bacteria(such as Eubacterium and Butyricicoccus).However,IDO1expression is dependent on gut microbiota,especially those associated with butyrate production.In addition to inhibiting phosphorylation activation and nuclear translocation of IDO1’s transcription factor STAT1,butyrate can also induce IDO1’s ubiquitination degradation pathway.This may be related to aryl hydrocarbon receptor(AHR)/suppressor of cytokine signaling 3(SOCS3)signaling activation.Therefore,does Ang Ⅱ induce a decrease in butyric acid production by inducing dysregulation of intestinal flora,thereby weakening the inhibition of STAT1/IDO1?And whether proper supplement of butyric acid can reverse the myocardial damage caused by Ang Ⅱ is also a problem to be solved in this subject.Therefore,this study proposed a scientific hypothesis:Ang Ⅱ induces IDO1expression through phosphorylation activation of STAT1,and the latter promotes ANGPTL4 expression through non-enzymatic inhibition of Notch signal transduction,thereby transcriptional activation of gp91 phox and aggravating myocardial oxidative damage.On the other hand,Ang Ⅱ induced intestinal flora dysbiosis and decreased butyric acid and related flora,resulting in weakened inhibitory effect of butyric acid on STAT1/IDO1,thus promoting myocardial oxidative damage induced by Ang Ⅱ.ObjectiveThrough Ang Ⅱ-induced mouse cardiomyopathy model and AC16 cardiomyocyte oxidative injury model,this study aims to explores the non-enzymatic function of IDO1in promoting cardiomyopathy via Notch1/ANGPTL to activate gp91 phox,and the mechanism of Ang Ⅱ promoting IDO1 non-enzymatic reaction to aggravate myocardial injury through gut microbiota was analyzed.To evaluate the preventive and therapeutic effect of butyrate supplementation targeting STAT1 inhibition and AHR/SOCS3-mediated IDO1 ubiquitination degradation on Ang Ⅱ-induced myocardial injury.This study provides new ideas and new targets for the prevention and treatment of Ang Ⅱ-related myocardial injury and oxidative stress-related cardiomyopathy.Methods1.Healthy male C57BL/6J mice aged 8 weeks were selected,and after 1 week of adaptive feeding,a small dose of Ang Ⅱ(0.5 mg/kg)was injected subcutaneously every other day for 2 months,and then observed until 6 months after drug withdrawal.The control group was injected with the same dose of normal saline to establish the myocardial oxidative injury model.After 6 months,cardiac function was measured by echocardiography.Myocardial tissue was collected,and protein and RNA were extracted to detect oxidative stress indicators(4-HNE and MDA),apoptosis indicators(cleaved Caspase3),and IDO1-related pathway molecules(STAT1,IDO1,Notch1,HES1,ANGPTL4,FAK and gp91 phox).Respectively the intestinal contents and serum were collected for 16S r RNA sequencing and short chain fatty acid content determination.To establish the Ang Ⅱ-induced myocardial oxidative damage model,and observe the change of STAT1/IDO1/Notch1/ANGPTL4 pathways,gut microbiota and butyrate content.2.Human AC16 cardiomyocytes were treated with 100 nM AngⅡfor 48 h to establish the oxidative damage model of cardiomyocytes,and STAT1 phosphorylation inhibitor(fludarabine)and IDO1 inhibitor(epacadostat)were given.Oxidative damage indicators such as 4-HNE,cleaved Caspase3,MDA and ROS were detected to explore the promoting effect of IDO1 on oxidative damage of cardiomyocytes and the mechanism of Ang Ⅱ inducing IDO1 expression through the activation of STAT1.3.In cardiomyocyte oxidative injury model,the expression of ANGPTL4 was observed with IDO1 inhibition.AC16 cardiomyocytes line with low expression of ANGPTL4 was constructed to detect the above oxidative damage indicators and explore whether IDO1 induces cardiomyocyte oxidative damage through the activation of ANGPTL4.4.IDO1 inhibitor(epacadostat)and IDO1 enzyme function activator(kynurenine)were applied to detect IDO1 phosphorylation level and cleaved Notch1 protein and m RNA expression of its target gene HES1;Notch1/HES1 agonist(valproic acid)was used to detect the expression of ANGPTL4 in Ang Ⅱ-induced cardiomyocyte oxidative injury model.Cistrome Date Browser and JASPAR database were used to predict the binding sites of HES1 and ANGPTL4 gene promoter region.The targeting relationship between HES1 and ANGPTL4 was verified by chromatin immunoprecipitation(Ch IP)experiment,and the mechanism of IDO1 up-regulation of ANGPTL4 expression by inhibiting Notch1/HES1 signal through non-enzymatic action,thus activating gp91phox to promote oxidative damage was discussed.5.In cardiomyocyte oxidative injury model,STAT1 phosphorylated agonist(2-NP)was used and treated with 1 m M butyrate to explore whether butyrate can inhibit IDO1/ANGPTL4 pathway by STAT1 and alleviate Ang Ⅱ-induced oxidative damage of cardiomyocytes.6.In cardiomyocyte oxidative injury model,AHR/SOCS3 signal was inhibited by small molecule drug CH-223191 and treated with 1 m M butyrate.Western blot and immunoprecipitation(IP)experiments were performed to verify whether butyrate induced IDO1 ubiquitination degradation by activating AHR/SOCS3 signal and thus played a protective role in cardiomyocyte oxidative damage.Results1.Low dose and long-term effects of AngⅡinduced left ventricular dilation,myocardial hypertrophy,cardiac insufficiency,and myocardial oxidative damage in mice;STAT1 and FAK phosphorylation increased,IDO1,ANGPTL4 and gp91 phox expression up-regulated,Notch1/HES1 pathway activity down-regulated;gut microbiota dysbiosis,community structure composition changed,and butyrate production related flora abundance and serum butyrate content decreased.2.In vitro Ang Ⅱ-induced cardiomyocyte oxidative damage model,administration of IDO1 inhibitor epacadostat significantly inhibited the expression of gp91 phox,and the oxidative injury of cardiomyocytes was alleviated.After treatment with fludarabine to inhibit STAT1 phosphorylation,IDO1 m RNA and protein expression decreased significantly.3.In vitro Ang Ⅱ-induced cardiomyocyte oxidative damage model,ANGPTL4expression increased,and inhibition of IDO1 could significantly reduce ANGPTL4expression;while knockdown of ANGPTL4 had no effect on IDO1 expression in AC16cardiomyocytes,but significantly reduced FAK phosphorylation and gp91 phox expression,and alleviated cardiomyocyte oxidative damage.4.In vitro Ang Ⅱ-induced cardiomyocyte oxidative damage model,cleaved Notch1 protein and HES1 m RNA expression were decreased,that is,the Notch1/HES1pathway was inhibited;however,the inhibitory effect of epacadostat on AngⅡ-induced IDO1 activation can restore the activity of Notch1/HES1 pathway,but kynurenine supplementation to improve the enzymatic function of IDO1,could not restore the inhibitory effect on Notch1/HES1 pathway.After activating Notch1/HES1 pathway with valproic acid,the expression of ANGPTL4 decreased.Ch IP experiment further confirmed that HES1 could directly bind to the promoter region of ANGPTL4 gene,and the enrichment of HES1 in the promoter region of ANGPTL4 gene in the AngⅡtreatment group was significantly lower than that in the control group.It was suggested that HES1 transcription inhibited ANGPTL4 expression.5.In vitro treatment with butyrate inhibited the phosphorylation of STAT1 and FAK,decreased the expression of IDO1/ANGPTL4/gp91 phox,increased the activity of Notch1/HES1 pathway,and inhibited the oxidative damage of cardiomyocytes induced by AngⅡ;activation of STAT1 phosphorylation with 2-NP reversed the protective effect of butyrate.6.In vitro butyrate treatment induced the increase of AHR/SOCS3 protein and IDO1 ubiquitination level in AC16 cardiomyocytes,and IDO1 protein level decreased,while CH-223191 was used to inhibited AHR/SOCS3 signal,IDO1 ubiquitination level decreased and its protein expression increased.Conclusion1.Long-term,low-dose of AngⅡstimulation causes myocardial oxidative damage and cardiac dysfunction,and the gut microbiota is disturbed,and butyrate and its related flora are reduced.2.Ang Ⅱ promotes the expression of IDO1 by activating STAT1,IDO1 inhibits Notch1/HES1 pathway through non-enzymatic action,to upregulate the expression of ANGPTL4,and thus activates gp91 phox to increase the production of ROS and promote the myocardial oxidative damage induced by Ang Ⅱ.3.In vitro supplementation of butyrate can reduce IDO1 expression by inhibiting STAT1 and inducing IDO1 ubiquitination degradation via activating AHR/SOCS3,thus alleviate Ang Ⅱ-induced cardiomyocyte oxidative damage.更多还原