Cyclic adenosine monophosphate (cAMP) plays a major role in normal and pathologic signaling in the heart. Phosphodiesterase 4 (PDE4) is a major PDE degrading cAMP in the heart. There are inconsistencies concerning the roles of the PDE4 isoforms 4B and 4D in regulation of cardiac function. Cardiac PDE4B overexpression is beneficial in remodeling and heart failure (HF), however, the effect of PDE4D and PDE4 inhibitor in HF remains unclear. We generated global and conditional cardiac-specific heterozygous PDE4D knockout mice and adeno-associated virus serotype 9-PDE4D overexpression to determine the role of PDE4D in cardiac hypertrophy and HF. PDE4D upregulation was observed in failing hearts from human and isoproterenol injection and TAC mice. In vitro, isoproterenol stimulation increased PDE4D expression via PKA but had no effect on PDE4B expression in cardiomyocytes. PDE4D overexpression per se induced oxidative stress, mitochondrial damage and cardiomyocyte hypertrophy by decreasing PINK1/Parkin-mediated mitophagy through inhibiting cAMP-PKA-CREB-Sirtuin1 (SIRT1) signaling pathway, while PDE4B overexpression did not affect CREB-SIRT1 pathway and mitophagy but exhibited a protective effect on isoproterenol-induced oxidative stress and hypertrophy in cardiomyocytes. PDE4D silencing or inhibition with PDE4 inhibitor roflumilast ameliorated isoproterenol-induced mitochondrial injury and cardiomyocyte hypertrophy. In vivo, ISO injection or TAC inhibited cardiac mitophagy and caused cardiac hypertrophy and HF, which were ameliorated by roflumilast or cardiac-specific PDE4D haploinsufficiency. Conversely, cardiac PDE4D overexpression suppressed cardiac mitophagy and abolished the protective effects of global PDE4D haploinsufficiency on TAC-induced cardiac hypertrophy and HF. In conclusion, these studies elucidate a novel mechanism by which sustained adrenergic stimulation contributes to cardiac hypertrophy and HF by increasing PDE4D via cAMP-PKA signaling, which in turn reduces cAMP-PKA activity, resulting in cardiomyocyte hypertrophy and mitochondrial injury via inhibition of CREB-SIRT1 signaling-mediated mitophagy. PDE4D inhibition may represent a novel therapeutic strategy for HF.

目的:建立缺血性脑卒中动物模型后,动态观察动物模型疾病发生后不同时间窗的证候,明确模型动物气虚证出现的时间窗.方法:首先依据临床意义相似性,将临床脑卒中及气虚证的诊断指标进行等效转化,建立动物模型诊断指标,再采用线栓法建立缺血性脑卒中动物模型后,动态采集动物气虚证诊断相关指标,对各阶段模型的气虚证特征数据进行分析、聚类以及检验.结果:动物模型气虚证诊断指标包括宏观表征、旷场、抓力及体质量.线栓法造模后模型组动物神经缺损严重程度评分均＞0分,大脑TTC染色可见模型组大脑出现明显白色梗死区域,脑卒中造模成功.疾病发生后第2、3周,模型组大鼠较假手术组体质量、抓力显著降低(P＜0.01),宏观表征见模型组大鼠毛发凌乱,精神不振,身体蜷缩,饮食饮水量减少,部分动物可见后肢颜色改变.术后第4周,模型组较假手术组体质量及抓力仍有显著降低(P＜0.05),但宏观表征可见模型组大鼠毛发稍凌乱,精神活跃,饮食饮水量较第2、3周模型组有所增加,但不及假手术组.结论:大脑中动脉线栓法制备的缺血性脑卒中大鼠模型术后第2、3周符合气虚证诊断,术后第4周模型组气虚表现减轻.

Ethnopharmacological relevance: Lonicerae japonicae flos (LJF) is a traditional Chinese medicine with the function of heat-clearing and detoxifying. And it is the sovereign drug in Si-Miao-Yong-An decoctions to treat heart failure in clinics. Though the pharmacological effects of LJF are reported widely, the protective effects of LJF on heart failure remain elusive. Emerging evidence exhibits that Myocardial fibrosis (MF) is a common pathological process in several cardiovascular diseases and is closely related to a prognosis of heart failure.Aim of the study: To explore the pharmacological effects of LJF and its main active ingredients Chlorogenic acid (CGA) on heart failure and the concrete mechanisms.Materials and methods: Isoproterenol hydrochloride (ISO) was used to induce MF in C57BL/6 mice. Transforming growth factor beta (TGFβ) induced neonatal rat-derived cardiac fibroblasts (CFs) were treated with different concentrations of CGA. The cardiac function was evaluated by echocardiography. Cardiac and CFs morphology were examined by pathological staining. RT-PCR and Western blot were used to detect the key regulators of the fibrosis regulatory pathway. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry.Results: (1) LJF significantly improved cardiac function in ISO-induced heart failure and cardiac fibrosis. The ejection fraction (EF) and fractional shortening (FS) in LJF (M) and the LJF (H) groups were increased compared with Model group. (2) CGA restrained CF migration and promoted apoptosis. (3) Both LJF and CGA significantly diminished the expression of Col 1, Col 3, α-SMA and inhibited the levels of p38 and TAK1.Conclusion: LJF and CGA could improve MF by inhibiting the TGFβ1-TAK1-p38 signaling pathway. These findings provided a mechanistic underpinning for the cardio protective association of LJF and CGA. More broadly, LJF and CGA may warrant further investigation as novel therapeutic targets in MF.

Psoriasis is a chronic inflammatory disease that affects approximately 1%–5% of the population worldwide. Considering frequent relapse, adverse drug reactions, and large costs of treatment, it is urgent to identify new medications for psoriasis. Keratinocytes play an essential role during psoriasis development, and they express high levels of β2-Adrenergic receptor (β2-AR), which increases intracellular cAMP levels when activated. Increased level of cAMP is associated with the inhibition of epidermal cell proliferation. In the present study, we observed the effect of salmeterol, a long-acting β2-AR agonist, on the proliferation and apoptosis of keratinocytes as well as imiquimod-induced psoriasis-like skin lesions in mice. As phosphodiesterase 4 (PDE4) inhibitors increases intracellular cAMP concentration by inhibiting its inactivation, we further explored the synergetic effect of a PDE4 inhibitor and salmeterol on psoriasis-like skin lesions in mice. Our results indicated that salmeterol effectively inhibited the proliferation of HaCaT cells induced by TNF-α and serum, and this effect was accompanied by significantly increased apoptosis and CREB phosphorylation, which were reversed by the PKA inhibitor, H89. Salmeterol ameliorated imiquimod-induced psoriasis-like skin lesions in mice, but salmeterol combined with a PDE4 inhibitor had no synergetic effect in improving skin lesions in mice. Of note, the synergistic effects of anti-proliferation and induction of apoptosis in HaCaT cells appeared by inhibiting ERK signaling. In summary, salmeterol, a long-acting β2-AR agonist, alleviates the severity of psoriasis via inhibiting the proliferation and promoting apoptosis of keratinocytes, partially by activating the cAMP/PKA signaling pathway.

Inflammation plays a central role in the development of heart failure. Prostaglandin E2 (PGE2) is a key mediator of the inflammatory process in the cardiovascular system. However, the role of PGE2 in heart failure is complex and controversial. A recent report suggested that PGE2 inhibits acute β adrenergic receptor (β-AR) stimulation-enhanced cardiac contractility. The aim of this study was to characterize the influence of PGE2 on chronic β-AR stimulation-induced heart failure. Male C57BL/6 J mice received isoproterenol (ISO) or vehicle for 4 weeks. PGE2 significantly reversed ISO-induced cardiac contractile dysfunction and remodeling. Mechanically, ventricular myocytes were found to be an important source of TGF-β1 in ISO-model and PGE2 ablated TGF-β1 synthesis in cardiomyocytes through inhibition of β-AR activated PKA-CREB signaling. Furthermore, PGE2 significantly suppressed TGF-β1-GRK2 crosstalk-induced pro-hypertrophy and pro-fibrotic signaling in cardiomyocytes and cardiac fibroblasts, respectively. Pharmacological inhibition of GRK2 also attenuated contractile dysfunction and cardiac hypertrophy and fibrosis in ISO-model. These studies elucidate a novel mechanism by which PGE2 reduces TGF-β1 synthesis and its downstream signaling in heart failure and identify PGE2 or TGF-β1-GRK2 crosstalk as plausible therapeutic targets for preventing or treating heart failure induced by chronic β-AR stimulation.

Ethnopharmacological relevance: Si-Miao-Yong-An decoction (SMYAD), a classical traditional Chinese medicine (TCM) formula, has been used to treat various cardiovascular diseases in clinics. Aim of the study: The aim of this study is to investigate the effective combinatorial components from SMYAD and its mechanism regarding the intervention on myocardial hypertrophy. Materials and methods: SMYAD constituents absorbed in rat plasma and heart were identified using UHPLC Q-Exactive-Orbitrap MS/MS. The identified constituents in SMYAD were further analyzed using ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. The effective constituents were identified using isoproterenol (ISO)-induced H9c2 cardiomyocyte hypertrophy, and neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid C (ICAC), angoroside C (AGDC), isochlorogenic acid A (ICAA), sweroside (SRD), and harpagide (HPD) in SMYAD extract were quantified by HPLC for compatibility. Finally, anti-hypertrophic activities of candidate effective combinatorial components, which were prepared according to the determined molar concentration ratio of effective constituents using reference substance solution, were analyzed using immunofluorescence staining and Quantitative real-time PCR. The expression levels of PI3K alpha, p-ERK, p-Akt, Akt, p-mTOR, mTOR and HIF-1 alpha were measured using Western blot. Results: 32 prototypes of SMYAD were identified from plasma and heart tissue of rat. Combining with ADMET prediction, 31 dominant constituents were focused. Based on HIF-1 pathway identified in preliminary result, 17 targets were focused, which were used to dock with 31 constituents. 27 constituents were therefore hit as the potential effective constituents of SMYAD in inhibiting myocardial hypertrophy. Bioactivity evaluation showed that NCA, CA, CCA, ICAC, AGDC, ICAA, SRD, and HPD significantly inhibited the increase of H9c2 cell surface area induced by ISO. Except for ICAA and AGDC, the remaining 6 effective constituents, showing a certain inhibitory effect on ISO-induced ANP mRNA overexpression at high and low concentrations, participated in compatibility based on the molar concentration ratio determined by HPLC. Effective combinatorial components composed of the 6 effective constituents (effective combinatorial components ABC) showed significant inhibitory effect on the increase of cell surface area, and the overexpression of ANP and beta-MHC mRNA in H9c2 cells induced by ISO. Moreover, effective combinatorial components ABC significantly inhibited the protein overexpressions of p-Akt, p-mTOR and HIF-1 alpha. Based on the results, we put forward the strategy of "Focusing constituents" and "Focusing targets" for the effective constituents research of TCM formula. Conclusion: Effective combinatorial components ABC composed of NCA, CA, CCA, ICAC, SRD and HPD from SMYAD inhibited ISO-induced cardiomyocyte hypertrophy and down-regulated expression of ANP and beta-MHC mRNA through the inactivation of Akt/mTOR/HIF-1 alpha pathway.

Chinese traditional medicine compound is the main form of Chinese medicine clinical application. The elucidation of the effective components of traditional Chinese medicine is one of the key scientific issues to promote the modernization of traditional Chinese medicine. At present, there are many research ideas on the effective components of traditional Chinese medicine compounds. By analyzing the current status and existing problems of existing research ideas, the author proposes a "double reduction network pharmacology"(2 R network pharmacology) research method based on "prediction of dominant components-potential target selection". Chemical components with good properties were selected by ADMET property prediction technology, and compared with the blood components and target organ components to determine the dominant components with potential therapeutic effect, that is "reducing constituents"; the potential core regulatory pathway of traditional Chinese medicine compound was enriched by RNA-Seq technology combined with network database, and then the target of traditional Chinese medicine compound was mined based on the signal pathway, that is "reducing targets". To improve the efficiency and accuracy of effective component screening, the network relationship of "component target" was established by the related technology of network pharmacology. The purpose of this study is to provide practical research ideas and methods for clarifying the effective components of traditional Chinese medicine, revealing the law of compatibility of traditional Chinese medicine and clarifying the target of drug action.

血小板生理病理性变化是造成心血管疾病主要因素之一,抗血小板的相关研究已经成为当前研究心血管疾病的热点.在血小板活化、聚集等过程中,与血栓和炎症反应息息相关,而且其调控机制复杂、药物的作用靶点繁多.临床上,中药的抗血小板作用显著,以及近年中药干预血小板活化与聚集的基础研究也取得了一定成果.文章中主要整理了血小板活化、聚集时常见的调控机制和传统的中药在以血小板为靶点时治疗心血管疾病的研究成果,其中主要为MAPK、PI3K、SFK等机制调控,调节血小板中药以川芎、人参、丹参等研究居多.

Chlorogenic acids (CGAs) have gained considerable attention as pervasive human dietary constituents with potential cardiovascular-preserving effects. The main sources include coffee, yerba mate, Eucommia ulmodies leaves, and Lonicerae Japonicae Flos. CGA consumption can reduce the risks of hypertension, atherosclerosis, heart failure, myocardial infarction, and other factors associated with cardiovascular risk, such as obesity and type 2 diabetes. This review recapitulates recent advances of CGAs in the cardiovascular-preserving effects, pharmacokinetics, sources, and safety. Emerging evidence indicates that CGAs exhibit circulatory guarding properties through the suppression of oxidative stress, leukocyte infiltration, platelet aggregation, platelet-leukocyte interactions, vascular remodeling, and apoptosis as well as the regulation of glucose and lipid metabolism and vasodilatory action in the cardiovascular system. CGAs exert these effects by acting on complex signaling networks, but the global mechanisms are still not clear. The oral bioavailability of CGA is poor, and there is a potential sensitization concern about CGA. The bioactive metabolites, systematic toxicity, and optimized structure are needed for further identification.

建立合理可重复的病证结合动物模型及规范客观的模型评价体系,对气虚血瘀型缺血性脑卒中的疾病研究及中医气虚血瘀证的证候研究都非常重要.笔者对目前国内外气虚血瘀型缺血性脑卒中的动物模型制作方法进行文献调查研究,汇总了目前该证型疾病动物模型的制作方法及相关评价检测方法,分析探讨各方法的利与弊,以便为气虚血瘀型缺血性脑卒中疾病的相关实验研究提供相关依据.

目的 探讨山奈酚和芒柄花黄素对缺氧损伤的H9c2心肌细胞超氧化物歧化酶(SOD)活性,丙二醛(MDA)含量和肌酸激西酶(CK)、乳酸脱氢酶(LDH)活性的影响.方法 将H9c2细胞分为对照组、缺氧模型组、阳性药夹竹桃组、山奈酚组、芒柄花黄素组,采用CCK8法检测细胞活力,采用试剂盒检测上清液中CK、LDH活性和细胞内SOD活性,MDA含量.结果 与正常组相比,模型组细胞活力显著降低(P＜0.01);山奈酚、夹竹桃和芒柄花黄素能升高模型组细胞活力(P＜0.01,P＜0.01,P＜0.05).与正常组相比,模型组CK、LDH释放量和MDA活性显著升高(P＜0.01),SOD活性下降(P＜0.05).山奈酚组CK、LDH释放量均明显低于模型组(P＜0.01),芒柄花黄素组CK、LDH释放量低于模型组(P＜0.01,P＜0.05).山奈酚与芒柄花黄素均能显著降低模型组MDA活性(P＜0.01),升高模型组SOD活性(P＜0.05).结论 山奈酚和芒柄花黄素能通过降低CK、LDH活性和MDA含量,升高SOD活性对缺氧损伤心肌细胞发挥保护作用.

Background: Diabetic cardiomyopathy (DCM) is a main cause of heart failure and death in diabetic patients. However, countermeasures to limit the development of this disease remain insufficient. Si-Miao-Yong-An decoction (SMYA), a Chinese herbal prescription, exhibits both lipid-lowering and cardiovascular preserving effects, and may have an effect on DCM management. Purpose: The current study is aimed to investigate the effects of SMYA on the cardiac function in diabetic mice and the underlying mechanisms involved. Methods: Streptozotocin-induced diabetic mice were fed intragastrically with SMYA every day for 15 weeks. Cardiac function was assessed by echocardiograph. Histopathological alterations in the heart were determined by hematoxylin/eosin, wheat germ agglutinin, Masson’s trichrome, Terminal dUTP nick end-labeling, Oil red O staining, and transmission electron microscopy. The potential involvements of GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α signaling pathways were investigated by western blot and/or immunohistochemical staining. Results: Treatment of diabetic mice with SMYA improved insulin sensitivity, and attenuated the increases of water consumption, food intake, blood glucose, and serum GLC. Furthermore, SMYA ameliorated cardiac systolic and diastolic functions, suppressed the myocardial hypertrophy, fibrosis, apoptosis, inflammation, and lipid accumulation as well as preserved the myofilaments arrangement and mitochondrial integrity. Finally, SMYA downregulated the expressions of GCGR, PGC-1α, PPARα and the phosphorylation of NF-κB, as well as upregulated the phosphorylation of AMPK in the hearts of diabetic mice. Conclusions: SMYA may ameliorate glucolipid metabolism and cardiac function through the regulation of GLC/AMPK/NF-κB and GLC/PPARα/PGC-1α signaling pathways in diabetic mice, suggesting that this prescription could provide a new source of drug candidates to protect against DCM.

溃疡性结肠炎(UC)是一种病因不明的直肠和结肠炎性疾病,临床表现为反复发作的腹泻、腹痛及黏液脓血便,又称为非特异性溃疡性结肠炎.选择合适的动物模型有助于进行UC的机制及治疗研究.UC造模方法众多,但因为模型制备手段、发病机制、持续时间等各不相同,文章就常用的葡聚糖硫酸钠(DSS)模型、三硝基苯磺酸(TNBS)-乙醇模型及免疫复合TNBS-乙醇模型进行介绍对比,以期为动物模型的合适选择及继续完善提供新思路.

目的 探讨中药防治化疗药物心脏毒性的一般规律.方法 检索1988年1月-2017年10月收录于中国期刊全文数据库(CNKI)的中药干预化疗药引起心脏毒性的相关文献,按照制定的纳入和排除标准筛选文献,采用频数统计的方法,归纳中药干预化疗药物心脏毒性的一般规律.结果 纳入文献279篇,包括中药126味,使用频次前10位为人参、黄芪、麦冬、丹参、甘草、五味子、附子、党参、三七、川芎;中药制剂剂型中注射剂和汤剂居多;常用中药组合为人参-麦冬、人参-麦冬-五味子、人参-黄芪、人参-附子、桂枝-甘草;根据中药的功效进行分类,功效频次前3位为补虚药(462次)、活血化瘀药(97次)、清热药(53次).结论 中药干预化疗药所致心脏毒性的治则以补虚为主,常用补虚、活血、清热、温里等药物.

目的 从疾病和证候2个维度建立溃疡性结肠炎(ulcerative colitis,UC)大鼠脾虚湿蕴证辨识方法,为中药药效评价及证候基础研究提供适宜动物模型.方法 将40只SD大鼠随机分为4周及8周的对照组与模型组,每组10只.采用免疫复合法造模,多时间点采集比较大鼠的宏观表征、抓力、毛发、旷场实验等方面数据,并分别于造模后28、56 d取材,HE染色分析结肠病理变化.将溃疡性结肠炎临床常见证候诊断标准等效转化,用于大鼠证候辨识.结果 在造模后1～3 d,模型组大鼠不欲饮食,体重下降明显,腹泻便血,抓取反抗变强,但活跃度、反应能力、自主活动、精神状态均正常,毛发较暗淡凌乱.4 d～2周,模型组大鼠表现为稀便黏附肛门,宏观表征、抓力等与对照组基本无差异.在3～4周,模型组大鼠排稀便,饮食量及抓力降低(P<0.05),毛发暗淡凌乱,较对照组活跃度降低,抓取反抗及反应能力减弱,病理符合疾病诊断.5～8周,模型组各指标逐渐恢复正常.结论 免疫复合法造模后3～4周模型动物符合溃疡性结肠炎脾虚湿蕴证.

Background: Myocardial fibrosis is an important pathological feature of pressure overload cardiac remodeling. Si-Miao-Yong-An decoction (SMYAD), a traditional Chinese formula, is now clinically used in the treatment of cardiovascular diseases in China. However, its mechanisms in the prevention of heart failure are not fully revealed. Purpose: To determine whether treatment with SMYAD for 4 weeks would lead to changes in collagen metabolism and ventricular remodeling in a mice model of heart failure. Methods: Mice were subjected to transverse aorta constriction to generate pressure overload induced cardiac remodeling and then were administered SMYAD (14.85 g/kg/day) or captopril (16.5 mg/kg/day) intragastrically for 4 weeks after surgery. Echocardiography and immunohistochemical examination were used to evaluate the effects of SMYAD. The mRNA of collagen metabolism biomarkers were detected. Protein expression of TGF-β1/Smad and TGF-β1/TAK1/p38 pathway were assessed by Western blot. Results: SMYAD significantly improved cardiac function, increased left ventricle ejection fraction, and decreased fibrosis area and αSMA expression. Moreover, SMYAD reduced proteins expression related to collagen metabolism, including Col1, Col3, TIMP2 and CTGF. The increased levels of TGF-β1, Smad2, and Smad3 phosphorylation were attenuated in SMYAD group. In addition, SMYAD reduced the levels of TGF-β1, p-TAK1 and p-p38 compared with TAC group. Conclusions: SMYAD improved cardiac fibrosis and heart failure by inhibition of TGF-β1/Smad and TGF-β1/TAK1/p38 pathway. SMYAD protected against cardiac fibrosis and maintained collagen metabolism balance by regulating MMP-TIMP expression. Taken together, these results indicate that SMYAD might be a promising therapeutic agent against cardiac fibrosis.

目的:根据网络药理学方法寻找苓桂术甘汤治疗心血管疾病的潜在靶点.方法:从在线的中医药生物信息学分析网站(BATMAN-TCM)筛查和预见苓桂术甘汤干预心力衰竭的可能的有效成分及其作用于心力衰竭的靶点.使用Cyto-scape软件建立苓桂术甘汤以及其中所含的单味药针对心力衰竭的靶点-成分网络图.采取韦恩图(Venny 2.1)进一步研究苓桂术甘汤中茯苓及甘草治疗心力衰竭的机制.结果:我们从结果里发现苓桂术甘汤在治疗心力衰竭方面有79个活性成分,有18个靶点与心力衰竭是相关的,且检索到的靶点和有效成分有紧密的关系,找到的18个靶点主要是:ACE;ADRA2A;ADRA2B等.甘草在苓桂术甘汤中的靶点数最多,占全方治疗心力衰竭总靶点数的94.4％.结论:苓桂术甘汤改善心力衰竭的机制可能与降低AngⅡ、抑制RAS的活性、调节交感神经系统等具有相关性,其中甘草在治疗心力衰竭方面是苓桂术甘汤中最突出药物.

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti-inflammatory, anti-oxidant and anti-apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)-induced heart failure mouse model, through mitigating the TNF-alpha-induced toxicity. We further used TNF-alpha-induced cardiac injury in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the underlying mechanisms. CGA pre-treatment could reverse TNF-alpha-induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF-kappa B/p65 and major mitogen-activated protein kinases (MAPKs) signalling pathways involved in TNF-alpha-induced apoptosis of hiPSC-CMs. Importantly, CGA can directly inhibit NI-kappa B signal by suppressing the phosphorylation of NF-kappa B/p65. As for the MAPKs, CGA suppressed the activity of only c-Jun N-terminal kinase (JNK), but enhanced extracellular signal-regulated kinasel/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF-kappa B and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.