A novel endothelin receptor antagonist CPU0213 improves diabetic cardiac insufficiency attributed to up-regulation of the expression of FKBP12.6, SERCA2a, and PLB in rats

The depressed sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and Ca2+-release channels (ryanodine receptor RyR2) are involved in the diabetic cardiomyopathy. However, an implication of a down-regulation of FK506-binding protein or calstabin-2 (FKBP12.6) is undefined. It was hypothesized that the down-regulation of FKBP12.6 and SERCA2a of the intracellular calcium handling system is closely related to an up-regulated endothelin (ET) system. An ET receptor antagonist CPU0213 is newly discovered and expected to ameliorate cardiac insufficiency which is mediated by the depressed FKBP12.6 and SERCA2a in diabetic rat heart. Diabetes was developed in male Sprague-Dawley rats 8 weeks after an injection of streptozotocin (60 mg/kg IP), and CPU0213 was instituted 30 mg/kg, SC in the last 4 weeks. The assessment of the cardiac function, cardiac calcium handling proteins, endothelin system, and redox enzyme system were conducted. The compromised cardiac function in diabetic rats was accompanied by a significant down-regulation of expression of FKBP12.6 as well as SERCA2a and phospholamban. These were closely linked with an increased ET-1 and up-regulation of endothelin converting enzyme, PropreET1, and inducible nitric oxide synthase mRNA in diabetic cardiomyopathy. After 4-week treatment, CPU0213 was capable to attenuate completely the down-regulated FKBP12.6 and SERCA2a, and up-regulated ET system in association with a recovery of the cardiac insufficiency of diabetic cardiomyopathy.     

Down-regulation of FKBP12.6 and SERCA2a contributes to acute heart failure in septic shock and is related to an up-regulated endothelin signalling pathway

Acute heart failure (AHF) critically affects morbidity and mortality in patients suffering from septic shock. It is hypothesized that AHF is linked to down-regulation of FKBP12.6 (calstabin 2) and SERCA2a (sarco/endoplasmic reticulum Ca2+ ATPase 2a), which may be mediated by an activated endothelin (ET) system in the myocardium. The aim of the study was to test whether an attenuation of septic AHF can be achieved by a novel dual endothelin receptor antagonist, CPU0213, in association with up-regulation of FKBP12.6 and SERCA2a in rats. AHF in septic shock was produced by faeces leak from a surgically punctured caecum for 72 h in rats. CPU0213 (30 mg kg(-1), s.c., every 12 h, for 3 days) was administered to rats 8 h after the operation. In the untreated model group, survival rate markedly decreased (P < 0.01), and the cardiac performance was seriously compromised (P < 0.01) relative to control. The AHF was characteristically associated with down-regulated mRNA and protein expressions of FKBP12.6, SERCA2a and PLB (phospholamban). Elevated ET-1 and mRNA abundances of the preproET-1, ECE (endothelin converting enzyme) and ET(A) and ET(B) receptors in the left ventricular tissue (P < 0.01) were found. All abnormalities were reversed significantly following CPU0213 administration. In conclusion, septic AHF is attributed to down-regulation of FKBP12.6 and SERCA2a, which is related to an activated ET system. An endothelin receptor antagonism of CPU0213 significantly improves the cardiac performance by blocking both ET(A) and ET(B) receptors.     

六味地黄汤对糖尿病大鼠心肌病变的影响

目的 通过研究六味地黄汤对糖尿病大鼠心肌细胞病理变化的影响,为其治疗糖尿病心脏并发症的药理作用提供实验依据.方法 随机将大鼠分为正常组、糖尿病组、六味地黄汤低、中、高剂量组(100、200和400mg.·kg-1·d-1)和罗格列酮组(100 mg·kg-1·d-1).除正常组外,其余各组采用腹腔注射链脲佐菌素法诱发糖尿病大鼠模型,造模成功后饲养4周,然后连续灌胃给药4周.于末次给药12 h后取材,观察大鼠心肌细胞的形态学变化及细胞凋亡指数.结果 与罗格列酮干预组相比,高剂量六味地黄汤能显著抑制糖尿病大鼠心肌细胞的形态学病变及细胞凋亡(P＜0.01),中剂量组亦具有一定抑制作用.结论 六味地黄汤对糖尿病心肌细胞的病理变化具有一定的抑制作用,可治疗该并发症.     

Sildenafil and FDP-Sr attenuate diabetic cardiomyopathy by suppressing abnormal expression of myocardial CASQ2, FKBP12.6, and SERCA2a in rats

Aim:

To study whether calcium-modulating proteins CASQ2, FKBP12.6 and SERCA2a participate in diabetic cardiomyopathy, and whether the beneficial actions of testosterone, sildenafil or fructose diphosphate Sr (FDP-Sr) in the treatment of diabetic cardiomyopathy result from suppressing these molecules.

Methods:

Fifty male Sprague-Dawley (SD) rats were divided into five groups. Except for the normal group (non-diabetic), the other four groups were injected with streptozotocin (STZ, 60 mg/kg, ip) to induce diabetes. Four weeks after STZ injection, the four groups received sildenafil (12 mg·kg^-1·d^-1, ig, for 4 week), FDP-Sr (200 mg/kg, ig, for 4 week), testosterone propionate (4 mg·kg^-1·d^-1, sc, for 4 week), or no treatment, respectively.

Results:

In the diabetic rats, blood glucose, free fatty acids, triglycerides, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) were significantly increased, while high-density lipoprotein cholesterol (HDL-C) was significantly reduced, as compared to the non-diabetic rats. Cardiac dysfunction and myocardial hypertrophy of the diabetic rats were associated with increased mRNA and protein expression of iNOS, OBRb, and PKCɛ, while expression of CASQ2, SERCA2a, and FKBP12.6 was significantly down-regulated. Sildenafil and FDP-Sr, but not testosterone, significantly attenuated the biomarker abnormalities, without changing the metabolic abnormalities.

Conclusion:

CASQ2, FKBP12.6 and SERCA2a were down-regulated in diabetic cardiomyopathy. Sildenafil and FDP-Sr, but not testosterone, attenuated the cardiac dysfunction in diabetic cardiomyopathy, without changing the metabolic abnormalities, which may results from inhibiting oxidative and inflammatory cytokines and improving calcium homeostasis.     

CPU228, a derivative of dofetilide,relieves cardiac dysfunction by normalizing FKBP12.6, NADPH oxidase and protein kinase C ε in the myocardium

Objectives The aim of this study was to determine if CPU228, a derivative of dofetilide, is more effective than dofetilide in attenuating isoproterenol‐induced heart failure by recovering downregulated FK506 binding protein (FKBP12.6), and suppressing oxidative stress, upregulated NADPH oxidase and protein kinase C ε (PKCε) hyperphosphorylation in the myocardium. Methods Heart failure was induced by isoproterenol (1 mg/kg s.c. for 5 days) in male Sprague‐Dawley rats. Intervention with either CPU228 or dofetilide (2 mg/kg on Days 3–5) was then conducted in vivo and in vitro. Key findings Isoproterenol produced compromised left ventricular systolic pressure, left ventricular pressure rise (dp/dt_max) and fall (dp/dt_min), and left ventricular end‐diastolic pressure, associated with oxidative stress, abnormal FKBP12.6, NADPH oxidase p67phox and PKCε in the myocardium. CPU228 was more effective in attenuating these changes than dofetilide in vivo. Dofetilide produced a prolonged QTc to replace a shortened one. In primary neonatal cardiomyocytes, cultured with isoproterenol and treated with either CPU228 or dofetilide at 10^?8, 10^?7 and 10^?6 mol/l, isoproterenol produced a hyperadrenergic state characterized by downregulated FKBP12.6, upregulated NADPH oxidase p67phox and PKCε in vitro. CPU228 was more effective than dofetilide in recovering these changes in a dose‐dependent manner without a prolonged QTc. Conclusions CPU228 was more effective than dofetilide in attenuating heart failure by normalizing isoproterenol‐induced changes, including downregulation of FKBP12.6, upregulation of NADPH oxidase and PKCε hyperphosphorylation in vivo and in vitro.     

Effect of the endothelin receptor antagonist CPU0213, and its modulation by rifampin, on cardiac and vascular tissue following chronic isoproterenol treatment

1. The aim of the present study was to investigate the effects of the endothelin (ET) receptor antagonist CPU0213 on cardiac and vascular tissues after impairment by chronic isoproterenol treatment. Because rifampin reduces plasma concentrations of CPU0213, the modulation of the effects of CPU0213 by rifampin was also investigated. 2. Thirty rats were randomly divided into five groups as follows: (i) control; (ii) isoproterenol treated (1 mg/kg, s.c., for 10 days); (iii) isoproterenol treated with a single injection of CPU0213 (30 mg/kg, s.c., on Day 11); (iv) isoproterenol treated with a single injection of rifampin (50 mg/kg, p.o., on Day 11); and (v) isoproterenol treated with rifampin gvien 3 h before CPU0213 on Day 11. Serum concentrations of CPU0213, haemodynamic and biochemical parameters, mRNA and protein expression levels of the ET(A) receptor (ET(A)R), calstabin 2 (FKBP12.6) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), and vasoactivity of the thoracic aorta were determined. 3. Haemodynamic parameters, serum creatine phosphokinase, lactate dehydrogenase and malondialdehyde levels, mRNA and protein expression of FKBP12.6, SERCA2a and vascular responses were altered following isoproterenol treatment for 10 days. These effects were significantly reversed by CPU0213. Rifampin caused a reduction in serum concentrations of CPU0213 to 36% of control values. However, this reduction in the serum concentrations of CPU0213 did not affect its effects on the heart, but did eliminate its beneficial action on vascular responses. Rifampin alone had no effect these paramters. 4. The data suggest that isoproterenol acts on the myocardium to cause cardiac insufficiency by upregulating ET(A)R and downregulating FKBP12.6 and SERCA2a. These effects were ameliorated by CPU0213, but were resistant to rifampin-induced decreases in plasma CPU0213 concentrations. In vascular tissue, the pathological effects of isoproterenol were ameliorated by CPU0213; however, lowering plasma CPU0213 concentrations with rifampin did partly eliminate the amelioration in vascular activity in respones to CPU0213.     

Abrupt changes in FKBP12.6 and SERCA2a expression contribute to sudden occurrence of ventricular fibrillation on reperfusion and are prevented by CPU86017

Aim： The occurrence of ventricular fibrillation （VF） is dependent on the deterioration of channelopathy in the myocardium. It is interesting to investigate molecular changes in relation to abrupt appearance of VF on reperfusion. We aimed to study whether changes in the expression of FKBP12.6 and SERCA2a and the endothelin （ET） system on reperfusion against ischemia were related to the rapid occurrence of VF and whether CPU86017, a class III antiarrhythmic agent which blocks Ikr.IKs. and ICa.L, suppressed VF by correcting the molecular changes on reperfusion. Methods： Cardiomyopathy （CM） was produced by 0.4 mg/kg sc L-thyroxin for 10 d in rats, and subjected to 10 min coronary artery ligation/reperfusion on d 11. Expressions of the Ca^2＋ handling and ET system and calcium transients were conducted and CPU86017 was injected （4 mg/kg, sc） on d 6-10. Results： A high incidence of VF was found on reperfusion of the rat CM hearts, but there was no VF before reperfusion. The elevation of diastolic calcium was significant in the CM myocytes and exhibited abnormality of the Ca^2＋ handling system. The rapid downregulation of mRNA and the protein expression of FKBP12.6 and SERCA2a were found on reperfusion in association with the upregulation of the expression of the endothelin-converting enzyme 0ECE） and protein kinase A （PKA）, in contrast, no change in the ryanodine type 2 receptor （RyR2）, phospholamban （PLB）, endothelin A receptor （ETAR）, and iNOS was found. CPU86017 removed these changes and suppressed VE Conclusion： Abrupt changes in the expression of FKBP12.6, SERCA2a, PKA, and ECE on reperfusion against ischemia, which are responsible for the rapid occurrence of VF, have been observed. These changes are effectively prevented by CPU86017.     

基于网络药理学和分子对接探讨六味地黄丸治疗糖尿病肾病的作用机制研究

目的 运用网络药理学和分子对接方法探讨六味地黄丸治疗糖尿病肾病（diabetic nephropathy，DN））的作用机制。方法 通过检索多个数据库及查阅文献筛选出六味地黄丸活性成分和对应靶点，借助Genecard、OMIM和Drugbank数据库收集DN作用靶点并通过Venny 2.1软件筛选出药物-疾病共同作用靶点。随后，利用STRING和Cytoscape软件分析和构建蛋白质-蛋白质相互作用网络并使用CytoNCA插件进行拓扑分析刷选出核心靶点。然后，利用ClueGO插件进行GO （GO Ontology）功能富集分析和KEGG-（Kyoto Encyclopedia of Genes and Genomes）通路分析。最后，使用AutoDock软件通过分子对接验证活性化合物与核心靶点的结合能力。结果 六味地黄丸40个活性成分通过调控128个DN相关靶点，尤其是通过调控28个核心靶点，参与调控体内基因转录、细胞凋亡和增殖、信号转导、炎症反应及蛋白质磷酸化等生物学过程，干预肿瘤、AGE-RAGE、IL-17、MAPK、HIF-1、Toll样受体等信号通路发挥治疗DN的作用。分子对接验证了5种活性化合物与其作用的关键靶点具有可靠的亲和力。结论 推测六味地黄丸治疗DN具有多成分-多靶点-多途径的特点，为进一步研究六味地黄丸治疗DN的分子机制提供了新思路和新方向。     

Protective effect of Liuwei Dihuang decoction on early diabetic nephropathy induced by streptozotocin via modulating ET-ROS axis and matrix metalloproteinase activity in rats

We aimed to investigate the effects of Liuwei Dihuang decoction (LW) on the endothelin-1-reactive oxidative species (ET-ROS) system and matrix metalloproteinases (MMPs) in the early diabetic nephropathy induced by streptozotocin (STZ) in rats. Rats were divided into six groups as follows: the control group, the untreated model group, the treated groups with the LW (5, 10 and 15 g kg(-1), p.o.) and the aminoguanidine-treated group (100 mg kg(-1), orally). The treatment was performed for 4 weeks, beginning on the fifth week after one intraperitoneal injection of STZ (65 mg kg(-1)). In the untreated model group, increased blood glucose, decreased plasma insulin level and an impaired renal function were observed. There was an altered redox system shown by an increased malondialdehyde and decreased activity of glutathione peroxidase and superoxide dismutase in the renal cortex. An enhanced inducible nitric oxide synthetase, total nitric oxide synthase and constitutive nitric oxide synthase and a declined nitric oxide were found. An increased extracellular matrix was indicated by an abnormality of MMP-2 and MMP-9 activities and an increase in hydroxyproline. An up-regulated ET-1 level and increased mRNA expression of endothelin-converting enzyme, preproET-1 and ET( A) receptor were presented in the affected renal cortex, but no change in ET(B) receptor mRNA. The LW was most effective in reversing these changes in diabetic rats and was as effective as aminoguanidine. The benefits of the extracts in relieving the abnormalities in early diabetic nephropathy are likely to be mediated by suppression of the renal ET-ROS system and escalating the activity of MMPs.     

Upregulation of leptin pathway correlates with abnormal expression of SERCA2a,phospholamban and the endothelin pathway in heart failure and reversal by CPU86017

Emerging evidence indicates that leptin may be a potential new target in chronic heart failure (CHF) treatment. We hypothesized that hyperleptinemia may correlate with abnormal expression of SERCA2a, PLB (phospholamban), and the endothelin (ET) pathway in CHF. An activated ET pathway is involved in CHF that is suppressed by CPU86017 (p-chlorobenzyltetrahydroberberine chloride), a complex class III antiarrhythmic agent with an antioxidant effect. Thus, relief of CHF may be mediated by a reversal of abnormalities of the leptin system, the ET-reactive oxygen species (ROS) pathway, SERCA2a, and PLB by CPU86017. CHF was produced by coronary artery ligation for 6 weeks in rats. The rats were divided into 3 groups: sham, CHF untreated, and CHF+CPU86017 (4 mg/kg per day, s.c.). Hemodynamic changes, cardiac morphology, serum biochemistry, messenger ribonucleic acid (mRNA) and protein expression of the leptin pathway, ET pathway, and redox were measured. In CHF rats, hemodynamic abnormalities, cardiac remodeling, and histological changes with features of cardiac failure were associated with hyperlipidemia accompanied by oxidative stress and upregulated OB-Rb, ECE, pp-ET-1, ET(A)R, and ET(B)R mRNA expression in the myocardium. Protein expression of leptin and ET(A)R in the myocardium was markedly increased in CHF rats. An activated leptin pathway was associated with downregulation of SERCA2a and upregulation of PLB in mRNA and protein expression in CHF. CPU86017 downregulated the leptin system and reversed the above changes in the myocardium. An activated leptin pathway correlates with abnormal expression of SERCA2a and PLB and an activated ET-ROS system in the affected myocardium. The multi-ion-channel-blocking and antioxidative effects of CPU86017 downregulate the leptin pathway and ET system, resulting in reversal of the abnormalities of expression of SERCA2a and PLB and cardiac performance in CHF.     

Endothelin receptor antagonist CPU0213 and vitamin E reverse downregulation of FKBP12.6 and SERCA2a: a role of hyperphosphorylation of PKCepsilon

Downregulation of FKBP12.6 and sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) contributes to sudden cardiac death and heart failure. We aimed to test the hypothesis that (i) downregulation of FKBP12.6 and SERCA2a can be taken as molecular markers for drug interventions and (ii) such downregulation is produced by crosstalk between endothelin-reactive oxygen species and beta-adrenoceptors stimulation, mediated by hyperphosphorylation of protein kinase Cvarepsilon (PKCvarepsilon). Rat cardiomyocytes were incubated with isoproterenol (1 microM), endothelin-1 (0.1 microM) or hydrogen peroxide (10 microM) for 18 h, resulting in downregulation of mRNA and protein of FKBP12.6 and SERCA2a, as well as upregulation of PKCvarepsilon mRNA and phosphorylated PKCvarepsilon protein. These changes were reversed by an application of either propranolol (1 microM), endothelin receptor antagonist CPU0213 (1 microM) or vitamin E (1 microM). As indicated by the fluorescent dye Fluo3, diastolic [Ca(2+)](i) in rat ventricular myocytes was increased after incubation with isoproterenol (0.1 microM). The increased [Ca(2+)](i) in diastole was dramatically decreased by CPU0213. Thus, the downregulation of FKBP12.6 and SERCA2a, and hyperphosphorylation of PKCvarepsilon, appear to be related to crosstalk between over-activated endothelin-reactive oxygen species and a beta-adrenoceptor pathway. CPU0213 is beneficial in treating cardiac insufficiency and preventing cardiac arrhythmias possibly by normalizing hyperphosphorylation of PKCvarepsilon and abnormal FKBP12.6 and SERCA2a. The antioxidant activity of vitamin E was sufficient to normalize the levels of FKBP12.6 and SERCA2a and phosphorylation of PKCvarepsilon. Thus by testing with biomarkers FKBP12.6 and SERCA2a, we have shown that the endothelin receptor antagonist CPU0213 and the antioxidant vitamin E may relieve risk of lethal arrhythmias and heart failure by suppressing PKCvarepsilon.     

Depressed calcium-handling proteins due to endoplasmic reticulum stress and apoptosis in the diabetic heart are attenuated by argirein

Diabetic cardiomyopathy (DC) is a unique disease frequently complicated to diabetes mellitus, manifesting endoplasmic reticulum (ER) stress and depressed calcium-handling proteins. We hypothesized that the abnormal FKBP12.6, SERCA2a, and CASQ2 are consequent to ER stress and apoptosis that are likely due to an entity of inflammation. These abnormalities may be attributed to reactive oxygen species genesis from activated NADPH oxidase which could respond to argirein (AR) through its anti-inflammatory activity. Sprague Dawley rats were randomly divided into six groups. Except the normal group, rats were injected with streptozotocin (STZ; 60 mg/kg, i.p.) once. During weeks 5 to 8 following STZ injection, rats were treated (in milligrams per kilogram per day, i.g.) with aminoguanidine (AMG, 100; an inducible nitric oxide synthase and AGEs inhibitor) or three doses of AR (50, 100, and 200). FKBP12.6, SERCA2a, and CASQ2 and ER stress chaperones Bip and PERK and apoptotic molecules were monitored in vivo and in vitro. Impaired cardiac performance and downregulated FKBP12.6, SERCA2a, and CASQ2 were significant in DC in vivo, and abnormal calcium-handling proteins were also found in high-glucose-incubated myocytes in vitro. ER stress manifested by upregulated Bip and PERK was predominant in association with DNA ladder and upregulated Bax and downregulated BCL-2 in vivo and in vitro. AR is effective to attenuate these abnormalities compared to AMG. Diabetic myocardium has inflammatory entity expressed as ER stress contributing to downregulated calcium-handling proteins. AR has potential in managing DC through attenuating depressed calcium-handling proteins, activated ER stress, and apoptosis in the myocardium.     

Effects of Endothelin B Antagonist RES−701−1 on Endothelin-induced Contractile Responses In-vivo and In-vitro in Guinea-pigs

The effects of an endothelin (ET)-receptor B-specific antagonist, RES−701−1, on ET-induced contraction of guinea-pig trachea and on ET-induced bronchoconstriction in anaesthetized guinea-pigs were investigated. In the epithelium-removed tracheal preparation, 1 times 10⁻⁵ M RES−701−1 inhibited contractions induced by the ET_B-specific agonist sarafotoxin S6c (pK_B = 6ṁ10). In the epithelium-intact tracheal preparation, RES−701−1 (1 times 10⁻⁵ M) inhibited the ET−3−induced contraction (pK_B = 5ṁ27), but enhanced the ET−1−induced contraction significantly and shifted the concentration-response curve to the left. The maximal responses of ET−1- and ET−3−induced contraction were augmented by epithelium removal by 1ṁ5 and 1ṁ8−fold, respectively. Against ET−3−induced contraction in the tracheal preparation without epithelium, RES−701−1 (0ṁ3−10 times 10⁻⁶ M) antagonized the contraction in a concentration-dependent manner (pA₂ = 5ṁ9). On the other hand, RES−701−1 (1 times 10⁻⁵ M) did not affect ET−1−evoked responses in the trachea without epithelium. The intravenous administration of ET−1 (1ṁ5nmol kg⁻¹) or ET−3 (1ṁ5 nmol kg⁻¹) evoked a biphasic, fast and sustained bronchoconstriction in anaesthetized guinea-pigs pretreated with propranolol (1ṁ0 mg kg⁻¹). When administered intravenously, RES−701−1 (0ṁ3 or 1ṁ0 mg kg⁻¹) showed significant reduction in both phases of bronchoconstriction induced by ET−3. As in the case of ET−1−induced bronchoconstriction, RES−701−1 augmented the sustained phase although a significant reduction of the fast phase was observed. These results indicate that RES−701−1 can inhibit the ET−3−induced airway responses not only in-vitro but also in-vivo.     

六味地黄丸对顺铂诱导大鼠急性肾损伤的保护作用

目的评价六味地黄丸对顺铂诱导的大鼠肾脏损伤模型的保护作用及其作用机制。方法雄性Wistar大鼠随机分为对照组、模型组、六味地黄丸(0.54、1.08 g/kg)组。注射用顺铂7.5 mg/kg单次腹腔注射建立模型,六味地黄丸ig 10 d。采用肌氨酸氧化酶法检测各组血清中肌酐(Cr)、尿素氮(BUN)含量,采用WST-1法检测肾组织中超氧化物歧化酶(SOD),采用化学比色法检丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)水平,采用ELISA方法检测肾损伤因子-1(Kim-1)浓度。检测肾组织中核转录因子E2相关因子2(Nrf2)、醌氧化还原酶1(NQO1)、血氧合酶1(HO-1)、Kelch样环氧氯丙烷相关蛋白1(Keap1)的m RNA相对表达量。结果六味地黄丸可减轻顺铂诱导的急性肾损伤大鼠肾小管损伤。六味地黄丸(0.54、1.08 g/kg)组肾脏系数显著低于模型组(P0.05)。予六味地黄丸后,Cr、BUN明显低于模型组(P0.05)。Kim-1值明显降低(P0.05);SOD、GSH-Px活力显著升高,MDA含量明显下降(P0.05);Nrf2、NQO1、HO-1的m RNA相对表达量高于模型组,Keap-1的m RNA表达受到抑制,表达量低于模型组(P0.05),且1.08 g/kg较0.54 g/kg差异更明显(P0.05)。结论六味地黄丸对顺铂诱导的大鼠急性肾损伤有显著的保护作用,其机制与激活Nrf2通路有关。     

六味地黄汤及“补泻”药对对肾阴虚模型小鼠下丘脑-垂体-性腺轴功能的影响

目的 观察六味地黄汤及其3个“一补一泻”药对对肾阴虚模型小鼠下丘脑-垂体-性腺（HPG）轴功能及性腺组织形态的影响。方法 将60只小鼠随机分为空白对照组、模型组、六味地黄汤组、山茱萸-牡丹皮组、熟地黄-泽泻组、山药-茯苓组共6个试验组;连续9 d分别灌胃给予以上对应药物;第5~9天,除空白对照组灌胃蒸馏水外,其他各组按50 mg·kg^-1体质量每天灌胃氢化可的松注射液建立模型,用ELISA法检测血浆环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）含量,用放射免疫分析法检测促卵泡素（FSH）、雌二醇（E2）及睾酮（T）含量,并用HE染色法观察性腺（睾丸、子宫）病理切片。结果 与空白对照组比较,肾阴虚模型小鼠血浆cAMP含量升高,血浆cGMP及血清FSH、E2、T含量降低,有显著性差异（P<0.01）,性腺组织受损;与模型组比较,六味地黄汤组及山茱萸-牡丹皮药对组均能上调肾阴虚小鼠血浆cGMP及血清FSH、E2、T含量,下调血浆cAMP含量,有显著性差异（P < 0.05,P < 0.01）,修复性腺组织,其中山茱萸-牡丹皮药对药效略低于六味地黄汤,而熟地-泽泻药对及山药-茯苓药对对肾阴虚小鼠基本无影响。结论 六味地黄汤能改善肾阴虚证,并对其引起的HPG轴功能紊乱有调节作用,其中仅山茱萸-牡丹皮药对的作用更近似于全方,可能是全方改善肾阴虚证的主要药效组成部分;说明3个“一补一泻”药对的协同作用是六味地黄汤调节HPG轴功能的重要基础,从而产生了六味地黄汤的综合作用,体现了其全方配伍高度的科学内涵。     

Does Bosentan Protect Diabetic Brain Alterations in Rats? The Role of Endothelin‐1 in the Diabetic Brain

Diabetes mellitus (DM) is a major problem all over the world, affecting more people in recent years. Individuals with diabetes are more prone to disease than non‐diabetics, especially vascular complications. The aim of this study was to examine the roles of the endothelin (ET)‐1 in brain damage formed in a streptozocin (STZ)‐induced diabetes model, and the effect of bosentan, which is the non‐specific ET1 receptor blocker in the prevention of the diabetes‐induced brain damage. To examine the effects of bosentan (50 mg/kg and 100 mg/kg) in this study, the rats were given the drug for 3 months. The rats were divided into four groups: the sham group (n = 10), the diabetic control group (n = 10), the group of diabetic rats given bosentan 50 mg/kg (n = 10) and the group of diabetic rats given bosentan 100 mg/kg (n = 10). Diabetes was induced in the rats by STZ (60 mg/kg i.p.). On day 91, all rats were killed. Brain tissues of the rats were measured by molecular, biochemical and histopathological methods. Antioxidant levels in the therapy groups were observed as quite near to the values in the healthy group. In this study, while the brain eNOS levels in the diabetic groups decreased, the ET1 and iNOS levels were found to be increased. However, in the diabetes group, hippocampus and cerebellum, pericellular oedema and a number of neuronal cytoretraction were increased in neuropiles, whereas these results were decreased in the therapy group. Based on all of these results, ET1 will not be ignored in diabetes‐induced cerebral complications.     

六味地黄丸联合氯沙坦钾治疗早期糖尿病肾病的临床研究

目的观察六味地黄丸联合氯沙坦钾治疗早期糖尿病肾病的临床疗效。方法选择2010年12月—2014年8月滦平县中医院收治的早期糖尿病肾病患者100例,随机分为治疗组与对照组,每组50例。对照组口服氯沙坦钾片50 mg/次,1次/d。治疗组患者在对照组的基础上口服六味地黄丸,9 g/次,2次/d。两组均连续治疗8周。观察两组患者的临床疗效,同时比较两组患者收缩压、舒张压、平均动脉压、血肌酐(Scr)、血尿素氮(BUN)、尿微量蛋白、尿白蛋白排泄率(UAER)、C反应蛋白(CRP)的变化。结果治疗后,对照组和治疗组的总有效率分别为70.0%、92.0%,两组总有效率比较差异有统计学意义(P0.05)。治疗后,两组患者收缩压、舒张压、平均动脉压、Scr、BUN、尿微量蛋白、UAER、CRP均较治疗前显著降低,同组治疗前后差异有统计学意义(P0.05);且治疗后治疗组这些观察指标(除Scr、BUN外)的改善程度优于对照组,两组比较差异有统计学意义(P0.05)。结论六味地黄丸联合氯沙坦钾治疗早期糖尿病肾病具有较好的临床疗效,可改善肾功能,有效降低尿微量蛋白,具有一定的临床推广价值。     

羟苯磺酸钙对早期糖尿病肾病大鼠肾脏的保护作用

用腹腔注射链脲佐菌素（streptozotocin,STZ）的方法制备大鼠糖尿病模型,观察羟苯磺酸钙对大鼠早期糖尿病肾病的肾脏保护作用。雄性Wistar大鼠随机分为正常对照组、DN空白对照组、羟苯磺酸钙75、150及300 mg·kg^-1组、培哚普利0.4 mg·kg^-1组。给药8周。动态检测各组大鼠血糖、24 h尿白蛋白,末次给药后测定大鼠糖化血红蛋白（HbA1c）,用放射免疫法测定血浆及肾皮质内皮素（ET）含量,用免疫组化技术检测肾皮质纤溶酶原激活物抑制剂-1（PAI-1）、基质金属蛋白酶-9（MMP-9）的表达,光镜、电镜下观察肾脏病理形态的改变。结果显示：羟苯磺酸钙能降低早期DN大鼠24 h尿白蛋白及血浆和肾皮质ET水平,降低肾脏局部PAI-1表达,升高MMP-9表达。羟苯磺酸钙可以保护血管内皮,抑制肾脏纤维化,减轻肾脏病理损伤,从而保护大鼠肾脏功能。     

FKBP12 Depletion Leads to Loss of Sarcoplasmic Reticulum Ca2+ Stores in Rat Vas Deferens

The immunophilin 12-kDa FK506 binding protein (FKBP12) stabilizes intracellular Ca²⁺ release channel (CRC) activity in different tissues. In this work, the presence of FKBP12 in rat vas deferens (RVD) and its possible contribution to RVD function was investigated. Treatment under appropriate pH, temperature, and ionic conditions was used to strip FKBP12 from CRC binding sites; Western blotting revealed FKBP12 in control but not in treated homogenates. Disruption of the FKBP12-CRC complex in RVD decreased the Ca²⁺ content of sarcoplasmic reticulum (SR) by increasing Ca²⁺ leakage through the ryanodine receptor (RyR3 isoform) but not through 1,4,5-iNOSitol trisphosphate receptors (IP₃R1 and IP₃R3 isoforms). The decrease of SR Ca²⁺ content was not related to inhibition of SERCA ATPase. It seems that dissociation of FKBP12-RyR leads to conformational changes in RyR that make it difficult for ryanodine to access its binding site. Rapamycin, which is commonly used as a pharmacological tool to disrupt the FKBP12-RyR complex, decreased phenylephrine-induced contractions in RVD epididymal halves. The data suggest that FKBP12 is expressed in RVD in a labile association with RyR3. Disruption of the FKBP12-RyR3 complex may lead to modifications of RVD physiology and in consequence may compromise male fertility.     

AQP4 knockout mice manifest abnormal expressions of calcium handling proteins possibly due to exacerbating pro-inflammatory factors in the heart

We tested the hypothesis that aquaporin-4 (AQP4) knockout (KO) mice might exhibit abnormal Ca²⁺ modulating proteins resulting from the exacerbation of pro-inflammatory factors in the heart. Downregulation of FKBP12.6, SERCA2a, and CASQ2 and calcium leak in diastole have been recognized as endpoints for assessing cardiac failure and arrhythmias. The AQP4 KO mice and wild-type (WT) mice were randomly divided into 3 groups, such as control, isoproterenol (ISO, β-receptor agonist) injected (1 mg/kg, sc, 5 d), and treated with aminoguanidine (AMG, 100 mg/kg, po, a selective inhibitor of the iNOS) during the last 3 d. RT-PCR, western blot and calcium transient measurements were conducted. The results demonstrated that the cardiac weight index was increased in AQP4 KO mice and further increased following treatment with ISO. The expression levels of FKBP12.6, SERCA2a, and CASQ2 were downregulated and diastolic calcium concentrations were elevated in the AQP4 KO mice, indicative of a calcium leak. In the myocardium, expressions of pro-inflammatory biomarkers, including ET_A, pPKC?, NADPH oxidase p67^phox were upregulated and associated with downregulation of Cx43. The aforementioned changes were exacerbated in response to ISO medication and were attenuated by AMG; however, its treatment effectiveness was less in the AQP4 KO mice. We concluded AQP4 KO caused abnormalities of calcium modulating proteins leading to an exacerbation of risk for cardiac arrhythmias and failure. These changes are likely due to an increase in pro-inflammatory factors which are exacerbated by stress. Therefore, AQP4 KO mice are prone to cardiac failure and arrhythmias through exacerbating pro-inflammatory factors in the myocardium.