Effects of diabetes and evening primrose oil treatment on responses of aorta,corpus cavernosum and mesenteric vasculature in rats

Diabetes causes endothelial dysfunction, with deleterious effects on nitric oxide (NO) mediated vasodilatation. However, in many vessels other local vasodilators such as endothelium-derived hyperpolarizing factor (EDHF), prostacyclin, epoxides or endocannabinoids are also important. Several of these factors may be derived from omega-6 essential fatty acids via arachidonate metabolism. Diabetes inhibits this pathway, a defect that may be bypassed by diets enriched with omega-6 gamma-linolenic acid-containing oils such as evening primrose oil (EPO). The aim was to examine the effects of preventive EPO treatment on endothelium-dependent and neurally mediated vasorelaxation. Diabetes was induced by streptozotocin in rats; duration was 8 weeks. Vascular responses were examined in vitro on thoracic aorta, corpus cavernosum and perfused mesenteric bed preparations. Diabetes caused 25% and 35% deficits, respectively, in aorta and corpus cavernosum NO-mediated endothelium-dependent relaxation to acetylcholine that were largely unaffected by EPO treatment. Moreover, a 44% reduction in maximum corpus cavernosum vasorelaxation to nitrergic nerve stimulation was not prevented by EPO. However, for the mesenteric vascular bed, a 29% diminution of responses to acetylcholine, mediated by both NO and EDHF, was 84% attenuated by EPO treatment. When the EDHF component was isolated during NO synthase inhibition, a 76% diabetic deficit was noted. This was completely prevented by EPO treatment, which also caused supernormal EDHF responses in nondiabetic rats. EPO treatment prevented the development of deficits in endothelium-dependent relaxation in diabetic rats. Effects were particularly marked on the resistance vessel EDHF system, which may have potential therapeutic relevance for diabetic microvascular complications.     

ATP-sensitive potassium channels are altered in ventricular myocytes from diabetic rats

Hypoxia-induced shortening of the action potential duration, attributed to activation of the ATP-sensitive potassium (K_ATP) channels, occurs to a much greater extent in ventricular cells from diabetic rats. This study examined whether the K_ATP channels are altered in streptozotocin-diabetic myocardium. In inside-out patches from ventricular myocytes (with symmetrical 140 mM [K+]), inward K_ATP currents (at potentials negative to the K+ reversal potential) were similar in amplitude in control and diabetic patches (slope conductances: 69 and 74 pS, respectively). However, outward single-channel currents were larger for channels from diabetic heart cells than from control cells (e.g., at +75 mV the diabetic channel currents were 3.7 ± 0.3 pA vs. 2.7 ± 0.1 pA for control currents, p < 0.05), due to reduced inward rectification of diabetic channel currents. There was no difference in open and closed times between control and diabetic channels. The IC₅₀ for ATP inhibition of the K_ATP channel single-channel currents was 11.4 M for control currents and 4.7 M for diabetic channel currents. Thus, the major difference found between K_ATP channels from control and diabetic hearts was the greater outward diabetic single-channel current, which may contribute to the enhanced sensitivity to hypoxia (or ischemia) in diabetic hearts.     

成年大鼠阴茎海绵体cajal间质细胞的分离、培养与鉴定

目的:探索成年大鼠阴茎海绵体内cajal间质细胞(ICCs)的分离、培养和鉴定方法,为进一步研究其在阴茎海绵体中的作用提供条件.方法:取大鼠阴茎海绵体组织,采用酶消化法分离细胞,差速贴壁法相对纯化ICCs,将纯化后的细胞悬液接种于DMEM培养基中进行培养.通过倒置显微镜下观察细胞贴壁和形态,并用c-Kit特异性抗体标记细胞,免疫荧光法鉴定ICCs.结果:培养24小时后ICCs贴壁良好,细胞形态学观察显示ICCs呈纺锤状,有两个或多的突起,免疫荧光检验可见ICCs呈c-Kit抗体染色阳性.结果:用酶消化法可成功分离和培养大鼠阴茎海绵体ICCs,大鼠海绵体组织内ICCs的生理学功能有待进一步研究.     

An in vitro study of corpus cavernosum and aorta from mice lacking the inducible nitric oxide synthase gene

Nitric oxide (NO), produced by NO-synthase (NOS), serves as an important vasodilator and inhibitory neurotransmitter. Inducible NOS (iNOS) is expressed in response to cytokine stimulation and is therefore not ordinarily present in healthy tissue. However, iNOS has been identified in certain organs, including the penis. The development of mice deficient in the iNOS gene (iNOS -/-) has provided a useful tool for the study of iNOS function. Therefore, an in vitro examination of vascular and nerve-mediated responses of corpus cavernosum (CC) and vascular responses of aorta from iNOS -/- mice and their wild-type controls was undertaken. Tissues were mounted in organ baths for agonist- and/or electrical field stimulation (EFS)-induced responses under isometric tension. CC from iNOS -/- mice developed increased sensitivity to phenylephrine (PE) and an increased maximum EFS-induced noradrenergic contraction of approximately 31%. Following PE precontraction, maximum relaxation to acetylcholine was reduced by approximately 39%; conversely, there was a 23% increase in relaxation to the NO-donor sodium nitroprusside. EFS-induced non-adrenergic, non-cholinergic (NANC) nerve-mediated relaxation was unaltered compared to control. Agonist-induced responses of aorta did not significantly differ between iNOS -/- and control mice. These results suggest that iNOS-derived NO may play a role in modulating erectile function and confirm that iNOS does not play a significant role in macrovascular function under normal physiological conditions.     

Antidiabetic effects of quercetin in streptozocin-induced diabetic rats

Effects of the intraperitoneal injection of quercetin in streptozocin-induced diabetic and normal rats were investigated and compared. Although quercetin had no effect on plasma glucose level of normal animals, it significantly and dose-dependently decreased the plasma glucose level of streptozocin-induced diabetic rats. Glucose tolerance tests of the diabetic animals approached those of normal rats, their plasma cholesterol and triglycerides were reduced significantly, while their hepatic glucokinase activity was significantly increased upon quercetin treatment. In normal rats, quercetin did not affect the glucose tolerance test, but resulted in an increase of plasma cholesterol and triglycerides and a decrease in hepatic glucokinase activity. No significant pathologic changes were noted in hepatocytes or kidney tubules and glomeruli, while the number of pancreatic islets significantly increased in both treated normal and diabetic groups. It is concluded that quercetin, a flavonoid with antioxidant properties brings about the regeneration of the pancreatic islets and probably increases insulin release in streptozocin-induced diabetic rats; thus exerting its beneficial antidiabetic effects. However, it may be of little value in normoglycemic animals.     

替米沙坦改善糖尿病大鼠肾脏功能机制研究

目的研究替米沙坦对糖尿病大鼠24 h尿蛋白、血肌酐、肌酐清除率(Ccr)和血清尿素氮(BUN)等相关代谢指标的影响,且应用基因芯片探讨替米沙坦改善肾功能的机制。方法 30只SD大鼠,其中随机选取10只为正常对照组(给予等体积生理盐水)。选用20只大鼠,采用STZ法制备糖尿病模型,而后将16只造模成功的糖尿病大鼠随机分为替米沙坦治疗组(给予10 mg/kg/d的替米沙坦,n=8)和糖尿病模型组(给予等体积生理盐水,n=8)。三组大鼠均连续灌胃12周。每4周测定大鼠空腹血糖(FBG)和体重。12周末测定大鼠24h尿蛋白、尿肌酐、血肌酐和BUN水平。12周末处死大鼠,取肾脏组织进行基因芯片实验,并运用real time PCR进行验证。结果糖尿病模型组24h尿蛋白(P<0.01)、血肌酐(P<0.05)和BUN(P<0.01)比对照组显著升高,Ccr较对照组显著降低(P<0.05)。替米沙坦能改善糖尿病大鼠24h尿蛋白、血肌酐、Ccr和BUN水平。基因芯片结果显示替米沙坦组较糖尿病模型组有1541个基因发生显著改变,其中554个上调,987个下调。基因富集分析显示这些差异表达基因集中在氧化磷酸化通路和PPAR通路。Real time PCR证实替米沙坦组较糖尿病模型组ATP合成酶β亚基(Atp5b)、细胞色素c氧化酶亚基VIc(Cox6c)和NADH脱氢酶(辅酶Q)铁硫蛋白3(Ndufs3)基因显著下调。结论替米沙坦能有效改善糖尿病大鼠肾脏功能。替米沙坦的肾脏改善作用可能是通过线粒体氧化磷酸化通路和PPAR-γ通路调节。     

Effects of chelator treatment on aorta and corpus cavernosum from diabetic rats.

Transition-metal catalyzed reactions contribute to oxidative stress, which has been implicated in the pathogenesis of diabetic complications. The aim was to evaluate the effects of treatment with the transition metal chelator trientine on endothelium-dependent relaxation of aorta and corpus cavernosum from streptozotocin-induced diabetes of 8 weeks duration in rats. Effects on cavernosum autonomic innervation were also examined. Diabetes caused a 30.1 +/- 3.8% reduction in maximum aorta endothelium-dependent relaxation to acetylcholine (ACh), which was markedly attenuated (72.7 +/- 10.6%) by trientine treatment. Reversal treatment (4 weeks untreated diabetes, 4 weeks trientine) did not effect endothelium-dependent relaxation compared with aortas from rats with 4 weeks of diabetes, however, there was a 22.5 +/- 6.2% improvement compared with 8 weeks of diabetes. Eight weeks of diabetes caused a 41.5 +/- 6.6% reduction in corpus cavernosum endothelium-dependent maximum relaxation to ACh that was 70.1 +/- 16.9% prevented by trientine. Cavernosum nonadrenergic, noncholinergic (NANC) nerve stimulation caused frequency-dependent relaxation to a maximum of 40.9 +/- 2.4%, which was reduced by diabetes to 24.2 +/- 2.1%. Trientine partially prevented this deficit, maximum relaxation being 31.9 +/- 2.3%. Thus, metal chelator treatment has beneficial effects on aorta and cavernosum endothelium-dependent relaxation and on cavernosum NANC innervation.     

丁咯地尔和α-硫辛酸对糖尿病大鼠肾功能的保护作用研究

目的：研究丁咯地尔和α-硫辛酸（ALA）对糖尿病（DM）大鼠肾功能的保护作用。方法：雄性SD大鼠用链脲佐菌素（60mg/kg）腹腔注射诱导DM。大鼠随机分为健康组（NC）、DM组（DMC）、DM＋ALA组（ALA组）、DM＋丁咯地尔组（丁咯地尔组）。ALA组和丁咯地尔组每天给予ALA100mg／kg灌胃和丁咯地尔（0．1g／kg）腹腔注射,NC组和DMC组大鼠给予等量生理盐水每日灌胃。干预4周后,测观察治疗后大鼠血糖、尿素氮、血肌苷,内生肌苷清除率、24小时尿蛋白排泄率,比色法检测血清抗氧化酶和丙二醛（MDA）含量。结果：①丁咯地尔和α-硫辛酸降低糖尿病大鼠的尿素氮、血肌苷、24h尿白蛋白排泄率、丙二醛,增加超氧化物歧化酶②两种药物联合用药具有协同作用。结论：丁咯地尔和ALA可能通过抑制机体氧化应激水平,减低早期DM大鼠肾脏损害。     

Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats

AimsThe effect of exercise training (ET) on vascular responsiveness in diabetes mellitus has been largely well studied. However, limited studies have investigated the effects of ET on functional responses of the corpus cavernosum (CC) in diabetic animals. Therefore, the aim of this study was to investigate whether prior ET prevents the impairment of erectile function in streptozotocin-induced diabetic rats.Main methodsRats were exercised for four weeks prior to the induction of diabetes, and then again for another 4 weeks thereafter. Concentration–response curves to acetylcholine, sodium nitroprusside, Y-27632, BAY 412272 and phenylephrine (PE) were obtained in CC. The excitatory and inhibitory effects of electrical-field stimulation were also evaluated.Key findingsPlasma SOD levels were markedly decreased in the sedentary diabetic group (D-SD) as compared to control sedentary animals (C-SD), approximately 53% (P < 0.05) and this reduction was restored in trained diabetic animals. Physical training restored the impairment of endothelium-dependent and -independent relaxation responses seen in the D-SD group. The potency values for Y-27632 in the CC were significantly reduced in the D-SD group, which was reversed by physical training. The impairment of electrical-field stimulation (EFS)-induced relaxation seen in the D-SD group was restored by physical training. On the other hand, both EFS-induced contractions and concentration–response curves to PE in cavernosal strips were not modified by either diabetes or physical training.SignificancePractice of regular physical exercise may be an important approach in preventing erectile dysfunction associated with diabetes mellitus by re-establishment of the balance between NO production and its inactivation.     

Comparison of amitriptyline metabolism in hepatocytes from streptozocin-induced diabetic rats and from non-diabetic rats

Biotransformation of amitriptyline (AMI) was studied at different intervals in freshly isolated hepatocytes from healthy or streptozocin-induced diabetic rats in order to investigate the influence of the diabetic state. Levels of free and conjugated AMI, demethylated and hydroxylated metabolites, were assessed by HPLC analysis. In hepatocytes isolated from diabetic rats, AMI was less completely metabolized and the demethylation reaction became more important than in non-diabetic rat hepatocytes. Although the proportions of hydroxylated metabolites decreased in diabetic rats, it always remained predominant. Furthermore, glucuronidation of metabolites was greater, especially for (Z)-10-hydroxynortriptyline in diabetic animals.     

Sodium molybdate improves the phagocytic function in alloxan-induced diabetic rats

Diabetes mellitus is a disease, which virtually affects all the systems in the body including the immune system. Bacterial infections are important causes of increased morbidity and mortality in diabetic patients. In the present study, we assessed the effect of molybdate on immune responses of diabetic rats. The phagocytic potency and nitroblue tetrazolium dye reduction capacity were found to be considerably lowered whereas soluble immune complex formation enhanced significantly in diabetic rats when compared with control rats. From our findings, it is suggested that dietary intake of molybdate may have a vital role in enhancing immune functions during diabetic mellitus.     

Oral glycine administration attenuates diabetic complications in streptozotocin-induced diabetic rats

Diabetes mellitus is a disease characterized by impaired glucose metabolism that leads to retinopathy, brain micro-infarcts and other complications. We have previously shown that oral glycine administration to diabetic rats inhibits non-enzymatic glycation of hemoglobin and diminishes renal damage. In this work, we evaluated the capacity of the amino acid glycine (1% w/v, 130 mM) to attenuate diabetic complications in streptozotocin (STZ)-induced diabetic Wistar rats and compared them with non-treated or taurine-treated (0.5% w/v, 40 mM) diabetic rats. Glycine-treated diabetic rats showed an important diminution in the percentage of animals with opacity in lens and microaneurysms in the eyes. Interestingly, there was a diminished expression of O-acetyl sialic acid in brain vessels compared with untreated diabetic rats (P<0.05). Additionally, peripheral blood mononuclear cells isolated from glycine-treated diabetic rats showed a better proliferative response to PHA or ConA than those obtained from non-treated diabetic rats (P<0.05). Glycine-treated rats had a less intense corporal weight loss in comparison with non-treated animals. Our results suggest that administration of glycine attenuates the diabetic complications in the STZ-induced diabetic rat model, probably due to inhibition of the non-enzymatic glycation process.     

Hypoglycemic benefit and potential mechanism of a polysaccharide from Hericium erinaceus in streptozotoxin-induced diabetic rats

In this study, the hypoglycemic effect and possible mechanism of a polysaccharide, HEP-C, isolated from the fruit body of Hericium erinaceus were evaluated in streptozotoxin (STZ)-induced diabetic rats. Compared with the untreated STZ-induced diabetic rats, the supplements with HEP-C (150 and 300 mg/kg body weight [BW]) could significantly and dose-dependently relieve BW loss and organ injures, reduce fasting blood glucose, enhance glucose tolerance, alleviate hepatic function and serum lipid metabolism, elevate antioxidant enzyme activities, and suppress lipid peroxidation, which contributed to its potent hypoglycemic benefit. Liver histopathological observation revealed that HEP-C could effectively attenuate the deteriorated hepatic lesions in STZ-induced diabetic rats. HEP-C with potent hypoglycemic effect positively mediated glycogen synthesis by activating the phosphatidylinositol-3-kinase/protein kinase B signaling pathway. In summary, these results suggested that HEP-C, as a new dietary functional food or therapeutic agent, exhibited great potential for the prevention and treatment of diabetes mellitus and its complications.     

The antidiabetic effects of cysteinyl metformin, a newly synthesized agent, in alloxan- and streptozocin-induced diabetic rats

In this paper, the antidiabetic effects of cysteinyl metformin (CM), a newly synthesized agent, were investigated to evaluate the hypoglycemic/hypolipidemic effects by measuring blood glucose, triglyceride and insulin levels in CM- and metformin-treated diabetic rats. Two diabetic models were used: (1) an alloxan-induced model in which diabetes was produced by alloxan (200 mg/kg, i.p.), then rats were treated with CM (300, 100 and 33 mg/kg) for 14 days; (2) a streptozocin-induced model in which diabetes was produced by streptozocin (30 mg/kg, i.p.) and a sustained high lipid diet, then rats were treated with CM for 8 weeks. The hypoglycemic effect of CM exceeded that of metformin while the hypolipidemic effect was similar. In addition, CM increased the blood insulin level of the alloxan-induced experimental animals (which had an insulin deficiency), but reduced the insulin level of the streptozocin-induced animals (which had an insulin excess), suggesting that CM improves pancreatic beta-cell function. The effects of CM, metformin and cysteine on the antioxidant defense system in alloxan-induced rats were also studied. The serum malondialdehyde (MDA) level was determined to provide evidence for lipid peroxidation, All the groups of animals given CM, metformin and cysteine exhibited less severe oxidative stress than the diabetic group. Then, several key antioxidants such as superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) and the pancreatic exocrine enzyme amylase (AMS) were measured. CM restored the activity of all these agents to nearly normal values while metformin and cysteine merely restored the activity of SOD. At the end of our study, the animals were sacrificed by decapitation and the liver, kidney and pancreas were weighed to allow investigation of organ edema. The results obtained showed that CM corrected the organ edema of the diabetic rats. All these findings suggested that CM has a protective effect on the antioxidant defense system and beta-cell dysfunction in alloxan-induced diabetic rats. All these results suggest that CM is a potential candidate for the future treatment of both type 1 and type 2 diabetes.     

Effects of ghrelin on gastric distention sensitive neurons in the arcuate nucleus of hypothalamus and gastric motility in diabetic rats

This study was performed to observe the effects of ghrelin on the activity of gastric distention (GD) sensitive neurons in the arcuate nucleus of hypothalamus (Arc) and on gastric motility in vivo in streptozocin (STZ) induced diabetes mellitus (DM) rats. Electrophysiological results showed that ghrelin could excite GD-excitatory (GD-E) neurons and inhibit GD-inhibitory (GD-I) neurons in the Arc. However, fewer GD-E neurons were excited by ghrelin and the excitatory effect of ghrelin on GD-E neurons was much weaker in DM rats. Gastric motility research in vivo showed that microinjection of ghrelin into the Arc could significantly promote gastric motility and it showed a dose-dependent manner. The effect of ghrelin promoting gastric motility in DM rats was weaker than that in normal rats. The effects induced by ghrelin could be blocked by growth hormone secretagogue receptor (GHSR) antagonist [d-Lys-3]-GHRP-6 or BIM28163. RIA and real-time PCR data showed that the levels of ghrelin in the plasma, stomach and ghrelin mRNA in the Arc increased at first but decreased later and the expression of GHSR-1a mRNA in the Arc maintained a low level in DM rats. The present findings indicate that ghrelin could regulate the activity of GD sensitive neurons and gastric motility via ghrelin receptors in the Arc. The reduced effects of promoting gastric motility induced by ghrelin could be connected with the decreased expression of ghrelin receptors in the Arc in diabetes. Our data provide new experimental evidence for the role of ghrelin in gastric motility disorder in diabetes.     

GLP-1 expression in von Ebner's gland of diabetic rats

GLP-1, a peptidergic endocrine hormone, which associate with appetite control, glucose homeostasis and satiety. It might play an important role in the gustatory system. We tried to investigate the expression of GLP-1 in von Ebner's gland of diabetic and control rats, and the ultrastructure changes on von Ebner's gland of diabetes rats. GLP-1 expression in von Ebner's gland was evaluated by immunohistochemistry. The number of GLP-1 positive cells in diabetic rat von Ebner's gland was significantly higher than that in normal controls. Electron micrographs showed that a series of pathologic changes in von Ebner's gland of diabetes rats. The results suggest that GLP-1 have some effects within the gustatory systems, and elevated von Ebner's gland GLP-1 expression may be one cause of diabetic taste impairment.     

格列本脲对大鼠皮层扩散性抑制中脑血管的调节作用

皮层扩散性抑制(corticalspreadingdepression,CSD)是研究偏头痛、脑梗塞等疾病的重要病理模型.已有研究表明,在普遍被观察到的CSD过程中软脑膜动脉血管大幅度舒张之前,还存在一个较小幅度的软脑膜动脉扩张和收缩.但其中的脑血管调节机制尚不清楚.采用550nm的内源信号光学成像(opticalintrinsicsignalimaging,OISI)监测ATP敏感钾离子通道(ATP-sensitivepotassiumchannels,KATP)的阻断剂格列本脲(glibenclamide,glyb),对针刺诱导的大鼠CSD过程中软脑膜动脉血管舒缩过程的影响.实验中观测到软脑膜血管的初始小收缩(initialslightconstriction,ISC)相对于对照组显著减弱,其中应用10μmol/Lglyb时,74.5%的ISC被完全抑制,100μmol/Lglyb时则有96.2%的ISC完全消失.相对于CSD发生前,软脑膜动脉血管大幅舒张(largedilation,LD)的峰值也分别显著增强了(53.8±19.3)%和(59.8±19.6)%.结果表明,可能是神经元上的KATP在CSD过程中被glyb阻断从而抑制了软脑膜动脉血管的收缩.     

17beta-Estradiol and/or progesterone protect from insulin resistance in STZ-induced diabetic rats

Recent clinical and experimental evidences suggest that sex steroids protect from insulin resistance associated with diabetes. Therefore, we have assessed the influence of E2 and/or P4 on insulin sensitivity by euglicaemic-hyperinsulinaemic clamp in ovariectomized streptozotocin-induced diabetic rats, focusing on key proteins of insulin signaling in skeletal muscle. Although low plasma levels of E2 (days 6 and 11) increased Glut-4 plasma membrane content and subsequent improved insulin sensitivity, they could not fully reverse hyperglycaemia negative effects on p85alpha-IRS-1 association and IRS-1 content during 11 days. However, high plasma levels of E2 (day 16) could reverse hyperglycaemia effects not only on Glut-4 plasma membrane content but also on p85alpha-IRS-1 association and IRS-1 protein content level. In contrast, P4 treatment only improved insulin sensitivity when its plasma concentration was low (days 6 and 11) and its effects were not associated with any proteins study in this paper. The combined therapy had a synergic effect on insulin sensitivity when their plasma levels were low (day 6) or high (day 16), that could be associated with Glut-4 plasma membrane content modulation, p85alpha-IRS-1 association and IRS-1 amount. These new findings improve our understanding of biochemical basis of insulin resistance due to hyperglycaemia and could open up new possibilities of treatment in uncontrolled type 1 DM.     

中医病证相符的糖尿病慢性皮肤溃疡大鼠造模方法初探

目的观察不同造模方法致糖尿病大鼠慢性皮肤溃疡创面形态及愈合时间的影响。方法 50只SD大鼠随机分为5组:皮肤缺损组(QS组:剪皮),糖尿病组(DM组:STZ+剪皮),糖尿病加金黄色葡萄球菌组(DMJJ组:STZ+剪皮+金葡菌),糖尿病加激素组(DMJS组:STZ+剪皮+激素注射),糖尿病加激素加异物组(DMYW组:STZ+剪皮+激素注射+异物埋置)。糖尿病模型稳定后每周测量血糖1次,每日称量体重、观察疮面情况、测量创面面积。12 d后处死,石蜡包埋肉芽组织观察其组织病理形态。结果 DMJJ组前5d愈合速度快于其余组(P0.01);DMYW组的愈合时间延长,DMYW组愈合率显著偏低,与其余组比较有统计学意义(P0.01)。造模12 d其余组愈合率无统计学差异,DMYW组愈合率显著低于其余组(P0.01)。结论注射激素大鼠表现出中医"阴证"证型特点,糖尿病加激素注射加异物埋置复合因素造模法能使大鼠创面表现出与临床相似的"阴证"证型特点。