Dipyridamole inhibits lipopolysaccharide-induced cyclooxygenase-2 and monocyte chemoattractant protein-1 via heme oxygenase-1-mediated reactive oxygen species reduction in rat mesangial cells

Dipyridamole contributes to its beneficial effects on inflammatory responses in many cell types. The anti-inflammatory mechanisms of dipyridamole on glomerular mesangial cells are mostly uncharacterized. In this study, we monitored the influence of dipyridamole on the expression levels of cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (MCP-1) in rat mesangial cells stimulated with lipopolysaccharide. Dipyridamole was found to inhibit lipopolysaccharide-induced COX-2 and MCP-1 expression, and reduced lipopolysaccharide-induced reactive oxygen species generation in rat mesangial cells. This inhibitory effect of dipyridamole is independent on cyclic AMP and cyclic GMP increase. Tin protoporphyrin IX (SnPP), a heme oxygenase-1(HO-1) inhibitor, blocked the inhibitory effect of dipyridamole on lipopolysaccharide-induced COX-2 and MCP-1 expression. By applying specific inhibitors in rat mesangial cells, ERK1/2 and p38 MAPK signaling pathways were demonstrated to be involved in the lipopolysaccharide-induced inflammatory responses, and were inhibited by SnPP and N-acetylcysteine treatment. Additionally, dipyridamole was also found to upregulate HO-1 in rat mesangial cells. Therefore, our data suggest that dipyridamole inhibits the expression of COX-2 and MCP-1 in lipopolysaccharide-treated rat mesangial cells via HO-1-mediated reactive oxygen species reduction.     

Influence of bradykinin B1 and B2 receptors in the immune response triggered by renal ischemia-reperfusion injury

Bradykinin B1 receptors are exclusively expressed in inflamed tissues. For this reason, they have been related with the outcomes of several pathologies. Ischemia-reperfusion injury is caused by the activation of inflammatory and cytoprotective genes, such as macrophage chemoattractant protein-1 and heme oxygenase-1, respectively. This study was aimed to analyze the involvement of bradykinin B1 and B2 receptors (B1R and B2R) in tissue response after renal ischemia-reperfusion injury. For that, B1R (B1-/-), B2R (B2-/-) knockout animals and its control (wild-type mice, B1B2+/+) were subjected to renal bilateral ischemia, followed by 24, 48 and 120 h of reperfusion. At these time points, blood serum samples were collected for creatinine and urea dosages. Kidneys were harvested for histology and molecular analyses by real-time PCR. At 24 and 48 h of reperfusion, B1-/- group resulted in the lowest serum creatinine and urea levels, indicating less renal damage, which was proved by renal histology. Renal protection associated with B1-/- mice was also related with higher expression of HO-1 and lower expression of MCP-1. In conclusion, the absence of B1R had a protective role against inflammatory responses developed after renal ischemia-reperfusion injury.     

全反式维甲酸抑制核因子 E2相关因子 2和血红素加氧酶 1加重大鼠肾脏缺血再灌注损伤表达

目的观察用全反式维甲酸( all-trans retinoic acid,ATRA)抑制核因子 E2相关因子 2(Nrf2)和血红素加氧酶 1(HO-1)是否会加重大鼠肾脏缺血再灌注损伤( IRI)表达。方法 2020年 10月至 2022年 2月选取 24只健康成年雄性 SD大鼠切除右肾,并按随机数字表法分为 3组( n=8),即假手术组( Sham)、肾脏缺血再灌注( I/R)组、全反式维甲酸( I/R+ATRA)组。其中 Sham组和 I/R组给予腹腔注射玉米油( 1 mL·kg?1·d?1)1周, ATRA组则给予腹腔注射溶于玉米油的 ATRA(10 mg·kg?1·d?1)1周后, 3组大鼠用 10%的水合氯醛( 0.3 mL/100 g)进行麻醉后固定四肢,将其在 37 ℃条件下沿腹中线打开腹腔并分离出左肾动脉。其中 Sham组切除右肾,不予以夹闭左肾动脉; I/R组和 ATRA组采用右肾切除联合左肾动脉夹闭 45 min后再灌注 24 h的方法建立大鼠肾脏 I/R模型。实验结束后收集 3组大鼠血清及肾组织标本,用比色法检测血清肌酐(Scr)、血尿素氮(BUN)浓度; HE染色法观察肾组织病理改变; TUNEL法进行肾组织细胞凋亡的检测;二氢乙啶( DHE)荧光染色评估肾组织活性氧的表达情况;通过比色法检测肾组织丙二醛( MDA)浓度、超氧化物歧化酶( SOD)活性;用蛋白质印迹法分别对 Nrf2、HO-1的表达进行检测。结果与 Sham组相比, I/R组大鼠肾组织 Nrf2、HO-1蛋白表达量均增加( P<0.01)活性氧表达量增加, SOD活性下降, MDA含量、血清 Scr、BUN浓度升高( P<0.01)肾小管损伤评分及凋亡细胞表达较高(P<0.05),其中 Nrf2蛋白和 HO-1蛋白分别为 0.52±0.09和 0.37±0.79,高于Sham组的0.06±,0.01和 0.02±0.01。与 I/R组相比, I/R+ATRA组大,鼠肾组织 Nrf2、HO-1蛋白显著减少( P<0.01)活性氧表达量明显增加, SOD活性严重下降, MDA含量、血清 Scr、BUN浓度明显升高( P<0.01)肾小管损伤评分显著增加,凋亡细胞表达亦增高( P<0.05)其中 I/R+ATRA组 Nrf2蛋白和 HO-1蛋白分别为 0.29±0.04.15±0.03,显著低于 I/R组。结论 Nrf2/HO-1通路参与肾脏缺灌注损伤过程, ATRA可能通过抑制 Nrf2/HO-1通路,加重氧化应激,进一步加重肾肾脏,和0,血再,     

Characterisation of bradykinin B1 and B2 receptors using rat isolated vas deferens

The actions of bradykinin and its metabolite des-Arg(9) bradykinin are mediated through activation of bradykinin B(2) and B(1) receptors, respectively. The aim of the present study was to characterize native bradykinin receptors focusing on induction and desensitization using rat isolated vas deferens. Tissues were mounted in organ baths for isometric recordings and neurogenically mediated contractions were evoked by electrical stimulation. Des-Arg(9) bradykinin enhanced the magnitude of the electrically evoked contractions and this effect (which was sensitive to blockade by the peptide bradykinin B(1) receptor selective antagonist B9858, Lys-Lys-(Hyp(3),Cpg(5),D-Tic(7),Cpg(8))des-Arg(9) bradykinin) was only observed following a pre-incubation period and was greatest following 5 h of pre-incubation. Bradykinin also potentiated neurogenically evoked contractions and this effect was sensitive to blockade by Hoe 140 (D-Arg(Hyp(3),Thi(5),D-Tic(7),Oic(8))bradykinin, a peptide bradykinin B(2) receptor antagonist) and was present without pre-incubation but was increased by pre-incubation and reached maximum at the 5-h incubation time point. Responses to bradykinin were larger than those to des-Arg(9) bradykinin. Bradykinin responses did not show desensitization on repeated agonist stimulation. These data confirm in rat isolated vas deferens bradykinin B(2), but not B(1), receptors are constitutively expressed, that both receptor populations are inducible and B(2) receptors do not exhibit desensitization.     

单核细胞趋化蛋白-1-2518A／G基因多态性与肺癌分型关系的研究

目的：（1）研究贵州地区汉族人群中单核细胞趋化蛋白‐1（MCP‐1）‐2518A／G位点是否存在基因多态性;（2）探讨MCP‐1‐2518A／G基因多态性与肺癌及其分型的相关性。方法收集2013年3－12月在我科住院治疗的无血缘关系的肺癌患者（实验组）90例,根据肿瘤组织病理学类型分为非小细胞肺癌（NSCLC ）和小细胞肺癌（SCLC）,同时收集同期健康体检者（对照组）32例。通过运用聚合酶链式反应‐限制性片段长度多态性（PCR‐RFLP）技术对MCP‐1‐2518A／G的基因型进行检测。结果（1）贵州地区汉族人群中MCP‐1‐2518A／G存在基因多态性,健康人群中三种基因型（AA、AG、GG）的分布频率分别为：6．2％、46．9％、46．9％;实验组为：26．7％、46．6％、26．7％;（2）实验组和对照组中MCP‐1‐2518A／G 三种基因型分布差异具有统计学意义,AA基因型个体患肺癌的相对风险度高（OR＝5．455,P＝0．015）,GG基因型个体患肺癌的相对风险度低（OR＝ 0．412,P＝0．035）,携带A等位基因的个体患肺癌的相对风险是携带G等位基因个体的2．368倍;（3）从组织学类型来看,NSCLC患者AA基因型和GG基因型分布频率在两组间差异具有统计学意义,AA基因型个体患NSCLC的相对风险度高（OR＝7．174,P＝0．004）,GG基因型个体患病的相对风险度低（OR＝0．408,P＝0．043）,SCLC患者各基因型患病的相对风险度与对照组差异无统计学意义。结论（1）贵州地区汉族人群中存在MCP‐1‐2518A／G基因多态性;（2）MCP‐1‐2518A／G基因多态性与肺癌的发病有相关性,A等位基因可能是肺癌发病的易感基因;（3）MCP‐1‐2518A／G基因多态性与NSCLC的发病有相关性,与SCLC的发病无相关。     

替米沙坦对AGEs诱导人内皮细胞表达VCAM-1及MCP-1的作用和机制

目的 通过观察替米沙坦对晚期糖基化终末产物（AGEs）诱导的人脐静脉内皮细胞表达血管细胞黏附因子-1（VCAM-1）和单核细胞趋化蛋白-1（MCP-1）的影响,研究替米沙坦对AGEs所致动脉粥样硬化（AS）的干预作用和机制。方法 采用胶原酶消化法获取人脐静脉内皮细胞,分为4组：空白对照组,牛血清白蛋白（BSA）对照组,AGEs诱导组（10^-4~10^-1mg/mL）,AGEs+替米沙坦组（1、10、100 nmol/L）。活性氧检测试剂盒检测及倒置荧光显微镜观察细胞内活性氧含量,RT-PCR检测VCAM-1、MCP-1及晚期糖基化终末产物受体（RAGE）的mRNA。结果 AGEs组人内皮细胞内活性氧荧光强度增强,替米沙坦干预后降低;AGEs呈浓度依赖性地增强人内皮细胞对VCAM-1和MCP-1基因的转录,与空白对照组相比,AGEs（10^-4mg/mL）组VCAM-1和MCP-1 mRNA表达水平显著增高（0.24±0.01 vs 0.07±0.02;0.25±0.01 vs 0.18±0.03,P<0.05）;替米沙坦呈浓度依赖性地抑制人内皮细胞对VCAM-1和MCP-1基因的转录,与AGEs诱导组相比,替米沙坦（10 nmol/L）组人内皮细胞的VCAM-1和MCP-1基因转录水平显著降低（0.23±0.01 vs 0.85±0.11;0.62±0.10 vs 1.05±0.04,P<0.05）;与AGEs诱导组相比,替米沙坦（1 nmol/L）组人内皮细胞RAGE基因表达水平显著降低（0.64±0.03 vs 1.18±0.10,P<0.05）。结论 AGEs增强人内皮细胞表达VCAM-1和MCP-1;替米沙坦可能通过抑制RAGE表达来抑制AGEs诱导的人内皮炎性损伤。     

Losartan inhibits monocytic adhesion induced by ADMA via downregulation of chemokine receptors in monocytes

Objective  Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, can induce the adhesiveness of monocytes to vascular endothelium, and chemokines play an important role in this process. The present study was carried out to test whether the inhibitory effect of losartan on ADMA-induced monocytic adhesion is mediated by chemokine receptors. Methods  Human monocytoid cells (THP-1) were incubated with exogenous ADMA (30 μM) for 4 or 24 h in the absence or presence of losartan. The monocytic adhesion, the levels of chemokines, and the expression of chemokine receptors were determined. The possible signal pathway was also explored. Results  In cultured monocytes, ADMA (30 μM) markedly increased monocytic adhesion to endothelial cells, elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), and upregulated the mRNA expression of chemokine receptors CCR₂ and CXCR₂. Exposure to ADMA (30 μM) significantly induced the generation of intracellular reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB. Pretreatment with AT₁ receptor blocker (ARB) losartan (1, 3, 10 μM) attenuated monocytic adhesiveness elicited by ADMA and downregulated the expression of CCR₂ and CXCR₂ mRNA, accompanied by a significant decrease in ROS generation and NF-κB activity and expression. Conclusion  The present study suggests that the inhibitory effect of losartan on ADMA-induced monocytic adhesion may be related to downregulation of chemokine receptors by inhibiting the ROS/NF-κB pathway.     

Discovery of BMS-753426: A Potent Orally Bioavailable Antagonist of CC Chemokine Receptor 2

To improve the metabolic stability profile of BMS-741672 (1a), we undertook a structure–activity relationship study in our trisubstituted cyclohexylamine series. This ultimately led to the identification of 2d (BMS-753426) as a potent and orally bioavailable antagonist of CCR2. Compared to previous clinical candidate 1a, the tert-butyl amine 2d showed significant improvements in pharmacokinetic properties, with lower clearance and higher oral bioavailability. Furthermore, compound 2d exhibited improved affinity for CCR5 and good activity in models of both monocyte migration and multiple sclerosis in the hCCR2 knock-in mouse. The synthesis of 2d was facilitated by the development of a simplified approach to key intermediate (4R)-9b that deployed a stereoselective reductive amination which may prove to be of general interest.     

二氧化碳气腹对大鼠肝脏造成的缺血再灌注损伤及异丙酚促进HO-1表达的保护作用

目的探讨实验室二氧化碳(CO_2)模拟腹腔镜高气腹压力对大鼠肝脏造成的缺血再灌注损伤以及异丙酚的保护作用和机制。方法选用280~320 g的Wistar成年雄性大鼠,实验前各组大鼠禁食12 h,可自由饮水。选取雄性大鼠48只,随机分成4组,每组12只:对照组(A组)、缺血组(B组)、再灌注组(C组)、异丙酚组(D组),观察各组天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、超氧化物歧化酶(SOD)、丙二醛(MDA)以及血红素氧合酶1(HO-1)表达的变化。结果 B组、C组与A组比较,MDA逐渐增高,而SOD逐渐降低,HO-1表达反应性逐渐增加,HE染色和免疫组化提示损伤逐渐加重;D组与B组、C组比较,MDA指标明显降低,SOD明显增高,HO-1表达继续增加,HE染色和免疫组化可见损伤明显减轻。结论 CO2气腹可以造成大鼠肝脏缺血再灌注损伤;缺血和再灌注是不同的损伤过程,即:缺血状态解除后,对大鼠肝脏损伤并未停止,反而由于再灌注过程而进一步加重;异丙酚可通过促进HO-1的表达机制而发挥其保护性作用。     

Clemaichinenoside protects renal tubular epithelial cells from hypoxia/reoxygenation injury in vitro through activating the Nrf2/HO-1 signalling pathway

Renal ischaemia/reperfusion (I/R) is a major cause of acute renal failure with increased morbidity, mortality, and prolonged hospitalizations. Clematichinenoside (AR), a triterpenoid saponin isolated from the roots of Clematis chinensis, was reported to possess a protective effect against I/R injury. However, the effect of AR on renal I/R injury has not been evaluated. This study aims to examine the effect of AR on an in vitro I/R model in human proximal tubular epithelial cells HK-2. HK-2 cells were subjected to hypoxia/reoxygenation (H/R) stimulation to mimic I/R in vitro. The results showed that AR improved cell viability of H/R-stimulated HK-2 cells. AR pretreatment resulted in decreased production of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as increased in superoxide dismutase (SOD) activity in H/R-stimulated HK-2 cells. In addition, AR also presented an anti-inflammatory activity, as evidenced by decreased secretion of pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α. Moreover, apoptotic rate was markedly decreased in HK-2 cells pretreated with AR. The bax expression was decreased, while bcl-2 expression was increased by AR pretreatment. Furthermore, AR enhanced the H/R-stimulated activation of the Nrf2/HO-1 signalling pathway in HK-2 cells. In conclusion, these findings indicated that AR protected HK-2 cells from H/R-induced cell injury via regulating the Nrf2/HO-1 signalling pathway.     

Role of serum and glucocorticoid-regulated kinase-1 in the protective effects of erythropoietin during renal ischemia/reperfusion injury

Erythropoietin (EPO) protects the kidneys from ischemia/reperfusion (I/R) injury; however, the exact signalling mechanisms are not fully understood. The serum and glucocorticoid-regulated kinase 1 (SGK1) is an anti-apoptotic protein kinase regulated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway by cellular stimuli, hormones and growth factors. The objective of the present study was to examine the role of SGK1 in the renoprotective effects of EPO in renal I/R injury.In vitro, cultures of HEK293 cells were exposed to 16 h hypoxia. Incubation with EPO at a doses of 400 U/ml exerted a protective effect on cell death assessed by LDH release and Annexin V FACS analysis. This was paralleled by up-regulation of SGK1 expression, as well as phosphorylation. Downregulation of SGK1 expression by small interfering RNA technique ameliorated the anti-apoptotic effect of EPO treatment.In an in vivo rat model of unilateral renal I/R injury, rats were treated with 500 U/kg EPO 24 h prior to ischemia. EPO resulted in less severe tissue injury and ameliorated the elevation in creatinine and urea nitrogen levels 24 h after reperfusion. Furthermore, SGK1 expression and phosphorylation were higher in EPO compared to vehicle-treated rats as demonstrated by real-time PCR, Western blot and immunofluorescence technique.We conclude that EPO protects from renal I/R injury and SGK1 might contribute to the mediation of EPO effects under ischemic conditions.     

Role of angiotensin II in the response to endothelin-1 of goat cerebral arteries after ischemia–reperfusion

As angiotensin II may underlie the deleterious effects of some vascular diseases, we have examined the role of this peptide on the cerbrovascular endothelin-1 action after ischemia–reperfusion. In anesthetized goats, 1 hour-occlusion followed by 1 hour-reperfusion of the left middle cerebral artery (MCA) was induced, and then segments 3-mm in length from branches of the right MCA (control) and the left MCA (ischemic) were obtained for isometric tension recording. Endothelin-1 (10^− 11–10^− 7 M) produced a contraction that was higher in ischemic than in control arteries, and in control but not in ischemic arteries this contraction was potentiated by angiotensin II (10^− 7 M). Losartan (3 × 10^− 6 M), antagonist of AT₁ receptors, did not affect the response to endothelin-1 in control arteries, but reduced it both in ischemic arteries and angiotensin II-treated control arteries. PD123,319 (3 × 10^− 6 M), antagonist of AT₂ receptors, or the inhibitor of nitric oxide synthesis l-NAME (10^− 4 M) did not alter the arterial effects of endothelin-1. Therefore, angiotensin II may potentiate the constriction to endothelin-1 in normal cerebral arteries by activating AT₁ receptors. The observed cerebrovascular increased response to endothelin-1 after ischemia–reperfusion might be related in part to activation of AT₁ receptors under this condition.     

Tadalafil alleviates cisplatin-induced reproductive toxicity through the activation of the Nrf2/HO-1 pathway and the inhibition of oxidative stress and apoptosis in male rats

Male reproductive toxicity is a well-known adverse effect of cisplatin (CIS), an important antineoplastic agent used to control several types of cancers. Tadalafil (TDF), is a long-acting phosphodiesterase-5 (PDE5) inhibitor commonly used as treatment for erectile dysfunction. The aim of this work was to study the possible protective effect of TDF against CIS-induced testicular toxicity in rats and the possible involvement of Nrf2/HO-1 pathway, which demonstrates antioxidant and inflammatory activities utilizing zinc protoporphyrin-IX (ZnPP) as HO-1 inhibitor. Results revealed that TDF attenuated the CIS-induced disturbances in sperm count and activities, normalized the serum testosterone level, improved the CIS-induced changes in epididymal and testicular weights and restored the normal structure of testicular tissues. In addition, TDF upregulated the gene expression levels of Nrf2 and HO-1 and the activity of HO-1 whereas, it reduced the CIS-induced changes in testicular oxidative stress markers and the levels of inflammatory mediators (TNF-α and iNOS). Furthermore, TDF antagonized the CIS-induced increase in testicular gene expression of apoptotic markers caspase-3 and Bax, and the decrease in Bcl-2. However, ZnPP co-administration significantly attenuated all TDF-mediated improvements in CIS-induced testicular toxicity, biochemical changes, and apoptosis. In conclusion, TDF exerts a protective effect against CIS-induced reproductive toxicity in males, through different mechanisms, besides its inhibitory action to PDE5, possibly mediated by the upregulation of Nrf2/HO-1, along with its antioxidant, anti-inflammatory, and anti-apoptotic effects. Hence, the use of TDF represents a promising therapeutic approach to protect the male reproductive system from the harmful toxic effects of CIS.     

Rupatadine effectively prevents the histamine-induced up regulation of histamine H1R and bradykinin B2R receptor gene expression in the rat paw

BackgroundActivation of histamine H₁ receptor (H₁R) is a well-known hallmark of allergic and inflammatory pathology. Both types of bradykinin receptors (B₁R and B₂R) are also known to contribute significantly to the latter and some sort of functional interaction between them and H₁R has been alluded to in the past. Here we use an experimental model of rat paw oedema formation to examine the effect of exogenously added histamine on the gene expression of H₁R and bradykinin receptors B₁R and B₂R, alone or in combination to rupatadine, a second generation antihistamine agent.MethodsHistamine-induced oedema formation was monitored with a plethysmometer. The gene expression of H₁R, B₁R and B₂R was analyzed with both conventional and real-time PCR. Rupatadine fumarate was used in pure form and administered intraperitoneally, prior to histamine injection into the paw. Microscopy of haematoxylin and eosin-stained sections of paw tissue was used to examine effects on tissue architecture.ResultsHistamine injection into the paw resulted in significant up regulation of H₁R and B₂R without inducing significant cellular infiltration, but appears to affect less the expression of B₁R. Rupatadine was, under the conditions used in this study, very effective in preventing this effect and in suppressing oedema formation through its antihistamine action.ConclusionRupatadine has a suppressing effect on H₁R and B₂R gene expression which could add to its efficacy towards allergy and allergy-like conditions.     

The effects of sildenafil on the functional and structural changes of ileum induced by intestinal ischemia–reperfusion in rats

There is evidence demonstrating the protective effect of cGMP-specific phosphodiesterase type 5 (PDE5) inhibitors against ischemic injury in certain tissues. In this study, sildenafil, a potent inhibitor of PDE5, was tested for its beneficial effects in the prevention of disrupted ileal contractility and damage to tissue caused by intestinal ischemia–reperfusion in rats. Male Sprague–Dawley rats were divided into four groups: sham-operated; sham-operated with sildenafil pretreatment; ischemia–reperfusion with vehicle pretreatment; and ischemia–reperfusion with sildenafil pretreatment. The superior mesenteric artery was occluded for 45 min to induce ischemia. The clamp was then removed for a 60 min period of reperfusion. Sildenafil (1 mg/kg, i.v.) or saline was administered prior to the surgical procedure in the ischemia–reperfusion and sham-operated groups. Isometric contractions of the ileal segments in response to acetylcholine or electrical field stimulation (120 V, 2 ms pulse for 5 s, 1–20 Hz) were recorded. Additionally, levels of thiobarbituric acid reactive substances and myeloperoxidase activity were measured in addition to a histopathological examination of the ileal tissue. The contractions induced by both acetylcholine and electrical field stimulations were markedly inhibited after ischemia–reperfusion. Sildenafil pretreatment (1 mg/kg, i.v.) abolished the inhibition of responses to acetylcholine. The increased levels of thiobarbituric acid reactive substances and myeloperoxidase activity caused by ischemia–reperfusion were reversed to control levels with sildenafil pretreatment. Intestinal ischemia–reperfusion caused severe ischemic injury in rat ileum, which was prevented by sildenafil. These results suggest that sildenafil pretreatment has a protective effect against ileal dysfunction and damage induced by intestinal ischemia–reperfusion in the rat.