Cdk5抑制剂Roscovitine对糖尿病大鼠肾功能及nestin表达的影响

<正>糖尿病肾病(diabetic nephropathy,DN)是糖尿病最常见的微血管并发症之一。研究显示,足细胞损伤在糖尿病肾病的进展中发挥了重要作用[1-2]。Nestin是属于第Ⅵ类中间丝的细胞骨架蛋白,对维持足细胞的形态和功能起着重要作用[3-4]。     

胆碱能M受体调控细胞周期蛋白依赖性激酶5及其在敌敌畏诱导SH-SY5Y细胞毒性损伤中的作用

目的研究胆碱能M受体(主要为M1受体)参与SH-SY5Y细胞周期依赖性蛋白激酶5(Cdk5)调控及其在敌敌畏(DDVP)诱导SH-SY5Y细胞毒性损伤中的作用。方法①应用免疫荧光结合共聚焦显微镜检测胆碱能M1受体及Cdk5在SH-SY5Y细胞中的分布和表达。②细胞对照组、氧化震颤素(Oxo-M)组(1×10^-4mol·L^-1作用48 h)、罗考唯亭(Rosc)组(培养结束前1 h加入Rosc 1×10^-4mol·L^-1)、Rosc+Oxo-M组(Rosc 1×10^-4mol·L^-1预处理1 h后,再加入Oxo-M 1×10^-4mol·L^-1作用至48 h),应用MTT比色法检测细胞存活率,Western印迹法检测Cdk5表达。③细胞对照组、DDVP组(1×10^-5mol·L^-1作用48 h)、Rosc组(培养结束前1 h加入1×10^-4mol·L<...     

Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞增殖及细胞周期的影响

目的观察Roscovitine对TNF-α诱导的大鼠血管平滑肌细胞(VSMC)增殖及细胞周期的影响。方法组织贴块法培养大鼠VSMC细胞,采用TNF-α诱导其增殖,加入不同浓度的Roscovitine预处理15 h,将细胞分为:对照组、TNF-α组、Roscovitine 5、10、15、30μmol·L~(-1)组。MTT比色法检测细胞增殖活性;Western blot检测增殖细胞核抗原(PCNA);流式细胞仪检测细胞周期;荧光定量RT-PCR及Western blot检测细胞周期蛋白(Cyclin A、Cyclin B、Cyclin D、Cyclin E)、细胞周期蛋白依赖激酶(CDK4、CDK5)、细胞周期抑制蛋白(p53、p21、p27)的表达。结果 Roscovitine能抑制VSMC增殖;抑制细胞周期从G_0/G_1期向S期转化。与TNF-α组比较,Roscovitine 5、10、15、30μmol·L~(-1)组能降低细胞周期蛋白Cyclin A、Cyclin B、Cyclin D、Cyclin E蛋白表达,降低细胞周期蛋白依赖激酶CDK4、CDK5蛋白表达,升高细胞周期抑制蛋白p53、p21、p27蛋白表达(P<0.05)。结论 Roscovitine可抑制大鼠VSMC细胞周期进程及增殖活性。     

氯沙坦钾逆转血管紧张素Ⅱ诱导人肾脏足细胞损伤的作用及其机制研究

目的探讨体外实验中氯沙坦钾对血管紧张素Ⅱ(AngⅡ)诱导的人足细胞损伤的保护作用及其机制。方法体外培养人足细胞,按照干预方式将其分为4组:正常足细胞组,AngⅡ刺激组,血管紧张素受体阻滞剂(ARB)干预组,蛋白激酶C(PKC)抑制剂干预组。通过噻唑蓝(MTT)试验、乳酸脱氢酶(LDH)释放试验观察不同浓度AngⅡ对足细胞生长的影响;通过苏木精-伊红染色(HE)和Gimsa染色观察足细胞形态变化;蛋白印迹法检测PKC活性改变;流式细胞术检测细胞裂隙隔膜蛋白nepnephrin和podocin表达变化。结果本研究成功培养人足细胞,同时发现在AngⅡ的作用下,足细胞生长受限,形态学也发生了显著改变,胞膜PKC/胞质PKC比例明显升高,nephrin和podocin表达减少,且平均荧光强度明显降低;而给予ARB及PKC抑制剂干预后上述改变有所恢复。结论 AngⅡ介导了人肾脏足细胞的损伤,给予其受体阻断剂可通过阻断PKC的表达从而一定程度上逆转损伤。     

PPAR-α激动剂对PAN诱导肾小球足细胞损伤的保护作用

目的探讨PPAR-α激动剂非诺贝特对氨基核苷嘌呤霉素(PAN)诱导肾小球足细胞损伤的作用及其机制。方法将18只8周龄SD♀大鼠随机分为3组(n=6)。PAN模型组和非诺贝特组1次尾静脉注射PAN 65 g·g-1,空白对照组注射生理盐水,PAN注射后d1,非诺贝特组灌胃非诺贝特40 mg·kg-1·d-1),空白对照组和PAN模型组灌胃等体积溶剂。分别于d 0、6、10收集24 h尿样,采用Bradford法测定大鼠24 h尿蛋白含量。PAN注射后10 d将大鼠安乐死,收集肾小球样本。利用Western blot和Real-Time PCR检测肾小球足细胞损伤标记物的表达和凋亡相关基因、骨架蛋白以及裂隙膜蛋白基因转录活性。结果注射PAN后d10,24 h尿蛋白含量较d 0明显上升,足细胞损伤标记物desmin基因水平和蛋白表达明显增加,线粒体凋亡通路、TGF-β/Smad通路和p38通路转录水平明显上调,足细胞骨架蛋白和裂隙膜蛋白的转录活性明显增加。非诺贝特处理能够明显降低PAN引起的尿蛋白,改善PAN对足细胞的损伤作用;明显抑制凋亡通路的转录活性;降低骨架蛋白和裂隙膜蛋白的转录活性。结论非诺贝特能够改善PAN诱导的肾小球足细胞损伤,其作用机制与抑制凋亡通路有关。     

黄芪甲苷抑制内质网应激及CHOP信号通道改善衣霉素诱导的系膜细胞凋亡

目的 观察黄芪甲苷对衣霉素诱导的系膜细胞凋亡、内质网应激标志蛋白和CHOP信号通路表达的影响,从系膜细胞凋亡角度探讨黄芪甲苷治疗早期糖尿病肾病的可能机制.方法 采用衣霉素诱导大鼠系膜细胞产生内质网应激,分为模型对照组、黄芪甲苷小剂量组(AS-IVL,50 μg·mL-1)、黄芪甲苷大剂量组(AS-IVH,100 μg·...     

马钱苷对糖基化终末产物诱导足细胞损伤的保护作用

目的探讨山茱萸环烯醚萜苷特征性成分马钱苷对糖基化终末产物(AGEs)诱导肾脏足细胞损伤的保护作用及其机制。方法体外培养小鼠肾小球足细胞,分为空白对照组、模型组(AGEs组)、马钱苷组,并设氨基胍组作为阳性对照。MTT法检测马钱苷对足细胞存活率的影响;Hoechst33342/PI双染观察足细胞凋亡情况,流式细胞仪检测细胞凋亡率;Western blot法检测足细胞AGEs受体(RAGE)、足细胞损伤标志蛋白desmin以及凋亡相关蛋白bax、bcl-2、cleaved caspase-3的表达。结果马钱苷能够抑制AGEs导致的足细胞损伤,下调足细胞Desmin、RAGE蛋白的表达,明显降低AGEs诱导的足细胞凋亡率,降低足细胞Bax/Bcl-2的比值和促凋亡蛋白cleaved caspase-3的表达。结论马钱苷能够改善AGEs诱导的肾小球足细胞损伤,其作用机制与降低RAGE蛋白表达,抑制凋亡通路有关。     

氧化应激在糖尿病肾病足细胞凋亡中的作用

氧化应激在抗肾小球基底膜滤过屏障中肾小球足细胞具有极其重要的作用,高血糖引起的氧化应激可经过多种途径参与足细胞的损伤。足细胞是一种终末分化细胞,损伤后病变不可逆转,因此在治疗足细胞损伤的最近研究中,预防足细胞的损伤尤为重要。对于阐明糖尿病肾病发病机制的研究中,氧化应激对于足细胞的损伤变成新的研究方向,为疾病的防治开辟全新的思路。也为糖尿病肾病患者的病理生理机制研究和早期防治带来新展望。     

Intrathecal administration of roscovitine attenuates cancer pain and inhibits the expression of NMDA receptor 2B subunit mRNA

Cancer pain is one of the most severe chronic pains. The mechanisms underlying cancer pain are still unclear. Because of the pain-relieving effects of Cdk5 (Cyclin-dependent kinase 5) antagonist roscovitine in inflammation pain models, we tested whether roscovitine would induce antihyperalgesia in cancer pain. Our previous study showed that the NR2B (N-methyl-d-aspartate receptor 2B) in the spinal cord participates in bone cancer pain in mice. In this study, we used a mouse model of bone cancer pain to investigate whether roscovitine could attenuate bone cancer pain by regulating the expression level of NR2B mRNA in spinal cord. C3H/HeJ mice were inoculated into the intramedullary space of the right femur with Osteosarcoma cells to induce ongoing bone cancer pain behaviors. At day 14 after operation, inoculation of Osteosarcoma cells significantly enhanced mechanical allodynia and thermal hyperalgesia, which was attenuated by intrathecal administration of different doses of roscovitine. Correlated with the pain behaviors changes, RT-PCR experiments in our study revealed that there was a marked increase in the expression of NR2B mRNA in spinal cord after operation, which was attenuated by intrathecal administration of roscovitine. These results suggest that roscovitine may be a useful adjunct therapy for bone cancer pain, and NR2B in spinal cord may participate in this effect.     

Crocin and quercetin prevent PAT‐induced apoptosis in mammalian cells: Involvement of ROS‐mediated ER stress pathway

Patulin (PAT) is a secondary metabolite produced by several species of the genera of Penicillium, Aspergillus, and Byssochlamys that can be found in rotting fruits, especially in apples and apple‐based products. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. The mechanism underlying such toxicity has been linked to the induction of apoptosis which occurred with reactive oxygen species production and endoplasmic reticulum (ER) stress induction. This study aimed to evaluate the effect of the two common dietary compounds Quercetin (QUER), a natural flavonoid, and Crocin (CRO), a natural carotenoid, on PAT‐induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We showed that antioxidant properties of QUER and CRO help to prevent ER stress activation and lipid peroxidation as evidenced by the reduction in GRP78 and GADD34 expressions and the decrease in malondialdehyde production. Furthermore, we demonstrated their ability to re‐establish the loss of the mitochondrial membrane potential to inhibit caspase 3 activation and DNA fragmentation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1851–1858, 2016.     

愤怒诱导大鼠肝损伤中内质网应激相关蛋白的表达

目的:研究慢性愤怒诱导的大鼠肝损伤中内质网应激(ERS)相关蛋白的表达.方法:选择20只雄性SD大鼠,随机分为两组,各10只.实验组大鼠予应激刺激造模,对照组正常饲养,2周后同时处死两组大鼠.检测肝脏形态学变化及肝组织中GRP78、Caspas-3、LC3-Ⅱ、Beclin-1的mRNA和蛋白表达水平.结果:与对照组相...     

FoxO1在同型半胱氨酸诱导的肾脏足细胞损伤及凋亡中的作用研究

摘要：目的　探讨叉头状转录因子O1（FoxO1）在同型半胱氨酸（Hcy）诱导的肾脏足细胞损伤及凋亡中的作用。方法　采用高蛋氨酸饮食喂养胱硫醚β-合成酶（Cbs）基因正常Cbs+/+小鼠和单基因敲除Cbs+/-小鼠各10只。8周后处死,收集肾组织,PAS染色观察小鼠肾小球形态学变化。体外培养足细胞,分为Control组和Hcy组（用含80 μmol/L Hcy的细胞培养液干预48 h）。将携带绿色荧光蛋白（GFP）的过表达FoxO1腺病毒（Ad-FoxO1）和干扰FoxO1腺病毒（Sh-FoxO1）转染足细胞,分为对照组、Ad-GFP组、Ad-FoxO1组、Sh-NC组、Sh-FoxO1组、Ad-GFP+Hcy组、Ad-FoxO1+Hcy组、Sh-NC+Hcy组、Sh-FoxO1+Hcy组。Western blot检测小鼠肾组织和足细胞裂隙膜蛋白（Podocin和Nephrin）、FoxO1及凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、半胱氨酸蛋白酶12（Caspase12）蛋白表达;qPCR检测小鼠肾组织和足细胞中FoxO1 mRNA的表达。结果　PAS染色结果显示,Cbs+/+组小鼠肾小球结构正常,基底膜清晰且分布均匀,而Cbs+/-组小鼠肾小球基底膜则呈现节段性增厚,系膜基质增多;与Cbs+/+组小鼠比较,Cbs+/-组小鼠肾组织中Podocin、Nephrin和FoxO1蛋白、FoxO1 mRNA的表达明显降低（P＜0.01）。与Control组比较,Hcy组FoxO1 mRNA和蛋白表达显著降低（P＜0.01）;过表达FoxO1后,与Ad-GFP+Hcy组相比,Ad-FoxO1+Hcy组Podocin和Nephrin蛋白表达均明显升高,Bax/Bcl-2比值、Caspase12蛋白表达均明显降低（P＜0.05）;而干扰FoxO1后,与Sh-NC+Hcy组相比,Sh-FoxO1+Hcy组Podocin和Nephrin蛋白表达均明显降低,Bax/Bcl-2比值、Caspase12蛋白表达均明显增高（P＜0.05）。结论　FoxO1可减轻Hcy诱导的小鼠肾脏足细胞损伤及凋亡。     

Arsenite‐induced endoplasmic reticulum‐dependent apoptosis through disturbance of calcium homeostasis in HBE cell line

Calcium (Ca²⁺) is a ubiquitous cell signal responsible for multiple fundamental cellular functions, including apoptosis. Whether the homeostasis of Ca²⁺ is involved in arsenite‐induced apoptosis remains unclear. In this study, we observed that arsenite significantly elevated the intracellular Ca²⁺ concentration in a dose‐ and time‐dependent manner. By using the Ca²⁺‐ATPase inhibitor, thapsigargin, and the inositol 1,4,5‐ trisphosphate receptors (IP3Rs) inhibitor, heparin, we further confirmed that the disturbance of endoplasmic reticulum (ER) Ca²⁺ homeostasis caused Ca²⁺ overload in the cells. Moreover, loss of ER Ca²⁺ homeostasis also led to ER stress, mitochondrial dysfunction, and NF‐κB activation. Importantly, pretreatment of cells with heparin remarkably attenuated the elevated cell apoptosis induced by arsenite, but inhibition of ER Ca²⁺ uptake with thapsigargin exacerbated arsenite‐induced cell damage significantly. Together, we demonstrated for the first time that arsenite disturbed the Ca²⁺ homeostasis in ER, which subsequently led to ER stress, mitochondrial dysfunction, and NF‐κB nuclear translocation, and thus consequently triggering cell apoptosis. Our findings indicate regulation of disrupted Ca²⁺ homeostasis in ER may be a potential strategy for prevention of arsenite toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 197–216, 2017.     

Pitavastatin suppresses hyperglycaemia‐induced podocyte injury via bone morphogenetic protein‐7 preservation

Podocytes form the essential components of the glomerular filtration barrier and play a critical role in diabetic nephropathy. Recent evidence suggests that HMG‐CoA reductase inhibitors (statins) exert renoprotective effects. We investigated whether pitavastatin directly suppresses hyperglycaemia‐induced podocyte injury using cultured podocytes and, if so, the mechanism of the beneficial effects. Cultured podocytes were exposed to media containing normal (NG; 5 mmol/L) or high (HG; 25 mmol/L) glucose for 1 week. HG increased the lethal injury of podocytes and disruption of F‐actin fibers, and reduced the mRNA expression of novel podocyte markers, synaptopodin and Wilms tumor‐1 (WT‐1), in association with decreased bone morphogenetic protein‐7 (BMP‐7) expression. Pitavastatin (100 nmol/L) reduced podocyte injury and restored the mRNA expression of synaptopodin and WT1; however, these protective effects were abolished by BMP‐7 siRNA. Additionally, pitavastatin suppressed HG‐induced Rho kinase activation, as assessed by the phosphorylation level of myosin phosphatase targeting subunit 1 (MYTP1), and C3 exotoxin, a Rho inhibitor, mimicked the effect of pitavastatin on BMP‐7 preservation. Pitavastatin attenuates hyperglycaemia‐induced podocyte injury via Rho‐Rho kinase‐dependent BMP‐7 preservation.     

Inhibition of cyclin-dependent kinase 5 by roscovitine impairs memory consolidation and reconsolidation in the day-old chick

Cyclin-dependent kinase 5 (CDK-5) is reported to phosphorylate the NMDA receptor prior to the induction of long-term potentiation (LTP), among its many other effects. Application of CDK-5 inhibitors disrupts LTP and results in impaired task acquisition in behaving animals. In this study, we investigated the effect of exogenously applied roscovitine, a potent CDK-5 inhibitor, on consolidation and reconsolidation processes in day-old male chicks. New HampshirexWhite leghorn cockerels were trained using a modified version of the passive avoidance learning task. Intracranial injections of roscovitine (2.5 microM) administered immediately after training induced a memory deficit that evolved from 5-minute post-training and persisted until at least 24 h following training. Injections of roscovitine (2.75 microM) administered immediately after the reminder trial induced a memory deficit observed by 30-minute post-reminder which had resolved by 24 h following the reminder. The comparison between consolidation and reconsolidation demonstrates differences both in the time of the onset of the memory deficit as well as in the permanence of this deficit. The results suggest an important, although different role for CDK-5 in consolidation and reconsolidation processes following passive avoidance learning.     

17-甲氧基-7-羟基-苯并呋喃查尔酮对大鼠心肌缺血再灌注损伤的保护作用

目的观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对心肌缺血再灌注(I/R)大鼠的影响。方法 SD大鼠50只随机分为5组,每组10只:假手术组,模型组,溶媒组,YLSC低、高剂量(2.50,5.00 mg/kg)组。缺血30 min再灌注60 min复制大鼠I/R模型,观察大鼠血清肌酸激酶(CK)、乳酸脱氢酶(LDH)、天门冬氨酸氨基转移酶(AST)含量的变化,伊文思蓝和氯化三苯四氮唑(TTC)双重染色确定心肌梗死面积,TUNEL法检测心肌细胞凋亡率,Western blot法检测心肌蛋白葡萄糖调节蛋白78(GRP78)和caspase12的表达。结果与模型组相比,YLSC能显著减少心肌梗死面积和CK、LDH、AST的漏出,降低心肌细胞凋亡率,并抑制内质网应激标志蛋白GRP78和caspase12的表达。结论 YLSC能减少大鼠I/R损伤,其心肌保护作用可能与减轻内质网应激有关。