Influence of edaravone on growth arrest and DNA damage-inducible protein 34 expression following focal cerebral ischemia-reperfusion in rats

Objective:To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34(GADD34).Methods:A total of 108 healthy male Sprague-Dawlcy rats were randomly divided into sham operation group,model group and edaravone.group(36 cases for each group).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h followed by reperfusion in Sprague-Dawlev rats.Then.GAOD34 expression was measured with immunohistochemistry at different time-points after reperfusion in the peri-infarct regions of all rats.Results:The GADD34 expression was detected in the peri-infaret regions of rats 1 h after reperfusion,which reached its peak 24 h after reperfusion.And edaravone could significantly down-regulate the GAOD34 expression.Conclusions:Edaravon could down-regulate GADD34 expression,which suggests that edaravone may exert an important function in inhibiting endoplasmic reticulum stress reaction by scavenging free radicals in the upper stream.

Gadd45α对绒毛外滋养细胞迁移和侵袭能力影响的相关研究

目的:研究人生长阻滞和DNA损伤45α(growth arrest and DNA damage 45 alpha,Gadd45α)基因对滋养细胞HTR8/SVneo增殖、凋亡、迁移和侵袭等生物学功能的影响,探讨其在子痫前期(preeclampsia,PE)发生发展中的可能作用。方法:构建Gadd45α短发夹干扰RNA,以阴性对照组作为参照,转染人滋养细胞HTR8/SVneo,即分为实验组(si-Gadd45α)和阴性对照组(si-NC)进行实验;应用流式细胞仪检测转染效率,并应用q RT-PCR和Western blot检测转染后各组细胞中Gadd45αm RNA和蛋白表达水平;采用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)显色法检测转染后各组细胞增殖能力;应用流式细胞仪对转染后各组细胞进行细胞凋亡分析;采用Transwell法检测转染后各组细胞迁移和侵袭能力;采用早孕绒毛外植体培养模型观察敲除Gadd45α基因后对绒毛外滋养细胞外生性迁移能力的影响;收集各组细胞培养上清液,明胶酶谱法检测基质金属蛋白酶(matrix metalloproteinases,MMPs)的表达,蛋白免疫印迹检测基质金属蛋白酶组织抑制因子(tissue inhibitors of MMPs,TIMPs)的表达。结果:流式细胞仪检测转染效率约为90%;转染后实验组较对照组Gadd45αm RNA的表达量约降低80%,蛋白表达水平约下降70%,差异均有统计学意义(P<0.01),证明干扰Gadd45α表达成功。MTT法和流式细胞仪检测细胞增殖和凋亡,结果显示实验组和对照组细胞增殖和凋亡均无统计学差异(P>0.05)。Transwell实验表明干扰Gadd45α后HTR8/SVneo侵袭能力明显增加,约为对照组的1.65倍;迁移能力也明显增加,约为对照组的2倍,均有统计学差异(P<0.01)。早孕绒毛外植体培养结果显示:与阴性对照组的绒毛相比,Gadd45α干扰片段处理的绒毛外滋养细胞外生性迁移的距离明显增加,差异有统计学意义(P=0.005)。明胶酶谱实验结果显示干扰Gadd45α后基质金属蛋白酶-2(matrix metalloproteinases,MMP-2)和MMP-9的活性增强,而Western blot检测发现其抑制因子TIMP-1和TIMP-2相应地下降(P<0.01)。结论:推测Gadd45α可能是通过调控蛋白酶的活性来抑制滋养细胞的迁移和侵袭,从而参与PE的发生发展。

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

Gadd45α基因敲减对缺氧/复氧下人绒毛外滋养细胞迁移侵袭能力的影响

目的·探讨沉默生长阻滞和DNA损伤45α(Gadd45α)基因对缺氧/复氧下人绒毛外滋养细胞迁移侵袭能力的影响。方法·用靶向Gadd45α基因的sh RNA慢病毒感染人绒毛外滋养细胞HTR8/SVneo,敲减Gadd45α的基因表达;采用氧化应激模型体外模拟子痫前期,观察细胞生物学功能的改变。实验分成4组:对照组、缺氧/复氧组、Gadd45α敲减+缺氧/复氧组和慢病毒阴性对照+缺氧/复氧组。采用早孕绒毛外植体实验观察Gadd45α基因敲减对氧化应激下人绒毛外滋养细胞生物学功能的影响,Western blotting法检测各组细胞Gadd45α蛋白水平变化,Transwell实验观察细胞侵袭和迁移能力,明胶酶谱法检测细胞培养上清液中基质金属蛋白酶2/9的活性。结果·缺氧/复氧可使HTR8/SVneo细胞中Gadd45α表达增加,进而细胞的迁移和侵袭能力下降;而Gadd45α干扰可提高基质金属蛋白酶2/9的活性进而提高氧化应激下绒毛外滋养细胞的迁移和侵袭能力。结论·Gadd45α基因敲减对氧化应激下人绒毛外滋养细胞迁移侵袭能力具有促进作用。

GADD45g基因在急性髓系白血病细胞中的低表达及其抑癌特性

目的:检测生长阻滞和DNA损伤诱导蛋白45g(growth arrest and DNA damage inducible protein 45g,GADD45g)基因在急性髓系白血病(acute myeloid leukemia,AML)患者骨髓标本和细胞系中的表达情况及其表达水平与AML患者预后的关系,探究过表达GADD45g对AML细胞增殖、凋亡、衰老、周期、分化和药物敏感性的影响。方法:选取中国医学科学院血液病医院2013年1月至2016年12月初次诊断为AML患者的骨髓标本共27例,用Real-time PCR和Western blotting检测AML患者、正常对照骨髓单个核细胞以及AML细胞系中GADD45g mRNA和蛋白水平的表达情况。在两个相互独立的数据集GSE10358和GSE425-GPL317中分析GADD45g表达与AML患者的总生存率(overall survival,OS)和无事件生存率(event-free survival,EFS)的相关性。构建GADD45g过表达慢病毒并感染AML细胞系U937、THP-1和Molm-13,通过生长曲线、集落形成、β-半乳糖苷酶染色、流式细胞术分析Annexin V/7AAD染色和PI染色等方法分析GADD45g过表达对AML细胞增殖、克隆形成、衰老、凋亡、周期、分化和化疗药物敏感性的影响。结果:GADD45g在AML患者和AML细胞系中的表达水平显著低于正常对照(均P<0.01)。低表达GADD45g的AML患者的OS(P<0.05)和EFS较高表达患者显著缩短(均P<0.05)。在AML细胞系中过表达GADD45g能显著抑制细胞增殖、集落形成,显著促进细胞的凋亡、衰老、周期阻滞和分化,增强了细胞对化疗药物敏感性(P<0.05或P<0.01)。结论:GADD45g基因在AML患者骨髓组织和AML细胞系中低表达,对AML细胞系有显著的全方位的抑制作用,其表达水平对AML具有预后判断价值。

GADD45α、CXCL14蛋白在胃癌组织中的表达及其与临床特征的关系

目的探讨生长停滞DNA损伤可诱导蛋白α(GADD45α)、趋化因子CXCL14蛋白在胃癌组织中的表达及其与临床特征的关系。方法收集术后胃癌组织标本60例(胃癌组)及其对应的癌旁组织标本60例(对照组),采用免疫组化染色法检测两组标本中的GADD45α、CXCL14蛋白表达情况,并分析GADD45α、CXCL14蛋白与胃癌的分化程度、浸润深度、TNM分期、淋巴结转移的关系。结果胃癌组标本的GADD45α、CXCL14蛋白阳性表达率分别为71.67%、78.33%,均明显高于对照组标本的30.00%、36.67%,差异均有统计学意义(P﹤0.01)。TNM分期为Ⅲ+Ⅳ期、发生淋巴结转移的胃癌组织的GADD45α蛋白阳性表达率均高于TNM分期为Ⅰ+Ⅱ期、未发生淋巴结转移的胃癌组织,差异均有统计学意义(P﹤0.05);不同肿瘤分化程度、浸润深度的胃癌组织的GADD45α蛋白阳性表达率比较,差异均无统计学意义(P﹥0.05)。低分化、浸润深度为T3+T4、TNM分期为Ⅲ+Ⅳ期的胃癌组织的CXCL14蛋白阳性表达率均高于高中分化、浸润深度为T1+T2、TNM分期为Ⅰ+Ⅱ期的胃癌组织,差异均有统计学意义(P﹤0.05);是否发生淋巴结转移的胃癌组织的CXCL14蛋白阳性表达率比较,差异无统计学意义(P﹥0.05)。结论胃癌组织中GADD45α、CXCL14蛋白表达上调,并且与患者临床特征具有一定的关系。

Gadd45α在干细胞DNA损伤中的作用

基因组完整性对于细胞和组织功能至关重要,这种稳态会不断地受到内源性和外源性应激刺激的影响。干细胞对这些应激刺激十分敏感,其DNA会发生不同程度的损伤,诱导干细胞内固有的DNA修复机制。组织特异性干细胞是局部环境中的多能群体,在其整个生命过程中负责维持组织或者系统的完整性。组织特异性干细胞在受到应激刺激之后,能通过某些反应修复损伤或激活程序性凋亡,从而减少由DNA损伤导致潜在的突变。生长停滞和DNA损伤诱导蛋白45α(growth arrest and DNA damage-45 alpha,Gadd45α)是细胞周期调控因子和生长抑制因子,其表达受到多种因素的调节,参与多种细胞DNA损伤反应。Gadd45α蛋白在组织特异性干细胞DNA损伤中发挥重要的作用。Gadd45α蛋白通过结合其他相关蛋白质控制细胞周期检测点、参与DNA修复和调控信号转导,是维持基因组稳定性的重要组成部分。该文综述了Gadd45α在胚胎干细胞、神经干细胞、造血干细胞、肠干细胞等干细胞DNA损伤中的作用以及涉及的信号通路,为进一步探讨Gadd45α在细胞中潜在的作用以及利用Gadd45α进行疾病治疗提供理论依据。

The Apoptotic Effect of the Methanol Extract of <i>Polygonum cuspidatum</i>through Up-Regulation Death Receptor 5 and CHOP in HSC-2 Human Oral Cancer Cells

Polygonum cuspidatum is used as a traditional medicinal herb for the therapy of various diseases including several types of cancers. In the present study, we focused on addressing the anti-cancer activity and molecular mechanism of methanol extract of Polygonum cuspidatum (MEPC) in HSC-2 human oral cancer cells. The effect of MEPC on oral cancer cells was estimated by 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis. MEPC inhibited the cell viability and induced apoptosis through the induction of death receptor (DR) 5. MEPC also increased the expression of C/EBP homologous protein/growth arrest and the DNA damage-inducible gene 153 (CHOP), a transcription factor induced by ER stress. Thus, we concluded that the induction of CHOP leading to DR5 up-regulation is required for the anti-cancer activity of MEPC in HSC-2 cells and MEPC may be a promising drug candidate for oral cancer.

放射线对体外培养棘球蚴及其Gadd45α mRNA表达的影响

目的观察放射线对体外培养棘球蚴的杀伤作用及对其生长阻滞和DNA损伤45α(growth arrest and DNA damage 45 alpha,Gadd45α)mRNA表达的影响。方法体外培养自然感染的羊肝脏中的棘球蚴,分为7组,分别以0(对照)、20、40、60、80、100、120 Gy的6兆电子线(6 MeV)进行1次放射。放射线照射后培养1、3、5、7 d时,光镜下观察棘球蚴的生长情况;采用反转录-聚合酶链反应(RT-PCR)法测定放射线照射后培养7 d时,对照,20、40、60 Gy组棘球蚴Gadd45αmRNA表达水平。结果各剂量组放射线照射后1、3、5、7 d时,光镜下棘球蚴结构崩解,勾突脱落,且棘球蚴死亡率与放射线照射剂量呈正相关(r=0.81,P<0.05)。放射线照射后培养7 d时,与对照组(100.00±0.00)比较,20、40、60 Gy组棘球蚴Gadd45αmRNA表达水平明显升高(279.74±80.08、759.38±160.98、1666.68±316.36,P均<0.01),且与放射线剂量呈正相关(r=0.93,P<0.01)。结论放射线照射对体外培养的棘球蚴有一定的杀伤作用,且与放射线的剂量存在一定的量效关系;Gadd45α基因可能参与放射线所致体外杀伤棘球蚴的分子机制。