Inhibition of the proliferation,invasion, migration,and epithelial-mesenchymal transition of prostate cancer cells through the action of ATP1A2 on the TGF-β/Smad pathway

BackgroundProstate cancer (PC) is one of the major male malignancies worldwide. Because Na⁺-K⁺-ATPase is widely involved in various pathological processes, but the action of its α2 subtype (ATP1A2) in PC is unclear, we investigated the role of ATP1A2 in the invasion and migration of PC cells.MethodsWe measured the expression levels of ATP1A2 in human normal prostate epithelial cell line (RWPE-1) and PC cell lines (PC-3 and DU145) by quantitative real-time PCR (qRT-PCR) and western blot. Cell proliferation, apoptosis, migration, and invasion of PC-3 and DU145 cells were investigated through clone formation assay, EdU assay, flow cytometry and transwell assay, respectively. The effect of ATP1A2 on a tumor-inhibitory pathway [transforming growth factor-β (TGF-β)/Smad] was assessed using western blot. In addition, tumor formation was detected using in vivo xenograft model in male BALB/c nude mice.ResultsThe Cancer Genome Atlas (TCGA) analysis showed that ATP1A2 expression was reduced in PC patients (P<0.05), and patients with low ATP1A2 expression had a lower survival rate (P<0.05). ATP1A2 levels were significantly reduced in PC-3 and DU145 cells, compared with RWPE-1 cells (P<0.01). We also demonstrated that overexpression of ATP1A2 significantly inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of PC-3 and DU145 cells (P<0.01) and promoted apoptosis (P<0.01). However, silencing ATP1A2 had the opposite effect (P<0.01). In addition, overexpression of ATP1A2 significantly inhibited the TGF-β/Smad pathway (P<0.01), whereas silencing ATP1A2 activated the TGF-β/Smad pathway (P<0.01). Meanwhile, the effect of ATP1A2 silencing on the proliferation, apoptosis, migration and invasion was reversed by TGF-β/Smad pathway inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY364947","term_id":"1257906561","term_text":"LY364947"}}LY364947). Furthermore, ATP1A2 inhibited tumor growth in vivo.ConclusionsATP1A2 inhibited proliferation, apoptosis, migration, invasion, and EMT in PC by inhibiting the TGF-β/Smad pathway.     

复合凋亡通路介导的水飞蓟宾抑制膀胱癌的动物实验研究

【摘要】 目的 探讨水飞蓟宾(Silibinin)对荷人膀胱癌细胞5637裸鼠皮下移植瘤的生长抑制作用及其机制。方法 取培养于体外的对数生长期的人膀胱癌细胞5637接种于裸鼠右后腿背侧皮下,建立裸鼠移植瘤模型,通过口服给予不同剂量的水飞蓟宾(0、50、100、200 mg/kg)后定期观察并测量各组肿瘤体积大小,给药一定时间后处死裸鼠,取出肿瘤组织,称重。采用流式细胞术检测肿瘤细胞凋亡率,采用Western blot法及免疫组织化学法检测肿瘤组织内凋亡相关因子Caspase-3、Cleaved PARP、Bax、Bcl-2及细胞色素C(Cyt-C)的蛋白表达情况并运用统计学方法进行比较分析。结果 50只裸鼠建模成功42只,随机选取40只进入实验,实验过程中无裸鼠死亡。通过测量实验过程中肿瘤体积算得在32 d观察结束时,水飞蓟宾低、中、高剂量组肿瘤生长抑制率分别为10.08%、47.64%、63.14%(F=52.79,P<0.01);对照组、低、中、高剂量组肿瘤质量分别为(1.54±0.25)、(1.46±0.22)、(0.94±0.15)和(0.72±0.16)g,差异具有统计学意义(F=39.90,P<0.01);肿瘤细胞凋亡率(%)对照组、低、中和高剂量组分别为(6.70±1.34)、(8.08±1.19)、(26.11±1.91)、(44.87±2.96),差异有统计学意义(F=794.17,P<0.01),但低剂量组与对照组相比凋亡率没有明显差别(P=0.14);各组间Caspase-3、Cleaved PARP、Bax、Bcl-2和Cyt-C的蛋白表达均有明显差异(P<0.05),但水飞蓟宾不同剂量组间及与对照组两两进行比较,结果差异性不尽一致。结论 水飞蓟宾在体内环境中可以显著抑制膀胱癌组织的生长,并且这种生长抑制作用随水飞蓟宾剂量增高而增强,这种抑制作用可能与Caspase介导的细胞凋亡,线粒体介导的细胞凋亡及Bcl-2家族蛋白表达下调/Bax家族蛋白表达上调有关。     

The effects of modified RNA-binding proteins HuR on the biological behavior of the bladder cancer T24 cell line

BackgroundIn tumors, the role of human antigen R (HuR) includes regulating tumor cell proliferation, differentiation, apoptosis, angiogenesis, and lymphangiogenesis. Previous studies have revealed that the expression of HuR can be detected in bladder cancer, and is related to the biological behavior of malignancy.MethodsT24 cells were transfected by HuR overexpression and HuR knockdown vectors, and divided into the control group, the overexpression-HuR group, and the cas9-HuR group. Cell viability was detected after 48 h by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, apoptosis was detected by Annexin V-allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) double staining, cell migration was detected by Transwell assays, and the expression levels of HuR, cyclin D1, and apoptosis-related factors [i.e., B-cell lymphoma 2 (Bcl-2)] were detected by fluorescence quantitative polymerase chain reaction (PCR) and Western blot.ResultsCompared to the control group, cell viability after 48 h increased significantly in the overexpression-HuR group, and decreased significantly in the cas9-HuR group (P<0.05). The number of migrating cells increased significantly in the overexpression-HuR group, and decreased significantly in the cas9-HuR group (P<0.05). The apoptosis rate was significantly decreased in the overexpression-HuR group, and significantly increased in the cas9-HuR group (P<0.05). The messenger ribonucleic acid and protein expression levels of HuR, cyclin D1, and Bcl-2 were significantly increased in the overexpression-HuR group, and significantly decreased in the cas9-HuR group (P<0.05).ConclusionsHuR promotes the proliferation and migration of T24 cells, and inhibits cell apoptosis. The mechanism may be related to the expression of cyclin D and the apoptosis-related protein, Bcl-2.     

厚朴酚对胃癌细胞增殖及凋亡的影响及机制研究

目的探讨厚朴酚对胃癌细胞增殖及凋亡的影响及机制研究。 方法0(对照组), 20, 40, 80 μmol/L的厚朴酚作用SGC－7901细胞24、48、72 h,MTT法检测细胞活力,流式细胞术检测细胞凋亡及细胞周期,蛋白质免疫印迹(Western blot)检测细胞中B淋巴细胞瘤－2(Bcl－2)、Bcl－2相关X蛋白(Bax)、天冬氨酸蛋白水解酶－3(cleaved caspase 3)、细胞周期蛋白D1(CyclinD1)、CyclinE及细胞周期依赖性蛋白激酶2(CDK2)蛋白的表达。所有数据均用SPSS 17.0软件进行统计分析,细胞增殖抑制率、细胞凋亡及细胞周期、蛋白表达量等用( ±s)表示,组间差异采用t检验进行分析。P<0.05表示差异具有统计学意义。 结果20, 40, 80 μmol/L的厚朴酚较对照组细胞活力降低(P<0.01),细胞凋亡率提高(P<0.01),细胞周期阻滞于G1期(P<0.01),Bcl－2,CyclinD1,CyclinE及CDK2表达量下调(P<0.01),Bax、cleaved caspase 3表达量上调(P<0.01)。 结论厚朴酚可能通过调控细胞周期蛋白及细胞凋亡相关蛋白抑制SGC－7901细胞增值,并诱导细胞凋亡。     

他莫昔芬与γ-干扰素联合抗人乳腺癌细胞株的作用及其机制研究

目的: 探讨他莫昔芬(TAM)与 γ-干扰素(γ-IFN)联合抗乳腺癌细胞株的作用及其机制。方法:在体外培养条件下，分别或联合应用γ-IFN,TAM或雌二醇（E2）作用于ER阳性的MCF-7人乳腺癌细胞株，用MTT比色法分析细胞生长抑制作用;用流式细胞仪（FCM）检测细胞周期分布、凋亡率及用药前后Bcl-2,Bax,Fas,FasL及Caspase-8蛋白的变化;荧光分光光度仪检测Caspase-3活性。结果:TAM能抑制ER阳性乳腺癌细胞的生长，阻滞细胞周期于G0/G1期，并可诱导细胞凋亡;同时，TAM能拮抗外源性雌激素对MCF-7细胞的促生长作用。γ-IFN预处理细胞24h后，TAM抗乳腺癌细胞的作用增强。联用γ-IFN与TAM后，细胞Bcl-2蛋白表达下调，Caspase-8表达上调;但药物处理前后，细胞Bax，Fas，FasL蛋白表达水平及Caspase-3活性未见明显变化。结论:体外条件下，TAM通过影响细胞周期、诱导细胞凋亡而发挥抗ER阳性乳腺癌细胞作用;γ-IFN能加强TAM的抗乳腺癌作用。两者作用机制可能系通过下调Bcl-2表达和上调Caspase-8的表达。     

白藜芦醇对七氟醚诱导神经细胞损伤的影响

目的探讨白藜芦醇(resveratrol,RES)对七氟醚诱导的离体神经细胞损伤的影响及其机制。方法取SD孕鼠的胎鼠海马组织,分离培养原代神经细胞,然后将细胞随机分为四组:对照组(C组),正常培养的原代神经细胞;3%七氟醚组(S组),3%七氟醚混合气体处理的细胞;3%七氟醚+RES组(SR组),用RES处理细胞6h后,再用3%七氟醚混合气体处理的细胞;RES组(R组),RES处理的细胞。流式细胞仪检测神经细胞的凋亡;CCK-8试剂盒检测神经细胞的增殖;Western blot检测Caspase-3、Bax、Bcl-2和Bace-1的表达水平;ELISA检测APP和Aβ的表达水平。结果 S组的细胞凋亡率、Caspase-3、Bax、Bace-1、APP及Aβ的表达明显高于C组(P0.05);细胞增殖能力和Bcl-2的表达明显低于C组(P0.05)。SR组的细胞凋亡率、Caspase-3、Bax、Bace-1、APP及Aβ的表达明显低于S组(P0.05);细胞增殖能力、Bcl-2的表达明显高于S组(P0.05)。结论白藜芦醇通过减少细胞凋亡,促进细胞增殖以缓解七氟醚诱导的离体神经细胞损伤,这可能与Aβ的表达下调,从而降低神经毒性有关。     

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca²⁺ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G₀/G₁ stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.     

Apoptotic stress pathway activation mediated by iron on endothelial cells in vitro.

BACKGROUND: Iron sucrose (Fe-S) and low-molecular-weight iron dextran (Fe-D) have been used successfully in the treatment of anaemia in chronic kidney disease patients. However, some side effects, such as endothelial cell dysfunction have been reported. Mechanisms by which iron can induce endothelial cell damage have not been completely understood. This study was designed to examine the effect of Fe-S and Fe-D on bovine aortic endothelial cells in vitro. METHODS: Cell proliferation was determined by [3H] thymidine incorporation, cytotoxicity by lactate dehydrogenase, pro-Caspase-3 by immunoblotting; and Caspase-3 activity using a colorimetric assay. Expression of the apoptosis stress pathway proteins Bcl-2 and Bax and cycle arrest proteins p53 and p21WAF/CIP1 were examined by immunoblot. Cell apoptosis was tested by terminal deoxynucleotidyltransferase-mediated nick-end labelling (TUNEL) and DNA fragmentation. RESULTS: Both iron preparations inhibited cell proliferation. This effect was more important and occurred at lower concentrations in Fe-S than Fe-D cultured cells. Expression of p53 and p21WAF/CIP1 increased in cells incubated with Fe-S, but not with Fe-D. Bcl-2 expression was significantly down-regulated in cells incubated with Fe-S in comparison with Fe-D, while Bax expression was not modified by the iron compounds. Pro-Caspase-3 expression and Caspase-3 activity increased only in cells treated with Fe-S. Apoptosis was present in cells treated with Fe-S. CONCLUSIONS: Our results demonstrate that Fe-S exerts a greater inhibitory effect on endothelial cell proliferation than Fe-D. The mechanisms involved in this process may be related, at least in part, to over expression of proteins related to the cell cycle arrest and apoptosis stress pathway.     

MiR-486-5p enhances cisplatin sensitivity of human muscle-invasive bladder cancer cells by induction of apoptosis and down-regulation of metastatic genes

ObjectivesCisplatin is one of the common chemotherapy drugs for bladder cancer, and resistance to this drug is one of the major obstacles to effective chemotherapy. MicroRNAs (miRNAs) are a category of small noncoding RNAs that can regulate the expression of numerous genes. Recent studies showed that miRNAs can act as a powerful regulator of chemo-sensitivity in cancer cells. Hence, this study aimed to investigate the effects of miRNA-486-5p on cisplatin-sensitivity of different bladder cancer cells.Material and MethodsThe 5637 and EJ138 cancer cells were treated with miRNA-486-5p and cisplatin, individually or in combination.ResultsAfterward, the cytotoxicity effects of these treatments were determined by MTT assay and the increased cisplatin-sensitivity observed in both cell lines, especially, 5637 cells. Moreover, subG1 phase cell cycle arrest, changes in the expression of caspase-9, caspase-3, P53, SIRT1, OLFM4, SMAD2, and Bcl-2 genes and nuclear fragmentation also revealed the induction of apoptosis in all treatments, which increased in combination groups. Also, the combination of miRNA-486-5p with cisplatin significantly down-regulated the expression of migration associated genes including ROCK, CD44, and MMP-9 as compared with cisplatin alone.ConclusionAltogether, these results indicated that the miRNA-486-5p could induce apoptosis and inhibit cell migration ability of the cells. It seems that pre-electroporation of cells with miRNA-486-5p has useful results in the enhancement of cisplatin sensitivity of 5637 and EJ138 cancer cells and this combination may be a promising treatment strategy for bladder cancer therapy.     

Roscovitine对增生期肝癌细胞周期的影响

目的 探讨细胞周期蛋白依赖激酶抑制剂Roscovitine对增生期肝癌细胞SMMC-7721细胞周期的影响.方法 采用体外培养的肝癌细胞SMMC-7721,经过Roscovitine作用后,对SMMC-7721细胞的形态、生长情况、细胞周期时相的分布、凋亡以及CDK2、Caspase-3、bcl-2 mRNA的表达情况进行观察.结果 MTT提示:Roscovitine对SMMC-7721细胞的增生有抑制作用,作用效果呈时间、剂量依赖性,并促进细胞的凋亡;流式细胞仪发现G0期、G1期的比例增加,细胞出现凋亡;R-T PCR显示CDK2 mRNA表达水平降低,凋亡相关基因bel-2表达降低,Caspase-3 mRNA水平表达升高.结论 Roscovitine可以抑制增生期肝癌细胞的生长、增生,阻滞细胞周期于G0/G1期,诱导细胞的凋亡,凋亡机制与bcl-2、Caspase-3的表达改变有关.     

矢车菊素-3-葡萄糖苷诱导人胃癌细胞株SGC7901凋亡及其机制

目的 观察矢车菊素-3-葡萄糖苷(C3G)在体外诱导人胃癌SGC7901细胞凋亡的生物学效应,并初步探讨其分子机制.方法 分别采用不同浓度梯度的C3G处理SGC7901胃癌细胞,MTT比色法检测细胞生长率;激光共聚焦显微镜观察细胞形态改变;TUNEL法分析细胞凋亡率;Western blot法检测凋亡相关基因Bcl-2、Bax、Caspase-3、ICAD蛋白表达的情况.结果 C3G在体外能明显抑制人胃癌SGC7901细胞的生长增殖,且呈浓度、时间依赖性(P＜0.01).激光共聚焦显微镜下可见Hoechst33258荧光染色细胞呈凋亡特征性改变;TUNEL法检测实验组细胞凋亡率均明显高于阴性对照组(P＜0.01);C3G可下调Bcl-2蛋白表达,上调Bax蛋白表达,降低Bcl-2/Bax蛋白的比值,促使Caspase-3酶原蛋白活化,下调ICAD蛋白表达(P＜0.01).结论 C3G具有在体外抑制胃癌细胞增殖的作用,并能诱导其凋亡,其机制可能与Bcl-2/Bax降低导致Caspase-3活化、ICAD蛋白表达下降相关.     

AT9283对基底样乳腺癌细胞侵袭及转移的影响

目的研究Aurora激酶抑制剂AT9283对基底样乳腺癌细胞（BLBC）系MDA-MB-231细胞运动能力和凋亡的影响，探讨AT9283降低BLBC侵袭和转移的作用机制。 方法取不同浓度（0、0.01、0.1、1、10 μmol/L）AT9283处理MDA-MB-231细胞，MTT法检测细胞增殖抑制率；PI染色流式细胞术检测多倍体细胞形成，Annexin V/PI双染流式细胞术、荧光染料显色观察不同浓度下AT9283诱导MDA-MB-231细胞凋亡情况；Western blotting检测凋亡相关蛋白的表达变化和Aurora激酶、Cofilin-1及磷酸化水平的改变；划痕试验及趋化试验观察AT9283对细胞迁移、趋化能力的影响。 结果AT9283（10 μmol/L）处理MDA-MB-231细胞24 h后，细胞增殖抑制率为（89.17±0.03）%，荧光染色可见AT9283处理的胞核绿染、碎裂；流式细胞术检测1 μmol/L AT9283作用于乳腺癌细胞48 h形成多倍体细胞，0.1、1、10 μmol/L AT9283作用下MDA-MB-231细胞凋亡率分别为（13.4±2.5）%、（29.5±3.4）%、（52.8±6.7）%，与对照组的（0.7±0.1）%相比，均明显升高（P<0.05）。同时，抗凋亡蛋白Bcl-XL表达减少，凋亡相关蛋白Caspase-3和PARP蛋白剪切增加。AT9283可显著延长划痕愈合时间，减少趋化穿膜细胞个数（P<0.05），并有效抑制Aurora激酶及Cofilin-1的磷酸化水平。 结论AT9283可通过抑制Aurora激酶的磷酸化及Cofilin-1磷酸化水平而诱导BLBC细胞系凋亡，并抑制其运动，从而抑制侵袭及转移。     

姜黄素对人脊索瘤细胞系CM-319凋亡和放射敏感性影响的实验研究

目的探讨姜黄素对人脊索瘤细胞系CM-319凋亡和放射敏感性的影响及作用机制。方法体外培养CM-319细胞,分为姜黄素0μmol/L组、姜黄素5μmol/L组、姜黄素10μmol/L组、姜黄素20μmol/L组、si-NC组、si-UHRF1组、姜黄素+pcDNA组、姜黄素+pcDNA-UHRF1组,流式细胞仪检测细胞凋亡,Western Blot检测细胞中Bcl-2、Bax、Cleaved Caspase-3和UHRF1蛋白表达,qRT-PCR检测UHRF1 mRNA表达,克隆形成实验检测CM-319细胞对放射线敏感性。结果姜黄素0μmol/L组CM-319细胞凋亡率7.24%±0.73%,姜黄素5、10、20μmol/L组CM-319细胞凋亡率分别为12.61%±1.57%、18.42%±1.54%、29.37%±2.65%,差异有统计学意义(t=9.305;t=19.680;t=24.153,均P<0.05);Bcl-2蛋白相对表达量为0.76±0.07,余3组分别为0.62±0.06、0.51±0.05、0.33±0.03(t=4.556;t=8.719;t=16.939,均P<0.05);Bax相对表达量为0.26±0.03、0.41±0.04、0.55±0.05、0.69±0.06(t=9.000;t=14.920;t=19.230,均P<0.05);Cleaved Caspase-3相对表达量为0.24±0.03、0.37±0.04、0.49±0.05、0.62±0.06(t=7.800;t=12.862;t=16.994,均P<0.05),UHRF1mRNA相对表达量为1.01±0.09、0.82±0.08、0.67±0.07、0.42±0.04(t=4.734;t=8.946;t=17.972,均P<0.05),UHRF1蛋白相对表达量为0.83±0.08、0.61±0.06、0.48±0.05、0.31±0.0(t=6.600;t=11.130;t=18.258,均P<0.05),且呈剂量依赖性,增敏比分别为1.433、1.708和2.183。Si-NC组CM-319细胞中UHRF1蛋白相对表达量为0.79±0.08,si-UHRF1组表达降低为0.35±0.04(t=14.758,P<0.05);CM-319细胞凋亡率分别为8.24%±0.83%和21.49%±2.11%(t=17.531,P<0.05);Bcl-2蛋白表达水平分别为0.71±0.07、0.34±0.03(t=14.575,P<0.05);Bax相对表达量为0.25±0.03、0.62±0.06(t=16.547,P<0.05);Cleaved Caspase-3相对表达量为0.23±0.03、0.58±0.05(t=18.007,P<0.05),增敏比为1.727。姜黄素+pcDNA组CM-319细胞凋亡率为28.31%±3.01%,姜黄素+pcDNA-UHRF1组为13.59%±1.25%(t=13.549,P<0.05);Bcl-2相对表达量分别为0.31±0.03、0.63±0.06(t=14.311,P<0.05);Bax相对表达量分别为0.71±0.07、0.42±0.04(t=10.791,P<0.05);Cleaved Caspase-3相对表达量分别为0.65±0.05、0.38±0.04(t=12.650,P<0.05),增敏比为0.539。结论姜黄素可诱导CM-319细胞凋亡并增强CM-319细胞的放射敏感性,其机制与下调UHRF1表达有关。     

Filamin A (FLNA) regulates autophagy of bladder carcinoma cell and affects its proliferation,invasion and metastasis

Purpose

This research intended to explore the effect of FLNA on cell proliferation, invasion and migration in bladder carcinoma (BC).

Methods

Microarray analysis was performed with the TCGA data, and the results were confirmed on 20 paired BC tissues and adjacent tissues using qRT-PCR and immunohistochemistry. Transmission electron microscope (TEM) and cell fluorescence assay were used to observe the quantity of autophagosomes. The expression of autophagy-related protein (LC3-I/II, p62) was detected by western blot. Cell proliferation was detected using CCK-8 and colony formation. The invasion and migration ability of the cell were tested by transwell and wound-healing assay. Tumor xenograft in BALB/c nude mice and HE staining were utilized to probe into the effects of FLNA-induced regulation of volume, weight and metastasis of tumors.

Results

We confirmed that FLNA was down-regulated in BC tissues. TEM and fluorescence analysis proved that FLNA overexpression promoted autophagy in BC cells. Colony formation assay and CCK-8 experiment showed that FLNA overexpression suppressed the proliferation of BC cells. In addition, FLNA blocked cell cycle and promoted apoptosis of BC cell. Transwell assay and wound-healing assay further proved that FLNA suppressed invasion and migration ability in BC cell. Meanwhile, in vivo study indicated that FLNA inhibited the tumor growth.

Conclusion

Overexpression of FLNA suppressed the proliferation, migration and invasion of the BC cell, suggesting the potential role of FLNA in clinical treatment.

     

The protective effects of exogenous spermine on renal ischemia-reperfusion injury in rats

BackgroundTo investigate the protective effects of exogenous spermine on renal ischemia-reperfusion injury in rats.Methods(I) Different doses of spermine were injected into rats to determine the safe dose on the kidneys. Kidney toxicity was assessed by hematoxylin and eosin (HE) staining of kidney tissue and enzyme-linked immunosorbent assay (ELISA) detection of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) in the venous blood. (II) A rat model of renal ischemia-reperfusion injury was established. Different doses of spermine were injected into the rats through the tail vein 30 minutes before and 3 days after the establishment of the model. Blood samples and kidney tissues were collected and renal injury was assessed via HE staining of the renal tissue, detection of apoptosis using the TUNEL assay, and detection of NGAL and KIM-1 in blood samples using ELISA. (III) Human HK-2 renal tubular epithelial cells were cultured under hypoxia/reoxygenation conditions. To evaluate the protective effects of spermine, apoptosis was assessed by flow cytometry and TUNEL assay. The mechanisms underlying the effects of spermine were studied using Western blot analyses.ResultsAt spermine concentrations below 200 µM (2 mL/kg body weight), no significant damage to the kidney was observed by HE staining, and there was no significant difference in NGAL and KIM-1 levels between rats treated with spermine and control rats (P<0.05). At spermine doses below 200 µM, HE staining showed that the degree of renal ischemia-reperfusion injury was gradually alleviated with increasing doses of spermine. TUNEL assays demonstrated that spermine reduced the apoptosis of renal tissue, and increasing doses of spermine gradually decreased the levels of NGAL and KIM-1 in the blood compared with the control group (P<0.05). Western blot analysis revealed that spermine increased the expression of pro-caspase9, phosphorylated protein kinase B (p-Akt), hypoxia-inducible factor 1 alpha (HIF-1α), B cell lymphoma 2 (Bcl-2), and Bcl2 interacting protein 3 (BNIP3), and decreased the expression of cleaved caspase-3, Bax and cytochrome C compared to control cells.ConclusionsExogenous spermine exerted a protective effect on renal ischemia-reperfusion injury in rats by inhibiting the apoptosis of renal tubular epithelial cells.     

DHA对人肝癌细胞增殖与凋亡的影响及其机制的研究

目的：探讨二十二碳六烯酸（DHA）对人肝癌细胞株Bel-7402增殖和凋亡的影响及其机制。方法：用不同浓度的DHA（0,25,50,100,200 μmol/L）分别作用人肝癌Bel-7402细胞24,48,72 h后,用MTT法检测细胞的增殖情况、Western blot检测Bcl-2和Bax蛋白的表达,并分别用流式细胞仪、real-time PCR和caspase-3活性检测试剂盒检测上述梯度浓度的DHA作用 Bel-7402细胞48 h后的凋亡情况、Bim基因表达和caspase-3活性。结果：不同浓度、不同时间DHA作用后,Bel-7402细胞增殖明显抑制,呈浓度和时间依赖性（均P<0.05）;细胞Bax蛋白表达增加、Bcl-2蛋白表达降低,呈明显浓度依赖性（均P<0.05）,但无明显时间依赖性（均P>0.05）。不同浓度DHA作用48 h后,Bel-7402细胞凋亡率、Bim基因表达和caspase-3的活性均明显增加,且均呈浓度依赖性（均P<0.05）。结论：DHA可抑制人肝癌Bel-7402细胞的增殖并诱导细胞凋亡,其机制可能与激活线粒体凋亡通路有关。