p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2 +, p21/WAF1?, while hidden infected cells were usually MDM2?, p21/WAF1 +. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53 +, p21/WAF1 +, MDM2?, or p53 +, p21/WAF1?, MDM2 + protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53 +, p21/WAF1?, MDM2 + profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2 + cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.     

黄芪桂枝五物汤加减对糖尿病大鼠坐骨神经细胞凋亡相关Bax和Caspase-12的影响

目的 研究黄芪桂枝五物汤加减对糖尿病大鼠坐骨神经细胞凋亡相关B细胞淋巴瘤-2相关X蛋白（Bax）和胱天蛋白酶-12（Caspase-12）蛋白与mRNA表达的影响,以探究黄芪桂枝五物汤加减治疗糖尿病周围神经病变的作用机制。方法 选用动物实验方法进行研究,将60只雄性SD大鼠通过高糖高脂饲料喂养联合链尿佐菌素（STZ）腹腔注射诱导成糖尿病大鼠动物模型,连续3 d随机血糖≥16.7 mmol·L^-1者为糖尿病大鼠造模成功,将48只造模成功的糖尿病大鼠随机分为模型组、α-硫辛酸组（0.026 8 g·kg^-1·d^-1）、中药高、低剂量组（2.5、1.25 g·kg^-1·d^-1）,每组各12只,并设正常组10只。监测大鼠体质量和随机血糖水平;干预16周末通过Key point肌电采集系统检测大鼠坐骨神经传导速度;分别通过蛋白免疫印迹法（Western blot）和实时荧光定量聚合酶链式反应（Real-time PCR）检测大鼠坐骨神经中Bax和Caspase-12蛋白与mRNA的表达。结果 与正常组比较,模型组大鼠体质量显著下降（P<0.01）,随机血糖水平显著升高（P<0.01）;干预16周,与模型组比较,中药高剂量组大鼠体质量明显升高（P<0.05）,其他给药组体质量变化差异无统计学意义;各给药组随机血糖水平均显著降低（P<0.01）。与正常组比较,干预16周,模型组大鼠运动和感觉神经传导速度显著降低（P<0.01）;与模型组比较,各给药组大鼠运动和感觉神经传导速度均明显升高（P<0.05,P<0.01）。与正常组比较,模型组大鼠坐骨神经Bax和Caspase-12蛋白表达显著升高（P<0.01）;与模型组比较,各给药组大鼠坐骨神经Bax和Caspase-12蛋白表达均显著降低（P<0.01）。与正常组比较,模型组大鼠坐骨神经Bax和Caspase-12 mRNA表达显著升高（P<0.01）;与模型组比较,α-硫辛酸组、中药高剂量组大鼠坐骨神经Bax mRNA表达明显降低（P<0.05,P<0.01）,中药低剂量组坐骨神经Bax mRNA表达降低有下降趋势;各给药组大鼠坐骨神经Caspase-12 mRNA表达显著降低（P<0.01）。结论 黄芪桂枝五物汤加减可能通过抑制坐骨神经细胞凋亡来改善和修复糖尿病大鼠坐骨神经损伤。     

薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响及机制

目的：探究薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响,并探讨其相关的细胞凋亡机制。方法：不同质量浓度(0.5、1、2、4、6、8、10、12、14、16 g·L^-1 )薏苡附子败酱散干预结肠癌细胞24、48、72 h,细胞增殖与活性检测-8(CCK-8)法检测细胞体外增殖的影响;设空白组、卡培他滨组(1.8 g·L^-1 )和薏苡附子败酱散组(6、10、14 g·L^-1 ),分别处理48 h,采用流式细胞技术检测细胞凋亡率,Hochest 33342荧光染色观察细胞凋亡形态,线粒体红色荧光探针(Mito-Tracker Red CMXRos)分析线粒体膜电位(MMP)变化,蛋白免疫印迹法(Western blot)检测线粒体凋亡途径相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、胱天蛋白酶(Caspase)-9、Caspase-3、活化的(cleaved) Caspase-9、cleaved Caspase-3的表达水平,实时荧光定量聚合酶链式反应(Real-time...     

Effects of Dietary Oat Beta-Glucans on Colon Apoptosis and Autophagy through TLRs and Dectin-1 Signaling Pathways—Crohn’s Disease Model Study

Background: Crohn’s disease (CD) is characterized by chronic inflammation of the gastrointestinal tract with alternating periods of exacerbation and remission. The aim of this study was to determine the time-dependent effects of dietary oat beta-glucans on colon apoptosis and autophagy in the CD rat model. Methods: A total of 150 Sprague–Dawley rats were divided into two main groups: healthy control (H) and a TNBS (2,4,6-trinitrobenzosulfonic acid)-induced colitis (C) group, both including subgroups fed with feed without beta-glucans (βG−) or feed supplemented with low- (βGl) or high-molar-mass oat beta-glucans (βGh) for 3, 7, or 21 days. The expression of autophagy (LC3B) and apoptosis (Caspase-3) markers, as well as Toll-like (TLRs) and Dectin-1 receptors, in the colon epithelial cells, was determined using immunohistochemistry and Western blot. Results: The results showed that in rats with colitis, after 3 days of induction of inflammation, the expression of Caspase-3 and LC3B in intestinal epithelial cells did not change, while that of TLR 4 and Dectin-1 decreased. Beta-glucan supplementation caused an increase in the expression of TLR 5 and Dectin-1 with no changes in the expression of Caspase-3 and LC3B. After 7 days, a high expression of Caspase-3 was observed in the colitis-induced animals without any changes in the expression of LC3B and TLRs, and simultaneously, a decrease in Dectin-1 expression was observed. The consumption of feed with βGl or βGh resulted in a decrease in Caspase-3 expression and an increase in TLR 5 expression in the CβGl group, with no change in the expression of LC3B and TLR 4. After 21 days, the expression of Caspase-3 and TLRs was not changed by colitis, while that of LC3B and Dectin-1 was decreased. Feed supplementation with βGh resulted in an increase in the expression of both Caspase-3 and LC3B, while the consumption of feed with βGh and βGl increased Dectin-1 expression. However, regardless of the type of nutritional intervention, the expression of TLRs did not change after 21 days. Conclusions: Dietary intake of βGl and βGh significantly reduced colitis by time-dependent modification of autophagy and apoptosis, with βGI exhibiting a stronger effect on apoptosis and βGh on autophagy. The mechanism of this action may be based on the activation of TLRs and Dectin-1 receptor and depends on the period of exacerbation or remission of CD.     

The Mechanisms of the Anti-Inflammatory and Anti-Apoptotic Effects of Omega-3 Polyunsaturated Fatty Acids during Methotrexate-Induced Intestinal Damage in Cell Line and in a Rat Model

Background: The aim of this study was to examine the anti-inflammatory and anti-apoptotic patterns of omega-3 polyunsaturated fatty acids (n-3 PUFAs) during methotrexate (MTX) induced intestinal damage in cell culture and in a rat model. Methods: Non-treated and treated with MTX HT 29 and HCT116cells were exposed to increasing doses of n-3 PUFAs and cell viability was evaluated using PrestoBlue^® assay. Male Sprague-Dawley rats were divided into 4 experimental groups: Control rats, CONTR+n-3 PUFA rats that were treated with oral n-3 PUFA, MTX rats were treated with MTX given IP, and MTX+n-3 PUFA rats were treated with oral n-3 PUFA before and following injection of MTX. Intestinal mucosal parameters and mucosal inflammation, enterocyte proliferation and apoptosis, TNF-α in mucosal tissue and plasma (ELISA), NF-κB, COX-2, TNF-α, Fas, FasL, Fadd, Bid, Bax and Bcl-2gene and protein levels were determined 72 h following MTX injection. Results: Exposure of HT 29 and HCT116cells to n-3 PUFA attenuated inhibiting effects of MTX on cell viability. MTX-n-3 PUFA rats demonstrated a lower intestinal injury score and enhanced intestinal repair. A significant decrease in enterocyte apoptosis in MTX+n-3 PUFA rats was accompanied by decreased TNF-α, FAS, FasL, FADD and BID mRNA levels. Decreased NF-κB, COX-2 and TNF-α levels in mucosa was accompanied by a decreased number of IELs and macrophages. Conclusions: n-3 PUFAs inhibit NF-κB/COX-2 induced production of pro-inflammatory cytokines and inhibit cell apoptosis mainly by extrinsic pathway in rats with MTX-induced intestinal damage.     

心房纤颤与心肌细胞凋亡的关系

目的　探讨心房纤颤患者的心房肌细胞是否存在细胞凋亡。方法　利用EdUTP缺口末端标记技术染色和透射电镜观察心房纤颤患者的心房肌组织。结果　心房纤颤患者心房肌少数细胞核不规则 ,核膜有皱褶 ,染色质凝集成颗粒状 ;细胞器密度增加 ,肌原纤维固缩或坏死 ,线粒体脱嵴 ,溶酶体和心钠素颗粒增多 ;有些细胞膜有皱褶。心肌细胞间胶原纤维和纤维细胞增多 ,有些纤维细胞的染色质在核膜下凝集明显。结论　心房纤颤状态下 ,有少部分心肌细胞和纤维细胞有凋亡现象 ,这可能不是房颤的病因而是房颤的结果。     

细胞凋亡在阿霉素大鼠肾病模型中的作用

目的：探讨细胞凋亡在阿霉素肾病中的作用及其机制。方法：将30只体重250－300g的Wistar雄性大白鼠随机分为3组,给模型组和SOD组一次性尾静脉注射阿霉素7．0mg/kg,SOD组于注射阿霉素30min后,每天尾静脉注射超氧化物歧化酶（SOD)1．8mg/kg,对照组注射等量生理盐水,于实验第7天,第14天,第28天分别用代谢笼留取24h尿液,测定尿蛋白,第28天末处死动物,取贤皮质做常规病理检查,电镜检查,并用原位末端标记法检测肾小球,肾小管细胞凋亡情况,肾皮质匀浆丙二醛（MDA）及谷胱甘肽过氧化物酶（GSH－Px)水平分别用硫代巴比妥酸比色法,还原型谷胱甘肽消耗法测定。结果：模型组肾小球,肾小管细胞凋亡数及肾皮质MDA水平明显高于对照组（P＜0．01）,GSH－Px水平则明显低于对照组（P＜0．01）,SOD组肾小球,肾小管,细胞凋亡数及肾皮质MDA水平明显低于模型组（P＜0．01）,GSH－Px水平则明显高于模型组（P＜0．01）,结论：阿霉素肾病鼠发病与病鼠肾小球,肾小管细胞亡有密切关系,而病鼠肾小球,肾小管细胞凋亡与氧自由基（OFR）有关。     

APP17肽对卵巢去势大鼠海马神经元凋亡相关因子表达的影响

为研究卵巢去势大鼠海马神经元内凋亡相关因子Fas、Fas L、NFκB、c fos、c Jun的表达及APP1 7肽对这些因子表达的影响 ,将健康雌性Wistar大鼠行双侧卵巢切除手术造模 ,并用APP1 7肽治疗 ,分别用免疫组化方法观察Fas、Fas L、NFκB、c fos、c Jun的表达 ,用TUNEL方法检测神经元的凋亡。结果发现 :卵巢去势大鼠海马神经元Fas、Fas L、c fos、c Jun的表达增高 ,而NFκB的表达则减少 ;使用APP1 7肽治疗后 ,上述蛋白质的表达恢复到正常水平 ,TUNEL检测未发现凋亡神经元。提示卵巢去势大鼠发生了海马神经元的凋亡相关蛋白的改变 ,可能是神经元处于凋亡前状态 ;APP1 7肽可改善卵巢去势大鼠海马神经元内凋亡相关蛋白的表达 ,维持神经元的正常功能     

红细胞免疫作用对胃癌细胞凋亡影响的初步研究

目的探讨红细胞免疫作用对胃癌BGC - 82 3细胞凋亡的影响 ,以及淋巴细胞的协同作用。方法应用红细胞免疫粘附肿瘤细胞花环试验、流式细胞术及放射免疫等方法 ,采用正常人外周血红细胞及红细胞 -淋巴细胞免疫粘附人胃腺癌BGC - 82 3细胞 ,检测红细胞及红细胞 -淋巴细胞免疫粘附胃癌细胞花环率 -胃癌细胞凋亡率、凋亡相关基因Fas、Bcl- 2以及凋亡相关信号传导物质cAMP、PKC的变化。结果红细胞组肿瘤红细胞花环率为 (2 8.41± 5 .0 1) % ,与对照组比较 ,红细胞组胃癌细胞凋亡率、Fas基因的表达、胃癌细胞内cAMP水平显著升高 ,蛋白激酶C(proteinkinaseC ,PKC)水平显著降低 (P <0 .0 1) ;与红细胞组相比 ,红淋混合组总花环率、胃癌细胞凋亡率、胃癌细胞内cAMP水平进一步增高 ,Bcl- 2基因的表达及PKC水平降低更为显著 (P <0 .0 1)。结论正常人红细胞可免疫粘附体外培养的胃癌BGC - 82 3细胞 ,通过红细胞的免疫作用 ,尤其是免疫粘附 ,可增加凋亡相关基因Fas的表达 ,增高胃癌细胞内cAMP水平 ,降低PKC水平 ,从而可能在诱导胃癌细胞凋亡方面起到某种作用 ;自身淋巴细胞有不同程度的促进和协同作用。     

大鼠心肌缺血再灌注时心肌细胞凋亡情况及缺血预处理对其影响

目的探讨缺血再灌注及缺血预处理对大鼠缺血再灌注心肌细胞凋亡的影响.方法制备大鼠缺血预处理(IP)、缺血再灌注损伤(I/R)模型,采用末端脱氧核苷酸转换酶介导的生物素平移缺口末端标记技术(TUNEL)检测心肌细胞凋亡情况;同时检测心肌梗死范围.结果I/R组细胞凋亡率(43.37±4.82%)高,IP组虽然也有一定的心肌细胞凋亡率(24.53±2.95%),但较I/R组明显降低(P＜0.001).IP组心肌梗死范围较I/R组明显减小.结论心肌缺血再灌注损伤可诱发或加重心肌细胞凋亡,IP能明显减少缺血再灌注诱导的心肌细胞凋亡的发生率,能明显减少心肌梗死范围,减轻缺血再灌注损伤;IP能减少心肌梗死范围、减轻缺血再灌注损伤的机理可能与其能明显减少心肌细胞凋亡有关.