Damaging effect of cigarette smoke extract on primary cultured human umbilical vein endothelial cells and its mechanism

Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10^4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability,proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.     

氧自由基致大鼠心肌线粒体损伤及维生素C对其保护作用的实验研究

目的：在大鼠心肌线粒体自由基损伤的体外实验中,观察维生素C的保护作用,为治疗心肌缺血再灌注损伤提供实验依据。方法：采用黄嘌呤-黄嘌呤氧化酶系统产生氧自由基,对分离的心肌线粒体进行体外损伤实验,通过电镜形态学和生物化学及同位素方法检测大鼠心肌线粒体病理变化、脂质过氧化物含量及摄钙能力。结果：黄嘌呤-黄嘌呤氧化酶系统产生的氧自由基对心肌线粒体有损伤作用,电镜可见形态学改变,线粒体悬液中脂质过氧化物含量明显升高,线粒体摄钙能力明显降低。维生素C可减少大鼠心肌线粒体的形态学损伤,改善线粒体的摄钙能力。结论：氧自由基可在体外条件下对大鼠心肌线粒体产生损伤,这种损伤表现在电镜形态学改变,线粒体摄钙能力的减弱;维生素C能保护氧自由基损伤的大鼠心肌线粒体,保护机制是能直接减少氧自由基的生成。     

大蒜活性成分二烯丙基三硫化物调控Wnt通路抑制香烟烟雾增强的胃癌干细胞干性

目的:探讨大蒜活性成分二烯丙基三硫化物(diallyl trisulfide,DATS)能否干预香烟烟雾促进的SGC-7901源胃癌干细胞干性,并探究Wnt通路在其中的作用.方法:用不同浓度(0％、0.25％和0.50％)的香烟烟雾悬液和DATS单独或联合处理SGC-7901源胃癌干细胞7 d,显微镜下观察胃癌干细胞球...     

山葡萄多酚对大鼠心肌线粒体氧化损伤的保护作用

目的:在大鼠心肌线粒体（Mt）自由基损伤的体外实验中观察山葡萄多酚(PVAR)对心肌线粒体氧化损伤的保护作用,为治疗心肌缺血再灌注损伤提供实验依据。方法: 实验分5组,正常线粒体组、诱导损伤线粒体组和预先加25、50、100 mg•L^-1PVAR再诱导损伤线粒体组;利用Fe²⁺/维生素C（VitC）系统产生氧自由基,体外对Wistar大鼠心肌Mt诱导损伤,观察各组Mt心磷脂和MDA含量、膜流动性、ATPase活性和肿胀度的变化。结果：与损伤组比较,三种剂量PVAR组心磷脂含量明显增高（P<0.05),MDA明显减少（P<0.01),膜流动性显著增大(P<0.01),ATPase活性增高（P<0.05),100 mg•L^-1PVAR组线粒体肿胀度与正常组比较差异无显著性。结论:氧自由基引起Mt损伤;PVAR对氧自由基引起的大鼠心肌Mt损伤具有保护作用,保护机制可能是直接减少氧自由基的生成。     

香烟烟雾提取物对气道平滑肌细胞增殖及内皮素释放的影响

探讨气道平滑肌细胞（ＡＳＭＣ）自分泌的内皮素（ＥＴ）是否介导了吸烟对ＡＳＭＣ的作用。方法在体外培养的家兔ＡＳＭＣ上加入香烟烟雾提取物（ＣＩＳＥ）测定细胞存活率，乳酸脱氢酶（ＬＤＨ）水平，并用Ｈ－ＴｄＲ掺入法及放射免疫方法观察ＣＳＥ对ＡＳＭＣ的增殖作用，及对ＥＴ－１释放的影响。用３Ｈ－ＴｄＲ掺入实验，观察低浓度（５％）ＣＳＥ对ＡＳＭＣ增殖的影响及与ＥＴ的关系。结果１０％和３０％ＣＳＥ对ＡＳＭＣ呈现明显毒性作用，表现为细胞存活率降低，脂质过氧化终产物丙二醛（ＭＤＡ）含量增加和细胞ＬＤＨ漏出随时间进行性增加。５％ＣＳＥ呈时间依赖地刺激ＡＳＭＣ释放ＥＴ－１并促进ＡＳＭＣ（３Ｈ－ＴｄＲ）掺入（较对照组增加５９．９％，Ｐ＜０．０１）。内皮素Ａ受体（ＥＴＡ受体）拮抗剂ＪＫＣ－３０１呈浓度依赖地抑制５％ＣＳＥ促ＡＳＭＣ增殖作用。结论吸烟可以刺激ＡＳＭＣ释放ＥＴ－１且通过ＥＴＡ受体介导ＡＳＭＣ增殖     

替普瑞酮对香烟提取物引起的气道平滑肌细胞凋亡中p-JNK表达的影响

目的:探讨替普瑞酮(GGA)诱导的热休克蛋白70(HSP70)在香烟提取物(CSE)引起的气道平滑肌细胞(ASMCs)凋亡中对C-Jun N-terminal激酶(JNKs)的影响。方法:在体外培养Wistar大鼠的气道平滑肌细胞中,给予GGA处理前后,给予CSE刺激3 h。用流式细胞仪(FCM)检测细胞凋亡情况,Western blot的方法检测HSP70和p-JNK的表达。结果:CSE诱导ASMCs凋亡随CSE浓度的增加而逐渐增加,两者相比有显著性差异(P<0.05);GGA预处理后再加入不同浓度的CSE处理的气道平滑肌细胞的凋亡较单纯CSE组相比有显著性差异(P<0.05)。在给予CSE刺激后,各组中的HSP70的表达较对照组有明显的降低,而且随CSE浓度越高降低越明显(P<0.05);p-JNK的表达随CSE浓度的增高而增多(P<0.05)。GGA预处理后的各组细胞的HSP70的表达与对照组比较有明显的增多(P<0.05)。在给予GGA预处理后,再加入CSE处理的气道平滑肌细胞中,与对照组比较HSP70的表达有所降低但无显著差异,p-JNK的表达在GGA+CSE组中的表达较单纯CSE组中有所下降(P<0.05)。结论:较高浓度的CSE能引起气道平滑肌细胞的凋亡并且伴随p-JNK的表达的增多。GGA处理气道平滑肌细胞后引起HSP70表达的增加,并且抑制较高浓度CSE所致的平滑肌细胞的凋亡,这可能与HSP70下调p-JNK的活性有关。     

白藜芦醇促进Ca2+介导的线粒体Ca2+转运发生

目的为探讨白藜芦醇（Tesveratrol，Res）诱导细胞凋亡过程中线粒体的作用，Res对线粒体通透性的影响。研究了Res对线粒体通透性转变孔道及Ca^2＋诱导的线粒体内Ca^2＋释放（CICR）发生的影响。方法提取大鼠肝线粒体。通过紫外分光光度仪检测Res作用下线粒体的膨胀，借此测定线粒体PTP的开放状态；采用双波长双光束紫外分光光度仪检测Res作用下测试体系内Ca^2＋浓度的变化引起的D（λ）值的变化，以反映线粒体Ca^2＋的转运情况（即CICR）。结果Res能促进Ca^2＋诱导的鼠肝线粒体PTP开放；并且Res可以加速Ca^2＋介导的线粒体Ca^2＋的转运（CICR）：而Ca^2＋通道抑制剂tdfluoperazine和钌红（RR）能分别部分或完全抑制Res的这种促进作用。结论Res能促进Ca^2＋诱导的鼠肝线粒体CICR过程。并且Res对PTP的作用依赖于Res对线粒体CICR的影响，Res促进线粒体膜的通透性可能是Res诱导细胞凋亡的一种途径。     

烟雾凝聚物对人正常支气管上皮细胞中p16转录的抑制作用及其机制

目的：通过体外模拟吸烟过程来检测抑癌基因p16表达的变化,探讨p16基因在吸烟导致肺癌发生过程中的作用。方法：常规正常支气管上皮细胞（HBE）分为实验组和对照组,分别接受烟雾凝聚物和DMSO处理。利用免疫印迹和Realtime RT-PCR分别检测P16蛋白表达和mRNA的转录水平。构建p16的基因组启动子载体,利用荧光素酶分析烟雾凝聚物对其转录活性的影响。结果：不同浓度（0,10,25,50,100 mg•L^-1）的烟雾凝聚物处理HBE后,其p16的mRNA和P16蛋白表达水平明显降低,并且表现出剂量依赖性。大于25 mg•L^-1的烟雾凝聚物能明显抑制P16蛋白表达和mRNA的转录水平,与对照组比较差异有统计学意义（P<0.05）,其中100 mg•L^-1烟雾凝聚物表现出最大的抑制效应。烟雾凝聚物也能以剂量依赖方式抑制p16启动子的转录活性,大于25 mg•L^-1的烟雾凝聚物能显著抑制p16启动子的荧光素酶活性,与对照组比较差异有统计学意义（P<0.05）,其中100 mg•L^-1的烟雾凝聚物表现出最大的抑制效应。结论：烟雾凝聚物能够抑制p16启动子的转录活性,进而抑制P16蛋白的表达;P16蛋白表达水平的降低可能是吸烟导致肺癌发生的一种分子机制。     

香烟烟雾促进膀胱癌干细胞的自我更新和干性基因的表达

目的: 探讨香烟烟雾对膀胱癌干细胞自我更新能力和干性基因表达的影响。方法: 用不同浓度（0%、0.1%和0.25%）的香烟烟雾悬液处理人膀胱癌细胞UM-UC-3来源的膀胱癌干细胞4 d,显微镜下观察膀胱癌干细胞球大小及数量,蛋白质印迹检测干性基因CD44、ALDH1-A1、OCT-4、Nanog等的蛋白表达。结果： 0.1%和0.25%的香烟烟雾悬液能够促进膀胱癌干细胞细胞球的形成,香烟烟雾处理组的细胞球明显大于对照组;蛋白质印迹结果显示,0.1%和0.25%的香烟烟雾悬液处理的膀胱癌干细胞CD44、ALDH1-A1、OCT-4、Nanog表达显著增加。结论： 香烟烟雾能够促进膀胱癌干细胞的自我更新和干性基因的表达。     

细胞内游离Ca2+在发育中大鼠皮层神经元无镁诱导惊厥性损伤中的作用

目的: 观察细胞内游离Ca2+([Ca2+]i)在培养的不同发育阶段皮层神经元无镁诱导惊厥性损伤中的作用,探讨惊厥性脑损伤年龄依赖性的可能机制.方法:体外培养6 d、17 d的胚胎大鼠皮层神经元用无镁细胞外液处理3 h,或于无镁处理前用NMDA(N-甲基-D-门冬氨酸)受体拮抗剂或Ca2+通道阻滞剂预处理,用MTT代谢率测定的方法检测神经元损伤,以Fluo-3作标记用激光共聚焦显微镜扫描的方法检测[Ca2+]i.结果:体外培养6 d、17 d的神经元单纯无镁组MTT代谢率较同期对照组降低.应用MK-801 10 μmol*L-1、AP-5 50 μmol*L-1、尼莫地平10 μmol*L-1预处理后再给无镁处理,培养6 d、17 d的神经元MTT代谢率均不同程度高于同期单纯无镁组.培养6 d、17 d的神经元相对荧光强度之间差异有显著性,两者与基线荧光强度比较差异亦有显著性.应用上述各种拮抗剂后,[Ca2+]i改变的峰值均明显低于同期单纯无镁组.结论: 在体外不同发育阶段的神经元,短暂无镁处理诱导惊厥样放电所引起的神经元线粒体功能损伤以及[Ca2+]i改变程度不同.这种[Ca2+]i改变的年龄依赖性可能是惊厥导致神经元损伤的年龄依赖性的机制之一.NMDA受体-Ca2+通道激活是导致这种[Ca2+]i改变及神经元损伤的关键环节.     

腺病毒介导MnSOD基因转染对香烟烟雾提取物致内皮细胞氧化损伤及凋亡的影响

目的探讨香烟烟雾提取物(CSE)对内皮细胞氧化损伤及凋亡的影响,腺病毒介导锰超氧化物岐化酶(MnSOD)基因转染内皮细胞后,能否部分抑制CSE致内皮细胞的氧化损伤及凋亡。方法体外培养人脐静脉内皮细胞株(ECV304),质粒转染MnSOD基因后将内皮细胞分为3组:对照组、空质粒组及MnSOD组,同时分别给予0%、5%及10%CSE刺激。收集不同CSE浓度刺激后的培养上清液和细胞爬片,TBA法检测上清丙二醛(MDA)浓度,TUNEL法检测细胞凋亡率。结果(1)CSE刺激对照组内皮细胞,可致MDA浓度增加和细胞凋亡,存在CSE浓度和时间依赖性。(2)与空质粒组比较,CSE刺激后MnSOD组MDA含量和凋亡减少,有统计学意义(P<0.05)。结论(1)CSE能导致内皮细胞氧化损伤及凋亡。(2)腺病毒介导MnSOD基因可以部分抑制香烟烟雾提取物致内皮细胞氧化损伤及凋亡。     

香烟及矽尘对肺泡巨噬细胞超微结构的影响

观察香烟、矽尘单独或共同作用对肺泡巨噬细胞(Am)超微结构的影响,结果发现香烟作用于Am使其粗面内质网(RER)、滑面内质网(SER)、高尔基复合体(GO),线粒体(Mit)及溶酶体(Ly)均有增加。各因素单独或共同作用均使超微结构出现不同程度损伤,主要表现为线粒体肿胀,嵴变短或消失甚至空泡变,SER扩张以及含有微丝的细胞数减少。本文根据以上发现从形态学角度解释香烟、矽尘作用后Am的功能变化。     

Effects of vitamin E on receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in rats treated with nicotine

Vitamin E is found to reverse the effects of nicotine on bone and this study aimed to determine its mechanism. Male Sprague Dawley rats were divided into four groups and treated for 3 months: Group 1 was the control group (RC). Groups 2 (N), 3 (N+TT) and 4 (N+ATF) received nicotine 7 mg/kg throughout the treatment period. In addition, groups 3 and 4 received tocotrienol 60 mg/kg and alpha-tocopherol 60 mg/kg respectively during months 2 and 3. Parameters measured were serum osteoprotegerin (OPG), serum receptor activator of nuclear factor kappa B ligand (RANKL), femoral and lumbar bone calcium content and body weight. Nicotine did not affect OPG or RANKL levels but reduced bone calcium content suggesting the calcium loss is not due to increase osteoclastogenesis. OPG was increased in N+ATF while RANKL was slightly increased in N+TT. Both vitamin E supplements restored bone calcium loss induced by nicotine. Nicotine impaired weight gain in all treatment groups starting week 4 however, N+TT group was comparable to RC from week 6 onwards. Bone protective effects of ATF, but not TT, may be partly due to inhibition of osteoclastogenesis.     

Effects of cigarette smoking,hypoxia and vasoactive mediators on the production of PGI2 and TXA2 in cultured pulmonary artery endothelial cells

Summary Effects of cigarette smoke extract (CSE) and some vasoactive mediators on the production of PGI₂ and TXA₂ in normoxic and hypoxic pulmonary artery endothelial cells (PAECs) in culture were studied. The production of PGI₂ in PAECs was inhibited by hypoxia or verapamil, but promoted by angiotensin II (A II), noradrenaline (NE) or platelet activating factor (PAF), while that of TXA₂ slightly increased except when treated with PAF. The effect of A II, NE, PAF and verapamil, however, was not influenced by hypoxia. CSE inhibited the production of PGI₂ in normoxic PAECs but did not further reduce 6-keto-PGF_1α in hypoxic PAECs medium. The results suggested that a) the production of PGI₂ during hypoxia might be stimulated by vasoactive mediators produced during hypoxia, not by hypoxia directly; b) the production and release of PGI, were related to intracellular calcium, c) the augmented production of PGI₂ might be one of the mechanisms in the pulmonary vasodilating role of PAF: and d) prostaglandin production might be associated with the alteration of hypoxic pulmonary vasoreactivity after cigarette smoking.     

Nicotine replacement in smoking cessation. Absorption of nicotine vapor from smoke-free cigarettes

Nicotine replacement is a promising new approach to aid smoking cessation, and various methods of delivery are being developed. One new device is a smoke-free cigarette (Favor) that has been test-marketed in several US states. Without lighting up, it delivers nicotine vapor and is free of other harmful products of tobacco smoke. To examine its therapeutic potential, we measured plasma nicotine concentrations before, during, and after its use in eight male subjects. Very little nicotine was absorbed when it was puffed like a conventional cigarette. However, with an intensive schedule of puffing at four further smoke-free cigarettes over a 20-minute period, plasma nicotine concentrations were increased by an average of 17.3 ng/mL (107.3 nmol/L) (range, 10.9 to 30.4 ng/mL [67.6 to 188.5 nmol/L]). Heart rate and blood pressure also increased significantly. The rate of nicotine absorption was slow and resembled that obtained from nicotine chewing gum, suggesting that most of the nicotine was deposited in the mouth, throat, and large airways and did not reach the lung alveoli. Despite the slow absorption, the plasma nicotine levels produced could be of therapeutic value as an aid to smoking cessation.     

Effects of Cigarette Smoke Extract on E-cadherin Expression in Cultured Airway Epithelial Cells

E--cadherin (ECD) is an important cell--cell adhesion molecule which maintains the polarity and..integrity of respiratory eplthelial cellsLlj. Smokingmay cause chronic bronchitis and emphysema, thenchronic ocr pulmonale[']. The early pathologicalchange of chronic bronchitis is characterized by thedamage to cilla--mucus transport system, degeneration, necrosis and shedding of the epithelial cells.In this study, we cultured porcine airway epithelialcells (AECs ) to investigate whether cigaret…     

缺血再灌注损伤对兔心肌线粒体钙稳态的影响

目的:观察心肌在高钾停搏液保护下经历缺血再灌注损伤时,其线粒体钙稳态的变化.方法:采用改良Langendorff离体灌注兔心脏模型,用高钾停搏液停跳停止灌注1 h后再灌注30 min.电镜观察线粒体的变化,分离心肌线粒体并负载钙离子敏感荧光试剂Fura 2-AM,测定线粒体基质游离钙浓度以及线粒体在高钙环境中摄钙能力和加入钠离子后的释钙能力.结果:心肌线粒体基质钙离子浓度明显升高,但摄钙和释钙能力基本已恢复正常.电镜下大量线粒体中可见高密度的钙盐沉积,少量线粒体肿胀、嵴消失.结论:在再灌注初期线粒体钙超载一方面是由于生理性的摄钙过程启动,但也有病理性的摄钙过程参与,30 min以后已没有病理性摄钙过程参与,摄钙释钙能力基本正常.具体的病理生理机制仍有待进一步的研究.     

香烟烟雾提取物对人肺动脉平滑肌细胞增殖的影响及蛋白激酶C的相关作用

目的 探讨香烟烟雾提取物(CSE)对人肺动脉平滑肌细胞(PASMC)增殖的影响及蛋白激酶C(PKC)信号转导途径在其中所起的作用.方法 ①不同浓度的CSE作用于人PASMC 24 h,四甲基偶氮唑盐(MTT)比色法检测PASMC增殖,锥虫蓝排斥法检测活细胞率.②PASMC与PKC激活剂PMA预孵24 h或与PKC抑制剂Ro31-8220(简称Ro)预孵30 min,然后暴露于5%CSE 24 h,采用MTT法、流式细胞术、增殖细胞核抗原(PCNA)免疫细胞化学染色等方法观察细胞增殖的变化.③5%CSE作用于PASMC 24 h,采用RT-PCR和Western blot方法分别检测PKC-α mRNA和蛋白的表达.结果 ①20%和30%的CSE对PASMC有细胞毒性作用;5%和10%CSE不影响PASMC的活细胞率;MTT检测结果示5%CSE组PASMC的吸光度(A)值较对照组明显增高.②5%CSE组PASMC 的MTT A值、增殖指数(PI)、S期细胞分数(SPF)、PCNA阳性表达率增高,与对照组比较,差异有显著性意义(P<0.05).PMA 5%CSE组和Ro 5%CSE组以上指标均降低,与5%CSE组比较,差异有显著性意义(P<0.05),与对照组比较,差异无显著性意义(P>0.05).③5%CSE组PKC-α mRNA和蛋白表达量增加,与对照组比较,差异有显著性意义(P<0.05).结论 高浓度的CSE对PASMC有细胞毒性作用,低浓度(5%)的CSE则能促进PASMC的增殖.PKC特别是PKC-α可能在CSE促人PASMC增殖的信号转导机制中起重要作用.     

Cigarette smoke extract promotes human pulmonary artery smooth muscle cells proliferation through protein kinase C alpha-dependent induction of cyclin D1

Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha （PKCα） and cyclin D1 in the cigarette smoke extract （CSE）-induced HPASMCs proliferation.Methods Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyttetrazolium bromide （MTT） assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA （siRNA） was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1protein levels were detected by Western blotting.Results Low concentrations of CSE （1%-10%） stimulated proliferation of HPASMCs, with its maximal effect at 5%.CSE （5%） led to PKCα activation. Inhibition of PKCα activity using G（o） 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cellproliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, G（o） 6976 or PKCα siRNA significantlysuppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.Conclusion PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.