金属离子Cu2+和Zn2+诱导β-淀粉样蛋白的聚集

为探讨金属离子Cu²⁺和Zn²⁺对β-淀粉样蛋白 (Αβ) 聚集的影响, 用紫外光谱、荧光光谱和透射电镜等方法, 从Aβ40聚集体的形态、大小和细胞毒性等多个角度研究了Cu²⁺和Zn²⁺对Aβ40聚集的作用。结果表明, Cu²⁺和Zn²⁺都能促进Aβ40的聚集, 并且两者能改变Aβ40聚集体的形态和大小。Zn²⁺诱导Aβ40聚集产生纤维状聚集体, 而Cu²⁺诱导Αβ40聚集产生纤维状和无定形聚集体。此外, Aβ40还原Cu²⁺产生了H₂O₂。本研究结果分析了金属离子与Αβ40聚集的关系, 阐明了金属离子在阿尔兹海默病 (AD) 中的作用。     

灯盏乙素通过调控circ-ACVR2A表达对结肠癌增殖、凋亡和侵袭的影响

目的 观察灯盏乙素对结肠癌HT-29细胞株增殖、凋亡、转移影响与circ-ACVR2A表达的相关性.方法 将结肠癌HT-29细胞株分为阴性对照组,灯盏乙素10、20、40 μmol·L-1组,采用CCK-8法检测细胞增殖情况,AnnexinV-FITC/propidium iodide(PI)流式细胞仪检测细胞凋亡情况...     

灯盏乙素抑制过氧化氢诱导PC12细胞凋亡及机制

目的：观察灯盏乙素对过氧化氢（H2O2）诱导PC12细胞凋亡的抑制作用及其作用机制。方法：采用H2O2诱导PC12细胞凋亡模型，用碘化丙啶（PI）单染和PI／Annexin V双染检测细胞凋亡，用DNA琼脂糖凝胶电泳观察DNA断裂，并采用RT-PCR检测Bcl-2 mRNA表达、荧光光度法检测caspase-3活性。结果：灯盏乙素可以抑制H2O2诱导的膜磷脂酰丝氨酸外翻，增加Bcl-2 mRNA表达，减小caspase-3活性，并降低DNA片段化和细胞凋亡率。结论：灯盏乙素显著抑制H2O2诱导的细胞凋亡，其抗细胞凋亡作用可能与其增加Bcl-2表达、抑制caspase-3活化有关。     

灯盏乙素对缺氧缺血性新生大鼠脑缺氧诱导因子-1α和水通道蛋白9表达水平的影响

目的：研究灯盏乙素对缺氧缺血性脑损伤（HIBD）新生大鼠脑缺氧诱导因子-1α（HIF-1α）和水通道蛋白9（APQ9）表达水平的影响。方法：建立HIBD新生大鼠模型,将新生7日龄SD大鼠随机分为假手术组（sham组）、缺氧缺血组（HIBD组）、灯盏乙素高、中和低剂量组、地塞米松阳性对照组。于缺氧缺血7 d后,观察大鼠体质量增长率,脑组织HE染色观察脑组织病理改变,测定脑含水量,免疫荧光法检测脑HIF-1α蛋白表达,逆转录-多聚酶链反应（RT-PCR）法检测脑HIF-1α、APQ9mRNA表达,ELISA法检测脑caspase-3活性。结果：灯盏乙素能显著升高HIBD大鼠体质量增长率,降低脑组织含水量,抑制脑组织HIF-1α和AQP9mRNA表达,降低脑caspase-3活性。结论：灯盏乙素可减轻HIBD大鼠脑组织水肿,降低HIF-1α和AQP9表达,抑制神经细胞凋亡,发挥脑保护作用。     

醒脑益智组分中药对β-淀粉样蛋白（1-40）诱导SH-SY5Y细胞损伤的保护作用

目的：探讨醒脑益智组分中药对β-淀粉样蛋白（Aβ_1-40）诱导SH-SY5Y细胞损伤的影响。方法：以SH-SY5Y细胞体外培养为研究对象,Aβ_1-40损伤细胞建立模型,石杉碱甲（6 μg·mL^-1）和不同浓度（70,35,17.5 μg·mL^-1）药物干预,四甲基偶氮唑盐（MTT）法检测细胞存活率;细胞膜损伤测定乳酸脱氢酶（LDH）漏出率;酶联免疫吸附法检测脑源性神经营养因子（BDNF）和肿瘤坏死因子（TNF-α）含量;Hochest 33258染色观察药物对损伤细胞的作用;AnnexinⅤ-FITC/PI双染流式细胞术检测细胞凋亡情况。结果：醒脑益智组分中药能明显改善Aβ_1-40（15 μmol·L^-1）诱导的神经细胞存活率,降低LDH漏出率,改善细胞内BDNF含量,降低TNF-α水平,并抑制细胞的凋亡。结论：醒脑益智组分中药可以改善Aβ_1-40诱导的SH-SY5Y细胞损伤,并抑制其凋亡,其机制可能与改善BDNF水平,调节免疫及相关凋亡基因的表达有关。     

二氢杨梅素对Cu2+诱导的β淀粉样蛋白聚集和细胞毒性的影响

目的研究二氢杨梅素(DMY)对Cu2+诱导的β淀粉样蛋白片段(Aβ1-40)聚集和细胞毒性的影响。方法采用硫磺素T荧光标记研究DMY对Cu2+诱导Aβ1-40聚集的影响,MTT法检测SH-SY5Y细胞活力的变化。结果 DMY能明显抑制Cu2+诱导的Aβ1-40聚集,对已形成的Cu2+-Aβ1-40聚集物有解聚作用,且能有效防止Cu2+和Aβ1-40共同诱导的SH-SY5Y细胞活力的下降。结论 DMY对阿尔茨海默病具有潜在的防治作用。     

益智仁盐制前后组成缩泉丸对下丘脑-垂体-肾上腺皮质系统与β3-AR、M3R表达的比较

目的：观察益智仁盐制前后组成缩泉丸对下丘脑-垂体-肾上腺皮质系统（HPA）的调节与膀胱β肾上腺素能受体（β₃-AR）与胆碱能神经受体（M₃R）mRNA和蛋白表达的影响,拟阐明盐制入肾-肾主水的机制。方法：采用酶联免疫吸咐法（ELISA）测定血浆中促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ATCH）和cAMP的浓度,RT-PCR测定β₃-AR与M₃R mRNA的表达,Western blot测定β₃-AR与M₃R的蛋白表达。结果：与模型组比较,缩泉丸中益智仁盐制后能提高肾阳虚多尿大鼠血浆中CRH、ATCH和cAMP的浓度（P<0.05）,增加β₃-AR mRNA、蛋白表达（P<0.05）,降低M₃RmRNA、蛋白表达（P<0.05）,且比生品组作用增强（P<0.05）。结论：缩泉丸中益智仁盐制后对腺嘌呤所致肾阳虚多尿模型大鼠HPA轴有上调作用而调节体液平衡,同时也通过调节膀胱逼尿肌β₃-AR、M₃R mRNA和蛋白表达增强缩尿作用,证实了缩泉丸中用盐益智仁的科学性。     

五味子乙素抑制M146L细胞Aβ42生成的机制研究

目的研究五味子乙素抑制M146L细胞Aβ₄₂生成的机制。方法体外培养高效表达Aβ42的M146L细胞株，分别加入不同浓度的五味子乙素(1.67，5.00和15.00 μg·mL^-1)、 β分泌酶抑制剂(S4562，100.00 μg·mL^-1)和γ分泌酶抑制剂(S2188，13.68 μg·mL^-1)。用CCK-8(cell counting kit-8)比色法检测不同处理对M146L细胞活性的影响；用ELISA法测定M146L细胞所分泌的Aβ₄₂的变化；用Western blotting检测APP的β分泌酶剪切产物C99蛋白的含量变化，结合用β和γ分泌酶活性试剂盒，检测五味子乙素对这两种酶活性的影响。结果不同处理因素对M146L细胞的存活率均无影响，不具有细胞毒作用。中、高剂量的五味子乙素均不同程度地抑制M146L细胞分泌Aβ₄₂及γ分泌酶的活性，但都不改变M146L细胞C99蛋白的含量及β分泌酶的活性。结论五味子乙素可抑制γ分泌酶活性，其降低Aβ₄₂生成是通过抑制γ分泌酶的活性来实现的。     

灯盏花素对老龄小鼠学习记忆及脑组织β-淀粉样蛋白水平的影响

目的:观察灯盏花素对老龄小鼠学习记忆及脑组织β-淀粉样蛋白水平的影响。方法:选用雄性自然衰老KM小鼠,随机分成5组:空白对照组、灯盏花素0.06 g.kg-1组、灯盏花素0.12 g.kg-1组、灯盏花素0.24 g.kg-1组和阳性对照组,每组10只,灌胃给予等容积药物或纯化水,每日灌胃1次,连续30 d。用Morris水迷宫测试学习记忆能力;用生化法测定SOD、MDA和AchE;双抗体夹心法测定脑组织Aβ水平。结果:灯盏花素能明显缩短老龄小鼠游泳时间及游泳路程;显著提高脑组织SOD活性,降低MDA和Aβ含量,降低脑组织AchE活性。结论:灯盏花素能改善老龄小鼠的学习记忆功能,其机制可能与提高SOD活性、降低脑组织脂质过氧化物和Aβ的含量、抑制AchE活性有关。     

半枝莲黄酮对复合Aβ所致大鼠皮层细胞凋亡抑制作用及线粒体凋亡通路的调节机制

目的:探讨半枝莲黄酮(SBF)对淀粉样蛋白25-35(Aβ_25-35)联合三氯化铝(AlCl₃)和人类重组转移因子-β1(RHTGF-β1)(复合Aβ)所致大鼠皮层细胞凋亡及线粒体凋亡通路中细胞色素-C及其下游凋亡因子Apaf-1、Caspase-9和Caspase-3的调节机制。方法:雄性SD大鼠,脑室注射复合Aβ建立记忆障碍模型,术后第45天进行记忆障碍模型筛选,模型成功大鼠随机分为模型对照组和SBF 35,70,140 mg·kg^-1剂量组。SBF药物组大鼠连续灌胃给药36 d,模型和假手术组连续灌胃生理盐水36 d。吉姆萨染色(Giemsa)法检测皮层细胞凋亡情况;RT-PCR法检测皮层细胞胞液中细胞色素-C(Cyt-C)、凋亡蛋白酶激活因子-1(Apaf-1)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)mRNA水平;Western blotting检测皮层细胞胞液中半胱氨酸天冬氨酸蛋白酶(Caspase-3)蛋白水平。结果:大鼠脑室注射复合Aβ能够引起大鼠皮层细胞凋亡,伴随着胞液中Cyt-C、Apaf-1和Caspase-9 mRNA及Caspase-3蛋白表达的增加。而SBF可显著抑制复合Aβ所致大鼠皮层细胞凋亡、逆转胞液中Cyt-C、Apaf-1、Caspase-9和Caspase-3表达的增加(P<0.01)。结论:SBF通过减少线粒体对Cyt-C的释放及逆转Apaf-1、Caspase-9和Caspase-3的表达抑制复合Aβ所致大鼠皮层细胞凋亡。     

FAK抑制剂对人肝星状细胞Akt/GSK-3β/β-catenin信号通路的影响

目的：探讨FAK抑制剂（PF562,271）对人肝星状细胞（LX-2）Akt/GSK-3β/β-catenin信号通路的影响,为抗肝纤维化寻找新的作用靶点。方法：CCK-8法检测不同浓度范围（0~7 μmol·L^-1）FAK抑制剂（PF562,271）在12、24、48 h和72 h时对LX-2细胞增殖的影响,Real-time PCR分析PF562,271对LX-2细胞中α-SMA、Collagen Ⅰ mRNA表达水平的影响,Western-blot检测不同浓度PF562,271对LX-2细胞中Akt、p-Akt、GSK-3β、p-GSK-3β、β-catenin蛋白表达的影响,评价PF562,271对LX-2细胞中Akt/GSK-3β/β-catenin信号通路的影响。结果：PF562,271能够抑制LX-2细胞增殖,且该作用呈剂量和时间相关性;qRT-PCR检测到PF562,271能够抑制LX-2细胞中α-SMA、collagen ⅠmRNA表达,与对照组相比,表达量显著降低（P<0.05）;Western-blot结果显示PF562,271对Akt/GSK-3β的磷酸化表达水平显著降低（P<0.05）。结论：FAK抑制剂能够抑制LX-2细胞增殖,促进其凋亡,其作用机制可能是通过抑制Akt/GSK-3β/β-catenin信号通路从而缓解肝纤维化进程。     

Puerarin pretreatment attenuates cardiomyocyte apoptosis induced by coronary microembolization in rats by activating the PI3K/Akt/GSK-3β signaling pathway

Coronary microembolization (CME) is associated with cardiomyocyte apoptosis and cardiac dysfunction. Puerarin confers protection against multiple cardiovascular diseases, but its effects and specific mechanisms on CME are not fully known. Hence, our study investigated whether puerarin pretreatment could alleviate cardiomyocyte apoptosis and improve cardiac function following CME. The molecular mechanism associated was also explored. A total of 48 Sprague-Dawley rats were randomly divided into CME, CME + Puerarin (CME + Pue), sham, and sham + Puerarin (sham + Pue) groups (with 12 rats per group). A CME model was established in CME and CME + Pue groups by injecting 42 μm microspheres into the left ventricle of rats. Rats in the CME + Pue and sham + Pue groups were intraperitoneally injected with puerarin at 120 mg/kg daily for 7 days before operation. Cardiac function, myocardial histopathology, and cardiomyocyte apoptosis index were determined via cardiac ultrasound, hematoxylin-eosin (H&E) and hematoxylin-basic fuchsin-picric acid (HBFP) stainings, and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Western blotting was used to measure protein expression related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway. We found that, puerarin significantly ameliorated cardiac dysfunction after CME, attenuated myocardial infarct size, and reduced myocardial apoptotic index. Besides, puerarin inhibited cardiomyocyte apoptosis, as revealed by decreased Bax and cleaved caspase-3, and up-regulated Bcl-2 and PI3K/Akt/GSK-3β pathway related proteins. Collectively, puerarin can inhibit cardiomyocyte apoptosis and thus attenuate myocardial injury caused by CME. Mechanistically, these effects may be achieved through activation of the PI3K/Akt/GSK-3β pathway.     

Polygalasaponin F inhibits neuronal apoptosis induced by oxygen‐glucose deprivation and reoxygenation through the PI3K/Akt pathway

Cerebral ischaemia is a common cerebrovascular disease and often induces neuronal apoptosis, leading to brain damage. Polygalasaponin F (PGSF) is one of the components in Polygala japonica Houtt, and it is a triterpenoid saponin monomer. This research focused on anti‐apoptotic effect of PGSF during oxygen‐glucose deprivation and reoxygenation (OGD/R) injury in rat adrenal pheochromocytoma cells (PC12) and primary rat cortical neurons. OGD/R treatment reduced viability of PC12 cells and primary neurons. This reduced viability was prevented by PGSF, as shown by MTT assay. OGD/R insult decreased expression of Bcl‐2/Bax both in PC12 cells and primary neurons but elevated levels of caspase‐3 in primary neurons. However, PGSF may up‐regulate expression of Bcl‐2/Bax and down‐regulate caspase‐3 in these particular cells. Furthermore, Bcl‐2/Bax and the ratio between phosphorylated Akt and total Akt were decreased in PC12 cells treated with OGD/R, and both were increased by PGSF. Moreover, increase in the ratios of Bcl‐2/Bax and phosphorylated Akt/total Akt in PC12 cells was suppressed by phosphatidylinositol 3‐kinase (PI3K) inhibitor. Data suggest PGSF might prevent OGD/R‐induced injury via activation of PI3K/Akt signalling. The ability of PGSF to block the effects of OGD/R appears to involve regulation of Bcl‐2, Bax and caspase‐3, which are related to apoptosis.     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

Type 2 IP3 receptors are involved in uranyl acetate induced apoptosis in HEK 293 cells

Calcium released from endoplasmic reticulum through special calcium release channels – inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs) – serves as a main source of cytosolic calcium signaling in the majority of cell types in physiological state and also in pathological situations. In this work, we studied whether IP₃Rs can be involved in uranyl acetate induced nephrotoxicity. Using human embryonic kidney cell line (HEK293) as an experimental model we have found that uranyl acetate (5 and 50 μM) up-regulates both, mRNA and protein levels of the type 1 and type 2 IP₃ receptors in HEK293 cells. This increase was associated with elevated expression of proapoptotic factors Bax and Caspase 3 and also by higher extent of apoptosis. Vice versa, induction of apoptosis resulted in increased mRNA levels of IP₃R2 and also elevated levels of apoptotic markers. Therefore we propose that enhanced expression of the type 2 IP₃Rs can at least partially contribute to increased levels of apoptosis due to uranyl acetate treatment.     

FORCE AND INTRACELLULAR Ca2+ DURING NANC-MEDIATED RELAXATION OF RAT ANOCOCCYGEUS MUSCLE AND THE EFFECTS OF CYCLOPIAZONIC ACID

1. Simultaneous recordings of tension and [Ca²⁺]_i during NANC-mediated relaxation were made in the rat anococcygeus muscle under various conditions. 2. In muscles precontracted with guanethidine, nitrergic stimulations at 2 Hz produced a rapid decrease in both the tension and [Ca²⁺]_i. 3. The nitric oxide synthase inhibitor, N^G-nitro-L-Arginine (NOLA, 100 μmol/L) completely abolished the decreases in the [Ca²⁺]_i and force response of the NANC-mediated relaxation. 4. Noradrenergic-mediated contractions elicited by electrical field stimulation were potentiated by the addition of NOLA. In the absence of NOLA, the motor responses were larger in magnitude at 10 Hz stimulation than at 2 Hz. After NOLA, both the force response and the associated rise in [Ca²⁺]_i were substantially increased in comparison to the control stimulations. Proportionately the potentiation of the 2 Hz response was of a far greater magnitude than that of the 10 Hz response. 5. The guanylate cyclase inhibitor methylene blue (10 μmol/ L), partially inhibited the force and [Ca²⁺]_i response of the NANC relaxation. 6. Following exposure of the muscles to the sarcoplasmic reticulum Ca²⁺-ATPase inhibitor, cyclopiazonic acid, (10 μmol/ L) the responses to NANC stimulation were inhibited. The attenuated relaxation response displayed a bi-phasic timecourse and the Ca²⁺ change in comparison to that of the control was markedly smaller. In some cases, a relaxation was observed with no detectable change in the [Ca²⁺]_i. 7. The results suggest that part of the relaxation response observed with NANC-mediated relaxation in the rat anococcygeus is dependent on Ca²⁺ sequestration into the sarcoplasmic reticulum. However, other Ca²⁺ lowering mechanisms and possible Ca²⁺ independent mechanisms may also contribute to the NANC relaxation response.     

基于NLRP3-IL-1β-NF-κB信号轴研究野菊花总黄酮对 脂多糖诱导HK-2细胞炎性反应及自噬的影响

摘要：目的 探究野菊花总黄酮（TFC）对脂多糖（LPS）诱导HK-2细胞炎性反应及自噬的影响,并初步探究其可 能的作用机制。方法 体外培养人肾小管上皮HK-2细胞,将细胞分为阴性对照（Control）组、LPS处理（LPS）组、TFC 预处理（TFC）组、NLRP3激活预处理（4-MET）组、TFC联合4-MET预处理（TFC+4-MET）组。CCK-8法检测细胞增殖 情况;Hoechst 33342染色观察各组细胞形态变化;免疫印迹法检测各组细胞核苷酸结合域样受体蛋白3（NLRP3）、凋 亡相关斑点样蛋白（Asc）、半胱氨酸天冬氨酸蛋白酶1（caspase-1）、p-IκB、IκB、自噬相关蛋白（Beclin1）、微管相关蛋 白轻链3Ⅱ（LC3Ⅱ）、LC3Ⅰ、蛋白表达情况;ELISA法检测细胞中白细胞介素（IL）-1β、IL-18水平;透射电镜观察细胞 自噬情况。结果 与Control组相比,LPS组细胞增殖率降低,细胞核破碎情况加重,细胞凋亡率升高,NLRP3、Asc、 caspase-1蛋白表达升高、IκB蛋白磷酸化水平升高,IL-1β、IL-18水平升高,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低 （P＜0.05）。与LPS组相比,TFC组细胞增殖率升高,细胞核破碎减轻,细胞凋亡率降低,NLRP3、Asc、caspase-1蛋白 表达降低、IκB蛋白磷酸化水平降低,IL-1β、IL-18水平降低,细胞自噬程度增加,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达升 高;4-MET组细胞增殖率降低,细胞凋亡率升高,细胞核破碎情况加重,NLRP3、Asc、caspase-1蛋白表达升高、IκB蛋 白磷酸化水平升高,IL-1β、IL-18水平升高,细胞自噬程度降低,LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达降低（P＜0.05）。 TFC+4-MET组细胞增殖率、细胞自噬程度、LC3Ⅱ/Ⅰ水平、Beclin-1蛋白表达高于4-MET组,细胞核破碎情况减轻, 细胞凋亡率、NLRP3、Asc、caspase-1蛋白表达、IκB蛋白磷酸化、IL-1β、IL-18水平低于4-MET组（P＜0.05）。结论 野菊花总黄酮可能通过抑制NLRP3-IL-1β-NF-κB信号通路活化,进而抑制脂多糖诱导HK-2细胞炎性反应,诱导 细胞自噬     

白藜芦醇促进Ca2+介导的线粒体通透转变孔道开放

目的 为研究白藜芦醇(Res)抗癌及诱导细胞凋亡的机理,观察了Res对线粒体通透转变孔道(PTP)开放的影响。方法 提取大鼠肝线粒体。用氧电极法检测Res对离体线粒体氧化磷酸化的影响;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;采用荧光分光光度仪测定Res对线粒体膜电位的影响。结果白藜芦醇抑制线粒体的呼吸,降低线粒体的呼吸控制率;能剂量依赖性地促进Ca²⁺诱导的鼠肝线粒体PTP开放;并且Res可以加速Ca²⁺介导的线粒体膜电位的升高和消失。结论白藜芦醇诱发细胞凋亡及抗癌的可能途径之一是抑制线粒体的呼吸,促进线粒体内Ca²⁺的释放和PTP开放。     

Ca2+ mobilization induced by δ‐hexachlorocyclohexane in Madin Darby canine kidney cells

The effect of the pesticide δ‐hexachlorocyclohexane (δ‐HCH) were examined on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) using fura‐2 as a Ca²⁺ probe. δ‐HCH at concentrations of 5–200 mM increased intracellular free Ca²⁺ concentration ([Ca²⁺]_i) concentration‐dependently. The [Ca²⁺]_i increase comprised an immediate rise followed by a sustained phase within 5 min of measurement. External Ca²⁺ removal slightly reduced the [Ca²⁺]i increase. In Ca²⁺‐free medium, 150 μM δ‐HCH did not increase [Ca²⁺]_i after pretreatment with carbonylcyanide m‐chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum (ER) Ca²⁺ pump inhibitors, thapsigargin (1 μM) and cyclopiazonic acid (100 μM). Conversely, pretreatment with δ‐HCH prevented thapsigargin, cyclopiazonic acid, and CCCP from releasing more Ca²⁺, suggesting 150 μM δ‐HCH released Ca²⁺ from the ER and mitochondria. δ‐HCH (150 μM) activated Mn²⁺ quench of fura‐2 fluorescence, confirming that δ‐HCH induced Ca²⁺ influx. Addition of 3 mM Ca²⁺ induced a concentration‐dependent [Ca²⁺]_i increase after pretreatment with 100–200 μM δ‐HCH for 870 sec in Ca²⁺‐free medium. The δ‐HCH (150 μM)‐induced Ca²⁺ release was decreased by inhibiting phospholipase C with 1 μM U73122. Collectively, we have found that δ‐HCH increased [Ca²⁺]_i in MDCK cells by releasing Ca²⁺ from the ER and mitochondria, followed by capacitative Ca²⁺ entry. Drug Dev. Res. 50:186–192, 2000. © 2000 Wiley‐Liss, Inc.