Systemic responses to carotid occlusion in the anesthetized rat.

We evaluated the effects of four standard anesthetization regimens on the systemic cardiovascular responses to bilateral common carotid artery occlusion in 28 adult male rats. Rats were randomly assigned to anesthesia groups: thiopental sodium (PT; 100 mg/kg ip), alpha-chloralose (CH; 100 mg/kg iv), ketamine hydrochloride plus acepromazine (KA; 135 mg/kg and 1.5 mg/kg sc), and pentobarbital sodium (PB; 50 mg/kg ip). PT and PB animals had similar baseline heart rates (HR; 333 and 345 beats/min, respectively) and arterial pressures (MAP; 126 and 118 mmHg, respectively), whereas both were lower in CH and KA (314 and 288 beats/min, 92 and 85 mmHg). During bilateral carotid occlusion, PT demonstrated the largest change in MAP (dMAP; +27 mmHg) but the smallest change in HR (dHR; +8 beats/min). CH and PB demonstrated similar dHR (+24 and +16 beats/min) and dMAP (+20 and +19 mmHg). KA demonstrated a significant dHR (+14 beats/min), but the average dMAP was not statistically significant (+3 mmHg). Therefore, carotid occlusion in rats anesthetized with PT, PB, or CH consistently elicits a systemic arterial pressor response comparable with that reported for conscious animals. When the magnitude and stability of baseline HR and MAP are also considered, PT and PB anesthetization seem to be the most reliable for evaluation of the carotid occlusion pressor response in rats.     

瞬时受体电位香草酸亚型1激活释放的P物质在小鼠急性心肌梗死后凋亡中的保护作用

瞬时受体电位香草酸亚型1 (transient receptor potential vanilloid 1, TRPV1)在心肌缺血激活后可传导心绞痛信号和释放P物质(substance P, SP).SP是速激肽家族成员之一,主要通过结合并激活神经激肽1 (neurokinin 1,NK1)受体发挥作用. TRPV1和SP在缺血性心脏病中对心功能的恢复和重塑有一定保护作用,但对心肌梗死后凋亡的作用及具体机制尚不明确.本研究用TRPV1基因敲除(TRPV1-/- )小鼠和野生型(wide type, WT)小鼠建立心肌梗死模型,并外源性给予SP和NK1受体拮抗剂RP67580,用TTC染色法观察梗死的面积,TUNEL法检测心肌细胞凋亡指数,Western印迹方法检测caspase-3、Bcl-2、Bax、p53的蛋白表达.结果发现,心肌梗死24 h后,TRPV1-/-小鼠比WT小鼠梗死面积更大,凋亡指数和caspase-3活性更高,Bcl-2/Bax和p53蛋白表达更低. SP预处理可以明显缩小TRPV1-/-小鼠梗死面积,降低凋亡指数、caspase-3活性和升高Bcl-2/Bax比值,而在WT小鼠中改善不明显.外源性给予RP67580,阻断SP与NK1受体结合后,与相应对照组相比,WT小鼠梗死面积和凋亡指数更大,caspase-3蛋白表达更高,Bcl-2/Bax比值更低;TRPV1-/-小鼠与相应对照组比较,凋亡指数和caspase-3表达升高,Bcl-2/Bax比值降低.研究结果表明,SP可能介导了TRPV1在急性心肌梗死后凋亡中的保护作用.     

Sympathetic and angiotensin-dependent hypertension during cage-switch stress in mice

The goal of this study was to determine the dependence of the acute hypertensive response to a novel model of acute psychosocial stress on the sympathetic and renin-angiotensin systems. Baseline mean arterial pressure (MAP), heart rate (HR), and locomotor activity were measured with telemetry in mice for a 1-h period and averaged 98 +/- 1 mmHg, 505 +/- 3 beats/min, and 5 +/- 1 counts, respectively. Stress was induced by placing a mouse into a cage previously occupied by a different male mouse, and this increased MAP, HR, and activity in the control group by 40 +/- 2 mmHg, 204 +/- 25 beats/min, and 68 +/- 6 counts, respectively. Each variable gradually returned to baseline levels by 90 min after beginning cage switch. Pretreatment with terazosin (10 mg/kg ip) significantly reduced the initial increase in MAP to 12 +/- 6 mmHg, whereas MAP for the last 45 min was superimposable on control values. Atenolol (10 mg/ml drinking water) had no effect to blunt the initial increase in MAP but had a growing effect from 10 min onward, decreasing MAP all the way to baseline by 60 min after starting cage switch. Captopril (2 mg/ml drinking water) treatment caused a very similar response. All three treatments significantly decreased the area under the blood pressure curve, and the blood pressure effect could not be attributed uniformly to effects on HR or activity. These data suggest that our novel model of psychosocial stress causes an initial alpha(1)-receptor-dependent increase in MAP. The later phase of the pressor response is blocked similarly by a beta(1)-receptor antagonist and an ACE inhibitor, independent of HR, suggesting that the beta(1)-dependent blood pressure effect is due, in large part, to the renin-angiotensin system.     

Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.     

Persistence of circadian variation in arterial blood pressure in beta1/beta2-adrenergic receptor-deficient mice

The beta-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in beta1/beta2-adrenergic receptor-deficient mice (beta1/beta2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four-hour recordings of MAP, heart rate (HR), and locomotor activity were made in conscious 16- to 17-wk-old mice [wild-type, (WT), n = 7; beta1/beta2ADR-/-, n = 10] by telemetry. Both WT and beta1/beta2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24-h mean MAP was 10% lower (102.02 +/- 1.81 vs. 92.11 +/- 2.62 mmHg) in beta1/beta2ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and beta1/beta2ADR-/- mice adapted to changed salt intake without changed MAP. However, the beta1/beta2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT mice, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the beta-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in blood pressure in mice.     

Large-conductance Ca2+-activated K+ channel beta1-subunit knockout mice are not hypertensive

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ～10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (～5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.     

急性力竭运动后小鼠肾细胞凋亡水平的变化及牛磺酸的保护作用

为探讨急性力竭运动后小鼠(Mus musculus)肾细胞凋亡水平的时相性变化及牛磺酸对肾的保护作用,将56只雄性小鼠随机分为对照组、力竭运动组(分为运动后即刻组、12h组、24 h组和48 h组)及牛磺酸运动组(分为12h组和24 h组),每小组8只,一次性力竭游泳运动后检测肾细胞凋亡水平、Bcl-2和Bax蛋白表达、一氧化氮(NO)含量及结构型一氧化氮合酶(cNOS)、诱导型一氧化氮合酶(iNOS)活性的变化.结果显示,力竭运动后各组小鼠肾细胞凋亡水平呈先升高后下降的趋势,其中运动后24 h组的凋亡水平达峰值(P＜0.05).与对照组相比,运动各组Bax表达均显著增强(P＜0.05).除运动后即刻组外,运动各组Bcl-2表达显著减弱(P＜0.05).各组Bax/Bcl-2比值显著升高,并在运动后24 h达峰值(P＜0.01),后出现下降趋势.小鼠力竭游泳后24 h和48 h肾组织NO含量显著高于对照组(P＜0.05),同时iNOS活性升高(P＜0.01),cNOS活性无显著性变化.相比同时刻运动组,牛磺酸运动组小鼠肾细胞凋亡水平、Bax表达及Bax/Bcl-2比值、iNOS活性显著降低(P＜0.05),Bcl-2表达显著升高(P＜0.05).以上结果表明,急性力竭运动可导致肾细胞凋亡的发生,iNOS、Bax、Bcl-2水平及Bax/Bcl-2比值可能在肾细胞凋亡的发生过程中发挥重要的介导作用.牛磺酸可通过调控iNOS活性及Bax/Bcl-2比值,抑制急性力竭运动后小鼠肾细胞凋亡的发生.     

AChE deficiency or inhibition decreases apoptosis and p53 expression and protects renal function after ischemia/reperfusion

We recently reported that the expression of the synaptic form of acetylcholinesterase (AChE) is induced during apoptosis in various cell types in vitro. Here, we provide evidence to confirm that AChE is expressed during ischemia–reperfusion (I/R)-induced apoptosis in vivo. Renal I/R is a major cause of acute renal failure (ARF), resulting in injury and the eventual death of renal cells due to a combination of apoptosis and necrosis. Using AChE-deficient mice and AChE inhibitors, we investigated whether AChE deficiency or inhibition can protect against apoptosis caused by I/R in a murine kidney model. Unilateral clamping of renal pedicles for 90 min followed by reperfusion for 24 h caused significant renal dysfunction and injury. Both genetic AChE deficiency and chemical inhibition of AChE (provided by huperzine A, tacrine and donepezil) significantly reduced the biochemical and histological evidence of renal dysfunction following I/R. Activation of caspases-8, -9, -12, and -3 in vivo were prevented and associated with reduced levels of cell apoptosis and cell death. A further investigation also confirmed that AChE deficiency down-regulated p53 induction and phosphorylation at serine-15, and decreased the Bax/Bcl-2 ratio during I/R. In conclusion, our study demonstrates that AChE may be a pro-apoptotic factor and the inhibition of AChE reduces renal I/R injury. These findings suggest that AChE inhibitors may represent a therapeutic strategy for protection against ischemic acute renal failure.     

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long-term anoxia while the Bcl-2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO-1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro-survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.     

lncRNA PVT1沉默对高糖诱导的血管内皮细胞增殖、凋亡及氧化应激的影响*

目的: 探讨抑制lncRNA PVT1对高糖诱导的血管内皮细胞的增殖,凋亡和氧化应激的影响。方法: 体外培养人脐静脉内皮细胞(HUVECs),分为四组:对照组(5.5 mmol/L葡萄糖),高糖组(30 mmol/L葡萄糖),高糖+siNC组(30 mmol/L葡萄糖+siNC,细胞转染阴性对照组),高糖+siPVT1组(30 mmol/L葡萄糖+siPVT1,抑制lncRNA PVT1组)。采用荧光定量PCR的方法检测转染后PVT1的表达水平。MTT检测siPVT1(短片段干扰RNA PVT1)对高糖诱导的HUVECs细胞增殖能力的影响。流式细胞术检测siPVT1对高糖诱导的HUVECs细胞ROS和凋亡水平。Western blot检测HUVECs细胞中凋亡相关蛋白如Bax,Bcl-2和cleaved-caspase-3的表达水平。结果: 与对照组比较,转染siPVT1后,PVT1的表达水平显著降低(P＜0.05)。MTT结果显示,与对照组比较,培养24 h和48 h后高糖组中HUVECs细胞增殖活力均显著降低,与高糖+siNC组(阴性对照组)比较,培养24 h和48 h后,高糖+siPVT1组中的HUVECs细胞增殖活力显著增加(P＜0.05)。流式细胞术检测结果表明,与对照组比较,高糖组HUVECs细胞中ROS和凋亡率均显著增加;和高糖+siNC组比较,高糖+siPVT1组中HUVECs细胞中ROS和凋亡率均有减少(P＜0.05)。Western blot结果表明,与对照组比较,高糖组中cleaved-caspase-3和Bax表达水平均显著上调,Bcl-2的表达水平显著下调(P＜0.05,P＜0.01)。与高糖+siNC组比较,高糖+siPVT1组cleaved-caspase-3和Bax表达水平显著下调,Bcl-2的表达显著上调(P＜0.05,P＜0.01)。结论: 抑制lncRNA PVT1可以显著增加高糖诱导的HUVECs细胞增殖活力,减轻氧化应激,抑制细胞凋亡。     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

辛伐他汀对大鼠糖尿病所致心肌细胞凋亡的影响及其机制

目的：探讨辛伐他汀对糖尿病所致心肌损伤的保护作用及可能机制。方法：24只SD大鼠（180~220） g随机分为对照组（control组,n=8）和模型组（n=16）,模型组给予链脲佐菌素（STZ）腹腔注射建立糖尿病模型,然后随机将模型组分为糖尿病组（DM组,n=8）和糖尿病+辛伐他汀组（DM+S组,n=8）,DM+S组给予辛伐他汀40 mg/（kg·d）灌胃四周,其余两组给予的等量生理盐水。实验结束后,取心脏,分光光度测定法测定大鼠心肌组织脂质过氧化物丙二醛（MDA）含量、超氧化物歧化酶（SOD）的活性,HE染色观察大鼠心肌病理学改变,TUNEL法检测大鼠心肌细胞凋亡,免疫组织化学法测定大鼠p53的表达;Western blot检测心肌组织p53,p53-phospho-serine15,Bax,Bcl-2等蛋白的表达。结果：①与对照组比较,DM组大鼠心肌组织MDA含量显著升高（P<0.01）,SOD活性明显下降（P<0.01）,与DM组比较,DM+S组大鼠SOD活性显著增加（P<0.01）,MDA含量显著下降（P<0.01）。②HE染色结果表明,与对照组比较,DM组大鼠心肌细胞排列紊乱,结构不清晰,有大量炎性细胞浸润;与DM组比较,DM+S组的心肌细胞结构明显改善。③ TUNEL细胞凋亡检测结果表明,与control组相比,DM组心肌凋亡细胞阳性率显著增加（P<0.01）,给予辛伐他汀后细胞凋亡明显减少（P<0.01）;④免疫组织学检测结果显示,与对照组比较,DM组p53表达量显著增加,且p53在细胞质和细胞核中均有表达（P<0.01）;与DM组比较,DM+S组p53表达量明显下降,p53在细胞质和核中均有表达,但核内表达量减少（P<0.01）。⑤ Western blot检测结果表明,与对照组比较,DM组大鼠p53,p53-Phospho-Serine15,Bax表达量显著升高（P<0.01）,Bcl-2表达量减少（P<0.01）;与DM组比较,DM+S组p53（P<0.01）,p53-Phospho-Serine15（P<0.01）,Bax （P<0.05）表达量显著下降,Bcl-2（P< 0.05）表达量显著增加。结论：辛伐他汀通过改善心肌结构异常、抑制氧化应激和心肌细胞凋亡对糖尿病导致的心肌损伤起到保护作用,其机制与p53介导的细胞凋亡通路相关蛋白的调控有关。     

Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction

Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.     

SHR Y chromosome enhances the nocturnal blood pressure in socially interacting rats

Our objective was to test the hypothesis that nocturnal mean arterial pressure (MAP), heart rate (HR), and activity would be increased in 1) colony over individually caged rats and 2) the spontaneously hypertensive rat (SHR) Y chromosome strain (SHR/y colony) compared with Wistar-Kyoto (WKY) rats. MAP, HR, and activity were monitored using radiotelemetry. The nocturnal MAP rise expressed as the percentage change in MAP from light to dark was increased (P < 0.05) in the SHR/y colony. The SHR Y chromosome increased MAP in both the colony and caged groups compared with WKY (P < 0.001). The SHR/y colony animals spent 23% of a 24-h period at a MAP >120 mmHg, whereas the WKY colony animals spent 2% of a 24-h period in this range. The MAP of the SHR/y colony on clonidine was reduced (P < 0.001) to WKY baseline values. Activity but not HR was increased (P < 0.01) in the WKY and SHR/y colonies compared with caged animals. In conclusion, colony housing and the SHR Y chromosome increased MAP compared with individually caged housing.     

木犀草素对子痫前期大鼠滋养层细胞凋亡的影响及其机制

目的探讨木犀草素（LUT）对子痫前期（PE）大鼠滋养层细胞凋亡的影响及其机制。 方法取妊娠10 d SD大鼠,按随机数字表法随机分为对照组、模型组、20、40、60 mmol/L LUT （LUT-L、LUT-M、LUT-H）组,每组各12只,模型组和给药组大鼠皮下注射100 mg/（kg·d）亚硝基左旋精氨酸甲酯建立PE大鼠模型,对照组大鼠皮下注射等量生理盐水,每天1次,注射6 d。妊娠16 d的大鼠分别予以20、40、60 mmol/L LUT腹腔注射,对照组、模型组大鼠腹腔注射等量生理盐水,每天1次,注射5 d。测量各组大鼠妊娠10、16、21 d尾动脉血压及24 h尿蛋白水平;妊娠21 d,原位末端标记法（TUNEL）检测滋养层组织细胞凋亡情况,Western blot法检测滋养层组织B淋巴细胞瘤-2 （Bcl-2）、Bcl-2相关X蛋（Bax）、磷脂酰肌醇3-激酶（PI3K）、磷酸化PI3K （p-PI3K）、蛋白激酶B （Akt）、磷酸化AKT （p-Akt）、内皮型一氧化氮合酶（eNOS）和磷酸化eNOS （p-eNOS）蛋白表达量。多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。 结果妊娠10 d,各组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量差异无统计学意义;妊娠16 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、24 h尿蛋白含量升高（P均< 0.05）;妊娠21 d,与对照组比较,模型组、LUT-L组、LUT-M组、LUT-H组收缩压[（110.33±3.67）比（147.28±4.16）,（131.29±4.31）,（124.46±4.27）,（118.54±4.18）mmHg]、24 h蛋白尿、细胞凋亡率[（1.38±0.34）%,（43.45±3.72）%,（39.21±3.53）%,（27.86±3.41）%,（23.21±3.28）%]和Bax蛋白表达量均升高;Bcl-2、p-PI3K/PI3K （1.06±0.09比0.25±0.02,0.37±0.03,0.57±0.06,0.73±0.08）、p-Akt/Akt（0.87±0.08比0.11±0.01,0.23±0.03,0.56±0.07,0.78±0.06）和p-eNOS/eNOS蛋白表达水平（0.85±0.07比0.09±0.01,0.16±0.02,0.38±0.04,0.69±0.07）均降低（P均< 0.05）。与模型组比较,LUT-L组、LUT-M组、LUT-H组大鼠尾动脉收缩压、舒张压、滋养层组织细胞凋亡率和Bax蛋白表达量降低,Bcl-2、p-PI3K/PI3K、p-Akt/Akt和p-eNOS/eNOS蛋白表达量升高（P均< 0.05）。 结论LUT可抑制PE大鼠滋养层组织细胞凋亡,其机制可能与PI3K/Akt/eNOS信号通路激活,调控凋亡相关蛋白表达有关。     

镉对小鼠精子和生精细胞超微结构及生精细胞bcl-2、bax基因表达的影响

研究镉暴露对小鼠附睾精子和睾丸生精细胞超微结构的变化以及镉对生精细胞凋亡相关基因bcl-2、bax表达水平的影响。采用24只雄性ICR小鼠随机分为4组,每组6只,分别以0.183、0.915、1.83mg/kg氯化镉腹腔注射,每天1次,连续5次,设阴性对照生理盐水组。于第6天透射电镜观察附睾精子超微结构、睾丸生精细胞核和线粒体超微结构的变化,免疫组化方法检测生精细胞Bcl-2、Bax表达水平。透射电镜观察显示,0.183mg/kg组精子超微结构无显著性变化,0.915mg/kg组精子头部两侧膜与头部胞质间隙轻微扩大,线粒体嵴间腔扩大且轻度空泡化,但与对照组相比无统计学意义(P>0.05)。1.83mg/kg组头部两侧膜与胞质间隙扩大,与对照组相比有显著性差异(P<0.05),尾部线粒体嵴间腔扩大且轻度空泡化,与对照组相比有显著性差异(P<0.05)。3种剂量处理组睾丸生精细胞核超微结构异常发生率显著高于对照组(P<0.05),且随着处理浓度的升高异常发生率升高;1.83mg/kg组线粒体肿胀空泡化发生率显著高于对照组(P<0.05)。3种剂量实验组生精细胞Bcl-2表达水平(吸光度)显著低于对照组(P<0.01),0.915mg/kg组Bax表达水平显著高于对照组和0.183、1.83mg/kg组(P<0.01)。3种剂量实验组Bcl-2/Bax吸光度比值显著低于对照组(P<0.01);0.915mg/kg组Bcl-2/Bax比值显著低于1.83mg/kg组(P<0.01)。上述结果提示:高浓度镉诱导附睾精子超微结构改变,高中低浓度镉致睾丸生精细胞超微结构的改变,生精细胞超微结构发生凋亡现象。镉对Bcl-2、Bax表达水平的改变可能是生精细胞凋亡的分子机制之一。     

Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction

Increases in NADPH oxidase activity, oxidative stress, and myocyte apoptosis coexist in failing hearts. In cardiac myocytes in vitro inhibition of NADPH oxidase reduces apoptosis. In this study, we tested the hypothesis that NADPH oxidase inhibition reduces myocyte apoptosis and improves cardiac function in heart failure after myocardial infarction (MI). Rabbits with heart failure induced by MI and sham-operated animals were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 4 weeks. Left ventricular (LV) dimension and function were assessed by echocardiography and hemodynamics. Myocardial NADPH oxidase activity was measured by superoxide dismutase-inhibitable cytochrome c reduction assay, NADPH oxidase subunit p47phox expression by Western blot and immunofluorescence analysis, myocardial oxidative stress evaluated by 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, and myocyte apoptosis by TUNEL assay. MI rabbits exhibited LV dilatation and systolic dysfunction measured by LV fractional shortening and the maximal rate of LV pressure rise (dP/dt). These changes were associated with increases in NADPH oxidase activity, p47phox protein expression, 8-OHdG expression, 4-HNE expression, myocyte apoptosis, and Bax protein and a decrease in Bcl-2 protein. Apocynin reduced NADPH oxidase activity, p47phox protein, oxidative stress, myocyte apoptosis, and Bax protein, increased Bcl-2 protein, and ameliorated LV dilatation and dysfunction after MI. The results suggest that inhibition of NADPH oxidase may represent an attractive therapeutic approach to treat heart failure.     

PARP inhibition prevents oxidative injury of bladder induced by acute urinary retention and subsequent emptying

It has been demonstrated that increases in poly(ADP-ribose) polymerase (PARP) activity causes damage to several organs under ischemia/reperfusion (I/R) conditions. The aims of this study were to investigate whether inhibition of PARP could suppress apoptosis in the bladder following acute urinary retention (AUR) and subsequent bladder emptying. Twelve-week-old male Sprague Dawley rats were divided into a control group, saline treated group, and 3-aminobenzamide (3-AB, a specific PARP inhibitor)-treated group. Sixty minutes after the administration of saline and 3-AB, the saline and 3-AB-treated groups had 60 min of over-distension and followed by 2 h of drainage. The degree of bladder apoptosis, levels of malondialdehyde (MDA), ATP and nicotinamide adenine dinucleotide (NAD+); expression of poly(ADP-ribose) (PAR), phosphorylation of protein kinase B (Akt); and levels of Bcl-2, Bax, and caspase 3 activity in the bladder were determined. Molecular and histological analyses showed that bladder apoptosis was associated with increases in the amount of PAR and decreases in ATP and NAD+ levels in the saline treated group. In addition, phosphorylated Akt and Bcl-2/Bax ratio were significantly decreased. The activity of caspase 3 was significantly increased in the saline treated group. Inhibition of PARP significantly increased the levels of ATP and NAD+, phosphorylation of Akt, and Bcl-2/Bax ratio, and significantly reduced the activation of caspase 3. As a result, apoptosis in the bladder was attenuated. These results indicate that PARP activation may be involved in apoptosis in the bladder induced by AUR and subsequent emptying via energy depletion and suppression of Akt activity.     

Astragaloside IV, a novel antioxidant, prevents glucose-induced podocyte apoptosis in vitro and in vivo

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 μg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg(-1)·d(-1)) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.     

脂可平对实验性早期动脉粥样硬化内皮细胞凋亡及Bax、Bcl-2表达的影响

研究脂可平对大鼠血脂、早期动脉粥样硬化血管内皮细胞凋亡及Bax、Bcl-2表达的影响。应用高脂饲料复制SD大鼠早期动脉粥样硬化模型,分别给以脂可平、辛伐他汀片混悬液灌胃,实验10周后,全自动生化分析仪、流式细胞仪定量分别检测各组血脂、主动脉内皮细胞凋亡率及Bax、Bcl-2蛋白的表达;苏木素伊红(HE)染色观察主动脉组织形态学变化。两治疗组均可降低血脂,不同程度的改善了主动脉组织病理损伤,降低内皮细胞凋亡率,调控Bax、Bcl-2蛋白表达。本实验说明脂可平可降低血脂,干预动脉粥样硬化的始动环节及其发生并从蛋白水平调控早期动脉粥样硬化内皮细胞凋亡。