雌激素对慢性间歇性低氧大鼠颏舌肌收缩功能及超微结构的影响

目的:探讨雌激素对慢性间歇性低氧大鼠颏舌肌等长收缩功能及肌细胞超微结构的影响. 方法:健康雄性SD大鼠40 只,随机分为正常对照组(NC)、慢性间歇性低氧组(CIH)、低、中、高剂量雌激素干预组(LE、ME、HE).后4 组建立CIH大鼠模型(8 h/d,5 周),同时LE、ME、HE组分别予苯甲酸雌二醇0.1、0.2、0.3 mg/kg(2 次/周)肌肉注射,NC、CIH组予无菌橄榄油 0.2 ml/次肌肉注射.第5 周末电刺激法测定各组大鼠颏舌肌等长收缩功能的变化,透射电镜观察肌细胞超微结构的改变.结果:各组大鼠颏舌肌单刺激收缩、强直收缩最大张力无明显变化;诱导疲劳试验中各时间点颏舌肌抗疲劳性:CIH组显著低于NC组(P＜0.01),ME、HE组明显高于CIH组(P＜0.05,P＜0.01);颏舌肌细胞超微结构:CIH组肌原纤维结构紊乱,部分肌丝溶解、消失,线粒体水肿,嵴断裂,空泡变;ME、HE组肌原纤维病变减轻,部分线粒体水肿,嵴减少.结论:慢性间歇性低氧可破坏大鼠颏舌肌细胞超微结构,降低抗疲劳能力;中、高剂量雌激素可不同程度逆转低氧对肌细胞超微结构及抗疲劳能力的损伤,保护低氧大鼠颏舌肌的抗疲劳能力.     

雌激素对低氧大鼠颏舌肌肌型磷酸果糖激酶表达的影响

目的 探讨雌激素药物治疗对慢性间歇性低氧(chronic intermittent hypoxia,CIH)大鼠颏舌肌肌型磷酸果糖激酶(phosphofructokinase muscle-specific isoform,PFK-M)表达的影响,以期从能量代谢的角度探讨雌激素用于治疗阻塞性睡眠呼吸暂停低通气综合征(OSAHS)的可能机制.方法 选择2个月龄健康雄性SD大鼠50只,用随机排列表分为5组:正常对照组(NC)、慢性间歇性低氧组(CIH)、低剂量雌激素治疗组(LE)、中剂量雌激素治疗组(ME)、高剂量雌激素治疗组(HE),每组10只.后4组建立CIH模型(8 h/d,5周);后3组分别给予0.1、0.2、0.3 mg/kg的苯甲酸雌二醇肌肉注射,NC、CIH组给予无菌橄榄油肌肉注射.实时反转录聚合酶链反应(real-time RT-PCR)检测大鼠颏舌肌PFK-M mRNA表达;蛋白质印迹法分析PFK-M蛋白表达.结果 CIH组PFK-M mRNA和蛋白的表达量分别为(2.144±0.260)和(0.875±0.025),与NC组相比(1.000±0.259,0.413±0.013)均显著升高(P＜0.05).LE、ME、HE组PFK-M mRNA的表达量分别为(1.424±0.193)、(1.395±0.251)和(1.310±0.094),PFK-M蛋白的表达量分别为(0.638±0.015)、(0.576±0.017)和(0.505±0.021),与CIH组相比,3个雌激素治疗组的PFK-M mRNA和蛋白表达均显著降低(P＜0.05).3个雌激素治疗组间两两比较显示,HE组PFK-M蛋白表达量比LE组明显降低(P＜0.05),而PFK-M mRNA表达量的差异无统计学意义(P＞0.05).结论 CIH可导致大鼠颏舌肌内PFK-M转录和翻译水平的升高,而雌激素药物治疗可显著抑制PFK-M的过度表达,并呈现一定的剂量依赖趋势.     

不同雌激素对慢性间歇性低氧去卵巢大鼠颏舌肌功能的影响比较

目的:探讨不同雌激素对慢性间歇性低氧(CIH)去卵巢大鼠颏舌肌功能的影响。方法:将40只8周龄健康雌性SD大鼠去卵巢后随机分为5组:正常氧组(NC)、慢性间歇性低氧组(CIH)、CIH雌二醇干预组(E2)、CIH金雀异黄素干预组(GEN)、CIH白藜芦醇干预组(RES)。后4组建立CIH大鼠模型,建模最后4 d,E2组、GEN组、RES组分别给予雌二醇、金雀异黄素、白藜芦醇2.5 mg/kg/d腹腔注射,NC组、CIH组给予相应剂量DMSO腹腔注射。建模后麻醉大鼠,分离颏舌肌,电脉冲刺激舌下神经中央支诱发颏舌肌收缩,检测颏舌肌收缩功能。结果:①各组大鼠颏舌肌单刺激收缩最大张力(Pt)与强直收缩最大张力(Po)无明显差异(P>0.05);②各组大鼠抗疲劳性比较,E2组、GEN组、RES组高于CIH组(P<0.01),E2组稍低于NC组,但无统计学差异(P>0.05),GEN组、RES组低于NC组、E2组(P<0.01),RES组高于GEN组,但差异无统计学意义(P>0.05)。结论:植物雌激素能一定程度恢复慢性间歇性低氧造成的去卵巢大鼠颏舌肌肌功能下降,但效果不如雌二醇,其作用机制有待进一步探讨。     

金雀异黄素对慢性间歇性低氧大鼠颏舌肌内氧化应激水平的影响

目的：探讨金雀异黄素对慢性间歇性低氧（CIH）大鼠颏舌肌氧化应激水平及抗氧化酶活性的影响。方法：8周龄健康雌性SD大鼠50只,去势1周后随机分为正常对照组（NC组）、慢性间歇性低氧组、慢性间歇性低氧＋低、中、高剂量金雀异黄素治疗组,每组10只。后4组建立CIH模型（8h/d,5周）。DCFH-DA荧光探针、硫代巴比妥酸法及底物催化法检测不同组颏舌肌内ROS和MDA的水平及抗氧化酶（超氧化物歧化酶酶,SOD）、过氧化氢酶（CAT）及谷胱甘肽过氧化物酶（GPx））的活性。结果：CIH导致大鼠颏舌肌内ROS和MDA的水平升高（P〈0.05）,金雀异黄素降低其水平（P〈0.05）。CIH导致大鼠颏舌肌内SOD、GPx及CAT的活性降低（P〈0.05）,低、中、高剂量的金雀异黄素恢复GPx和CAT的活性至正常水平（P〈0.05）,高剂量的金雀异黄素恢复SOD的活性至正常水平（P〈0.05）。结论：CIH导致大鼠颏舌肌内氧化应激水平的升高,金雀异黄素通过增强抗氧化酶的活性降低其氧化应激水平。     

低氧环境下自噬对颏舌肌卫星细胞增殖与凋亡的影响

目的:探讨低氧环境下自噬对颏舌肌卫星细胞(Genioglossus muscle satellite cells,GG MuSCs)增殖能力与凋亡蛋白表达的影响。方法:体外培养大鼠颏舌肌卫星细胞, MTT法筛选自噬抑制剂氯喹(chloroquine,CQ)无毒剂量并检测细胞增殖能力。实验设常氧组、常氧+25 μmol/L氯喹组、单纯低氧组、低氧+5 μmol/L氯喹组、低氧+15 μmol/L氯喹组、低氧+25 μmol/L氯喹组。Western blot检测自噬标志蛋白LC3、促凋亡蛋白caspase-3及细胞色素c(cytochrome c,Cyt c)的表达水平。结果:1)Western blot结果显示:与常氧组相比,单纯低氧组LC3-Ⅱ/Ⅰ、caspase-3、Cyt c表达均增加;与单纯低氧组相比,低氧+氯喹组中随着给药浓度增加,LC3-Ⅱ/Ⅰ、caspase-3、Cyt c的表达水平呈递增趋势。2)MTT结果显示:与常氧组相比,单纯低氧组细胞增殖能力显著增强;而低氧+氯喹组中随着给药浓度增加,细胞增殖能力受到明显抑制。结论:低氧诱发的自噬促进颏舌肌卫星细胞存活,抑制自噬导致细胞增殖能力下降,凋亡蛋白表达显著增加。这提示自噬可能是颏舌肌卫星细胞抵御低氧损伤的重要机制。     

雌激素对雌性大鼠舌下神经核及颏舌肌的影响

目的:研究SD大鼠外周血不同雌激素水平对舌下神经核自发放电及颏舌肌功能的影响,探讨雌激素影响上气道稳定性的中枢途径.方法:将30只SD大鼠随机分为假手术组(sham operation group,Sham)、去卵巢组(ovariectomized group,OVX)及去卵巢+雌二醇回补组(ovariectomized+ estrogen covering group,OVX+E2),采用玻璃微电极记录神经元放电,记录舌下神经核自发放电频率和最大幅值.采用BL-420生物学系统,检测颏舌肌功能:颏舌肌肌电的平均频率、最大频率、积分幅度、最大幅度;电刺激诱发肌电幅度、临界融合频率、50％强直收缩时程及阈刺激强度.采用SPSS13.0软件包对数据进行单因素方差分析.结果:OVX组舌下神经核的自发放电频率和最大幅值低于Sham组和OVX+E2组(P＜0.05);OVX组颏舌肌肌电最大频率、平均频率、积分幅度和最大放电幅度、50％强直收缩时程低于Sham组和OVX+E2组(P＜0.05),OVX组阈刺激强度和临界融合频率均显著高于Sham组和OVX+E2组(P＜0.05),OVX+E2组和Sham组各项指标相比无显著差异(P＞0.05).OVX组、Sham组、OVX+E2组颏舌肌的电刺激诱发肌电幅度无显著差异(P＞0.05).结论:外周血雌激素水平影响舌下神经核的兴奋性,进而影响颏舌肌功能.雌激素可能影响颏舌肌的收缩功能及抗疲劳能力.     

ERα在雌激素调控颏舌肌功能中的作用研究

目的:探索ERα在雌激素调控SD大鼠颏舌肌功能中的作用。方法:将30只8周龄雌性SD大鼠(250±25) g,随机分为假手术组(SHAM组),去卵巢+雌二醇组(OVX+E₂组),去卵巢+雌二醇+ERα拮抗剂MPP组(OVX+E₂+MPP组)(n=10)。通过卵巢切除与雌激素回补，在外周血建立稳定的雌激素水平大鼠模型；使用玻璃微电极检测SD大鼠舌下神经核团放电频率，BL-420E+生物机能实验系统检测颏舌肌肌电频率及幅值。结果:(1)舌下神经核放电频率：OVX+E₂+MPP组明显弱于OVX+E₂组(P<0.05)。(2)颏舌肌电频率及幅值：OVX+E₂+MPP组明显弱于OVX+E₂组(P<0.05)。结论：雌激素可能通过与ERα受体结合，影响舌下神经核放电，进而改变颏舌肌肌电功能。     

雌激素对雌性大鼠颏舌肌肌质网Ca^2＋-ATPase活性及其基因表达的影响

目的:观察雌激素对雌性大鼠颏舌肌细胞肌质网(sarcoplasmic reticulum, SR )Ca2 -ATPase活性及其基因表达的影响,探索其影响颏舌肌功能的分子生物学机制.方法:将30 只成年雌性SD大鼠随机分为对照组(control)、去卵巢组(ovariectomy,OVX)、去卵巢回补激素组(ovariectomy with 17β-estradiol, OVX E2).术后6 周,处死动物,提取颏舌肌SR.采用定磷法检测颏舌肌SR Ca2 -ATPase活性,荧光定量RT-PCR测定SR Ca2 -ATP酶 mRNA表达的变化.结果:OVX组血清雌激素水平及子宫湿重较对照组明显降低(雌二醇P<0.01;子宫湿重P<0.01),而OVX E2组与对照组相比差别无统计学意义(P>0.05).OVX组颏舌肌SR Ca2 -ATP 酶活性较对照组明显降低(P<0.01);OVX E2组较OVX组有明显提高(P<0.01)、与对照组相比差别无统计学意义(P>0.05).OVX组颏舌肌 SR Ca2 -ATPase mRNA表达较对照组明显降低(P<0.01);OVX E2组较OVX组有明显提高(P<0.01)、与正常组相比差别无统计学意义(P>0.05).结论:成年雌性大鼠雌激素水平的变化可引起颏舌肌SR Ca2 -ATP 酶活性及其mRNA表达的变化,雌激素可能通过该途径改变颏舌肌功能.     

低氧对大鼠颏舌肌成肌细胞分化的影响

目的:通过观测低氧环境对体外培养大鼠颏舌肌成肌细胞分化及低氧诱导因子1α(HIF-1α)表达的影响,探讨低氧引起颏舌肌损伤的机制以及HIF-1α在其中的作用。方法:根据环境氧浓度不同,将体外培养的原代大鼠颏舌肌成肌细胞分为正常氧浓度组(NC)(21%)和低氧组(HG)(1%),分别诱导分化0 d、1 d、3 d、6 d。采用RT-PCR及Western blotting检测生肌调节因子(MyoD)、肌源性决定因子(myogenin)、肌球蛋白重链(MHC)以及HIF-1α的mRNA及蛋白表达;倒置显微镜下观察成肌细胞分化的形态变化。结果:在颏舌肌成肌细胞分化过程中,两种氧状态下HIF-1αmRNA表达均没有显著变化(P>0.05),但蛋白表达逐渐上调;低氧对MyoD、myogenin、MHC的mRNA(P<0.05)和蛋白表达有显著的抑制作用,致肌管形成延迟;低氧使HIF-1αmRNA(P<0.05)和蛋白显著上调。结论:低氧环境可能是通过上调HIF-1α表达抑制大鼠颏舌肌成肌细胞的分化,从而抑制颏舌肌损伤的修复。     

去卵巢雌性大鼠颏舌肌雌激素受体表达的研究

目的:检测分析去卵巢雌性大鼠颏舌肌内雌激素受体的表达及其因雌激素水平而变化的规律。方法:30只雌性SD大鼠随机分成3组:正常组、假手术组、去卵巢组。采用SP法检测各组大鼠颏舌肌细胞内雌激素受体ERα、ERβ的表达,并分析比较各组间差异。结果:正常雌性大鼠的颏舌肌细胞存在ERα和ERβ的阳性表达,ERβ的表达明显较ERα弱(P<0.05);去卵巢组大鼠颏舌肌细胞ERα的表达较正常组显著降低(P<0.001),但ERβ的表达无显著变化(P>0.05)。结论:雌性大鼠颏舌肌细胞内存在雌激素受体的表达,其中ERα的表达明显受雌激素水平的影响。     

Effects of phytoestrogens on genioglossus contractile properties in ovariectomized rats exposed to chronic intermittent hypoxia may be independent of their estrogenicity

Chronic intermittent hypoxia (CIH) is a frequent feature of obstructive sleep apnea/hypopnea syndrome (OSAHS), and it may alter upper airway muscle endurance. We have previously reported the positive effects of estrogen on genioglossus fatigue resistance in rats. Our present study was designed to evaluate the effects of two phytoestrogens - genistein and coumestrol - on genioglossus contractile function and estrogen receptor (ER) expression in female rats exposed to CIH. Eight-wk-old female rats were ovariectomized and exposed to CIH for 5 wk. Genistein and coumestrol, respectively, were administered by intraperitoneal injection, at a dose of 2.5 mg kg(-1) d(-1), during the last 4 d of exposure to CIH. The contractile properties of the genioglossus were measured. Real-time RT-PCR and western blotting were performed to determine the expression of ERs in the genioglossus. Phytoestrogens were found to significantly increase genioglossus fatigue resistance, the effect of genistein being more powerful than that of coumestrol. However, higher levels of ER mRNA and protein were detected in the coumestrol group than in the genistein group. We conclude that phytoestrogens, especially genistein, could improve the endurance of the genioglossus muscle in ovariectomized rats exposed to CIH, and this effect is, in part, not related to its estrogenic action.     

Effects of estrogen on genioglossal muscle contractile properties and fiber‐type distribution in chronic intermittent hypoxia rats

Obstructive sleep apnea‐hypopnea syndrome (OSAHS) is a highly prevalent disorder that is characterized by recurrent sleep‐induced collapse of the upper airway. Genioglossus is an important pharyngeal dilator muscle that helps to maintain the patency of the upper airway. The effect of female hormones on pharyngeal dilator muscle activity may be one possible explanation for the differences observed in the prevalence of OSAHS between genders. The aim of this research was to investigate the influence of estrogen on genioglossus activity in rats exposed to chronic intermittent hypoxia (CIH). Eight‐wk‐old female rats were ovariectomized or sham‐operated, received 5‐wk of estrogen replacement therapy, and/or were exposed to CIH. The contractile properties of the genioglossus were measured. ATPase staining was perfomed to determine the per cent fiber‐type distribution and to measure the cross‐sectional area (CSA) of muscle fibers. Myosin heavy chain phenotypes were determined by gel electrophoresis. Chronic intermittent hypoxia reduced the contractile properties of the genioglossus muscle, decreased the CSA of type IIA fibers, and decreased the proportion of myosin heavy chain IIA, and ovariectomy exacerbated this effect. However, estrogen replacement can partially reverse the effect of CIH in ovariectomized rats. It is concluded that a low female hormone level and CIH may increase fatigue and alter genioglossus structure and function, and may compromise the maintenance of upper airway patency, while estrogen may help to reverse this effect.     

Effects of oestrogen on sarcoplasmic reticulum Ca-ATPase activity and gene expression in genioglossus in chronic intermittent hypoxia rat

This study was designed to investigate the effects of oestrogen on sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity and gene expression in ovariectomised rats under the condition of chronic intermittent hypoxia (CIH). Thirty-two female Sprague-Dawley rats were randomly divided into four groups: the normal control group (NC), the CIH group (CIH), the CIH-ovariectomised group (CIH + OVX), and the group of CIH-ovariectomised rats receiving estradiol replacement (CIH + OVX + E₂). Rats in the latter three groups were exposed to CIH for 5 weeks. The animals were killed before genioglossus (GG) was rapidly excised, and their body and uterus mass were determined. Estradiol level was detected by radioimmunoassay. SR Ca²⁺-ATPase (SERCA) activity was observed by detecting inorganic phosphorus ion, and the SERCA mRNA level was measured using real-time quantitative polymerase chain reaction (real-time PCR). It was found that, compared with the NC group, the SERCA activity and mRNA level were remarkably reduced (p < .01) in the CIH group. And compared with the CIH group, the SERCA activity and mRNA level were also significantly reduced (p < .01) in the CIH + OVX group. Meanwhile, the SERCA activity and mRNA level significantly increased (p < .01) in the CIH + OVX + E₂ group compared with the CIH + OVX group, but lower than those in the NC group (p < .01). The results showed that CIH could reduce the SERCA activity and mRNA expression, and oestrogen-deficiency could exacerbate this effect; whilst estradiol replacement can partially reverse the effect of CIH in ovariectomised rats.     

Genistein attenuates genioglossus muscle fatigue under chronic intermittent hypoxia by down-regulation of oxidative stress level and up-regulation of antioxidant enzyme activity through ERK1/2 signaling pathway

Oral Diseases (2011) 17 , 677–684 Objective: This study aims to investigate the effects of genistein on contractile properties of genioglossus under chronic intermittent hypoxia (CIH) conditions and its relationship with oxidative stress, antioxidant enzyme, and ERK1/2 signaling pathway. Materials and Methods: Fifty female Sprague–Dawley rats were randomly divided into five groups 1 week after ovariectomy: the normal control group, the CIH group, the CIH with low‐dose, medium‐dose, and high‐dose genistein groups. Rats in the latter four groups were exposed to CIH for 5 weeks. Twitch tension, tetanic tension, and fatigue resistance of genioglossus were investigated. Malondialdehyde (MDA) and mitochondrial reactive oxygen species (ROS), enzymatic activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and ERK1/2 were detected. Results: Muscle fatigue resistance and enzymatic activity of GPx, CAT, and SOD were reduced after CIH exposure and improved by different doses of genistein at different degrees. CIH increased the level of ROS and MDA, and they were returned to normal by genistein. The expression of phospho‐ERK1/2 is opposite to the changes in muscle fatigue resistance. Conclusion: Chronic intermittent hypoxia decreases fatigue resistance of genioglossus, and genistein treatment reverses the fatigability of genioglossus by down‐regulation of oxidative stress level and up‐regulation of antioxidant enzymatic activity probably through ERK1/2 signaling pathway.     

不同酸性水解催化液对硅基质陶瓷与树脂粘接效果的影响

目的 研究使用新型双液型硅烷底涂剂(A、B液)对树脂-硅基质陶瓷粘接效果的影响.方法 将432个陶瓷实验体分为9组(每组48个),分别使用9种实验性底涂剂处理陶瓷表面.9种底涂剂中A液均为50 g/L甲基丙烯酰氧基丙基三甲氧基硅烷(γ-methacryloxypropyletrimethoxysilane,MPTS)的乙醇溶液,B液分别为0.10、0.05、0.01 mol/L盐酸、磷酸和醋酸水溶液与同体积99％乙醇配比的混合溶液.处理后陶瓷与树脂粘接剂(Link Max,GC,日本)粘接形成实验体.其后将每组分为3个亚组(每个亚组各16个实验体)分别在24h后、5000次和10000次冷热循环后测试剪切强度.结果 冷热循环前后最高剪切强度均在0.05 mol/L盐酸组,分别为(19.5±3.7)、(15.7±3.0)、(14.6±3.0) Mpa.10 000次冷热循环后,当酸性溶液浓度为0.10 mol/L时,盐酸组的(10.6±3.5)Mpa和磷酸组的(9.5±2.7)Mpa明显高于醋酸组的(6.3±2.5) Mpa (P＜0.05);当酸性溶液浓度为0.05 mol/L时,盐酸组的(14.6±3.0)Mpa明显高于磷酸组的(6.3±1.9) Mpa和醋酸组的(4.7±1.8) Mpa (P＜0.05);当酸性溶液浓度为0.01 mol/L时,盐酸组的(12.7±3.2) Mpa明显高于磷酸组的(2.3±1.2) Mpa和醋酸组的(1.5±1.2) Mpa (P＜0.05).结论 选择0.05 mol/L盐酸作为水解催化液的硅烷底涂剂可得到较高的硅基质陶瓷-树脂粘接强度及粘接耐久性.