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。     

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。     

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.     

间歇性完全鼻阻塞诱导幼年大鼠颏舌肌低氧诱导因子1α的表达

目的: 研究幼鼠在双侧间歇性鼻阻塞时低氧诱导因子1α(hypoxia inducible factor, HIF-1α)的表达变化, 探讨双侧鼻阻塞对生长发育期大鼠颏舌肌的影响。方法: 将30只4周龄SD大鼠(Sprague-Dawley rat, SD rat)分为3组。A组为双侧鼻阻塞组, 常氧条件下鼻孔双侧阻塞4 h(8:00 am-12:00 am), 分别阻塞21 d和55 d;B组为单侧鼻阻塞组, 常氧条件下鼻孔单侧阻塞4 h/d(8:00 am-12:00 am), 分别阻塞21 d和55 d;C组为对照组, 常氧条件下饲养。取出颏舌肌, 分别进行H-E染色和免疫组织化学染色, 检测颏舌肌中HIF-1α的表达, 采用SPSS13.0软件包对数据进行统计学分析。结果: 在双侧鼻阻塞组, 颏舌肌HIF-1α的表达随鼻阻塞时间的延长而增加, 单侧鼻阻塞组以及对照组中颏舌肌均无HIF-1α表达。结论: 在双侧鼻阻塞的未成年大鼠中, 颏舌肌表达HIF-1α, 并且随着鼻阻塞时间的延长, HIF-1α的表达不断增加。     

雌激素对慢性间歇性低氧大鼠颏舌肌线粒体细胞色素C氧化酶活性及其亚基表达的影响

目的 通过研究雌激素对慢性间歇性低氧(chronic intermittent hypoxia,CIH)大鼠颏舌肌线粒体细胞色素C氧化酶(cytochreme C oxidase,COX)活性及其亚基(COX Ⅰ、COXⅣ)表达的影响,探讨阻塞性睡眠呼吸暂停低通气综合征(OSAHS)发病中雌激素所起的作用.方法 3个月龄健康雄性SD大鼠48只,分为正常对照组(NC组)、慢性间歇性低氧组(CIH组)及慢性间歇性低氧+雌激素干预组(E+CIH组).后两组建立CIH模型,同时E+CIH组给予苯甲酸雌二醇0.2 mg/kg,NC组、CIH组给予无菌橄榄油0.2 ml/次,2次/周,肌肉注射.密度梯度离心法分离大鼠颏舌肌线粒体,极谱法测量COX活性;Western blotting分析COX Ⅰ和COXⅣ蛋白表达;实时聚合酶链反应(real-time polymerase chain action)法检测COX Ⅰ和COXⅣ的基因表达.结果 CIH组大鼠颏舌肌线粒体COX活性为(0.143±0.029)μkat/mg,与NC组[(0.273±0.058)μkat/mg]相比显著降低(P＜0.01),E+CIH组COX活性[(0.203±0.073)μkat/mg]较CIH组有所回升(P＜0.05),但仍显著低于NC组(P＜0.05).CIH组与E+CIH组COX Ⅰ蛋白表达分别为(10.789±8.144)和(25.593±11.108),与NC组(47.325±7.502)相比均显著降低(P＜0.01),且E+CIH组COX Ⅰ蛋白表达显著高于CIH组(P＜0.05).3组COXⅣ蛋白表达的差异无统计学意义(P＞0.05);3组COX Ⅰ、COXⅣmRNA表达量的差异无统计学意义(P＞0.05).结论 CIH可抑制大鼠颏舌肌线粒体COX蛋白表达及活性,而雌激素干预可部分恢复COX蛋白表达及活性.     

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。     

去卵巢对大鼠颏舌肌肌电及其肌纤维型的影响

目的检测雌性大鼠去卵巢后颏舌肌肌电及肌纤维组织化学特征变化,探索雌激素影响上气道稳定性的机制。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。手术6周后,采用插入式双极针电极引导大鼠颏舌肌肌电,检测各实验动物颏舌肌的肌电强度;采用颏舌肌肌球蛋白ATP酶组织化学染色法,检测各组大鼠颏舌肌肌纤维的分类及其面积,并分析比较各组间差异。结果假手术组颏舌肌的肌电强度与正常组相比无显著性差异（P>0.05）,去卵巢组颏舌肌的肌电强度较正常组明显降低（P<0.05）。正常雌性大鼠颏舌肌由Ⅱ型肌纤维构成,其中ⅡA为（40.27±1.01）%,ⅡB/ⅡX为（59.73±1.01）%;假手术组和正常组大鼠颏舌肌肌纤维构成和面积无显著性差异;去卵巢组颏舌肌肌纤维构成比例与正常组相比较无显著性变化（P>0.05）,但ⅡA型肌纤维面积较正常组有明显减小（P<0.05）。结论去卵巢后大鼠颏舌肌肌电减弱可能与其ⅡA型肌纤维减小有关,雌激素可能通过影响颏舌肌肌功能对上气道稳定性发挥保护作用。     

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.     

人牙髓干细胞条件培养基通过AMPK/PGC-1α途径改善氯化钴诱导的颏舌肌成肌细胞低氧损伤

目的： 研究氯化钴 (CoCl₂) 模拟的低氧对颏舌肌成肌细胞活性和氧化应激的影响,探讨人牙髓干细胞 （human dental pulp stem cells,hDPSCs）条件培养基 (conditioned medium,CM)保护作用的机制与AMPK/PGC-1α通路的关系。方法： 分离、培养及鉴定hDPSCs,通过超滤浓缩法提取条件培养基;分离、培养小鼠颏舌肌成肌细胞,分为对照组、CM组、CoCl₂组、CoCl₂+CM组。采用CCK-8法检测颏舌肌成肌细胞的活性,分别使用DCFH-DA和MitoSOX Red评估细胞内和线粒体活性氧 (reactive oxygen species,ROS) 水平,实时定量PCR分析NRF-1、NRF-2线粒体相关基因的表达,Western 印迹法检测PGC-1α、p-AMPK、总AMPK蛋白表达。采用SPSS 22.0软件包对数据进行统计学分析。结果： 颏舌肌成肌细胞经过200 μmol/L CoCl₂处理24 h后增殖显著下降（P<0.05）,细胞内和线粒体内ROS含量显著上升（P<0.05）;与CoCl₂组相比,加入hDPSCs-CM后,细胞活性显著增加（P<0.05）,胞内和线粒体内ROS含量显著降低 (P<0.05);hDPSCs-CM显著上调颏舌肌成肌细胞中pAMPK和PGC-1α蛋白的表达水平以及PGC-1α下游的线粒体效应物NRF-1、NRF-2的mRNA表达水平 (P<0.05)。结论： 人牙髓干细胞条件培养基能减轻CoCl₂诱导的颏舌肌成肌细胞低氧损伤,其机制可能与AMPK/PGC-1α通路的调节有关。     

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.     

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.     

慢性间歇性低氧条件下大鼠龈沟液中牙龈卟啉单胞菌的变化

目的:探讨间歇性低氧条件下大鼠龈沟液中牙龈卟啉单胞菌的变化.方法:将32只6周龄雄性SD大鼠随机分成4组(n=8):常氧对照组(A组)、常氧牙周炎组(B组)、间歇性低氧组(C组)、间歇性低氧合并牙周炎组(D组).用正畸结扎丝结扎双侧上颌第二磨牙颈部和高糖饮食方法建立牙周炎模型,常氧和低氧组分别在常氧和模拟阻塞性睡眠呼吸暂停低通气综合征条件下饲养8周,采集大鼠实验牙龈沟液标本,实时荧光定量PCR方法检测牙龈卟啉单胞菌的变化.结果:所有的大鼠均检出牙龈卟啉单胞菌.D组牙龈卟啉单胞菌的量明显高于其他3组(P＜0.05);B组牙龈卟啉单胞菌的量高于A组(P＜0.05).结论:慢性间歇性低氧可加重牙周炎病程,与龈沟液中牙龈卟啉单胞菌的增加有关.     

慢性间歇性缺氧小鼠下颌骨三维结构及组织形态

目的 探讨阻塞性睡眠呼吸暂停(obstructive sleep apnea hypoxia,OSAH)引起的长期慢性间歇性低氧(CIH)对下颌骨三维结构以及组织形态的影响。方法 12 只8 周C57BL/6雄性小鼠随机分成两组,CIH组以间歇性缺氧饲养,对照组(CON)以常氧饲养,4 周后麻醉处死小鼠取右侧下颌骨行micro CT检测观察其三维结构,苏木精-伊红染色观察下颌骨组织学变化。结果 造模4周后,micro CT观察两组小鼠的下颌骨三维结构,对其松质骨含量进行定量分析显示,缺氧组小鼠下颌骨感兴趣区域松质骨量比对照组小鼠显著减少(P<0.05)。苏木精-伊红染色显示,缺氧组小鼠下颌骨松质骨骨小梁数量和体积均变小,说明慢性间歇性缺氧小鼠下颌骨松质骨三维结构和骨组织微结构均发生显著改变。结论 慢性间歇性低氧抑制骨形成影响骨代谢。