慢性间歇性缺氧对大鼠颏舌肌内质网应激和炎症损伤的影响及脂联素的干预机制

发布时间：2018-05-14 16:05

本文选题：慢性间歇性缺氧 + 脂联素　；参考：《南京医科大学》2014年博士论文

【摘要】：目的慢性间歇性缺氧(CIH)是阻塞性睡眠呼吸暂停低通气综合征(OSAHS)的重要病理生理学特点,但其对机体全身系统和局部组织器官的影响机制尚未完全阐明。本研究课题旨在：1)探索CIH对上气道扩张肌的损伤及其潜在机制；2)研究CIH对全身炎症状况的影响；3)探讨脂联素对机体全身和上气道扩张肌的潜在保护作用及其可能的机制。从而为阐明OSAHS的发病机制及寻找新的治疗手段提供线索。方法将45只健康的雄性Sprague-Dawley大鼠(8周龄,体重180---200 g)随机分为三组：A组(空白对照,Control)组、B组(慢性间歇性缺氧,CIH)组和C组(慢性间歇性缺氧+脂联素,CIH+Ad)组,每组各15只。在完成总共35天、每天8小时的CIH造模后,采集所有大鼠的血液标本,以酶联免疫吸附法(ELISA)检测血清脂联素、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、 IL-6和C-反应蛋白(CRP)水平,同时检测代谢功能相关生化指标。采集所有大鼠的颏舌肌组织标本,以如下实验技术做相关检测：1)透射电子显微镜观察超微结构；2)蛋白免疫印迹技术(Western Blotting)检测内质网应激(ERS)一未折叠蛋白应答反应(UPR)及其相关性细胞凋亡信号通路的标志物蛋白分子表达水平；3)缺口原位标记(TUNEL)法检测组织细胞凋亡率；4)丙二醛(MDA)法检测脂质过氧化物水平；5)WST(?)法检测超氧化物歧化酶(SOD)活力；6)探针标记技术荧光显微镜观察检测活性氧成分(ROS)水平；7)实时定量一聚合酶链式反应(RT-qPCR)技术检测核因子(NF)-κB和脂联素受体(AdR)基因的转录水平；8)ELISA检测髓过氧化物酶(MPO)水平。数据分析以P0.05作为差异具有统计学意义。结果B组大鼠的颏舌肌组织中ERS-UPR及其相关性细胞凋亡信号传导通路中主要标志物蛋白分子,包括GRP78、p-PERK.p-eIF2α、p-IREla.XBPls.ATF6. CHOP、BAX/Bcl-2比值、caspase-3和caspase-12等的表达均显著高于A、C两组(所有P0.05)；后者两组之间差异没有统计学意义(所有P0.05)。B组大鼠颏舌肌组织细胞凋亡率((28.2±21.3)%)显著高于A组的((11.5±3.7)%,PBA=0.002)和C组的((15.9±10.3)%,PBC=0.019),后者两组之间差异没有统计学意义(PAC=0.390)。MDA在B组大鼠颏舌肌组织中的含量((8.05士4.53) nmol/mg prot)分别高于A组((5.18±3.03)nmol/mg prot,PBA=0.001)和C组((5.74±3.06)nmol/mg prot,PBC=0.002),A、C两组之间差异无统计学意义(PAC=0.691)。C组大鼠颏舌肌组织的SOD活力水平((42.42±23.17)U/mg prot)低于B组((61.77±36138)U/mg prot,PCB=0.015),但高于A组((18.62±11.67) U/mg prot,PCA=0.009).C组大鼠颏舌肌组织的ROS相对水平(1.94士1.01)低于B组(3.31±1.56,PCB=0.001),但高于A组(1.08±0.38,PCA=0.038)。B组大鼠颏舌肌组织中NF-κB的mRNA相对量高于A组和C组(所有P0.05),后者两组之间差异没有统计学意义(P0.05)。B组和C组大鼠的颏舌肌组织中MPO含量分别为(0.40±O.29)μIU/mg prot和(0.31±O.17)μIU/mg prot,都高于A组((0.17±0.08)μIU/mg prot,PBA=0.002,PCA=0.047),但B、C两组之间差异无统计学意义(P=0.240)。B组大鼠颏舌肌组织的AdR1 mRNA和AdR2 mRNA的相对量都分别低于A组和C组(所有P0.01),后者两组之间的差异都没有统计学意义(所有P0.05)。B组大鼠的血清TNF-α水平((70.87±35.16)pg/mL)分别高于A组((26.54±20.32)pg/mL,PBA0.01)和C组((29.50±22.54)pg/mL, PBC0.01),后者两组之间差异无统计学意义(PAC=0.777)。B组((30.54±12.25) pg/mL)和C组((23.04±13.85)pg/mL)大鼠的血清IL-6水平分别高于A组((14.10±8.83)pg/mL,.PBA0.01,PCA=0.045),但B、C两组的差异没有统计学意义(PCB=0.090)。B组大鼠的血清脂联素水平((4207.9±2238.5)ng/mL)分别低于A组((7051.2±2431.8)ng/mL,PBA=0.002)和C组((6404.5±2383.6)ng/mL,PBC= 0.015),后者两组之间差异无统计学意义(PAC=0.468)。结论CIH引起低脂联素血症的同时,也触发颏舌肌组织内质网应激,并激活下游的未折叠蛋白应答反应信号传导通路和诱发相关的细胞凋亡。此外,CIH还导致颏舌肌内氧化应激和炎症损伤。与此同时,还引起全身炎症反应。补充脂联素可改善内质网应激性细胞凋亡及氧化应激和炎症损伤,同时也部分的缓解全身炎症。
[Abstract]:Objective chronic intermittent hypoxia (CIH) is an important pathophysiological feature of obstructive sleep apnea hypopnea syndrome (OSAHS), but the mechanism of its influence on systemic systemic and local tissues has not been fully elucidated. 1) the aim of this study is to explore the damage of CIH to the upper airway dilator and its potential mechanism; 2) to study CIH The effect on the systemic inflammatory condition; 3) to explore the potential protective effect of adiponectin on the body and the upper airway dilator and its possible mechanism, thus providing clues for clarifying the pathogenesis of OSAHS and finding new treatment methods. Methods 45 healthy male Sprague-Dawley rats (8 weeks of age, and weight 180---200 g) were randomly divided into three groups Group A (blank control, Control), group B (chronic intermittent hypoxia, CIH) and C group (chronic intermittent hypoxia + adiponectin, CIH+Ad) group, 15 rats in each group. After completing a total of 35 days and 8 hours of CIH model each day, the blood samples of all rats were collected and serum adiponectin and tumor necrosis factor (TNF) - alpha were detected by enzyme linked immunosorbent assay (ELISA). The levels of interleukin (IL) -1 beta, IL-6 and C- reactive protein (CRP) were measured, and the biochemical indexes of metabolic function were detected. The genial tongue muscle specimens of all rats were collected, and the related tests were performed as follows: 1) transmission electron microscopy was used to observe the ultrastructure; 2) protein immunoblotting (Western Blotting) was used to detect endoplasmic reticulum stress (ERS). An unfolded protein response response (UPR) and its associated apoptotic signaling pathway marker protein molecular expression level; 3) the apoptosis rate of tissue cells was detected by the nick in situ labeling (TUNEL) method; 4) malondialdehyde (MDA) assay was used to detect the lipid peroxide level; 5) WST (?) method was used to detect the activity of superoxide dismutase (SOD); 6) probe labeling technique The level of reactive oxygen species (ROS) was detected by fluorescence microscopy; 7) real-time quantitative polymerase chain reaction (RT-qPCR) technique was used to detect the transcription level of nuclear factor (NF) - kappa B and adiponectin receptor (AdR) gene; 8) ELISA was used to detect myeloperoxidase (MPO) level. Data analysis was statistically significant in P0.05 as a difference. The main marker protein molecules in ERS-UPR and its associated apoptotic signal transduction pathway in the tongue muscle tissue, including GRP78, p-PERK.p-eIF2 alpha, p-IREla.XBPls.ATF6. CHOP, BAX/Bcl-2 ratio, caspase-3 and caspase-12, were significantly higher than A, C two group (all P0.05), and the difference between the two groups was not statistically significant (all P0.05). The apoptosis rate ((28.2 + 21.3)%) of the genial myocytes of group.B was significantly higher than that in group A ((11.5 + 3.7)%, PBA=0.002) and C group (15.9 + 10.3)%, PBC=0.019). The difference between the two groups was not statistically significant (PAC=0.390) in the group of genocutaneous muscles of the B group (8.05. 4.53) nmol/mg prot) higher than that of A group (5.18 + 3.03) nmol/mg. Prot, PBA=0.001) and group C ((5.74 + 3.06) nmol/mg prot, PBC=0.002), A, C two groups had no statistically significant difference (PAC=0.691).C group genocutaneous tissue SOD activity level ((42.42 + 23.17) U/mg) lower than that of the group (61.77 + 36138), 0.015), but higher than (18.62 + 11.67) group (18.62 + 11.67) genocutaneous group The relative level of ROS (1.94 se 1.01) was lower than that of group B (3.31 + 1.56, PCB=0.001), but the mRNA relative amount of NF- kappa B in the gennial muscle tissue of group A (1.08 + 0.38, PCA=0.038) was higher than that of A and C groups (all P0.05), the difference between the two groups was (0.40). IU/mg prot and (0.31 + O.17) mu IU/mg prot were all higher than those of A group ((0.17 + 0.08) mu IU/mg prot, PBA=0.002, PCA=0.047), but there was no significant difference between the two groups. The level of serum TNF- alpha ((70.87 + 35.16) pg/mL) in group.B rats ((70.87 + 20.32) pg/mL, PBA0.01) and C group (29.50 + 22.54) pg/mL, PBC0.01), respectively, the difference between the two groups was not statistically significant ((PAC=0.777) in.B group (30.54 + 12.25) and (23.04 + 13.85) (23.04 + 13.85) No higher than group A ((14.10 + 8.83) pg/mL,.PBA0.01, PCA=0.045), but the difference in B, C two groups was not statistically significant ((4207.9 + 2238.5) ng/mL) in group.B rats ((7051.2 + 2431.8) ng/mL, PBA=0.002) and (6404.5 + 2383.6) 0.015), and there was no statistical difference between the latter group (6404.5 + 2383.6). AC=0.468). Conclusion CIH causes hypo hyponatremia, triggers the stress of the genocolx muscle tissue endoplasmic reticulum, activates the downstream unfolded protein response signal transduction pathway and induces apoptosis related. In addition, CIH also causes internal oxidative stress and inflammatory damage in the genial tongue muscle. At the same time, it also causes systemic inflammatory response. It can improve endoplasmic reticulum stress cell apoptosis, oxidative stress and inflammatory injury, and partly alleviate systemic inflammation.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R766

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