α7nAChR在脂多糖诱导小鼠急性肺损伤的作用机制及GTS-21干预研究

发布时间：2018-03-24 13:41

本文选题：急性肺损伤　切入点：脂多糖　出处：《南华大学》2016年硕士论文

【摘要】：急性肺损伤(ALI)发病率高,其发病机制复杂。当急性肺损伤进一步加重则会发展为急性呼吸窘迫综合征(ARDS),其致死率高达40%。研究发现促炎细胞因子与抗炎细胞因子之间的失衡可能是ALI的主要发病机制,革兰阴性细菌感染和内毒素血症是ALI/ARDS的常见致病因素。胆碱能抗炎通路是近年来的研究热点,该通路由迷走神经及神经递质乙酰胆碱和胆碱能受体构成,具有调节全身性炎症反应的作用,主要通过迷走神经抑制炎症因子的合成和释放,从而抑制机体炎症反应。其中α7烟碱型乙酰胆碱受体(α7nAChR)在胆碱能抗炎通路中起重要的作用。大量动物实验结果表明使用α7nAChR激动剂能在创伤、败血症及类风湿关节炎等全身性炎症中抑制炎症因子的生成和释放。GTS-21是α7nAChR特异性激动剂,在脂多糖诱导的急性肺损伤是否发挥着抗炎作用及其作用机制,目前国内报道较少。目的观察α7nAChR与炎症因子IL-1β、IL-18、HMGB1在脂多糖诱导小鼠急性肺损伤模型中的动态表达,研究α7nAChR激动剂GTS-21对脂多糖诱导小鼠急性肺损伤模型的保护作用,探索GTS-21在急性肺损伤中的抗炎作用机制,为治疗急性肺损伤提供新策略。方法实验分为两个部分。第一部分:α7nAChR在急性肺损伤中的作用机制研究。将50只C57BL/6小鼠随机分成5组:正常对照组、急性肺损伤3小时组、急性肺损伤6小时组、急性肺损伤12小时组、急性肺损伤24小时组,每组10只。正常对照组腹腔注射等量生理盐水,1小时后断颈法处死小鼠;余组采用30mg/kg的LPS腹腔注射造模并在LPS注射相应时间后处死存活小鼠。每组选取8个标本留取肺组织检测小鼠左肺的湿/干(W/D)比重;HE染色,光镜下观察肺脏病理变化;运用ELISA分别检测右肺组织匀浆中的IL-1β、IL-18、HMGB1水平表达;采用Real-Time PCR检测肺组织α7nAChR m RNA的表达。第二部分,GTS-21靶向干预观察。将63只小鼠随机分成4组:正常对照组(NC组,n=9),急性肺损伤模型组(LPS组,n=9),药物对照组(GTS-21组,n=9),GTS-21干预组(LPS+GTS-21组,n=36)。NC组给予腹腔注射磷酸盐缓冲液(PBS);LPS组予以腹腔注射30mg/kg的LPS造模;GTS-21组在PBS注射前30分钟腹腔注射3 mg/kg的GTS-21;LPS+GTS-21组在LPS注射前30分钟腹腔注射3 mg/kg的GTS-21。正常对照组、急性肺损伤模型组、药物对照组均在PBS注射或LPS注射后6小时断颈处死小鼠。GTS-21干预组分别在LPS腹腔注射后3小时、6小时、12小时、24小时处死小鼠。每组选取8个标本送检,观察各组肺湿/干(W/D)比重、肺组织病理变化、肺组织匀浆中的IL-1β、IL-18、HMGB1水平表达及肺组织α7nAChR m RNA的表达。结果第一部分(1)与正常对照组比较,急性肺损伤组肺脏W/D比重明显增高(3h:4.49±0.16 vs 4.06±0.11,6h:5.57±0.18 vs 4.06±0.11,12h:5.32±0.14 vs 4.06±0.11,24h:5.25±0.20 vs 4.06±0.11,均p0.01);肺脏病理变化明显加重;IL-1β水平明显升高(3h:2875.28±169.95 vs555.73±132.89,6h:3136.51±130.56 vs 555.73±132.89,12h:2475.36±190.50 vs 555.73±132.89,24h:256.83±413.40 vs 555.73±132.89,均p0.01);IL-18水平明显升高(3h:176.72±20.86 vs 144.92±21.43,6h:200.96±21.16 vs 144.92±21.43,12h:183.82±7.76 vs 144.92±21.43,24h:159.59±16.09 vs 144.92±21.43,p0.05);HMGB1水平亦明显升高(3h:3.99±0.82 vs 1.88±0.73,6h:5.9±1.33 vs 1.88±0.73,12h:7.85±0.65 vs 1.88±0.73,24h:6.49±0.51 vs 1.88±0.73,p0.01);肺脏W/D比重、肺脏病理评分、肺组织匀浆中IL-1β、IL-18的水平在6小时达高峰,HMGB1在12小时达高峰,组间差异具有统计学意义(p0.05)。(2)与正常对照组比较,急性肺损伤组肺组织匀浆中α7nAChR m RNA表达呈逐渐降低趋势,差异均有统计学意义。(3h:1.57±0.16 vs 2.06±0.29,6h:0.79±0.13 vs 2.06±0.29,12h:0.65±0.12 vs 2.06±0.29,24h:0.19±0.04 vs 2.06±0.29,p0.01)。第二部分(1)与正常对照组比较,药物对照组小鼠的肺W/D比重及肺脏病理评分及IL-1β、IL-18、HMGB1水平及α7nAChR m RNA的表达的差异均无明显统计学意义(p0.05)。(2)与急性肺损伤组比较,GTS-21干预组的肺组织的α7nAChR m RNA表达明显增高(6h:1.23±0.41 vs 0.81±0.13,p0.05)。(3)与急性肺损伤组相对应时间点比较,GTS-21干预组的肺W/D比重明显改善(3h:4.20±0.13 vs 4.49±0.16,6h:5.21±0.11 vs 5.57±0.18,12h:5.17±0.13 vs5.32±1.14,24h:5.06±0.13 vs 5.25±0.20,p0.05);肺组织病理评分明显降低(3h:5.25±0.46 vs 6.00±0.75,6h:10.5±0.92 vs 12.37±1.18,12h:7.25±0.71 vs 8.50±1.19,24h:5.75±1.03 vs 7.25±1.58,p0.05);IL-1β水平明显降低(3h:2585.10±143.27 vs 2875.28±169.95,6h:1899.01±121.74 vs 3136.51±130.56,12h:1640.89±211.90 vs 2475.36±190.50,24h:1624.06±130.13 vs 2256.83±413.40,p0.01);IL-18水平明显降低(3h:155.73±13.90 vs 176.72±20.86,6h:164.05±15.20vs 200.96±21.16,12h:151.83±22.06 vs 183.82±7.76,24h:132.45±13.05 vs 159.59±16.09,p0.05);HMGB1水平明显降低(3h:3.16±0.61 vs 3.99±0.82,6h:4.07±1.40 vs 5.9±1.33,12h:5.37±1.03 vs7.85±0.65,24h:5.12±1.31 vs 6.49±0.51,p0.05)。结论(1)α7nAChR在脂多糖诱导的急性肺损伤中发挥重要作用。(2)GTS-21对脂多糖诱导的急性肺损伤起保护作用。
[Abstract]:Acute lung injury (ALI) has high incidence, its pathogenesis is complex. When the acute lung injury will further aggravate the development of acute respiratory distress syndrome (ARDS), the mortality rate as high as 40%. study found that the unbalance between pro-inflammatory cytokines and anti-inflammatory cytokines may be the main pathogenesis of ALI, gram negative bacterial infection and endotoxemia is a common pathogenic factor of ALI/ARDS. The cholinergic anti-inflammatory pathway is a hot research topic in recent years, the path of the vagus nerve and the neurotransmitter acetylcholine and cholinergic receptor, can regulate the systemic inflammatory response, inhibit the synthesis and release of inflammatory factors, mainly through the vagus nerve, thereby inhibiting the inflammatory reaction. The alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR) plays an important role in the cholinergic anti-inflammatory pathway. A large number of animal experiments show that the use of alpha 7nAChR agonists in a Injury, inflammation and sepsis, rheumatoid arthritis and systemic in inhibition of cytokine production and release of.GTS-21 alpha 7nAChR specific agonists in lipopolysaccharide induced acute lung injury or play an anti-inflammatory effect and mechanism, currently reported. Objective To observe the effect of alpha 7nAChR and inflammatory cytokine of IL-1, IL-18, HMGB1 in lipopolysaccharide the dynamic model of acute lung injury in mice induced by the expression of protective effect of alpha 7nAChR agonist GTS-21 on lipopolysaccharide induced acute lung injury model of mice, to explore the anti-inflammatory mechanism of GTS-21 in acute lung injury, for the treatment of acute lung injury and provide new strategies. Methods the experiment was divided into two parts. The first part: the mechanism research alpha 7nAChR in acute lung injury. 50 C57BL/6 mice were randomly divided into 5 groups: normal control group, 3 hours group, 6 hours group acute lung injury, acute lung injury, acute lung Injury 12 hours group, 24 hours group acute lung injury, 10 rats in each group. The normal control group were injected with saline, 1 hours after the mice were killed by cervical dislocation; more than 30mg/kg group by intraperitoneal injection of LPS and LPS in rats were sacrificed after injection of the corresponding time survival in mice. Each group selected 8 samples of lung specimens detection of mice left lung wet / dry weight (W/D); HE staining, to observe the pathologic changes under light microscope were detected by ELISA; the right lung tissue homogenates of IL-1 beta, IL-18, HMGB1 expression by PCR; expression of Real-Time in lung tissue was measured by alpha 7nAChR RNA m. In the second part, GTS-21 targeted intervention observation. 63 mice were randomly divided into 4 groups: normal control group (group NC, n=9), acute lung injury model group (group LPS, n=9), the drug control group (group GTS-21, n=9), GTS-21 group (group LPS+GTS-21, n=36).NC group were given intraperitoneal injection of phosphate buffer (PBS) LPS group was given intraperitoneal; Injection of 30mg/kg LPS model; GTS-21 group was injected PBS 30 minutes before the intraperitoneal injection of 3 mg/kg GTS-21; LPS+GTS-21 group was injected LPS 30 minutes before the intraperitoneal injection of 3 mg/kg GTS-21. normal control group, model group, acute lung injury, drug control group were injected into PBS or LPS were sacrificed 6 hours after injection of.GTS-21 in mice the intervention group were intraperitoneal injection of LPS after 3 hours, 6 hours, 12 hours, 24 hours. The mice were killed in each group were randomly selected 8 specimens were observed, lung wet / dry weight (W/D), the pathological changes of lung tissue, lung tissue homogenate of IL-1 beta, IL-18, HMGB1 expression level in pulmonary tissue and expression of alpha the 7nAChR m RNA. The results of the first part (1) compared with the normal control group, acute lung injury group W/D a significantly higher proportion of lung (3h:4.49 + 0.16 vs 4.06 + 0.11,6h:5.57 0.18 + vs 4.06 + 0.11,12h:5.32 0.14 + vs 4.06 + 0.11,24h:5.25 + 0.20 vs 4.06 + 0.11, P0.01); lung disease Physical changes significantly increased; IL-1 beta levels were significantly increased (3h:2875.28 169.95 + vs555.73 + 132.89,6h:3136.51 + 130.56 vs 555.73 + 132.89,12h:2475.36 190.50 + vs 555.73 + 132.89,24h:256.83 + 413.40 vs 555.73 + 132.89, P0.01); the IL-18 level was significantly higher in 3h:176.72 (+ 20.86 vs 144.92 + 21.43,6h:200.96 21.16 + vs 144.92 + 21.43,12h:183.82 7.76 + vs 144.92 + 21.43,24h:159.59 144.92 + 16.09 vs + 21.43, P0.05); the HMGB1 level also increased significantly (3h:3.99 0.82 + vs 1.88 + 0.73,6h:5.9 + 1.33 vs + 1.88 0.73,12h:7.85 + 0.65 vs + 1.88 0.73,24h:6.49 + 0.51 vs 1.88 + 0.73, P0.01); W/D lung weight, lung pathological score, lung tissue homogenates of IL-1 beta, IL-18 level reached the peak in 6 hours, HMGB1 reached the peak in 12 hours, the difference was statistically significant (P0.05). (2) compared with normal control group, lung tissue homogenates of alpha 7n in acute lung injury The expression of AChR m RNA was gradually decreased, the differences were statistically significant. (3h:1.57 + 0.16 vs 2.06 + 0.29,6h:0.79 0.13 + vs 2.06 + 0.29,12h:0.65 0.12 + vs 2.06 + 0.29,24h:0.19 + 0.04 vs 2.06 + 0.29, P0.01). The second part (1) compared with the normal control group, drug control group and the proportion of W/D in the lungs of mice lung pathological score and IL-1 beta, IL-18, HMGB1 and 7nAChR m level alpha RNA expression difference was statistically significant (P0.05). (2) compared with the acute lung injury group, GTS-21 intervention group lung tissue alpha 7nAChR m expression of RNA was significantly higher (6h: 1.23 + 0.41 vs 0.81 + 0.13, P0.05). (3) and acute lung injury group of corresponding time points, GTS-21 in the intervention group significantly improved the proportion of lung W/D (3h:4.20 + 0.13 vs 4.49 + 0.16,6h:5.21 + 0.11 vs + 5.57 0.18,12h:5.17 + 0.13 vs5.32 + 5.25 1.14,24h:5.06 + 0.13 vs + 0.20, P0.05); lung tissue pathological score of Ming Dynasty Significant decrease (3h:5.25 + 0.46 vs 6 + 0.75,6h:10.5 0.92 + vs 12.37 + 1.18,12h:7.25 0.71 + vs 8.50 + 1.19,24h:5.75 + 1.03 vs 7.25 + 1.58, P0.05); beta IL-1 levels were significantly lower (3h:2585.10 143.27 + vs 2875.28 + 169.95,6h:1899.01 121.74 + vs 3136.51 + 130.56,12h:1640.89 211.90 + vs 2475.36 + 190.50,24h:1624.06 + 130.13 vs 2256.83 + 413.40. P0.01); IL-18 significantly decreased (3h:155.73 + 13.90 vs 176.72 + 20.86,6h:164.05 200.96 + 15.20vs + 21.16,12h:151.83 + 22.06 vs 183.82 + 7.76,24h:132.45 + 13.05 vs 159.59 + 16.09, P0.05); HMGB1 significantly decreased (3h:3.16 + 0.61 vs 3.99 + 0.82,6h:4.07 1.40 + vs 5.9 + 1.33,12h:5.37 1.03 + vs7.85 + 0.65,24h:5.12 + 1.31 vs + 6.49 0.51, P0.05). Conclusion (1) alpha 7nAChR play an important role in acute lung injury induced by lipopolysaccharide. (2) GTS-21 on lipopolysaccharide induced acute lung injury It plays a protective role.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.8

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