高良姜素对支气管哮喘气道炎症的影响及机制研究

发布时间：2018-03-02 12:53

本文选题：哮喘　切入点：气道炎症　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分高良姜素对小鼠哮喘模型气道炎症的影响目的：研究高良姜素对小鼠哮喘模型气道炎症和气道高反应性的影响及其相关机制。方法：36只BALB/c小鼠按随机原则分成对照组(Control组)、哮喘组(OVA组)、DMSO组(OVA+DMSO组)、高剂量高良姜素组(OVA+GA15组)、低剂量高良姜素组(OVA+GA5组)和地塞米松组(OVA+DXM),每组6只。哮喘组及干预组小鼠于第0及第14天腹腔内注射20μg OVA致敏,第22、23、24天1%OVA连续雾化三天,每天一次,每次30分钟。干预组于OVA激发前一天开始每天腹腔内注射DMSO、不同剂量的高良姜素、地塞米松。末次激发24h后,使用小动物肺功能分析系统进行气道反应性测定,取肺组织病理切片行苏木精-伊红(HE)染色观察肺组织气管旁及血管旁炎性细胞浸润,PAS染色观察杯状细胞增生,用酶联免疫吸附试验(ELISA)分别检测各组小鼠血清OVA特异性IgE和肺泡灌洗液(BALF)中IL-4、IL-4和IL-13的水平。使用免疫组织化学法(IHC)及蛋白质印迹法(Western blot)检测肺组织中iNOS及VCAM的蛋白表达。使用Western blot检测肺组织细胞总蛋白IλBα和磷酸化的p65的蛋白表达,核蛋白和细胞质中p65的蛋白表达。结果：对小鼠肺阻力(RL)检测发现,随着氯化乙酰胆碱浓度的增加,OVA组小鼠RL明显增加,Control组小鼠RL轻度增加,各组小鼠的基础气道阻力没有差异(P0.05),当氯化乙酰胆碱浓度在30μg/kg及以上时,OVA组小鼠RL显著高于Control组小鼠(P0.05),高良姜素组和地塞米松组小鼠与OVA组小鼠相比有统计学意义(P0.05); OVA组小鼠BALF中炎性细胞总数、EOS细胞数和NEUT细胞数、LYM细胞数高于Control组小鼠(P0.05),高良姜素组和地塞米松组小鼠BALF中炎性细胞总数、EOS细胞数、NEUT细胞数和LYM细胞明显降低,与OVA组小鼠相比有统计学意义(P0.05); OVA组BALF中IL-4、IL-5和IL-13和血清OVA特异性IgE含量高于Control组(P0.05),高良姜素组和地塞米松组BALF中IL-4、IL-5和IL-13和血清OVA特异性IgE含量低于OVA组小鼠(P0.05)；OVA组小鼠气道粘液评分高于Control组小鼠(P0.05),高良姜素组和地塞米松组小鼠气道粘液评分低于OVA组小鼠(P0.05); OVA组小鼠肺组织iNOS和VCAM-1的蛋白表达高于Control组(P0.05),高良姜素和地塞米松组小鼠肺组织iNOS和VCAM-1的蛋白表达与OVA组相比降低(P0.05); OVA组小鼠肺组织细胞总蛋白IκBα的表达低于Control组(P0.05),高良姜素和地塞米松组小鼠肺组织细胞总蛋白IκBα的蛋白表达与OVA组相比增高(P0.05); OVA组小鼠肺组织细胞总蛋白磷酸化的p65表达高于Control组(P0.05),高良姜素和地塞米松组小鼠肺组织细胞总蛋白磷酸化的p65表达与OVA组相比降低(P0.05); OVA组小鼠肺组织核蛋白p65表达高于Control组(P0.05),高良姜素和地塞米松组小鼠肺组织核蛋白p65表达与OVA组相比降低(P0.05); OVA组小鼠肺组织胞浆蛋白p65表达低于Control组(P0.05),高良姜素和地塞米松组小鼠肺组织核蛋白p65表达与OVA组相比增高(P0.05)。结论：高良姜素可在一定程度上抑制哮喘模型小鼠的气道炎症,气道高反应性,这种抑制作用可能与抑制NF-κB的活化有关。第二部分高良姜素在人气道平滑肌细胞中的抗炎作用目的研究探讨高良姜素对肿瘤坏死因子-α (TNF-α)诱导的人气道平滑肌细胞(ASMCs)表达单核细胞趋化蛋白-1(MCP-1)、嗜酸性粒细胞趋化因子(eotaxin)、CXC趋化因子配体10(CXCL10)和血管细胞黏附分子-1(VCAM-1)的影响及可能的机制。方法体外培养原代人气道平滑肌细胞,Cell Counting Kit-8(CCK-8)法检测不同浓度的高良姜素对人气道平滑肌细胞的活性影响；实时荧光定量PCR(Real-time PCR)法检测高良姜素对TNF-α诱导人气道平滑肌细胞表达MCP-1、eotaxin、CXCL10、VCAM-1mRNA的影响；蛋白质印迹法(Western blot)检测高良姜素对TNF-α诱导人气道平滑肌细胞NF-κB活性的影响。结果研究发现高良姜素浓度为1μM～20～M时,对人气道平滑肌细胞的活性无明显影响,差异无统计学意义(P0.05),而高良姜素浓度为50μM、100μM时,人气道平滑肌细胞活性明显降低,差异有统计学意义(P0.05);10ng/mL TNF-α可以诱导人气道平滑肌细胞MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表达(P0.05);1μM、10μM高良姜素及10μNF-κB特异性抑制剂TPCA-1均能有效抑制MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表达(P0.05);1μM、10μM高良姜素均能抑制TNF-a诱导的人平滑肌细胞NF-κB p65亚单位的核易位(P0.05)。结论高良姜素能通过抑制NF-κB活性,下调TNF-α诱导的人气道平滑肌细胞MCP-1、Eotaxin、CXCL10和VCAM-1mRNA的表达,发挥其抗炎作用。
[Abstract]:Part 1 Effect of galangin on airway inflammation in asthmatic mice model
Objective: To study the effect of Takara Jiangso on airway inflammation and airway hyperresponsiveness in mice model of asthma and its mechanism.
Methods: 36 BALB/c mice were randomly divided into control group (Control group), asthma group (OVA group), DMSO group (OVA+DMSO group), high dose of galangin group (OVA+GA15 group), low dose of galangin group (OVA+GA5 group) and dexamethasone group (OVA+DXM), and the intervention group with 6 rats in each group. The asthma group mice at zeroth and 14 days by intraperitoneal injection of 20 g OVA sensitized 22,23,24 days 1%OVA continuous spray for three days, once a day, 30 minutes each time. The intervention group was treated with intraperitoneal injection of OVA activated DMSO the day before, different doses of galangin, dexamethasone. 24h after the last challenge. Determination of airway reactivity using small animal lung function analysis system, the lung pathological section with hematoxylin and eosin (HE) staining of tracheal and lung tissue adjacent to vascular inflammatory cell infiltration, goblet cell hyperplasia were observed with PAS staining, adsorption test by enzyme-linked immunosorbent assay (ELISA) were detected in mice Serum OVA specific IgE and bronchoalveolar lavage fluid (BALF) in IL-4, IL-4 and IL-13. Using immunohistochemistry method (IHC) and Western blotting (Western blot) expression of iNOS and VCAM in lung tissues was detected. The protein expression in lung tissue of total cell protein using Western blot detection I lambda B alpha and phosphate the expression of p65 protein, nuclear protein and p65 protein in the cytoplasm.
Results: the pulmonary resistance in mice (RL) showed that with the increase of acetylcholine chloride concentration, OVA mice RL mice in group Control increased significantly, RL increased slightly, no differences in baseline airway resistance in mice (P0.05), when the concentration of acetylcholine chloride at 30 g/kg and above, OVA group were significantly higher than that of Control RL groups of mice (P0.05), galangin group and dexamethasone group compared with OVA group were statistically significant (P0.05); the number of inflammatory cells in OVA mice BALF, EOS cell count and NEUT cell number, LYM cell number is higher than that of mice in group Control (P0.05), the number of inflammatory cells of galangin group and dexamethasone group in BALF mice, EOS cells, NEUT cells and LYM cells were significantly decreased, which was statistically significant compared with OVA group (P0.05); IL-4 OVA group BALF, IL-5 and IL-13 and serum OVA specific IgE were higher than group Control (P0.05), Gaoliangjiang IL-4 group and dexamethasone group BALF, IL-5 and IL-13 and serum OVA specific IgE was lower than that in OVA group (P0.05); OVA group of mice airway mucus was higher than that of mice in group Control (P0.05), the Takara Jiangso group and dexamethasone group mice airway mucus score was lower than that of mice in OVA group (P0.05); the expression of OVA in lung tissues of mice iNOS and VCAM-1 protein is higher than that of Control group (P0.05), galangin and dexamethasone in lung tissue of mice iNOS and VCAM-1 protein expression decreased compared with OVA group (P0.05); group the expression of OVA in lung tissues of mice with total cell protein I kappa B alpha was lower than that of Control group (P0.05), the expression of Takara Jiangso and dexamethasone in lung tissue of mice total cell protein I kappa B alpha protein compared with OVA group (P0.05); OVA group of mouse lung cell total protein phosphorylation of p65 was higher than that in group Control (P0.05), Takara Jiangso and dexamethasone in mice lung tissue cells The expression of protein phosphorylation of p65 decreased compared with OVA group (P0.05); nuclear protein p65 in lung tissue of mice in group OVA was higher than that in Control group (P0.05), the expression of nuclear protein p65 Takara Jiangso and dexamethasone group of mice lung tissue compared with the OVA group decreased (P0.05); OVA group of mice lung tissue cytosolic protein expression of p65 was lower than that of Control group (P0.05), nuclear protein p65 Takara Jiangso and dexamethasone group of mice lung tissue expression compared with OVA group (P0.05).
Conclusion: alpinin can inhibit airway inflammation and airway hyperresponsiveness to a certain extent, which may be related to inhibiting the activation of NF- kappa B.
The second part of the anti-inflammatory effect of galangin in human airway smooth muscle cells.
Objective to investigate the galangin on tumor necrosis factor alpha (TNF- alpha) induced human airway smooth muscle cells (ASMCs) expression of monocyte chemoattractant protein -1 (MCP-1), eosinophil chemotactic factor (eotaxin), CXC chemokine ligand 10 (CXCL10) and vascular cell adhesion molecule (-1 VCAM-1) the effects and possible mechanism.
Primary cultured human airway smooth muscle cells in vitro, Cell Counting Kit-8 (CCK-8) method was used to detect the effects of different concentrations of Takara Jiangso activity on human airway smooth muscle cells; real time fluorescence quantitative PCR (Real-time PCR) method for the detection of galangin on TNF- induced MCP-1 expression in human airway smooth muscle cells eotaxin, CXCL10, VCAM-1mRNA; protein Western blotting (Western blot) detection effect of galangin induced kappa B activity in human airway smooth muscle cells of NF- TNF- alpha.
Results galangin concentration is 1 ~ M to 20 ~ M, had no significant effect on human airway smooth muscle cells, there was no statistically significant difference (P0.05), and galangin concentration was 50 M, 100 M, the activity of human airway smooth muscle cells decreased significantly, the difference was statistically significant (P0.05 10ng/mL TNF-); alpha can induce airway smooth muscle cells MCP-1, Eotaxin, expression of CXCL10 and VCAM-1mRNA (P0.05); 1 M, 10 M and 10 NF- of galangin kappa B inhibitor TPCA-1 could effectively inhibit the MCP-1, Eotaxin, CXCL10 and VCAM-1mRNA expression (P0.05); 1 M the nuclear translocation of vascular smooth muscle cells, NF- kappa B subunit p65 10 M galangin can inhibit TNF-a induced by (P0.05).
Conclusion galangin can inhibit NF- kappa B activity, reduced human airway smooth muscle cells induced by TNF- MCP-1, Eotaxin, expression of CXCL10 and VCAM-1mRNA, exert its anti-inflammatory effect.

【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】：R562.25

【共引文献】