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目的应用小鼠慢性支气管哮喘(简称哮喘)模型,观察褪黑素对慢性哮喘小鼠肺组织中胶原沉积的影响及其机制。方法 96只 SPF 级雄性 BALB/c 小鼠按随机数字表法分为5组。对照组(21只)腹腔注射生理盐水;哮喘组(22只)腹腔注射生理盐水;褪黑素组(23只)腹腔注射褪黑素;地塞米松组(23只)腹腔注射地塞米松;褪黑素拮抗组(7只)腹腔注射褪黑素拮抗剂2-苯基-N-乙酰色胺。根据实验要求每组采用卵白蛋白致敏并反复雾化吸入2、4、8周时再分为2、4、8周亚组。对照组(每组均为7只);哮喘组(分别为7、7、8只);褪黑素组(分别为7、8、8只);地塞米松组(分别为7、8、8只);而褪黑素拮抗组只做2周组(7只)。实验造成慢性哮喘模型。用 Masson 三原色法染胶原纤维;用免疫组化方法标记蛋白;用 MetaMorph 软件测量单位长度基底膜周径上的胶原面积(Wcol/Pbm)和单位面积上基质金属蛋白酶9(MMP-9)及基质金属蛋白酶组织抑制剂1(TIMP-1)免疫组化阳性面积(PA/UA);用半定量逆转录-聚合酶链反应(RT-PCR)法测定肺组织相应蛋白 mRNA 水平。结果褪黑素组2、4、8周 Wcol/Pbm 分别为(11.8±1.3)、(12.3±1.1)、(12.7±1.4)μm~2/μm,哮喘2、4、8周组分别为(14.5±1.5)、(15.8±1.8)、(16.2±1.4)μm~2/μm,两组比较差异有统计学意义(t_d值分别为3.89、5.96、5.50,P 均<0.01);褪黑素组2、4、8周 MMP-9的 PA/UA 像素分别为(9.7±4.9)、(14.8+4.9)、(11.0±6.8)万,哮喘2、4、8周组分别为(15.7±6.1)、(26.2±6.9)、(24.6±6.0)万,两组比较差异有统计学意义(t_d 值分别为3.00、4.83、5.50,P 均<0.01);褪黑素组2、4、8周MMP-9 mRNA 水平表达分别为0.80±0.40、0.68±0.15、0.67±0.24,哮喘2、4、8周组分别为1.48±0.29、1.40±0.50、1.20±0.40,两组比较差异有统计学意义(t_d 值分别为3.92、4.50、3.29,P 均<0.01);地塞米松组2、4、8周 Wcol/Pbm(11.6±1.3、12.3±1.0、13.0±1.7)μm~2/μm、MMP-9蛋白[(12.5±5.6)、(14.0±4.7、13.6±4.8)万]和 mRNA 水平(0.69±0.11、0.61±0.16、1.10±0.40)均较哮喘2、4、8周组降低。褪黑素拮抗2周组与哮喘2周组以上各指标比较差异均无统计学意义(t_d 值=0.96,P>0.05)。结论早期使用褪黑素可抑制胶原沉积,其作用与地塞米松相当。褪黑素可能通过 MMP-9介导的途径抑制哮喘气道重塑。
Objective To study the effect and mechanism of melatonin on the collagen deposition in the lung tissue of chronic asthmatic mice by using mouse model of chronic bronchial asthma (asthma). Methods 96 SPF male BALB / c mice were randomly divided into 5 groups according to random number table. The rats in the control group (21 rats) were injected intraperitoneally with saline; the rats in the asthma group (22 rats) were injected intraperitoneally with normal saline; the rats in the melatonin group (23 rats) were injected intraperitoneally with melatonin; the dexamethasone group Melatonin antagonist group (7) intraperitoneal injection of melatonin antagonist 2-phenyl-N-acetyl tryptamine. According to the experimental requirements of each group using ovalbumin sensitization and repeated inhalation inhalation 2, 4, 8 weeks and then divided into 2, 4, 8 weeks subgroup. Control group (7 in each group); asthma group (7, 7, 8); melatonin group (7, 8 and 8, respectively); dexamethasone group 8); while melatonin antagonist group only 2 weeks (7). Experiment resulted in chronic asthma model. Masson’s three-primary method was used to stain collagen; protein was detected by immunohistochemistry; the per unit area length of collagen membrane (Wcol / Pbm) and per unit area of matrix metalloproteinase 9 (MMP-9) and matrix Immunohistochemistry positive area (PA / UA) of tissue inhibitor of metalloproteinase 1 (TIMP-1) was detected by enzyme linked immunosorbent assay (ELISA); mRNA of lung tissue was detected by semi-quantitative reverse transcription polymerase chain reaction (RT- Results The Wcol / Pbm concentrations at 2, 4 and 8 weeks in the melatonin group were (11.8 ± 1.3), (12.3 ± 1.1) and (12.7 ± 1.4) μm ~ 2 / μm, respectively 14.5 ± 1.5), (15.8 ± 1.8) and (16.2 ± 1.4) μm ~ 2 / μm respectively. There was significant difference between the two groups (t_d values were 3.89,5.96 and 5.50 respectively, P <0.01) The PA / UA pixels of MMP-9 in group 2, 4 and 8 were (9.7 ± 4.9), (14.8 ± 4.9) and (11.0 ± 6.8) ), (26.2 ± 6.9) and (24.6 ± 6.0) respectively. The differences between the two groups were statistically significant (t_d values were 3.00, 4.83 and 5.50, P all <0.01) The expression of MMP-9 mRNA were 0.80 ± 0.40,0.68 ± 0.15,0.67 ± 0.24 respectively in asthma group and 1.48 ± 0.29,1.40 ± 0.50,1.20 ± 0.40 in 2,4,8 weeks asthma group, the difference was statistically significant (t_d values were 3.92,4.50,3.29, P <0.01). Wcol / Pbm (11.6 ± 1.3,12.3 ± 1.0,13.0 ± 1.7) μm ~ 2 / μm, MMPs -9 protein [(12.5 ± 5.6), (14.0 ± 4.7,13.6 ± 4.8)] and mRNA levels (0.69 ± 0.11,0.61 ± 0.16,1.10 ± 0.40) were lower than those in 2,4,8 week asthma group. Melatonin antagonist 2 weeks group and asthma 2 weeks group the above indicators were no significant difference (t_d value = 0.96, P> 0.05). Conclusion Early use of melatonin can inhibit collagen deposition, its role and dexamethasone equivalent. Melatonin may suppress asthma airway remodeling through MMP-9-mediated pathway.