【摘要】 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。更多还原

【Abstract】 Objective To investigate the effect of melatonin on the regulation of collagen accumulation and the underlying mechanisms.Methods Ninety-six BALB/c mice were randomly divided into five groups.The mice were injected i.p.with normal saline in the control group(n=21)and the asthma group(n=92),melatonin in the melatonin group(n=23),dexamethasone in the dexamethasone group(n=24),and melatonin antagonist luzindole in the luzindole group(n=7)respectively.The mice of the control group,asthma group,melatonin group,dexamethasone group were sensitized and repeatedly challenged with ovalbumin for 2,4,8 weeks(the number of control group were 7,7,7 respectively at 2,4, 8 weeks;the number of asthma group were 7,7,8 respectively at 2,4,8 weeks;the number of melatonin group were 7,8,8 respectively at 2,4,8 weeks;the number of dexarnethasone group were 7,8,8 respectively at 2,4,8 weeks).The mice of the luzindole group(n=7)were sensitized and repeatedly challenged with ovalbumin for 2 weeks,to establish the murine model of chronic asthma.Masson staining was used to stain collagen fibers and immunohistoehemistry to stain protein expression.MetaMorph image analysis software was used to measure the positive area per micrometer perimeter of basement membrane (Wcol/Pbm),to evaluate collagen accumulation in lung tissue.The immuno-positve area per unit area(PA/ UA)was measured to evaluate the protein level of matrix metalloproteinase-9(MMP-9)and tissue inhibitor of matrix metalloproteinase-1(TIMP-1).Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)was used to evaluate the mRNA levels.Results The collagen area per unit perimeter of basement membrane(Wcol/Pbm)in the melatonin group was(11.8±1.3),(12.3±1.1),(12.7±1.4) μm~2/μm respectively at 2,4,and 8 weeks,which were decreased as compared with(14.5±1.5), (15.8±1.8),(16.2±1.4)μm~2/μm in the asthma group(t_d=3.89,5.96,5.50 respectively,all P< 0.01).The PA/UA of MMP-9 was(9.7±4.9)pixel,(14.8±4.9)pixel,(11.0±6.8)pixel respectively at 2,4,8 weeks in the melatonin group,(15.65±6.11)pixel,(26.2±6.9)pixel,(24.6± 6.0)pixel respectively at 2,4,8 weeks in the asthma group,the difference being statistically significant between the two groups(t_d=3.00,4.83,5.50 respectively at 2,4,8 weeks,all P<0.01).The MMP-9 mRNAlevel was 0.80±0.40,0.68±0.15,0.67±0.24 in the melatonin group at 2,4,8 weeks respectively,and was 1.48±0.29,1.40±0.50,1.20±0.40 in the asthma group at 2,4,8 weeks respectively(t_d=3.92,4.50,3.29 respectively at 2,4,8 weeks,all P<0.01).The Wcol/Pbm[(11.6± 1.3),(12.3±1.0),(13.0±1.7)μm~2/μm],protein[(12.5±5.6),(14.0±4.7),(13.6±4.8)pixel] and mRNA(0.69±0.11,0.61±0.16,1.10±0.40)level were reduced in the dexamethasone group as compared with the asthma group at 2,4,8 weeks respectively.The above mentioned parameters in the luzindole group were not statistically different as compared with the asthma group at 2 weeks.Conclusions The results suggest that early treatment with melatonin inhibits airway collagen accumulation,probably mediated by the inhibition of MMP-9.更多还原