熊果酸对香烟诱导肺气肿大鼠干预作用及其对肺组织MMP-9和TIMP-1表达的影响

Intervention effects and affect of ursolic acid on expression of MMP-9 and TIMP-1 in lung tissue on cigarette smoke induced emphysema in rats

目的通过观察熊果酸对香烟诱导大鼠肺气肿的干预作用及其对肺组织MMP-9和TIMP-1表达的影响,探讨熊果酸减轻肺气肿的作用机制。方法建立香烟诱导肺气肿大鼠模型,将40只SPF级雄性Wistar大鼠按随机数字表法分为健康对照组、模型组、熊果酸10mg/kg(UA10)组、熊果酸20 mg/kg(UA20)组以及熊果酸40 mg/kg(UA40)组,每组8只。取每只大鼠的右下叶肺组织,石蜡包埋切片,常规HE染色。对HE染色标本进行肺气肿评分即每视野的平均肺泡数和平均肺泡直径的测定。进行TIMP-1、MMP-9免疫组化染色,主要应用链霉素亲生物素蛋白-过氧化酶复合技术。对石蜡包埋的肺组织标本行MMP-9、TIMP-1相对含量的测定。结果与对照组比较,模型组大鼠肺泡腔扩大、部分肺泡间隔断裂、肺泡腔融合、肺气肿形成。熊果酸各组大鼠可见局部轻度肺气肿形成,主要表现为肺泡间隔略增宽,肺泡腔呈局限性轻度扩大;模型组大鼠肺泡平均内衬间隔(120.17±10.86)μm明显高于对照组(47.76±3.55)μm、平均肺泡数(14.25±10.23)/mm^(2)明显低于对照组(338.08±26.08)/mm^(2)、MMP-9相对阳性面积(18.16±4.62)%明显高于对照组(5.38±0.93)%、TIMP-1相对阳性面积(5.36±2.15)明显低于对照组(12.80±1.31)%,MMP-9/TIMP-1比值(3.38±0.79)明显高于对照组(0.42±0.16),均具有统计学差异(P<0.01);与模型组比较,熊果酸10 mg/kg、20 mg/kg、40 mg/kg组平均内衬间隔分别为(104.48±10.85)μm、(80.82±7.68)μm、(53.90±9.92)μm均显著减少,平均肺泡数分别为(165.12±16.65)/mm^(2)、(206.98±25.55)/mm^(2)、(279.94±29.53)/mm^(2)均显著增加,MMP-9蛋白表达含量分别为(14.31±1.90)%、(10.52±1.88)%、(6.90±0.43)%均显著减少,TIMP-1蛋白表达含量分别为(7.07±1.3)%、(9.86±1.80)%、(11.56±1.46)%均升高,MMP-9/TIMP-1比值分别为(2.02±0.54)、(1.06±0.37)、(0.52±0.23)均降低,均具有统计学差异(P<0.01)。结论熊果酸有减轻香烟所致大鼠肺气肿作用,且熊果酸可能通过调节蛋白酶/抗蛋白酶失衡减轻肺气肿。

Objective Though observing the intervention effects and affect of ursolic acid on the expression of MMP-9 and TIMP-1 in lung tissue on cigarette smoke induced emphysema in rats,investigate the mechanism of ursolic acid on the emphysema.Methods Preparation of animal grouping and ciagrate smoke induced emphysema in rats model.Forty healthy male rats were randomly assigned into five groups via random number method:control group,cigarette smoking group,UA 10 mg/kg group,UA 20 mg/kg group and UA 40 mg/kg group with 8 rats in each group.Process of pathological specimen and the morphological analysis:embedded the right lower lobe of each rat with paraffin and sliced the tissue for section and HE staining was performed.The mean alveolar intercept(MLI)and mean alveolus number(MAN)was measured on the HE staining section for morphological analysis.The protein expression of MMP-1 and TIMP-1 are analyzed by immunohistochemistry staining.Results Compared with the control group,alveolar expand,part of the alveolar septum rupture,alveolar fusion,emphysema were formation in the cigarette smoking group.Alveolar mild were expanded,alveolar septum were slightly widened,local mild emphysema were formation in the ursolic acid 10 mg/kg,20 mg/kg,40 mg/kg group.The average lining interval(120.17±10.86)μm in the cigarette smoking group were higher than the control group(47.76±3.55)μm,and the mean alveolar number(114.25±10.23)/mm^(2) in the cigarette smoking group were lower than the control group(338.08±26.08)/mm^(2);The expression of MMP-9 in the cigarette smoking group(18.16±4.62)were significantly higher compared with control group(5.38±0.93),the expression of TIMP-1 in the cigarette smoking group(5.36±2.15)were significantly lower compared with the control group(12.80±1.31),in addition the ratio of[respectively(14.31±1.90)%,(10.52±1.88)%,(6.90±0.43)%]MMP-9/TIMP-1 in the cigarette smoking group(3.38±0.79)was higher than that in the control group(0.42±0.16),they were all statistically different(P<0.01).Compared with the cigarette smoking group,mean lining interval(MLI)respectively(104.48±10.85)μm,(800.82±7.68)μm,(53.90±9.92)μm were lower in the ursolic acid 10 mg/kg,20 mg/kg,40 mg/kg group.The mean alveolar number respectively(165.12±16.65)/mm^(2),(206.98±25.55)/mm^(2),(279.94±29.53)/mm^(2) were higher in the ursolic acid 10 mg/kg,20 mg/kg,40 mg/kg group.And the expression of MMP-9(14.31±1.90)%,(10.52±1.88)%,(6.90±0.43)%were decresed,the expression of TIMP-1 respectively(7.07±1.3)%,(9.86±1.80)%,(11.56±1.46)%were increased,the ratio of MMP-9/TIMP-1 respectively(2.02±0.54),(1.06±0.37),(0.52±0.23)were decreased.They were all statistically different(P<0.05).Conclusion Ursolic acid have the fuction of relieving the cigarette smoke induced emphysema,and ursolic acid maybe relieve the emphysema through balancing the matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase.