小剂量多西环素对野百合碱诱导左肺切除大鼠肺动脉血管重构的影响研究

Effects of low-dose doxycycline on the development of monocrotaline-induced pulmonary arterial remodeling in left-pneumonectomized rats

目的 研究小剂量多西环素对大鼠左肺切除后注射野百合碱所造成肺动脉重构及肺动脉高压的作用影响.方法 将45只健康雄性SD大鼠随机分为正常组（N组）15只、模型组（PM组）15只、小剂量多西环素干预组（PMD组）15只.5周后观察各组的血流动力学指标、肺动脉管壁厚度及肌性化程度比、肺动脉新生内膜形成情况、肺动脉管壁细胞增殖度及凋亡蛋白Caspase-3表达变化情况;免疫组化和RT-PCR检测明胶酶（包括MMP-2、9）及TIMP-1在肺组织中的表达水平变化;明胶酶谱法检测肺组织匀浆中明胶酶活性的变化情况.结果 （1）PM组肺动脉平均压为（38.89±1.68） mmHg,高于N组的（18.28±1.52） mmHg和PMD组的（29.89±1.75） mmHg（F=79.246,P＜0.05）.（2）PM组肺动脉管壁厚度比为（51.98±8.49）％,均高于N组的（10.97±3.22）％和PMD组的（26.19±3.10）％ （F =58.439,P＜0.05）.（3）PM组凋亡蛋白Caspase-3的表达[（14.38±2.60） ×103]低于N组[（5.29±1.39） ×103]和PMD组[（25.81±6.14） ×103]（F=58.421,P＜0.05）.（4）PM组新生内膜增殖比为（75.69±3.98）％,高于PMD组的（30.38±5.22）％（t=10.236,P＜0.05）,N组未见新生内膜形成.（5）PM组TIMP-1在肺组织中的表达定量均高于N组和PMD组（P＜0.05）,PM组MMP-2免疫组化表达[（59.61±13.01）×103]均高于N组[（3.78±2.18） ×103]和PMD组[（25.95.±6.89） ×103]（F=76.484,P＜0.05）,PM组MMP-2 RT-PCR表达[（9.80±0.78）]均高于N组[（1.90±1.02）]和PMD组[（7.90±0.62）] （F =45.891,P＜0.05）.（6）PM组肺组织明胶酶活性测定[（4.89±0.29） ×103]均高于N组[（1.52±0.31） ×103]和PMD组[（2.87.±0.17） ×103]（F=47.659,P＜0.05）.结论 小剂量多西环素能有效降低胶原酶在肺组织中的表达及活性,从而减轻实验性大鼠肺动脉重构,并降低其肺动脉压力.其机制可能与降低肺动脉管壁细胞增殖并诱导增生细胞凋亡、限制肺动脉平滑肌细胞迁移有关.该研究为今后小剂量多西环素作为金属基质蛋白酶抑制剂用于肺动脉高压的治疗提供了参考依据.

Objective To investigate the effects of low-dose doxycycline on the development of monocrotaline-induced pulmonary hypertension and pulmonary arterial remodeling in left-pneumonectomized rats.Methods 45 healthy male SD rats were randomly divided into the three groups：normal control group （N）,model group （PM） and doxycycline intervention group （PMD）,each group with 15 cases.After five weeks,the rats were sacrifised to record the pulmonary arterial pressure（PAP） and measure the fulton index immediately.Histological and morphometric analysis were used to determine percent arterial wall thickness,muscularizition of non-musclarised peripheral pulmonary arterioles and neointima formation microscopically.Immunohistochemical stains of PCNA and Caspases-3 were used to investigate cellular proliferation and apoptosis,respectively.Expression of MMP-2,MMP-9 and TIMP-1 were assessed by both immunohistochemical stain and RT-PCR of relevant mRNAs.Meanwhile,the activity of MMPs was measured by gelatin zymography in the lung tissue.Results （1） Rats in PM group showed that mPAP obviously increased comparison with their PMD group and N group [PM group （38.89 ± 1.68）mmHg,PMD group （29.89 ± 1.75） mmHg,N group （18.28 ± 1.52） mmHg,F =79.246,P 〈 0.05].（2） The percent arterial wall thickness were significantly raised in PM group compared with PMD group and N group [PM group （51.98 ± 8.49）%,PMD group （26.19 ± 3.10） %,N group （10.97 ± 3.22） %,F =58.439,P 〈 0.05].（3） The expression of Caspases-3 was decreased in PM group compared to the PMD group significantly[PM group （14.38 ±2.60） × 103,PMD group （25.81 ± 6.14） × 103,N group （5.29 ± 1.39） × 103,F =58.421,P 〈 0.05].（4） Neointima was observed in PM group [（75.69 ± 3.98） %] and PMD group[（30.38 ± 5.22）%].Rats in group PM demonstrated more severe neointima hyperplasia than the group PMD （t =10.236,P 〈 0.05）,N group had no new neointima formation.（5）The expression of TIMP-1 in the lung tissue were higher in PM group compared with PMD group and N group（P 〈0.05）,and the expression of MMP-2 in the lung tissue were higher in PM group[（59.61 ± 13.01） × 103] compared with PMD group[（25.95.±6.89） × 103] and N group [（3.78 ± 2.18） × 103] as assessed by both the immunohistochemical stain and RT-PCR （F =76.484,P 〈 0.05）;The expression of MMP-2 by RT-PCR in PM group [（9.80 ± 0.78）] were higher than PMD group[（7.90 ±0.62）] and N group[（1.90 ± 1.02）] （F =45.891,P 〈 0.05）.（6） The activity of MMPs were inhibited by doxycycline effectively as assessed by gelatin zymography [PM group （4.89 ± 0.29） × 103,PMD group （2.87.± 0.17） ×l03,N group（1.52 ±0.31） ×103,F=47.659,P〈0.05].Conclusion Low-dose doxycycline attenuated pulmonary arterial remodeling in left-pneumonectomized rats which suffered from monocrotaline-injection simultaneously.The underlying mechanism of that is possiblly ascribled to the reduced VSMCs proliferation,suppress migration and promote cell apoptosis,which are thought to be associated to the inhibition of MMPs by the drug.These results provide using low-dose doxycycline as a new therapeutic strategy for the PAH in the future.