Effects of MMP-1 and MMP-7 on Pulmonary Hypertension in Rats
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摘要:
目的 探究MMP-1和MMP-7在肺动脉高压（pulmonary arterial hypertension，PAH）发展中的作用。 方法 采用ELISA检测PAH患者血清中的MMPs（MMP-1、MMP-2、MMP-7、MMP-9、MMP-10、MMP-14）的含量并选择出2种MMPs进行动物体内实验。收集临床信息并比较PAH患者和健康人的体重指数（body mass index， BMI）、NYHA心功能等级、6 min步行距离（6 min walking distance，6MWD）、脑钠肽（brain natriuretic peptide，BNP）、平均肺动脉压（mean pulmonary arterial pressure，mPAP）、甘油三酯（triglyceride，TGs）、低密度脂蛋白（low-density lipoprotein，LDL）、总胆固醇（total cholesterol，TC）、血红蛋白（hemoglobin，HB）、心率（heart rate，HR）等指标的差异。采用野百合碱诱导PAH大鼠模型，然后使用过表达MMP-1质粒和shMMP-7质粒分别上调和下调PAH大鼠中MMP-1和MMP-7的表达。通过苏木精-伊红染色法 （ hematoxylin-eosin staining，HE）观察肺动脉病理变化，Masson染色观察肌纤维和胶原纤维的变化，免疫荧光检测α-SMA和VWF，对左心室、右心室和室间隔进行称重后计算右心室肥厚指数。 结果 与健康人相比，PAH患者血清中的MMP-1（P < 0.0001）、MMP-2（P < 0. 001）、MMP-7（P < 0.01）、MMP-9（P < 0.001）、MMP-10（P < 0.001）、MMP-14（P < 0.01）含量升高，6MWD降低（P < 0.05），BNP含量升高（P < 0.001），NYHA分级升高（P < 0.0001）。与对照组相比，PAH大鼠的MMP-1（P < 0.0001）和MMP-7（P < 0.0001）在肺组织中蛋白表达升高，右心室肥厚指数增加（P < 0.0001），肺动脉血管增厚（P < 0.05），胶原纤维沉积增多（P < 0.05），α-SMA（P < 0.05）和VWF（P < 0.05）在血管中表达增多。与PAH组相比，在PAH大鼠中过表达MMP-1促进PAH发展，而干扰MMP-7则延缓其发展。 结论 MMPs（MMP-1、MMP-2、MMP-7、MMP-9、MMP-10、MMP-14）在肺动脉高压患者血清中升高。MMP-1和MMP-7促进肺动脉高压大鼠模型的肺动脉血管重塑、肺纤维化和右心室功能不全。 Abstract:Objective To explore the effects of MMP-1 and MMP-7 on pulmonary arterial hypertension （PAH）. Methods The contents of MMPs （MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, MMP-14） in the serum of PAH patients and healthy controls were detected and two MMPs were selected for in vivo experiments. Clinical information was collected and body mass index （BMI）, NYHA cardiac function class, 6 min walking distance （6MWD）, brain natriuretic peptide （BNP）, mean pulmonary arterial pressure （mPAP）, triglyceride （TGs）, low-density lipoprotein （LDL）, total cholesterol （TC）, hemoglobin （HB）, heart rate （HR） were compared between PAH patients and healthy controls. The PAH rat model was induced by monocrotaline, and then MMP-1 overexpression plasmid and shMMP-7 plasmid were used to up-regulate and down-regulate the expression of MMP-1 and MMP-7, respectively. Hematoxylin-eosin （HE） staining was used to observe the pathological of PAH, Masson staining was used to observe the changes of muscle fibers and collagen fibers, and immunofluorescence was used to detect the expression of α-SMA and VWF. The left ventricle, right ventricle and interventricular septum were weighed and the right ventricular hypertrophy index was calculated. Results The serum levels of MMP-1 （P < 0.0001）, MMP-2 （P < 0.001）, MMP-7 （P < 0.01）, MMP-9 （P < 0.001）, MMP-10 （P < 0.001） and MMP-14 （P < 0.01） in PAH patients were significantly higher than those in healthy controls. 6MWD was significantly decreased （P < 0.05）, BNP level was increased （P < 0.001）, NYHA class was increased （P < 0.0001）. Compared with the control group, the protein expression of MMP-1 （P < 0.0001） and MMP-7 （P < 0.0001） in lung tissue, right ventricular hypertrophy index （P < 0.0001）, pulmonary artery vascular thickness （P < 0.05）, and collagen fiber deposition （P < 0.05） were increased in PAH rats. The expressions of α-SMA （P < 0.05） and VWF （P < 0.05） in blood vessels were increased in PAH rats than control rats. Compared with PAH group, overexpression of MMP-1 in PAH rats promoted the development of PAH, while interference with MMP-7 delayed its development. Conclusions The serum levels of MMPs （MMP-1, MMP-2, MMP-7, MMP-9, MMP-10, MMP-14） are increased in patients with PAH. MMP-1 and MMP-7 promote pulmonary artery remodeling, pulmonary fibrosis and right ventricular dysfunction in PAH rats. -
Key words:
- MMP-1 /
- MMP-7 /
- Pulmonary arterial hypertension
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图 1 健康人和PAH患者临床指标的差异
A：比较健康人与PAH组的6MWD差异；B：比较健康人与PAH组的BMI差异；C：比较健康人与PAH组的BNP差异；D：比较健康人与PAH组的NYHA分级差异；E：比较健康人与PAH组的LDH差异；F：比较健康人与PAH组的TGs差异；G：比较健康人与PAH组的mPAP差异；H：比较健康人与PAH组的HR差异；I：比较健康人与PAH组的HB差异；J：比较健康人与PAH组的TC差异。*P < 0.05，***P < 0.001，****P < 0.0001。
Figure 1. Differences of clinical indicators between healthy people and patients with PAH
图 2 Ctrl组与PAH患者血清中MMPs的水平
A：ELISA试剂盒检测健康人和PAH患者MMP-1水平；B：ELISA试剂盒检测健康人和PAH患者MMP-2水平；C：ELISA试剂盒检测健康人和PAH患者MMP-7水平；D：ELISA试剂盒检测健康人和PAH患者MMP-9水平；E：ELISA试剂盒检测健康人和PAH患者MMP-10水平；F：ELISA试剂盒检测健康人和PAH患者MMP-14水平。**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 2. Concentration levels of MMPs in serum of patients with Ctrl group and PAH
图 3 筛选3个shMMP-7干扰质粒对MMP-7蛋白水平和mRNA水平的干扰效率
A和B：western blot检测比较3个shMMP-7干扰质粒对MMP-7蛋白表达的干扰效率； C：qRT-PCR检测比较3个shMMP-7干扰质粒对MMP-7 mRNA的干扰效率。*P < 0.05，**P < 0.01，***P < 0.001，****P < 0.0001。
Figure 3. Interference efficiency of three shMMP-7 interfering plasmids screened on MMP-7 protein level and mRNA level
图 4 MMP-1和MMP-7对PAH大鼠模型的右心室肥厚指数的影响
*P < 0.05，**P < 0.01，****P < 0.0001。
Figure 4. Impacts of MMP-1 and MMP-7 on right ventricular hypertrophy index in a PAH rat model
图 5 MMP-1过表达质粒和MMP-7干扰质粒对PAH的蛋白表达水平和病理作用
A-C：western blot检测过表达MMP-1质粒和干扰MMP-7质粒的转染效率； D:HE染色观察MMP-1和MMP-7对PAH的形态学影响（40×）；E:Masson染色观察MMP-1和MMP-7对PAH肌纤维和胶原纤维的影响（40×）。****P < 0.0001。
Figure 5. Protein expression levels and pathological effects of MMP-1 overexpression plasmid and MMP-7 interference plasmid on PAH
图 6 MMP-1和MMP-7对PAH中α-SMA和vWF蛋白的表达的影响（40×）
A:IF检测PAH中α-SMA的表达和分布；B：IF检测PAH中vWF的表达和分布。
Figure 6. Effects of MMP-1 and MMP-7 on α-SMA and vWF protein expression in PAH （40×）
表 1 临床PAH患者和健康人的基线资料表[
$ \bar x \pm s $ /n（%）]Table 1. Baseline data table of clinical PAH patients and healthy individuals [
$ \bar x \pm s $ /n（%）]项目 健康人（n = 16） PAH患者（n = 15） 年龄（岁） 31.69 ± 11.45 69.07 ± 11.11 性别（女性） 10（62.50） 4（26.67） BMI（cm/kg2） 22.13 ± 2.68 23.60 ± 5.49 NYHA心功能等级 0 16（100.00） 0（0.00） 1 0（0.00） 6（40.00） 2 0（0.00） 9（60.00） 6MWD（m） 593.00 ± 69.19 508.67 ± 102.102 BNP（pg/mL） 55.19 ± 15.71 139.13 ± 89.37 mPAP（mmHg） 19.62 ± 1.20 19.86.25 ± 3.46 HR（次/min） ≤80 11（68.75） 10（66.67） > 80 5（31.25） 5（33.33） TGs（mmol/L） 1.74 ± 0.50 1.45 ± 0.99 LDL（mmol/L） 2.71 ± 0.55 2.63 ± 0.70 TC（mmol/L） 3.87 ± 0.55 4.41 ± 0.94 HB（g/L） 135.56 ± 11.37 141.47 ± 20.43 
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