| [Backgroud]Pulmonary hypertension (PH) is a group of severe diseases characterized by a progressive increase in pulmonary vascular resistance, and pulmonary arterial hypertension is one of the most common type of PH. The remodeling of pulmonary vessels is a key point of PH. Now the therapeutic drugs can only improve the clinical symptoms of PH patients without inhibition or reversibility of pulmonary vascular remodeling. It has been proved recently that the decreased expression of peroxisome proliferator activated receptor y(PPARy) could play an important role in the pathogenesis of PH, and the ligands of PPARy can inhibit or reverse the development of PH. Rosiglitazone is a synthetic ligand of PPARy, which can produce effects on anti-inflammation, cell proliferation, apoptosis and differentiation. To determine the possible therapeutic effects of rosiglitazone on pulmonary hypertension, we investigated the effects of rosiglitazone on monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) in rats.[Objective]To study the therapeutic effects of rosiglitazone on established severe PAH through observation on changes of general condition, pulmonary hemodynamics and vascular morphology in rats.[Methods]1.40 male Sprague-Dawley rats were randomly divided into four groups:①Group C (n=10):the rats were injected subcutaneously with sodium chloride.②Group P (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received sodium chloride(1.5ml/d) by daily gavage from day 21st to day 34th after injection of MCT.③Group PRH (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (5mg/kg-d) by daily gavage from day 21st to day 34th after injection of MCT.④Group PRL (n=10):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (2.5mg/kg-d) by daily gavage from day 21st to day 34th after injection of MCT.2. Right ventricular systolic pressure (RVSP) and mean pulmonary arterial pressure (mPAP) were detected by right heart catheter on the day 35th. The right ventricle (RV) was separated from the left ventricle (LV) and septum (S), and weighed. Right ventriclular hypertrophy index (RVHI) was caculated.3. Histopathological changes and the tunica media thickness of small pulmonary arteries were evaluated by hematoxylin and eosin staining and Elastin Van Gieson staining (ET+VG)4. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Effects of rosiglitazone on general condition of rats injected with MCT. From the 14th day after MCT injection, some rats in Group P, Group PRH and Group PRL became weak, short of breath and presented a dull coat, and the condition got worse day by day. Until the day of sacrifice, the case fatality rate of Group C, Group P, Group PRH and Group PRL was 0,50%,50% and 30%, separately. Three weeks and five weeks after MCT injection, the mean body weight of rats in Group C were (386±14) g and (475±18) g, separately. Compared with Group C, the mean body weight of rats in Group P[(335v28)g, (316±28) g], Group PRH [(331±22)g, (320±43)g] and Group PRL [(327±30) g, (296±41) g] decreased extremely significantly (P<0.01). However, the mean body weight of rats among Group P, Group PRH and Group PRL showed no significant difference (P>0.05)2. Effects of rosiglitazone on hemodynamics and right ventricular hypertrophy of rats injected with MCT.The mPAP, RVSP and RVHI of rats in Group C were (21.66±2.43) mmHg, (43.76±3.64) mmHg and 0.28±0.02. Compared with Group C, the mPAP, RVSP and RVHI of rats in Group P[(61.57±7.28) mmHg, (108.65±9.97) mmHg,0.70±0.03] increased extremely significantly (P<0.01). Compared with Group P, the mPAP, RVSP and RVHI of rats in Group PRH[(58.83±9.47)mmHg,(106.61±11.67)mmHg,0.63±0.07] and Group PRL[(56.31±8.44) mmHg, (102.90±11.69) mmHg,0.63±0.07] decreased slightly without significant difference (P>0.05), and the mPAP, RVSP and RVHI of rats in Group PRH and Group PRL were still extremely significantly higher than those of Group C (P<0.01). There was no significant difference between Group PRH and Group PRL on mPAP, RVSP and RVHI (P>0.05)3. Effects of rosiglitazone on pulmonary arterial remodeling of rats injected with MCT.HE staining showed prominent tunica media hypertrophy, smooth muscle proliferation, stenosis in muscular pulmonary arteries and evident muscularization of pulmonary arterioles of rats in Group P, Group PRH and Group PRL. ET+VG staining showed a "wave-like" change on internal elastic lamina of rats in Group P, Group PRH and Group PRL. The tunica media thickness percentage of small pulmonary arteries (WT%)of rats in Group C was (8.82±3.23)%. Compared with Group C, WT% of rats in Group P [(55.22±5.72)%], Group PRH [(50.92±5.18)%] and Group PRL [(52.45±6.45)%] increased extremely significantly (P<0.01). Compared with Group P, WT% of rats in Group PRH and Group PRL decreased slightly without significant difference (P>0.05). No significant difference between Group PRH and Group PRL on WT% was found (P>0.05)[Conclusions]1. Rosiglitazone treatment can not lower established severely elevated mPAP and RVSP in pulmonary arterial hypertension rats induced by MCT.2. Rosiglitazone treatment can not reverse established pulmonary vascular remodeling and right ventricular hypertrophy in pulmonary hypertension rats induced by MCT. [Backgroud]The pathogenesis of pulmonary hypertension (PH) is extremely complicated and it has not been explored clearly till now. It has been proved recently that inflammatory mechanisms might play an important role in the pathogenesis and progression of PH. Rosiglitazone, a kind of thiazolidinedione (TZDs), is the syhthetic ligand of peroxisome proliferator activated receptor y (PPARy). It has been shown that rosiglitazone could exert immunomodulatory and anti-inflammatory effects on various animal diseases models. To determine the possible preventive effects of rosiglitazone on pulmonary hypertension, we investigated the protective effects of rosiglitazone intervention on monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) with focus on anti-inflammation and vascular remodeling.[Objective]1. To study the protective effects of rosiglitazone intervention on pulmonary arterial hypertension through observation on changes of general condition, pulmonary hemodynamics and vascular morphology in rats injected with MCT.2. To study the changes of pulmonary interleukin 6 (IL-6), tumor necrosis a (TNF-a) and monocyte chemotactic protein 1 (MCP-1) in rats injected with MCT, and explore possible mechanisms of the protective effects of rosiglitazone intervention.[Methods]1.32 male Sprague-Dawley rats were randomly divided into four groups:①Group N (n=8):the rats were injected subcutaneously with sodium chloride.②Group M (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received sodium chloride(1.5ml/d) by daily gavage from day 0 to day 20th after injection of MCT.③Group MRH (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (5mg/kg-d) by daily gavage from day 0 to day 20th after injection of MCT.④Group MRL (n=8):the rats were injected subcutaneously with MCT (60mg/kg) and received rosiglitazone (2.5mg/kg·d) by daily gavage from day 0 to day 20th after injection of MCT.2. On the day 21st, right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP) and mean arterial pressure (MAP) were detected by right heart catheter and internal carotid artery catheter, separately. The right ventricle (RV) was separated from the left ventricle (LV) and septum (S), and weighed. Right ventriclular hypertrophy index (RVHI) was caculated.3. Histopathological changes were evaluated by hematoxylin and eosin staining, and the tunica media thickness of small pulmonary arteries were evaluated by Elastin Van Gieson staining (ET+VG). The muscularization degree of non-muscularized pulmonary arterioles were evaluated by muscularization percentage and mean optical density through a-SMA immunohistochemical staining.4. Interleukin 6, tumor necrosis factor a and monocyte chemotactic protein 1 of lung tissue were detected by ELISA.5. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Effects of rosiglitazone intervention on general condition of rats injected with MCT.From the 14th day after MCT injection, some rats in Group M became weak, short of breath and presented a dull coat. The mean body weight of Group N on day 7th,14th,21st were (287±12) g, (331±15) g and (375±19) g. Compared with Group N, the mean body weight of Group M[(261±17) g, (276±24) g, (319±29) g], Group MRH [(268±8) g, (275±15)g, (328±14)g] and Group MRL[(261±8) g, (284±11)g, (329±12)g]on day 7th,14th,21st decreased significantly (P<0.01 or P<0.05). However, the mean body weight of rats in Group M, Group MRH and Group MRL showed no significant difference (P>0.05)2. Effects of rosiglitazone intervention on hemodynamic parameters and right ventricular hypertrophy of rats injected with MCT.Three weeks after injection of MCT, the mPAP, RVSP and RVHI of rats in Group N were (17.13±3.30) mmHg, (40.33±5.77) mmHg and 0.25±0.02, separately. Compared with Group N, the mPAP, RVSP and RVHI of rats in Group M [(37.00±4.98) mmHg, (67.04±4.87)mmHg,0.40±0.02]increased extremely significantly (P<0.01).Compared with Group M, the mPAP, RVSP and RVHI of rats in Group MRH [(26.88±3.55) mmHg, (50.09±5.61)mmHg,0.33±0.03] and Group MRL[(28.29±3.60)mmHg, (55.71±4.85) mmHg,0.33±0.02]decreased extremely significantly (P<0.01),but were still higher than those of Group N (P<0.01 or P<0.05). There was no significant difference between Group MRH and Group MRL on mPAP, RVSP and RVHI. No significant difference of MAP among Group N [(87.81±7.78) mmHg], Group M [(88.23±9.29) mmHg], Group MRH [(90.17±6.82) mmHg] and Group MRL [(89.88±9.08) mmHg] was found (P >0.05)3. Effects of rosiglitazone intervention on pulmonary arterial remodeling of rats injected with MCT.HE staining showed obvious tunica media hypertrophy, smooth muscle proliferation, stenosis in muscular pulmonary arteries and evident muscularization of pulmonary arterioles of rats in Group M, ET+VG staining showed a "wave-like" change on internal elastic lamina of rats in Group M. The the tunica media thickness percentage of small pulmonary arteries (WT%) of rats in Group N was (8.91±2.30)%. Compared with Group N, the WT% of rats in Group M [(45.52±5.48)%]increased extremely significantly (P<0.01). Compared with Group M, the WT% of Group MRH [(13.11±3.91)%] and Group MRL [(16.70±1.68)%] decreased extremely significantly (P<0.01), but the WT% of Group MRL was still higher than that of Group N (P< 0.05). Compared with Group MRL, the WT% of Group MRH decreased slightly without significant difference (P>0.05)a-SMA immunohistochemical staining showed that, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group N were [(19.91±2.27)%,0.22±0.04]. Compared with Group N, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group M [(67.55±2.95)%,0.49±0.03] increased extremely significantly (P<0.01).Compared with Group M, the muscularization percentage of pulmonary arterioles and a-SMA mean optical density of rats in Group MRH [(51.74±2.67)%,0.31±0.02] and Group MRL [(54.73±2.39)%,0.38±0.03] decreased significantly (P<0.01), but were still higher than those of Group N (P<0.01). Compared with Group MRL, the muscularization percentage of pulmonary arterioles of rats in Group MRH decreased slightly without significant difference (P> 0.05), and a-SMA mean optical density of rats in Group MRH decreased significantly (P<0.05)4. Effects of rosiglitazone intervention on perivascular inflammation of rats injected with MCT.Three weeks after injection of MCT, HE staining showed obvious inflammatory cells infiltration around the small pulmonary arteries. The perivascular inflammation scale of Group N was 0.40±0.16. Compared with Group N, the perivascular inflammation scale of Group M(3.24±0.41) increased extremely significantly(P<0.01). Compared with Group M, the perivascular inflammation scale of Group MRH (1.26±0.22)and Group MRL(1.22±0.36) decreased extremely significantly(P<0.01). There was no significant difference of perivascular inflammation scale between Group MRH and Group MRL (P>0.05)5. Effects of rosiglitazone intervention on pulmoanry IL-6, TNF-a and MCP-1 level of rats injected with MCT.The pulmonary IL-6, TNF-a and MCP-1 level of rats in Group N were (64.97±17.65) pg/ml, (56.51±14.92) pg/ml and (65.18±23.37) pg/ml, separately. Compared with Group N, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group M [(459.40±94.77)pg/ml, (436.46±62.20)pg/ml, (6265.53±2014.83)pg/ml] increased extremely significantly(P<0.01). Compared with Group M, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group MRH [(102.94±45.28) pg/ml, (244.50±41.01) pg/ml, (979.28±428.71) pg/ml] and Group MRL [(156.97±61.01) pg/ml, (249.92±61.23) pg/ml, (1107.56±408.32) pg/ml] decreased significantly (P<0.01), but the TNF-a and MCP-1 level were still higher than those of Group N (P<0.05 or P<0.01).Compared with Group MRL, the pulmonary IL-6, TNF-a and MCP-1 level of rats in Group MRL decreased slightly without significant difference (P>0.05)[Conclusions]1. Rosiglitazone intervention may delay the increase of mean pulmonary artery pressure and right ventricular systolic pressure in pulmonary arterial hypertension rats induced by MCT, and the improvement of hemodynamic parameters by rosiglitazone is related to drug dosage.2. Rosiglitazone intervention may delay the remodeling of pulmonary vessels and inhibit right ventricular hypertrophy in pulmonary arterial hypertension rats induced by MCT, and this effect is related to drug dosage.3. Rosiglitazone intervention may exert protective effect on MCT-induced pulmonary arterial hypertension partly by inhibiting the pervascular inflammation and the increase of IL-6, TNF-a and MCP-1 in lung tissue. [Backgroud]Remodeling of extracellular matrix (ECM) is a main pathologic feature of PH. Researches showed that the homeostasis and destruction of ECM were closely related to vascular remodeling. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are chief regulatory factors of ECM homeostasis. It has been proved that MMPs and TIMPs played an important role in pulmonary vascular remodeling in patients or animal models of PH. It is recognized recently that peroxisome proliferator activated receptor y (PPARy) is involved in the regulation of MMPs and TIMPs. To explore the underlying mechanisms of rosiglitazone intervention on PH, we investigated the changes of PPARy, MMP-2, MMP-9 and TIMP-1 expression in rats with monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) after rosiglitazone intervention.[Objective]1. To observe the influence of rosiglitazone intervention on PPARy expression of lung tissue in rats with MCT induced PAH.2. To explore the underlying mechanisms of rosiglitazone intervention on PH through observing the influence of rosiglitazone intervention on pulmonary MMP-2,MMP-9 and TIMP-1 expression and MMP-2,MMP-9 activity in rats with MCT induced PAH.[Methods]1. Grouping of experimental animals:see part 2 of this article.2. All of the animals were sacrificed on day 21st, PPARγ, MMP-2 and MMP-9 expression location on pulmonary histological sections of rats were observed through immunohistochemical staining.3. Expression of PPARy, MMP-2, MMP-9 and TIMP-1 mRNA in lung tissue of rats were detected through fluorescent quantitative PCR.4. The activity of MMP-2 and MMP-9 in lung tissue of rats were detected through gelatin zymography.5. Data were analyzed with the analysis of variance and the t test. Statistical significance was assigned for P<0.05, and extremely significant difference was assigned for P<0.01.[Results]1. Influence of rosiglitazone intervention on PPARy expression of lung tissue in rats injected with MCT.Immunohistochemical staining showed that the PPARy positive cells mostly located at the intima of the vascular wall, and presented a brown staining of nucleus and cytoplasm. PPARy partly expressed at the intima of small pulmonary arteries in rats of Group N, the PPARy expression at the intima of small pulmonary arteries in rats of Group M decreased significantly. The PPARy expression at intima of small pulmonary arteries in rats of Group MRH and Group MRL increased.Fluorescent quantitative PCR:PPARy mRNA expression of lung tissue in rats of Group N was 1.06±0.22. Compared with Group N, PPARy mRNA expression of lung tissue in rats of Group M (0.57±0.07) decreased significantly (P<0.05). Compared with Group M, PPARy mRNA expression of lung tissue in rats of Group MRH (1.98±0.39) and Group MRL (1.73±0.31) increased extremely significantly (P<0.01), and were higher than that of Group N (P<0.01). Compared with Group MRL, PPARy mRNA expression of lung tissue in rats of Group MRH increased slightly without significant difference (P>0.05)2. Influence of rosiglitazone intervention on MMP-2, MMP-9 and TIMP-1 expression of lung tissue in rats injected with MCT.Immunohistochemical staining showed that the MMP-2, MMP-9 positive cells mostly located in the intima and adventitia of the vascular wall, presented a brown staining of cytoplasm. MMP-2 and MMP-9 expression of small pulmonary arteries in rats of Group M increased significantly when compared with Group N. Compared with Group M, MMP-2 and MMP-9 expression of small pulmonary arteries in rats of Group MRH and Group MRL decreased.Fluorescent quantitative PCR:MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group N were 0.96±0.08,0.96±0.06 and 1.02±0.14, separately. Compared with Group N, MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group M (3.30±0.39,7.07±0.54,3.52±0.44) increased extremely significantly (P<0.01). Compared with Group M, MMP-2, MMP-9 and TIMP-1 mRNA expression of lung tissue in rats of Group MRH (2.00±0.20,3.89±0.65,2.20±0.34) and Group MRL (2.37±0.17,5.00±0.71,2.58±0.36) decreased extremely significantly (P<0.01), but were still higher than those of Group N(P<0.01). Compared with Group MRL, MMP-9 mRNA expression of lung tissue in rats of Group MRH decreased significantly (P< 0.05), but MMP-2 and TIMP-1 expression only decreased slightly (P>0.05)3. Influence of rosiglitazone intervention on MMP-2 and MMP-9 activity of lung tissue in rats injected with MCT.Gelatin zymography:the MMP-2 and MMP-9 activity of lung tissue in rats of Group N were 40669.68±4978.39 and 9511.13±3429.93. Compared with Group N, the MMP-2 and MMP-9 activity of lung tissue in rats of Group M (54893.89±8480.12, 23864.69±5121.83), Group MRH (52806.28±4759.47,14774.15±2392.96) and Group MRL (53884.35±4688.02,16199.92±2933.85) increased significantly (P<0.01 or P< 0.05). Compared with Group M, the MMP-9 activity of lung tissue in rats of Group MRH and Group MRL decreased significantly (P<0.01 or P<0.05), the MMP-2 activity of lung tissue in rats of Group MRH and Group MRL decreased slightly without significant difference (P>0.05). Compared with Group MRL, the MMP-2 and MMP-9 activity of lung tissue in rats of Group MRH decreased slightly without significant difference (P>0.05)[Conclusions]1. PPARy mRNA expression of lung tissue in rats injected with MCT decreased. Rosiglitazone can activate PPARy to exert protective effects to pulmonary vessels, and this effect is related to drug dosage.2. Rosiglitazone intervention can derease the MMP-2, MMP-9 and TIMP-1 expression of lung tissue in PAH rats injected with MCT, and this effect is related to drug dosage.3. Rosiglitazone intervention can derease the MMP-9 activity of lung tissue in PAH rats injected with MCT (but not MMP-2). It suggests that rosiglitazone may improve the remodeling of extracellular matrix to exert protective effects to pulmonary vessels through regulation of MMP-9 activity. |
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