Effects Of Rosiglitazone On The Expression And Action Of INOS In Pulmonary Artery Of Bleomycin-induced Pulmonary Hypertension In Rats

Objective:Bleomycin is a kind of anti-tumor drug whose main side effect is to induce pulmonary injury and fibrosis. The modle of pulmonary fibrosis induced by a single intratracheal instillation of blemycin (BLM) is generally accepted. Pulmonary fibrosis can result in pulmonary hypertension. Up to now, the abnormal changs of pulmonary artery, during BLM-induced lung fibrosis, has not been clear yet. Our previous study has shown that pulmonary hypertension and the disorder of pulmonary artery in rats on d 14 after a single intratracheal instillation of blemycin (BLM), and that rosiglitazone (RSG), a potent synthetic agonist of peroxisome proliferator-gamma (PPAR-γ), can effectively prevent the BLM-induced pulmonary hypertension. It is reported that inducible nitro oxide synthase ( iNOS ) participated in the development of pulmonary hypertension, but the effect of iNOS on the pathogenises of BLM-induced pulmonary hypertension remains unclear. In the present study, the effects of RSG on the expression and action of iNOS in pulmonary artery of BLM-induced pulmonary hypertension rats were investigated.PartⅠEffects of rosiglitazone on expression of iNOS in pulmonary artery of bleomycin-induced pulmonary artery hypertension rats.Methods:Thirty-six male Sprague-Dawley rats were randomly divided into four groups:①NS+NS group (n=6) : NS (0.5ml/kg) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;②BLM+NS group (n=18): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days;③BLM+RSG group (n=6): BLM (5mg·kg-1·0.5ml) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days;④NS+RSG group (n=6): NS (0.5ml/kg) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat, dissolved in 2ml NS, i.g) for 14 days.On day 14 after intratracheal instillation, All rats were killed, and then pulmonary tissue and pulmonary artery were collected for immunohistochemistry. Twelve pulmonary arteries of BLM+NS 14 day group were incubated in DMEM for 24 hours present or absent rosiglitazone (10um/L) under the condition of 37℃, and then were collected for immunohistochemistry.Data were analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogeneitic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference (LSD). Data of incubated pulmonary arteries were analyzed using independent-samples T test. The statistical significance level was set at P<0.05.Results: 1 RSG reduces the expression of iNOS in pulmonary artery induced by BLM. In NS+NS group, the expression of iNOS in pulmonary artery was slight and localized to the smooth muscle layer and endothelium (average optical density: 0.29±0.03, percentage of positive areas:6.80±1.80), and the pulmonary artery expression of iNOS in NS+RSG group was similar to that in NS+NS group. Compared with NS+NS,NS+RSG group, the expression of iNOS in pulmonary artery of BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 16.82±2.42) (P<0.01). Compared with NS+NS,NS+RSG group, the expression of iNOS in pulmonary artery of BLM+RSG group (average optical density: 0.33±0.02, percentage of positive areas: 11.03±2.06) were significiantly increased (P<0.05). Comparied with BLM+NS, the expression of iNOS protein in pulmonary artery of BLM+RSG group were singnificiantly reduced (P<0.05). In addition, sections showed that vascular endothelium was intact and continuous in NS+NS group; but vascular endothelial cells apeared losting in BLM+NS group; vascular endothelial cells were protected in BLM+RSG group. In incubation with DMEM present rosiglitazone group, the expression of iNOS was significantly reduced (average optical density: 0.32± 0.02,percentage of positive areas:11.09±1.58)compared with DMEM absent rosiglitazone (average optical density: 0.39±0.02,percentage of positive areas: 16.60±2.75) (p<0.05).2 Rosiglitazone reduce the expression of iNOS in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.05, percentage of positive areas: 5.27±0.44). The expression of iNOS in pulmonary arteries in NS+RSG group was similar with that in NS+NS group (P>0.05). Compared with NS+NS,NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.36±0.01, percentage of positive areas:17.91±3.07) (P<0.01). Compared with NS+NS,NS+RSG group, the expression of iNOS in pulmonary artery (average optical density: 0.33±0.01, percentage of positive areas: 10.32±3.55) in BLM+RSG group were singnificantly increased (P<0.05). Compared with BLM+NS group, the expression of iNOS in pulmonary artery in BLM+RSG group was singnificiantly reduced (P<0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of iNOS in pulmonary artery was slight (average optical density: 0.30±0.06, percentage of positive areas: 4.96±1.00). The expression of iNOS in pulmonary artery in NS+RSG group was similar to that in NS+NS group (P>0.05). Compared with NS+NS,NS+RSG group, the expression of iNOS in BLM+NS group increased significantly (average optical density: 0.37±0.02, percentage of positive areas: 16.71±5.00) (P<0.05 ) . Compared with BLM+NS group, the expression of iNOS in BLM+RSG group was markedly reduced (P<0.05). The expression of iNOS in BLM+RSG group was similar to that in NS+NS,NS+RSG group (P>0.05)The results suggested that strong expression of iNOS and injury of endothelium in pulmonary arteries of rats on day 14 after intratracheal instillation of BLM, and rosiglitazone could significantly reduce the above changes.PartⅡThe effects of RSG on reactivities of PARs in BLM-induced pulmonary hypertension rats and the roles of iNOSMethods: 67 male Sprague-Dawley rats were randomly divided into six groups:①NS+NS with endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;②NS+NS without endothelium group: NS (0.5ml/kg) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary arteries was removed;③BLM+NS with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and NS (2ml/d, i.g) for 14 days;④BLM+NS without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then NS (2ml/d, i.g) for 14 days. The endothelium of pulmonary artery was removed;⑤BLM+RSG with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days.⑥BLM+RSG without endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1per rat,dissolved in 2ml NS, i.g.) for 14 days. The endothelium of pulmonary artery was removed. The rats were sacrificed on day 14 after single intratracheal instillation. Changs of vascular tension of pulmonary artery rings (PARs) were detected in vitro. The contraction responses to phenyleyphrine (PE 10-6mol/L) were then tested separatedly to observe the stability of the PARs reactivity.When the contraction responses had become stable, PARs of all groups were detected:①the contraction responses of PARs to PE (10-6mol/L) ;②the relaxation responses of PARs to acetylcholine (Ach 10-6mol/L);③the contraction responses of PARs to PE (10-6mol/L) after preincubation with aminoguanidine (AG 10-4mol/L) for 20 min;④the contraction responses of PARs to PE (10-6mol/L) after preincubation with N (omega) -nitro-L-arginine methyl ester (L-NAME 10-4mol/L) for 20 min.The PARs responses to PE were expressed as g/mg·d·w, and vascular relaxtion responses to Ach were expressed as percentage reduction of initial vascular tension induced by PE.Sections were stained with immunohistochemistry for the expression of NT . Results: 1 Rosiglitazone improves PARs contraction responses to PE after BLM intracheal instillation Compared with the PARs in NS+NS group, the contraction reponses to PE of PARs in BLM+NS , either with endothelium or without endothelium, were significantly reduced (P<0.05). In BLM+RSG group, rosiglitazone administration reversed PARs contraction reponses to PE. The results suggested that PARs contraction responses to PE was damaged by BLM administratiom, and the damaged contraction reponses to PE was reversed by rosiglitazone.2 Rosiglitazone protects endothelial cells of pulmonary artery and improves the endothelial cells-dependedent- relaxtion. Compared with PARs in NS+NS group with endothelium, the relaxation reponses to Ach of PARs in BLM+NS with endothelium group were significantly decreased (0.93±0.08 vs 0.43±0.14) (P<0.01) . In BLM+RSG group, the relaxtion responses to Ach of PARs were markedly improved compared with those in BLM+NS group (0.69±0.18 vs 0.43±0.14,P<0.05), which indicated that rosiglitazone impoves the function of pulmonary artery endothelial cells and reduce the damage to pulmonary artery endothelium induced by BLM, this may be one of mechanisms involved in rosiglitazone reversing the pulmonary artery hypertension induced by BLM.3 Aminoguanidine (AG) has no effect on the PARs contraction reponses to PE in all groups. In all groups, the PARs contraction responses to PE were not changed after preincubation with AG(10-4mol/L), which indicated that in pulmonary artery hypertension induced by BLM NO derived by iNOS may not participate in the regulation of pulmoary artery vascular tension.4 L-NAME preincubation enhance the PARs contraction responses to PE in groups with endothelium. After incubation with L-NAME, the contraction responses of PARs to PE in NS+NS group with endothelium significantly increased (1.20±0.50 g/mg·d·w vs 1.73±0.57 g/mg·d·w) (P<0.01) , while the contraction reponses of PARs in BLM+NS group with endothlium did not changed after preincubation with L-NAME. In BLM+RSG with endothelium group the contraction responses to PE markedly increased (1.31±0.38 vs 1.81±0.40) (P<0.05) . The contraction responses to PE in all groups without endothelium had no siginificant differences.The result indicated that rosiglitazone protect pulmonary artery vascular endothelial cells.5 RSG reduces the expression of NT protein in pulmonary artery induced by BLM. In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas:6.87±0.56). The expression of NT protein of pulmonary artery in BLM+NS group was significantly increased (average optical density: 0.39±0.02, percentage of positive areas: 21.42±1.84) compared with that in NS+NS group, (P<0.01). The expression of NT protein of pulmonary artery in BLM+RSG group (average optical density: 0.34±0.01, percentage of positive areas: 9.80±0.94) was lower than that in BLM+NS (P<0.05), and still higher than that in NS+NS group (P<0.05).6 Rosiglitazone reduce the expression of NT in pulmonary artery in lungs induced by bleomycin. Small arteries in lung (diamerter<100um): In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 4.89±1.00). Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.40±0.02, percentage of positive areas:14.60±2.55) (P<0.01). The expression of NT in pulmonary artery in BLM+RSG group (average optical density: 0.31±0.01, percentage of positive areas: 5.84±1.02) was lower than that in BLM+NS group (P<0.01) and was similar to that in NS+NS group(P>0.05). Larger arteries in Lung (diamerter>100um) : In NS+NS group, the expression of NT in pulmonary artery was slight (average optical density: 0.31±0.01, percentage of positive areas: 5.52±0.83).Compared with NS+NS group, the expression of NT in BLM+NS group increased significantly (average optical density: 0.38±0.02, percentage of positive areas: 16.47±1.82) (P<0.01). The expression of NT in BLM+RSG group was lower than that in BLM+NS group (P<0.01), and was similar to that in NS+NS group (P>0.05).The results suggested that: (1) the contraction responses of PARs to PE and the endothlium-dependent relaxtion resposes of PARs to Ach were striking impaired by BLM, which led to PARs reactivties disorder; (2) the expression of NT in pulmonary arteries was markedly increased by BLM; (3) Rosiglitazone could reduce the expression of NT in pulmonary artery, partly protect the funtion of pulmonary artery endtholium, and reverse the abnormal funtion of smooth muscle induced by BLM.Conclusions: 1 The expression of iNOS and NT in all kinds of pulmonary arteries was significantly increased and endothelium of pulmonary artery was injured by bleomycin, which maybe implicated in the bleomycin induced pulmonary artery hypertension.2 Bleomycin induced the markedly impaired contraction to PE and the relaxtion to Ach of PARs, which had led to the disorder of pulmonary artery vascular reactivity. This might be one of the mechanisms of pulmonary hypertension in this model.3 Rosiglitazone could significantly reduce the expression of iNOS and NT in all kinds of pulmonary arteries and reverse the impaired contraction responses of PARs. In addition, rosiglitazone could protect the function of pulmonary artery endothelial cells and correct the disorder of PARs induced by BLM. All above may be one of the mechanisms that rosiglitazone reverse the pulmonary artery hypertension induced by bleomycin.