| ObjectivePulmonary Fibrosis(PF) can induce Pulmonary artery hypertension(PAH). Long-term PAH can induce pneumocardial disease(PHD), which is one of the most important lethal facors of PF. Up to now, the mechanism of PAH generated in PF has not been clear. And there is still no ideal method for prevention and therapy. Studies have shown that pulmonary artery endothelial cells were injured in the formation of PF induced by BLM. Other researches have indicated that intratracheal instillation of BLM can lead to the decrease of endothelium nitric oxide synthase (eNOS) expressed in pulmonary artery endothelial cells, which may be one of the mechanisms implicated in chronic PAH. Thus the effective protection of pulmonary artery endothelial cells may play an important role in the prevention and therapy of PAH induced by BLM.Rosiglitazone (RSG), is a kind of Thiazolidines (TZDs) drugs, was mainly used in the therapy of type II diabetes. Some researches show that RSG can activate peroxisome proliferator-activated receptor-γ(PPAR-γ). The activation of PPAR-γ, expressed abundantly both in blood vessels and heart, participates in the modulation of inflammation, cell apoptosis, migration of vascular smooth muscles etc. Previous researches have shown that PPAR-γexpressed in blood vessels can protect the endothelial cells and induce the decrease of the blood pressure by facilitate the release of endothelium derived NO(nitric oxide). But, whether RSG has the reversal effects on PAH and the injury of pulmonary artery endothelial cells induced by BLM has not been reported.Endothelial cells can generate many vasoactive substances, which are very important to the maintenance of the vascular tension. Among these vasoactive substances, endothelium derived NO has the effects on the relaxation of blood vessels and the inhibition of platelet aggregation. It is an important modulator of vascular reactivity under many physiologic or pathophysiologic conditions. Ling YL et al reported that the injury of pulmonary artery endothelial cells and the decrease of endothelium derived NO release, both generated in the PAH induced by lipopolysaccharide (LPS), led to the disorder of pulmonary artery vascular activity. Howerver, whether the injury of pulmonary artery endothelial cells in the PAH induced by BLM can lead to the disorder of pulmonary artery vascular reactivity has not been clear.Thus, in our research, we observed the effects of RSG on PAH generated in PF induced by BLM and the effects of pulmonary artery endothelial cells on the changes of pulmonary artery vascular reactivity induced by BLM.Rats model of PF were duplicated by intratracheal instillation of BLM. Right Cardiac Catheter technique was applied to detect rat mean pulmonary artery pressure(mPAP). Light microscope was used to observe the morphologic changes of pulmonary artery. The changes of PARs reactivity were observed utilizing the PARs technique.Part1 Effects of RSG on Pulmonary Artery Hypertension during Pulmonary Fibrosis Induced by BleomycinMethods:Sixty-four male Sprague-Dawley rats were randomly·divide -d into eight groups:①Control group(n=9): Normal rats;②B + NS14d group(n=10): BLM(5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then NS (2ml per rat) was administrated by intragastric gavage(i.g) every day of the following 14 days;③B+R14d group(n=12): BLM was administrated by single intratracheal instillation (5mg·0.5ml-1·kg-1) and then RSG (3mg·kg-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days;④R14d group(n=9): NS(0.5ml·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days;⑤B+NS28d group(n=6): BLM(5mg·kg-1) was administrated by single intratracheal instillation and then NS (2ml per rat, i.g) was administrated every day of the following 28 days;⑥B+R28d group(n=6): BLM(5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 28 days;⑦R28d group(n=7): NS (0.5ml·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 28 days;⑧B+R14d+N14d group(n=6): BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation and then RSG (3mg·kg-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days, followed by NS(2ml per rat, i.g) administrated every day of the following 14 days. RSG or NS was administrated 2h after BLM or NS intratracheal instillation. mPAP of each group was detected respectively 14 or 28 days after NS or BLM intratracheal instillation. Three rats in each(①,②,③) group were sacrified to observe the morphologic changes of the PARs.Results:1 Effects of RSG on PAH induced by BLM Compared with control group, the mPAP of B+NS14d group was significantly incresed(19.00±2.94 mmHg vs. 14.83±1.17mm Hg, P<0.05), the mPAP of B+R14d group had no significant differences, while the mPAP of B+R28d group and B+R14+N14 group were decreased significantly(11.33±2.07 mmHg vs. 14.83±1.17 mmHg, P<0.05; 11.83±1.33 mmHg vs. 14.83±1.17mmHg, P<0.05). Compared with B+NS14d group, the mPAP of B+R14d group was significantly decreased (13.11±1.96 mmHg vs. 19.00±2.94 mmHg, P<0.05).2. Effects of RSG on morphologic changes of PARs The LM results showed, vascular endothelial cells in control group were intact and continuous, with the smooth muscle layers arranged orderly; In B+NS14d group, vascular endothelial cells apeared losing. The structure of the elastic fiber beneath the endothelium was nearly normal. The structure of smooth muscles were well arranged. Compared with Control group, no significant change was observed in B+R14 group.The results suggested:Fourteen days after intratracheal instillation, BLM induced significant increase of rats mPAP. RSG may have the reversing function to the increase of rats mPAP induced by BLM. RSG can inhibit the pulmonary artery endothelial cells injury induced by BLM, which implied that RSG may decrease mPAP by protecting pulmonary artery endothelium.Part2 Reactivites of PARs during PAH Induced by BLM Methods:Twenty-four Sprague-Dawley rats were randomly divided into four groups (n=6 in each group):①Control with endothelium group: Normal rats;②Control without endothelium group: Normal rats. The pulmomary artery endothelium was removed;③B14d with endothelium group: BLM (5mg·0.5ml-1·kg-1) was administrated by single intratracheal instillation. Rats were sacrificed 14 days later;④B14d without endothelium group: BLM was administrated by single intratracheal instilled(5mg·0.5ml-1·kg-1). Rats were sacrificed 14 days later. The pulmonary artery endothelium was removed. Changes of vascular tension of pulmonary artery rings (PARs) were detected in vitro. PARs were carefully prepared. The contraction responses to phenylephrine(PE,10-6mol/L) were then tested separately to observe the stability of the PARs reactivity. When the contraction responses had become stable, the group with endothelium was detected:①recording curve of the contraction responses of PARs to PE(10-6mol/L);②recording curve of the relaxation responses of PARs to acetylcholine(ACh, 10-6mol/L);③recording curve of the 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 5 min. As to the groups without endothelium, when the contraction responses to PE(10-6mol/L) had become stable, the relaxation responses to ACh(10-6mol/L) were observed. The disappearance of the PARs relaxation responses to ACh(10-6mol/L) was the identification of the complete endothelium removing. Then recorded: the group without endothelium:①recording curve of the contraction responses of PARs to PE (10-6mol/L);②recording curve of the cumulative dose relaxation responses of PARs to sodium nitroprusside (SNP, 10-910-6mol/L);③recording curve of the contraction responses of PARs to PE(10-6mol/L) after preincubation with L-NAME(10-4mol/L) for 5 min. The PARs responses to PE were expressed as g/mg.dw, and vascular relaxation responses to ACh or SNP were expressed as percentage reduction of initial vascular tension induced by PE. Results: 1 The changes of PARs contraction responses to PE after BLM intratracheal instillation Compared with the PARs in control group with endothelium, the contraction responses to PE of PARs in B14d with endothelium group were significantly increased (0.73±0.17 g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05). Compared with the PARs in control group without endothelium, the contraction responses to PE of PARs in B14d without endothelium group were significantly increased (0.97±0.18 g/mg.d.w vs. 0.73±0.18g/mg.d.w, P<0.05). The results indicated that BLM enhanced the contraction responses of pulmonary smooth muscles to PE.2 The contraction responses of PARs endothelial cells to PE Compared with the PARs in control group with endothelium, the contraction responses to PE of PARs in control group without endothelium were significantly increased (0.73±0.18g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05). Compared with PARs in B14d with endothelium group, the contraction responses to PE of PARs in B14d without endothelium group were significantly increased(0.97±0.18g/ mg.d.w vs. 0.80±0.15g/ mg.d.w, P<0.05). The results indicated that endothelial cells injury can enhance the PARs contraction responses to PE.3 The injury effects of BLM on pulmonary artery endothelial cells Compared with PARs in control group with endothelium, the relaxation responses to ACh of PARs in B14d with endothelium group were significantly decreased (34.21±13.97% vs.83.63±6.57%, P<0.01), which indicated that BLM injured the pulmonary artery endothelium, and attenuated the relaxation responses of PARs to ACh.4 Effects of L-NAME preincubation on PARs reactivities After incubation with L-NAME, the contraction responses of PARs to PE in control with endothelium group increased significantly(0.71±0.42 g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05), while the contraction responses of PARs to PE in control without endothelium group, B14d with endothelium group and B14d without endothelium group had no significant differences.5 Effets of SNP cumulative dose on relaxation responses of PARs With the increase of SNP concentration, the relaxation responses of PARs increased gradually, but there was not significant difference between the control without endothelium group and B14d without endothelium group. The results indicated that the pulmonary artery relaxation responses to exogenous NO were independent on endothelial cells.The results suggested:BLM increased the contraction responses of PARs to PE and decreased the relaxation resonses of PARs to ACh, decreased the endothelium derived NO, made the function of endothelial cells and vascular smooth muscles abnormal, led to PARs reactivities disorder, which maybe one of the important mechanisms involved in the increase of pulmonary artery pressure induced by BLM during the initial period of PF in rats.Conclusion:1 BLM induced the increase of pulmonary artery pressure in initial period of PF in rats, which maybe have the relationship with the injured pulmonary artery endothelial cells, the decreased endothelium derived NO and the abnormal function of vascular smooth muscles that led to the PARs reactivities disorder.2 RSG can prevent and cure the increase of pulmonary artery pressure in initial period of PF in rats, which implied that RSG may decrease mPAP by protecting pulmonary artery endothelial cells.
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