Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hyp...Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hypertension （PAH） and discuss the underlying mechanisms preliminarily. Methods A classical PAH animal model was used, which was established by single injection of 50 mg · kg^-1 monocrotaline （MCT）. One week later, the rats were administrated with 1, 3, 10 mg · kg^-1 DL0805-2 via intraperitoneal injection for 18 days. At the end of the experiment, the body weight and survival rate were recorded. Meanwhile, the respiration function, heart function, blood pressure and pulmonary artery pressure were detected. Serum was collected for biochemical index analysis. The weight of vital organs was used to calculate the organ index. Histopathology examination was em-ployed to observe the subtle changes in hearts, vessels and lungs. Furthermore, the mechanisms were studied main- ly by the method of western blotting. Results DL0805-2 did not show significant influence on body weight of PAH rats. But the survival rate of PAH rats treated with 3 and 10 mg · kg^-1 DL0805-2 was increased up to 90. 9% com- pared with the model group （68.2%）. DL0805-2 improved the pulmonary artery blood flow especially the maximal -1 -1 velocity （PV max） from 397.2 cm · s^-1 to 506.5, 540. 1 and 574.0 cm · s^-1 respectively. The results of echocar- diography and electrocardiogram show that DL0805-2 had little effect on left ventricle and systemic circulation but attenuated right ventricle injury and decreased the right ventricle pressure from 73.73 mmHg to 47.80, 42.64 and 46.45 mmHg respectively after DL0805-2 intervention. Disease markers of PAH including NT-proBNP in serum and ET-1 in lung tissue homogenate and serum biochemical indicators, ALT, AST and LDH, were reduced by DL0805-2. DL0805-2 also relieved edema of lungs and decreased inflammatory cytokines production. Through the examination on histopathologic slide of pulmonary main artery, right ventricle and lung, DL0805 derivatives were found to have significant protection effect on structural changes of organs induced by pulmonary hypertension. Ac- cording to the preliminary study on the mechanisms of DL0805-2 in PAH, Rho/ROCK pathway was significantly in- hibited by DL0805 derivatives. In addition, DL0805 derivatives showed effect on BMPRII/p-Smad pathway and ap- optosis related pathway. Conclusion DL0805-2 has showed potent treatment effect on the PAH rats. And the un- derlying mechanisms studies also indicated that RhoA/ROCK and BMPRII pathways were involved. This work will provide basis experimental data for the further research and development of DL0805-2.展开更多
OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At...OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.展开更多
文摘Aim DL0805-2 is a novel Rho-kinases inhibitor which has been found to have potent cardiovascular effects. In the present research, we aimed to study the potential of DL0805-2 in the treatment of pulmonary arterial hypertension （PAH） and discuss the underlying mechanisms preliminarily. Methods A classical PAH animal model was used, which was established by single injection of 50 mg · kg^-1 monocrotaline （MCT）. One week later, the rats were administrated with 1, 3, 10 mg · kg^-1 DL0805-2 via intraperitoneal injection for 18 days. At the end of the experiment, the body weight and survival rate were recorded. Meanwhile, the respiration function, heart function, blood pressure and pulmonary artery pressure were detected. Serum was collected for biochemical index analysis. The weight of vital organs was used to calculate the organ index. Histopathology examination was em-ployed to observe the subtle changes in hearts, vessels and lungs. Furthermore, the mechanisms were studied main- ly by the method of western blotting. Results DL0805-2 did not show significant influence on body weight of PAH rats. But the survival rate of PAH rats treated with 3 and 10 mg · kg^-1 DL0805-2 was increased up to 90. 9% com- pared with the model group （68.2%）. DL0805-2 improved the pulmonary artery blood flow especially the maximal -1 -1 velocity （PV max） from 397.2 cm · s^-1 to 506.5, 540. 1 and 574.0 cm · s^-1 respectively. The results of echocar- diography and electrocardiogram show that DL0805-2 had little effect on left ventricle and systemic circulation but attenuated right ventricle injury and decreased the right ventricle pressure from 73.73 mmHg to 47.80, 42.64 and 46.45 mmHg respectively after DL0805-2 intervention. Disease markers of PAH including NT-proBNP in serum and ET-1 in lung tissue homogenate and serum biochemical indicators, ALT, AST and LDH, were reduced by DL0805-2. DL0805-2 also relieved edema of lungs and decreased inflammatory cytokines production. Through the examination on histopathologic slide of pulmonary main artery, right ventricle and lung, DL0805 derivatives were found to have significant protection effect on structural changes of organs induced by pulmonary hypertension. Ac- cording to the preliminary study on the mechanisms of DL0805-2 in PAH, Rho/ROCK pathway was significantly in- hibited by DL0805 derivatives. In addition, DL0805 derivatives showed effect on BMPRII/p-Smad pathway and ap- optosis related pathway. Conclusion DL0805-2 has showed potent treatment effect on the PAH rats. And the un- derlying mechanisms studies also indicated that RhoA/ROCK and BMPRII pathways were involved. This work will provide basis experimental data for the further research and development of DL0805-2.
基金The project supported by Central Public Scientific Research Institution Fundamental Project(2016CX09)by National Natural Science Foundation of China(81573645)
文摘OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.
