Effects Of Celastrol On Proliferation,migration,Apoptosis And Phenotype Of Pulmonary Artery Smooth Muscle Cells Induced By Hypoxia

Abnormal proliferation,hypertrophy,phenotypic change,and extracellular matrix increase of pulmonary artery smooth muscle cells are the basis of hypoxic pulmonary vessel remodeling.HPVR is the heart of the Hypoxic Pulmonary Hypertension.Therefore,inhibition of abnormal proliferation,hypertrophy and phenotypic transformation of PASMCs is an important strategy to slow down the occurrence and development of HPH.Celastrol,as one of the five traditional natural medicinal compounds most likely to be developed into modern drugs,has strong anti-oxidation,anti-tumor angiogenesis,anti-rheumatoid and other therapeutic effects.In addition,Celastrol also alleviated angiotensin-Ⅱ induced myocyte hypertrophy and fibrosis and improved myocardial remodeling;It plays an anticancer role by inhibiting the proliferation of various tumor cells.The results of our preliminary experiment showed that celastrol had certain inhibitory effect on the abnormal proliferation of PASMCs under hypoxia.In this experiment,the related indexes of proliferation,migration,phenotypic transformation and apoptosis of PASMCs induced by hypoxia were further observed.Objective:The effects of celastrol on the proliferation,apoptosis,migration and phenotypic transformation of PASMCs induced by hypoxia were observed,and its mechanism of action was preliminarily discussed,which provided an experimental basis for the potential application of celastrol in the treatment of hypoxic pulmonary hypertension.Methods:1.MTTmethod was used to detect the OD value of each group to reflect the cell viability.2.Cell cycle of each group was detected by flow cytometry.3.Cell migration was detected by scratch test and Transwell test.4.Use Western Blot to detect the intracellular smooth muscle in each group α-Actin and smooth muscle-22 α Protein.The level of osteopontin was detected by immunohistochemistry to explore the effect of celastrol on cell phenotype transformation.5.The level of reactive oxygen species in PASMCs under different conditions was detected by DCFH-DA fluorescence probe method,and the effect of celastrol on PI3K/AKT signal pathway was detected by Western Blot.6.The ratio of apoptotic cells in each group was detected by Annexin V/PI flow cytometry and TUNNELstaining.7.The mitochondrial membrane potential of each group was further detected by JC-1staining and the level of Bax/Bcl-2 in each group was detected by Western Blot.Results:1.MTT results showed that the OD value of pulmonary artery smooth muscle cells in the hypoxic group was significantly higher than that in the normoxic group;After treatment with celastrol,the OD value of PASMCs in the hypoxic group decreased significantly,while the OD value of the normoxic group did not change significantly.2.The results of the cell cycle showed that hypoxia 24 h promoted the proportion of cells in the S phase of PASMCs,and the treatment of celastrol significantly increased the proportion of cells in the G2/M phase of PASMCs under hypoxia.3.Scratch experiments showed that the migration area of hypoxia 6h,12 h and 24 h PASMCs increased significantly compared with the normox group,and celastrol could significantly reduce the migration area of PASMCs at 12 h and 24 h,but the effect on the migration area of PASMCs under normal oxygen was not obvious;Transwell experiments showed that the number of migrating cells increased significantly after 24 hours of hypoxic culture of PASMCs,while celastrol could reduce the migration of PASMCs induced by hypoxia,but had no obvious effect on the migration of PASMCs in normoxic culture.4.Detection of intracellular α-SMAand SM-22α by Western Blot,immunohistochemistry to detect the level of OPN,the results showed that the expression of intracellular α-SMA and SM-22α decreased and the expression of OPN increased;Celastrol alleviates hypoxia-induced decreased expression of intracellular α-SMAand SM-22α and increased OPN.5.By detecting intracellular ROS,it was found that the ROS level in PASMCs increased significantly when hypoxia,and celastrol can inhibit the production of hypoxia-induced ROS;The Western Blot results showed that the expression of P-PI3K/PI3 K,P-AKT/AKT in PASMCs increased in the hypoxic group,while the expression of P-PI3K/PI3 K,P-AKT/AKT was reduced by celastrol.6.The results of Annexin-V/PI double staining and TUNNEL staining showed that compared with the normox group,the proportion of PASMCs apoptotic cells in the hypoxic group was significantly reduced.However,the proportion of PASMCs apoptotic cells under low oxygen increased after treatment with celastrol,but there was no significant effect on the apoptosis of PASMCs under normal oxygen.7.Furthermore,JC-I staining showed that after 24 hours of hypoxia,the mitochondrial membrane potential of PASMCs decreased,while the mitochondrial membrane potential of PASMCs increased to a certain extent after treatment with celastrol.By detecting the level of Bax/Bcl-2 in cells by Western Blot,it was found that the ratio of Bax/Bcl-2 in PASMCs in the hypoxic group was significantly lower than that in the normox group,and celastrol could upregulate the level of Bax/Bcl-2 in PASMCs under low oxygen to a certain extent.Conclusion:1.Celastrol inhibits hypoxia-induced proliferation,migration and phenotypic transformation of pulmonary vascular smooth muscle cells.The mechanism may be related to the inhibition of ROS/PI3K/AKT signaling pathway.2.Celastrol induces PASMCs apoptosis under hypoxia,and its mechanism may be related to the activation of mitochondria-dependent apoptosis pathway.