Therapeutic Effect And Mechanism Of Targeted Autophagy Pathway In Pulmonary Hypertension

【Research Background】Pulmonary hypertension（PH）is a clinical and pathophysiological syndrome caused by a variety of causes and pathogenesis of pulmonary vascular structure or function changes,causing pulmonary vascular resistance and pulmonary artery pressure rise,with persistent pulmonary vascular contraction and pulmonary vascular remodeling as the main pathological characteristics.The continuous increase in pulmonary vascular resistance leads to increased load on the right heart,right ventricular hypertrophy,and eventually right heart failure or death.The mortality rate of the first group of PH——pulmonary arterial hypertension is high.The current therapeutic drugs and technical means can relieve the symptoms of patients to a certain extent,but can not prevent its progression.It is still a difficult task to explore effective therapeutic targets.Autophagy is involved in the occurrence and development of pulmonary hypertension.Studies have reported that autophagy activator rapamycin plays a favorable role in Su Hx-induced PH model,improving hemodynamics and vascular remodeling.and it has been reported that knocking out LC3B can induce pulmonary vascular remodeling in hypoxia-induced PH mice model.The role of autophagy in pulmonary hypertension remains unclear.ROC-325 is a novel autophagy inhibitor that contains the core motifs of hydroxychloroquine and thioanthrone.Basic studies have reported that chloroquine and hydroxychloroquine have certain therapeutic effects on PH,but their side effects are relatively large,while ROC-325 has a better autophagy inhibition effect.This study applied different PH experimental animal models to study the changes of autophagy in models and the effects of ROC-325 on PH experimental animal models and its mechanism of action.【Objectives】To explore the changes of autophagy in PH experimental animal model,and to evaluate the effect and mechanism of targeting autophagy pathway in pulmonary hypertension.【Methods】1.Monocrotaline-induced pulmonary hypertension（MCT-PH）,Sugen combined with hypoxia-induced pulmonary hypertension（Su Hx-PH）,Left pulmonary artery ligation-induced PH（LPAL-PH）rat model,and endothelial cell-specific knockout PHD2 spontaneous pulmonary hypertension(Phd2^ECKO-PH)mouse model were constructed.Hemodynamics,cardiac function,and HE staining of lung tissue were detected at different time points to evaluate the PH model.Western blot（WB）experiment was used to detect the expression changes of phenotype and autophagy-related proteins in lung tissue of several animal models,and immunofluorescence experiment was used to detect the phenotypic changes in lung tissue.2.ROC-325,a targeted autophagy inhibitor,was injected intraperitoneally to all PH animal models.After 2 or 4 weeks of treatment,the related indicators of pulmonary hypertension were detected.Western blotting was used to detect the expression changes of phenotypic and autophagy-related proteins in the lung tissue of the animal model,and immunofluorescence was used to detect the phenotypic changes in the lung tissue of the model.3.Pulmonary arterial smooth muscle cells（PASMCs）and Pulmonary arterial endothelial cells（PAECs）of rats were isolated in vitro for hypoxia exposure.PAECs were isolated from wild-type and Phd2^ECKO-PH mice and treated with autophagy inhibitor ROC-325.Western blotting and immunofluorescence were used to detect the expression of autophagy in PAECs,and flow cytometry was used to detect cell proliferation and apoptosis.【Results】1、Compared with the control group,the protein expression of LC3B-Ⅱwas increased and the protein expression of p62 was decreased in the lung tissues of MCT-PH model at 2,3weeks and 4 weeks.After intraperitoneal injection of ROC-325（5 mg/kg and 25 mg/kg）for 4 weeks,compared with the model group,the protein expression of LC3B-Ⅱin the lung tissue of the drug intervention group was decreased.2、Compared with the control group,the expression of LC3B-Ⅱand p62 protein was increased in the lung tissue of the Su Hx-PH model at 3 and 7 weeks.After intraperitoneal injection of ROC-325（25 mg/kg）for 4 weeks,compared with the model group,the protein expressions of LC3B-Ⅱand p62 in the lung tissue of the drug intervention group were decreased.3、Compared with the control group,the protein expression of LC3B-Ⅱwas increased,and the protein expression of p62 was decreased in the lung tissue of LPAL-PH rats at 3 and 5 weeks.After intraperitoneal injection of ROC-325（25 mg/kg）for 2 weeks,compared with the model group,the protein expression of LC3B-Ⅱin the lung tissue protein of the drug intervention group was decreased,and the protein expression of p62 was increased.4、Compared with the wild type,the protein expressions of LC3B-Ⅱand p62 in the lung tissue of Phd2^ECKO-PH mice were increased.After intraperitoneal injection of ROC-325（25 mg/kg）for 4 weeks,compared with the model group,the protein expression of LC3B-Ⅱin the lung tissue protein of the drug intervention group decreased,and the protein expression of p62 had no significant change.5、After intraperitoneal injection of ROC-325（25 mg/kg）in the LPAL-PH rat model and Phd2^ECKO-PH mouse model,right ventricular systolic pressure and contractility were decreased,right ventricular hypertrophy was improved,pulmonary artery pressure was decreased,pulmonary artery media thickening,musculization and smooth muscle cell layer proliferation were improved.6,The protein expression of LC3B-Ⅱincreased and p62 decreased after hypoxic treatment of r PAECs.After treatment with different concentrations of ROC-325,the protein expression of LC3B-Ⅱand p62 increased,and the higher the drug concentration,the more obvious the protein expression increased.NO release was increased in r PAECs treated with ROC-325.7.ROC-325 treatment of rat PASMCs after hypoxia exposure inhibited autophagy,reduced cell proliferation,decreased survival activity,and increased apoptosis.8.ROC-325 treatment of PAECs in Phd2^ECKO-PH mice increased the protein expression of LC3B-Ⅱand p62.After treatment with different concentrations of ROC-325,the protein expression of LC3B-Ⅱand p62 increased,and the higher the drug concentration,the higher the protein expression.ROC-325 treatment reduced PAECs apoptosis in Phd2^ECKO-PH mice.【Conclusion】There are different changes in autophagy in different stages of the four PH animal models.Autophagy was increased in the lung tissues of MCT-PH and LPAL-PH rats,while it was decreased in Su Hx-PH rats and Phd2^ECKO-PH mice.ROC-325,a targeted autophagy inhibitor,inhibited the occurrence of autophagy in the four models,and alleviated the elevation of pulmonary artery pressure,vascular remodeling and right heart remodeling to a certain extent.