The Role And Mechanism Of Nrf2 In Pulmonary Artery Endothelial Cells And Smooth Muscle Cells

Pulmonary hypertension（Pulmonary hypertension,PH）is a refractory disease in which the pulmonary blood vessels continue to constrict and pulmonary vascular resistance is progressively increased,leading to right heart failure and death.The pathogenesis of the disease is unknown,the treatment is very limited and the prognosis is usually poor.Therefore,a large amount of basic research is needed to assist drug development and clinical treatment.Nrf2 participates in a variety of cell activities through its regulatory role in oxidative stress,inflammation and other aspects,including plays an important regulatory role in various malignant phenotypes of cancer cells.During the development of pulmonary hypertension,pulmonary vascular cells exhibit excessive proliferation,migration and cellular metabolic reprogramming which were similar to cancer cells.To explore the role and mechanism of Nrf2 in pulmonary hypertension,Nrf2 expression was detected in two common PH rat models.Subsequently,the regulation of Nrf2 on Endothelial-mesenchymal transformation（Endothelial-mesenchymal transformation,End MT）in pulmonary artery endothelial cell（Rat pulmonary arterial endothelial cell,rPAEC）was studied.Finally,the regulation of Nrf2 on proliferation and migration of pulmonary artery smooth muscle cell（Rat pulmonary arterial smooth muscle cells,rPASMC）was detected,and the following results were obtained:（1）Detect the expression of Nrf2 in two PH rat models:MCT-PH and H-PH rat models were constructed and the protein and m RNA expressions of Nrf2 in rat lung tissues were detected by WB and RT-q PCR;The distribution of Nrf2 in lung tissues was detected by immunofluorescence.The results showed that the protein and mRNA expression levels of Nrf2 in animal model were significantly lower than those in control group.（2）Regulation of Nrf2 on（Endothelial-mesenchymal transformation,End MT）in rPAECs:We found that Nrf2 protein expression was down-regulated in ECs from human and rat PAECs under 5%O₂ hypoxia.After we knockdown Nrf2,rPAEC culture with TGF-β1 for 3-4d.It was found that the protein levels of mesenchymal cell markers in EC were significantly increased after TGF-β1 treatment,and having more serious up-regulation after Nrf2 knockdown.At the same time,after Nrf2 knockdown,the protein level of Snail1,an important transcription factor related to End MT,was also significantly up-regulated.（3）The regulation of Nrf2 on the proliferation of rPASMC:We found that the protein expression of Nrf2 was up-regulated in rPASMCs treated at different time gradients under 5%hypoxia.Then Nrf2 knockdown treatment was carried out,and the effect of Nrf2 knockdown on the proliferation of rPASMC was investigated by WB、EdU and CCK8 assaies.The results showed that knockdown Nrf2 under normoxic and hypoxic conditions could significantly inhibit the proliferation phenotype of rPSAMC.Through CCK8 test,treatment with Nrf2 activator Sulforaphane（SFP）in normoxic and hypoxic states promoted the proliferation of rPASMC.The regulation of Nrf2 on proliferative signaling pathway m TOR/P70S6K and PDGFR-α/ERK1/2 was investigated by WB test.We found that Nrf2 knockdown significantly inhibited the protein expression of PDGFR-α/p-ERK1/2 and p-m TOR/p-P70S6K,while SFP activated both pathways.（4）The regulation of Nrf2 on the migration of rPASMC:Through scratch experiment,it was found that under normoxic and hypoxic conditions,knockdown Nrf2could significantly inhibit the migration of rPASMC.Our RT-q PCR detection showed that knockdown Nrf2 down-regulated the mRNA level of MMP2/3/7.In addition,we verified that SFP can effectively promote the migration ability of PASMC through scratches.These results indicate that Nrf2 shows the dual regulating effect on phenotypes of rPAEC and rPASMC.Therefore,we believe that the effect of a single Nrf2 activator in the treatment of PH is limited,and related drug development should pay attention to eliminating the promoting effect of Nrf2 activation on the abnormal phenotypes of rPASMC.