ABSTRACT
Autophagy is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We aimed to investigate whether the p62-Keap1-Nrf2 pathway affects the development of PAH by mediating autophagy. A PAH rat model was established using monocrotaline (MCT). Pulmonary artery smooth muscle cells (PASMCs) were extracted, and the changes in proliferation, migration, autophagy, and oxidative stress were analyzed following overexpression or knockdown of p62. The impact of p62 on the symptoms of PAH rats was assessed by the injection of an adenovirus overexpressing p62. We found that the knockdown of p62 increased the proliferation and migration of PASMCs, elevating the oxidative stress of PASMCs and upregulating gene expression of NADPH oxidases. Co-IP assay results demonstrated that p62 interacted with Keap1. p62 knockdown enhanced Keap1 protein stability and Nrf2 ubiquitination. LC3II/I and ATG5 were expressed more often when p62 was knocked down. Treating with an inhibitor of autophagy reversed the impact of p62 knockdown on PASMCs. Nrf2 inhibitor treatment reduced the expression of Nrf2 and p62, while increasing the expression of Keap1, LC3II/I, and ATG5 in PASMCs. However, overexpressing p62 diminished mRVP, SPAP, and Fulton index in PAH rats and attenuated pulmonary vascular wall thickening. Overexpression of p62 also decreased the expression of Keap1, LC3II/I, and ATG5 and increased the nuclear expression of Nrf2 in PAH rats. Importantly, overexpression of p62 reduced oxidative stress and the NADPH oxidase expression in PAH rats. Overall, activation of the p62-Keap1-Nrf2 positive feedback signaling axis reduces the proliferation and migration of PASMCs and alleviates PAH by inhibiting autophagy and oxidative stress.
Subject(s)
Pulmonary Arterial Hypertension , Animals , Rats , Autophagy/physiology , Cell Proliferation , Kelch-Like ECH-Associated Protein 1/genetics , Kelch-Like ECH-Associated Protein 1/metabolism , Monocrotaline , Myocytes, Smooth Muscle/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Pulmonary Arterial Hypertension/pathology , Pulmonary Artery/metabolismABSTRACT
OBJECTIVE: To study the correlation analysis between the HPLC fingerprints of Astragali Radix extracts and the antifatigue effects. METHODS: Three types of extracts of Astragali Radix were obtained and arranged by uniform design for HPLC fingerprint analysis and the antifatigue effect experiment. The correlation analysis between the HPLC fingerprints of Astragali Radix extracts and the antifatigue effects were carried out with orthogonal signal correction-partial least squares (OSC- PLS) method. RESULTS: The antifatigue activity could be strengthened by chromatographic peaks in the fingerprints of flavonoids and other kinds of ingredients (including saponins), but could be weakened by Astragalus polysaccharides in Astragali Radix. Among total variables, there were 36 variables (including 35 peaks and a variable of Astragalus polysaccharides) that had important contribution to "fingerprint-efficacy" model. CONCLUSION: OSC-PLS can remove uncorrelated information between fingerprint and efficacy, simplify the structure of model and improve the interpretative ability of model, and could be a reference method to investigate chromatographic fingerprint-efficacy relationship of complex system of traditional Chinese medicine.
