ABSTRACT
This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensin â ¡(Angâ ¡) in vascular smooth muscle cell(VSMC) and its molecular mechanism. Primary rat VSMC were cultured using tissue block method, and VSMC were identified by α-actin immunofluorescence staining. Angâ ¡ at a concentration of 1×10~(-6) mol·L~(-1) was used as the stimulating factor, and Sprague Dawley(SD) rats were orally administered with Tianma Gouteng Decoction to prepare drug serum. Rat VSMC were divided into normal group, model group, Chinese medicine group, and inhibitor(3-methyladenine, 3-MA) group. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation activity. Bromodeoxyuridine(BrdU) flow cytometry was used to detect cell cycle. Transwell assay was used to detect cell migration ability. Enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of superoxide dismutase(SOD), catalase(CAT), and malondialdehyde(MDA) in VSMC. The intracellular reactive oxygen species(ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe. Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1), Parkin, p62, and microtubule-associated protein 1A/1B-light chain 3(LC3-â ¡) proteins in VSMC. The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8% could significantly inhibit VSMC growth after 48 hours of intervention. Compared with the normal group, the model group showed significantly increased cell proliferation activity and migration, significantly decreased levels of SOD and CAT, significantly increased levels of MDA, significantly enhanced ROS fluorescence intensity, significantly decreased expression of PINK1, Parkin, and LC3-â ¡ proteins, and significantly increased expression of p62 protein. Compared with the model group, the Chinese medicine group showed significantly reduced cell proliferation activity and migration, significantly increased levels of SOD and CAT, significantly decreased levels of MDA, significantly weakened ROS fluorescence intensity, significantly increased expression of PINK1, Parkin, and LC3-â ¡ proteins, and significantly decreased expression of p62 protein. Compared with the Chinese medicine group, the addition of the mitochondrial autophagy inhibitor 3-MA could block the intervention of Tianma Gouteng Decoction-containing serum on VSMC proliferation, migration, mitochondrial autophagy, and oxidative stress levels, with statistically significant differences. In summary, Tianma Gouteng Decoction has good antioxidant activity and can inhibit cell proliferation and migration. Its mechanism of action may be related to the activation of the mitochondrial autophagy PINK1/Parkin signaling pathway.
Subject(s)
Angiotensin II , Cell Proliferation , Drugs, Chinese Herbal , Muscle, Smooth, Vascular , Oxidative Stress , Protein Kinases , Rats, Sprague-Dawley , Ubiquitin-Protein Ligases , Animals , Drugs, Chinese Herbal/pharmacology , Oxidative Stress/drug effects , Rats , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Male , Cell Proliferation/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Protein Kinases/metabolism , Protein Kinases/genetics , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Reactive Oxygen Species/metabolism , Cell Movement/drug effects , Signal Transduction/drug effects , Cells, Cultured , Superoxide Dismutase/metabolismABSTRACT
We studied the effect of Tianma Gouteng Decoction on vascular aging in spontaneously hypertensive rats(SHRs) to explore the molecular mechanism of the decoction in improving arterial vascular aging by regulating the expression of mitofusin 2(MFN2).Twenty 64-weeks-old SHRs were randomly assigned into the aging group and the treatment group(Tianma Gouteng Decoction 5.48 mg·kg~(-1)).Wistar-Kyoto(WKY) rats of 64 weeks old were taken as the normal group and SHR rats of 14 weeks old as the young group.The intervention with Tianma Gouteng Decoction lasted for 12 weeks.We then employed HE staining and Masson staining to observe the morphology of thoracic aorta under an electron microscope and measured the malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, respiratory chain complex â ¢ level, and thioredoxin peroxidase(TPX) activity.The vascular aging was detected via the biomarker senescence-associated beta-galactosidase(SA-ß-Gal).The expression levels of MFN2 and aging marker proteins silent information regulator 1(SIRT1), Klotho, p21, and p53 in thoracic aorta were detected by immunohistochemistry/fluorescence, qRT-PCR, and Western blot.Compared with the young group and the normal group, the aging group had risen blood pressure, lumen stenosis caused by thickened intima of blood vessels, decreased SOD and TPX activities, increased MDA and mitochondrial respiratory chain complex â ¢ levels, down-regulated expression of MFN2, SIRT1, and Klotho, and up-regulated expression of p21 and p53(P<0.01 or P<0.05).The treatment with Tianma Gouteng Decoction significantly lowered blood pressure, mitigated vascular intimal thickening, increased SOD and TPX activities, and decreased MDA and mitochondrial respiratory chain complex â ¢ levels, thus alleviating vascular aging.At the same time, the decoction up-regulated the expression of MFN2, SIRT1, and Klotho, while down-regulated that of p21 and p53(P<0.01 or P<0.05).In summary, Tianma Gouteng Decoction can significantly delay the vascular aging in hypertension.Specifically, it may up-regulate the expression of MFN2 and regulate the expression of aging-related proteins to alleviate oxidative stress.