Abstract: BACKGROUND: Previous studies have shown that paeoniflorin has an ameliorative effect on fibrosis in liver and kidney organs, especially in hepatic fibrosis. However, the protective effect of paeoniflorin on angiotensin II-induced fibrosis in cardiac fibroblasts remains unclear.
OBJECTIVE: To investigate the protective effects of paeoniflorin on angiotensin II-induced extracellular matrix deposition in cardiac fibroblasts and molecular mechanisms.
METHODS: Angiotensin II (1 μmol/L) was added to primary isolated cultured SD rat mammary rat cardiac fibroblasts for 48 hours as the model group. The paeoniflorin low and high dose groups were pretreated with different doses of paeoniflorin (50 and 100 μmol/L) for 2 hours, and then treated with angiotensin II for 48 hours. The SIRT1 inhibitor group was treated with SIRT1 inhibitor EX527 10 μmol/L for 2 hours, followed by paeoniflorin (100 μmol/L) for 2 hours, and then co-incubation with angiotensin II for 48 hours. The cell viability was detected using the cell counting kit-8 method (CCK-8) assay. The cell migration ability was detected by Transwell. The level of intracellular reactive oxygen species was detected by DHA fluorescent probe. The level of oxidative stress markers was detected by relevant kits. Protein expression of fibrosis-related genes was detected by western blot assay. The mRNA expression levels of extracellular matrix and fibrosis-related genes were detected by qRT-PCR.
RESULTS AND CONCLUSION: (1) Compared with the control group, the proliferation and migration of cardiac fibroblasts were significantly increased after angiotensin II intervention; the intracellular content of reactive oxygen species and malondialdehyde content were elevated; the activities of superoxide dismutase and catalase were decreased, and the mRNA expression levels of α-smooth muscle actin, type I collagen, type III collagen, fibronectin, connective tissue growth factor, and matrix metalloproteinase 9 were increased. Compared with the model group, paeoniflorin could dose-dependently inhibit the above effect changes (P < 0.01). (2) Compared with the model group, paeoniflorin up-regulated the protein expression of SIRT1 in dose-dependent manner (P < 0.001). (3) Compared with the high-dose paeoniflorin group, the number of cell migration and the expression level of α-smooth muscle actin, type I collagen, and type III collagen were significantly increased in the SIRT1-inhibitor group (P < 0.01). All the experimental results show that paeoniflorin effectively attenuates the angiotensin II-induced changes in cardiac fibroblast fibrosis possibly through up-regulating the expression of SIRT1, dose-dependently inhibits cardiac fibroblast oxidative stress and extracellular matrix deposition, and has a protective effect on cardiac fibroblast fibrosis.

Key words: paeoniflorin, cardiac fibroblast, angiotensin II, extracellular matrix, fibrosis, oxidative stress