Abstract
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase (eNOS). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Elevated levels of ADMA lead to reduction in nitric oxide (NO) production, which is linked to endothelial dysfunction and atherosclerosis. Piper sarmentosum is an herb that has shown stimulation on endothelial NO production by increasing both expression and activity of eNOS. Thus, this study determined whether the positive effect of P. sarmentosum on NO production is related to its modulation on the DDAH–ADMA pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to tumor necrosis factor-α (TNF-α). HUVEC were divided into four groups: control, treatment with 250 µg/ml of aqueous extract of P. sarmentosum leaves (AEPS), treatment with 30 ng/ml of TNF-α, and concomitant treatment with AEPS and TNF-α for 24 h. After treatments, HUVEC were collected to measure DDAH1 messenger RNA (mRNA) expression using quantitative real-time polymerase chain reaction. DDAH1 protein level was measured using enzyme-linked immunosorbent assay (ELISA), and DDAH enzyme activity was measured using colorimetric assay. ADMA concentration was measured using ELISA, and NO level was measured using Griess assay. Compared to control, TNF-α-treated HUVEC showed reduction in DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05). Treatment with AEPS successfully increased DDAH1 mRNA expression (P < 0.05), DDAH1 protein level (P < 0.01), and DDAH activity (P < 0.05) in TNF-α-treated HUVEC. Treatment with TNF-α caused an increase in ADMA level (P < 0.01) and a decrease in endothelial NO production (P < 0.001). Whereas treatment with AEPS was able to reduce ADMA level (P < 0.01) and restore NO (P < 0.001) in TNF-α-treated HUVEC. The results suggested that AEPS promotes endothelial NO production by stimulating DDAH activity and thus reducing ADMA level in TNF-α-treated HUVEC.
Highlights
Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide (Benjamin et al, 2017)
Griess assay showed that treatment of tumor necrosis factor-α (TNF-α)-induced human umbilical vein endothelial cells (HUVEC) with 150, 250, and 300 μg/ml of AEPS increased nitric oxide (NO) production by HUVEC (P < 0.05) (Figure 2E)
The results showed that TNF-α decreased DDAH1 messenger RNA (mRNA) expression compared to control
Summary
Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide (Benjamin et al, 2017). The earliest stage of atherosclerosis development is endothelial dysfunction that is associated with other cardiovascular risk factors such as hypertension, hypercholesterolemia, hyperhomocysteinemia, diabetes mellitus, obesity, and systemic inflammation, contributing to the pathogenesis of cardiovascular diseases (Su, 2015). Endothelial dysfunction is defined as the loss of the homeostatic mechanisms that operate in healthy endothelial cells. It is characterized by decreased bioavailability of nitric oxide (NO), which promotes vascular dysfunction and atherosclerosis progression (Su, 2015). ADMA level was increased in individuals with hypercholesterolemia, hypertension, diabetes mellitus, and hyperhomocysteinemia, leading to increased risk of atherosclerosis (Jawalekar et al, 2013)
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