Shenfu Injection Promotes Vasodilation by Enhancing eNOS Activity Through the PI3K/Akt Signaling Pathway In Vitro

Abstract

Vasomotor dysfunction is one of the key pathological aspects of shock and heart failure (HF). Shenfu injection (SFI) has been widely used for the treatment of shock and HF in China. Pharmacological studies have suggested that SFI can reduce peripheral circulation resistance and improve microcirculation. However, whether it has a regulatory effect on macrovascular has not been elucidated. In this study, we used thoracic aorta rings isolated from Wistar rats and the human umbilical vein cell line (EA.hy926) to explore the vasodilative activity of SFI and its potential mechanisms. The relaxation due to SFI was measured after pre-treatment with selective soluble guanylate cyclase (sGC) inhibitor or cyclooxygenase (COX) inhibitor and compared with the vasodilation effect of SFI only treated with norepinephrine (NE). The contents of NO, endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS), COX-1, 6-K-PGF_1α, and caveolin-1 were evaluated respectively. Additionally, the level of eNOS mRNA and total eNOS and its phosphorylation were studied to investigate the potential mechanisms involved. Experimental results showed that SFI markedly attenuated NE-induced vasoconstriction but that this effect was significantly eliminated after pre-incubation with the selective sGC inhibitor 1-H-[1, 2, 4] oxadiazolo [4, 3-α] quinoxaline-1-one (ODQ), instead of the COX inhibitor indomethacin (INDO). SFI significantly increased the eNOS content and up-regulated the eNOS mRNA expression, while it did not affect the content of COX-1 and 6-K-PGF_1α. SFI also markedly increased NO content but significantly reduced the content of ET-1 and caveolin-1 in the cell supernatant. Furthermore, it promoted the expression of total eNOS and the phosphorylation of eNOS at serine (Ser) 1177 but inhibited the phosphorylation at threonine (Thr) 495, which was significantly reversed by PI3K-specific inhibitor LY294002. In conclusion, our study showed the vasodilation effect of SFI in thoracic aorta is mediated entirely by enhancing eNOS activity through the PI3K/Akt signaling pathway, providing novel knowledge on the effect of SFI on shock and HF for future clinical applications.

Keywords: PI3K/Akt signaling pathway; Shenfu injection; endothelial nitric oxide synthase (eNOS); nitric oxide (NO); vasodilation.

Figure 1

Effects of selective sGC inhibitor ODQ and COX inhibitor INDO on relaxations induced by SFI in isolated thoracic aorta rings contracted with NE. The inhibition of NE (1 μM)-pre-contracted rat thoracic aorta rings with intact endothelium in response to cumulative addition of SFI (0.1, 1, 2 and 10 µl/ml) in the presence and absence of selective sGC inhibitor ODQ (10 µM) and COX inhibitor INDO (10 µM). ODQ, instead of INDO, can obviously restrain the vasorelaxation of SFI. Values are expressed as the mean ± SD, n = 6. ^** P < 0.01 vs. Control (an equal volume K-H solution was added for the control group); ^## P < 0.01 vs. SFI.

Figure 2

Effect of SFI on eNOS content (A) and eNOS mRNA expression (B). 10, 20, and 40 μl/ml of SFI and mevastatin significantly increased eNOS content (A), and 10 μl/ml of SFI obviously up-regulated eNOS mRNA expression, as mevastatin (B). Values are expressed as the mean ± SD, n = 6. ^* P < 0.05, ^** P < 0.01 vs. Control.

Figure 3

Effect of SFI on extracellular (A) and intracellular (B, C) NO content. After treatment with different dosages (10, 20, and 40 μl/ml) of SFI for 24 h, all doses of SFI markedly increased NO production in EA.hy 926 cell supernatant (A), and 20μl/ml of SFI also increased the intracellular NO production (B, C), like sildenafil. Values are expressed as the mean ± SD, n = 6. ^* P < 0.05, ^** P < 0.01 vs. Control.

Figure 4

Effect of SFI on ET-1 content. After treatment with different dosages (10, 20, and 40 μl/ml) of SFI for 24 h, 40 μl/ml of SFI significantly reduced ET-1 content in EA.hy 926 cell supernatant. Values are expressed as the mean ± SD, n = 6. ^* P < 0.05 vs. Control.

Figure 5

Effect of SFI on the expression of total eNOS and the phosphorylation of eNOS at Ser1177 and Thr 495. Representative blot images demonstrating total eNOS, p-eNOS Ser 1177, p-eNOS Thr 495, and β-actin protein expression levels (A). SFI promoted the expression of total eNOS (B) and the phosphorylation of eNOS at Ser1177 (C) but inhibited the phosphorylation at Thr495 (D), which was significantly reversed by PI3K-specific inhibitor LY294002. Values are expressed as the mean ± SD, n = 3. ^** P < 0.01 vs. Control; ^## P < 0.01 vs. SFI (20 μl/ml).

Figure 6

Effect of SFI on the caveolin-1 content in the cell supernatant. SFI significantly reduced the caveolin-1 content in the cell supernatant, like mevastatin. Values are expressed as the mean ± SD, n = 6. ^* P < 0.05, ^** P < 0.01 vs. Control.

Figure 7

Scheme of the proposed mechanism by which SFI increases eNOS activity. Administration of SFI results in an increase in the gene and protein expression of eNOS and promotes the eNOS phosphorylation at Ser1177 but inhibits the eNOS phosphorylation at Thr 495 and decreases the Cav-1 content, ultimately resulting in enhanced NO production. This effect was antagonized by the PI3K/Akt signaling pathway inhibitor LY294002. The inhibitor used and its location of action is also indicated.