Salvianolic acid A promotes mitochondrial biogenesis and function via regulating the AMPK/PGC-1α signaling pathway in HUVECs

Abstract

Mitochondrial dysregulation is an important pathology that leads to endothelial dysfunction, and the occurrence and development of cardiovascular diseases. Salvianolic acid A (SAA) has been demonstrated to be effective in the treatment of vascular complications of type 2 diabetes mellitus. Limited information has been reported on the effects of SAA on mitochondrial function in endothelial cells. In the present study, the effects of SAA on mitochondrial biogenesis and the related underlying mechanisms were investigated in human umbilical vein endothelial cells (HUVECs). Mitotracker red staining and transmission electron microscopy were used to evaluate the effect of SAA on mitochondrial quality. The effect of SAA treatment on mitochondrial DNA/nuclear DNA ratio of HUVECs was detected by real-time quantitative PCR. Western blot was used to determine the protein expression levels of complex III and Complex IV of mitochondrial oxidative phosphorylation subunit, and ATP production was determined by ATP test kit. Real-time quantitative PCR and Western blot were used to determine the effects of SAA on the expression of peroxisome proliferator-activated receptor γ coactivator (PGC-1α) and its target genes nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) proteins and genes. Finally, in the presence of 5'AMP-activated protein kinase (AMPK) specific inhibitors, the expression of PGC-1α, NRF1 and TFAM proteins and the phosphorylation levels of AMPK and Acetyl CoA Carboxylase (ACC) were detected by Western blot or real-time quantitative PCR. The results showed that SAA treatment significantly promoted mitochondrial biogenesis and enhanced mitochondrial function of HUVECs. SAA significantly increased the expression levels of PGC-1α and its target genes NRF1 and (TFAM), a key regulator of mitochondrial biogenesis in HUVECs. These enhancements were accompanied by significantly increased phosphorylation of AMPK and ACC, and were significantly inhibited by specific AMPK inhibitors. These results suggest that SAA may promote mitochondrial biogenesis in endothelial cells by activating the AMPK-mediated PGC-1α/TFAM signaling pathway. These data provide new insights into the mechanism of action of SAA in treating diabetic vascular complications.

Keywords: 5'AMP-activated protein kinase; diabetic vascular complications; mitochondrial biogenesis; peroxisome proliferator-activated receptor γ coactivator 1-α/mitochondrial transcription factor A; salvianolic acid A.

Figure 1

SAA promotes mitochondrial biogenesis in HUVECs. HUVECs were treated for 24 h with SAA at concentrations of 10^-3, 10^-2 and 10^-1 µM. (A) Mitochondria were stained with Mitotracker red dye. The integrated fluorescence intensity was analyzed to assess the mitochondrial mass relative to the vehicle group. (B) Mitochondrial density was assessed using transmission electron microscopy (magnification, x6,000). (C) mtDNA/nDNA was determined using reverse transcription-quantitative PCR and is presented relative to the control cells. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. vehicle. SAA, salvianolic acid A; mtDNA/nDNA, mitochondrial/nuclear DNA.

Figure 2

SAA enhances mitochondrial function. (A) Western blotting of complex Ⅲ and complex Ⅳ protein expression levels following SAA treatment of the cells. (B) Intracellular ATP levels were assessed using colorimetry. ^*P<0.05, ^***P<0.001 vs. vehicle. SAA, salvianolic acid A.

Figure 3

SAA promotes the expression of PGC-1α, NRF1 and TFAM. (A) Following SAA treatment, western blotting was performed to assess PGC-1α, NRF1 and TFAM protein expression levels. (B) mRNA expression levels of PGC-1α, NRF1, and TFAM were determined using reverse transcription-quantitative PCR. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. vehicle. SAA, salvianolic acid A; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1-α; NRF1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A.

Figure 4

SAA promotes mitochondrial biogenesis via the AMPK signaling pathway. (A) Following SAA and compound C stimulation, western blotting was performed to assess PGC-1α, NRF1 and TFAM protein expression levels. (B) mRNA expression levels of PGC-1α, NRF1 and TFAM were determined using RT-qPCR. (C) mtDNA/nDNA was determined using RT-qPCR and is presented relative to vehicle. (D) Following treatment of the cells with SAA, compound C and AICAR stimulation, western blotting was performed for the detection of p-AMPK and p-ACC expression levels. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. vehicle; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. SAA treatment group. SAA, salvianolic acid A; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, 5'AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1-α; NRF1, nuclear respiratory factor 1; TFAM, mitochondrial transcription factor A; RT-qPCR, reverse transcription-quantitative PCR; mtDNA/nDNA, mitochondrial/nuclear DNA, p, phosphorylated; ACC, acetyl-CoA carboxylase.