FGF21 attenuates high uric acid‑induced endoplasmic reticulum stress, inflammation and vascular endothelial cell dysfunction by activating Sirt1

Abstract

Uric acid (UA) is the final oxidation product of purine metabolism. Hyperuricemia has been previously reported to contribute to vascular endothelial dysfunction and the development of cardiovascular diseases, metabolic syndrome and chronic kidney diseases. In addition, it has been reported that fibroblast growth factor 21 (FGF21) can exert regulatory effects on UA‑induced lipid accumulation. Therefore, the present study aimed to investigate the possible role of FGF21 in HUVEC cell injury induced by UA. The study used UA to induce HUVEC cell injury, inhibited sirtuin 1 (Sirt1) expression using EX527 and overexpressed FGF21 by transfection. Subsequently, reverse transcription‑quantitative PCR was performed to measure the mRNA expression levels of FGF21, Sirt1 and inflammatory cytokines TNF‑α, IL‑1β and IL‑6, whereas western blotting was performed to measure their corresponding protein expression levels including FGF21, Sirt1, NLR family pyrin domain containing 3, pro‑caspase1, apoptosis‑associated speck‑like protein containing a CARD, activating transcription factor 4, C/EBP homologous protein and eukaryotic initiation factor 2. Furthermore, dichloro‑dihydro‑fluorescein diacetate staining was performed to measure intracellular reactive oxygen species (ROS) generation in HUVECs. The levels of ROS and nitric oxide were also quantified using commercial assay kits. The results demonstrated that overexpression of FGF21 significantly inhibited UA treatment‑induced endoplasmic reticulum (ER) stress, inflammation and oxidative stress in HUVECs. Furthermore, overexpression of FGF21 significantly activated Sirt1. The sirt1 inhibitor, EX527, significantly abrogated the suppressive effects of FGF21 overexpression on ER stress, inflammation and oxidative stress in UA‑stimulated HUVECs. To conclude, results of the present study suggested that FGF21 may attenuate UA‑induced ER stress, inflammation and vascular endothelial cell dysfunction by activating Sirt1. Therefore, FGF21 may be a potential effective target for the future treatment of vascular endothelial cell dysfunction.

Keywords: fibroblast growth factor 21; hyperuricemia; sirtuin 1; uric acid.

Figure 1.

High UA levels inhibits FGF21 and Sirt1 expression levels and promotes endoplasmic reticulum stress in HUVECs. (A) mRNA expression levels of FGF21 and Sirt1 were determined using reverse transcription-quantitative PCR. (B) Protein expression levels of FGF21 and Sirt1 were determined using western blotting. (C) Protein levels of ATF4, CHOP and p-eIF2A/eIF2A were determined using western blotting. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 mg/dl UA control group. FGF21, fibroblast growth factor 21; UA, uric acid; Sirt1, sirtuin 1; CHOP, eIF2A, eukaryotic initiation factor 2; ATF4, activating transcription factor 4; p-, phosphorylated.

Figure 2.

Overexpression of FGF21 induces Sirt1 activation and suppresses endoplasmic reticulum stress in HUVECs. (A) FGF21 mRNA expression levels were determined using RT-qPCR. (B) Western blotting was performed to measure FGF21 protein expression levels. (C) RT-qPCR was performed to detect FGF21 mRNA expression levels. (D) Western blotting was performed to detect Sirt1 protein expression levels. (E) RT-qPCR was performed to detect Sirt1 mRNA expression levels. (F) Western blotting analysis was performed to detect the protein levels of ATF4, CHOP, p-eIF2A and eIF2A. ***P<0.001 vs. control; ^##P<0.01 and ^###P<0.001 vs. UA + Ov-NC. UA, uric acid; FGF21, fibroblast growth factor 21; Sirt1, sirtuin 1; RT-qPCR, reverse transcription-quantitative PCR; ATF4, activating transcription factor 4; eIF2A, eukaryotic initiation factor 2; p-, phosphorylated; NC, negative control; Ov, overexpressed.

Figure 3.

Overexpression of FGF21 attenuates endoplasmic reticulum stress in high UA-induced HUVECs by activating Sirtuin 1. Western blotting was performed to detect the protein levels of ATF4, CHOP, p-eIF2A and eIF2A. ***P<0.001 vs. control. ^###P<0.001 vs. UA + Ov-NC; ^$$$P<0.001 vs. UA + Ov-FGF21. FGF21, fibroblast growth factor 21; UA, uric acid; ATF4, activating transcription factor 4; eIF2A, eukaryotic initiation factor 2; Ov, overexpressed; p-, phosphorylated; NC, negative control.

Figure 4.

Overexpression of FGF21 attenuates the inflammatory response in high UA-induced HUVECs by activating Sirtuin 1. (A) Reverse transcription-quantitative PCR analysis was performed to detect the mRNA expression levels of TNF-α, IL-1β and IL-6. (B) Western blotting was performed to detect the protein expression levels of NLRP3, pro-caspase 1 and ASC. ***P<0.001 vs. control; ^###P<0.001 vs. UA + Ov-NC; ^$$P<0.01 and ^$$$P<0.001 vs. UA + Ov-FGF21. FGF21, fibroblast growth factor 21; UA, uric acid; NLPR3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a CARD; Ov, overexpressed; NC, negative control.

Figure 5.

Overexpression of FGF21 attenuates oxidative stress in high UA-induced HUVECs by activating sirtuin 1. (A) ROS production was assessed via dichloro-dihydro-fluorescein diacetate staining (magnification, ×200). (B) ROS levels were quantified using a ROS assay kit. ***P<0.001 vs. control; ^###P<0.001 vs. UA + Ov-NC; ^$$$P<0.001 vs. UA + Ov-FGF21. FGF21, fibroblast growth factor 21; UA, uric acid; ROS, reactive oxygen species; Ov, overexpressed; NC, negative control.

Figure 6.

Overexpression of FGF21 attenuates vascular endothelial cell dysfunction in high UA-induced HUVECs by activating Sirtuin 1. (A) NO levels were quantified using a NO assay kit. (B) Western blotting was performed to detect the protein levels of p-AKT, AKT, p-eNOS and eNOS. (C) Semi-quantification of p-AKT/AKT and p-eNOS/eNOS ratios. ***P<0.001 vs. control; ^###P<0.001 vs. UA + Ov-NC; ^$P<0.05, ^$$P<0.01 and ^$$$P<0.001 vs. UA + Ov-FGF21. FGF21, fibroblast growth factor 21; UA, uric acid; NO, nitric oxide; eNOS, endothelial NO synthase; Ov, overexpressed; NC, negative control; p-, phosphorylated.