Ozone alleviates MSU-induced acute gout pain via upregulating AMPK/GAS6/MerTK/SOCS3 signaling pathway

Abstract

Background: Gout pain seriously affects the quality of patients' life. There is still no effective treatment. The inflammatory response is the main mechanism of gout. Here, we found that ozone can reduce the inflammatory reaction in the joints of gouty mice and relieve gout pain, and we further explore its protective mechanism.

Methods: MSU was used to establish the gouty mice model. Nociception was assessed by Von Frey hairs. Cell signaling assays were performed by western blotting and immunohistochemistry. The mouse leukemia cells of monocyte macrophage line RAW264.7 were cultured to investigate the effects of ozone administration on macrophage.

Results: Ozone reduced inflammation, relieved gout pain and improved the paw mean intensity and duty cycle of the gouty mice. Ozone increased the phosphorylation of AMP-activated protein kinase (AMPK), induced suppressor of cytokine signaling 3 (SOCS3) expression and inhibited metallopeptidase 9 (MMP9) expression. In vivo, ozone activated AMPK to induce Gas6 release, and upregulated MerTK/SOCS3 signaling pathway to reduce inflammation in mouse macrophage line RAW264.7. Inhibitors of AMPK and MerTK, respectively abolished the analgesic and anti-inflammatory effects of ozone in vivo and in vitro. Gas6 knockout cancelled the protectively effects of ozone on gout pain and the paw mean intensity and duty cycle of gouty mice. Additionally, the level of Gas6 and protein S in plasma of patients with hyperuricemia was significantly higher than that of healthy contrast group.

Conclusion: Ozone reduces inflammation and alleviates gout pain by activating AMPK to up-regulate Gas6/MerTK/SOCS3 signaling pathway.

Keywords: AMPK; Gas 6; MMP9; MerTK; Ozone; SOCS3.

Fig. 1

Ozone suppressed MSU-induced gout pain in mice. Mice (n = 8) were treated with various doses of ozone (i.p) 10 min after the injection of MSU crystals (0.5 mg/10 μl). A Mechanical allodynia was performed to evaluate the effect of ozone. B Time course of changes in MSU-induced ankle swelling. C Representative photographs and hematoxylin- and eosin-stained sections of the ankle joints obtained 6 h after MSU injection. D Representative footprint images were recorded during the treatment of Ozone. The colored bands represent standing time of each foot and the right panels are footprints of the right hind paw reflected by green LED light. E, G, I The paw mean intensity was analyzed by CatWalk software. RH, right hind. F, H, J The duty cycle was analyzed by CatWalk software. RH, right hind. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. sham group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the MSU-treated group

Fig. 2

Ozone activated the AMPK/SOCS3 signal pathway and inhibited MMP9 expression to relieve gout pain. A After 6 h of indicated treatments, the MMP9 of mice 's plasma was tested using western blot. Albumin was used as a loading control. B MMP9 were measured with zymography assays. Blood cells (C, D) and ankle joints (E)with the indicated treatments were subjected to western blot assay of pAMPK, AMPK, SOCS3, MMP9 and β-actin. F–H Up/down-regulation of IL-1β, TNF-α and IL-6 mRNA were compared with the control group and MSU-treated group (n = 4). I Effect of ozone on MSU-induced mechanical withdrawal threshold combined with CC-treatment in GA mice. CC (i.p, 5 mg/kg) were primed 12 h before MSU injection. J Joint swelling gain at different time points. K, L Western blot analysis and the expression of MMP9 and SOCS3 in ankle joint. Representative western blot bands and a data summary are shown. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. MSU-treated group; ^&p < 0.05, ^&&p < 0.01 and ^&&&p < 0.001 vs. MSU + ozone group

Fig. 3

Ozone activated AMPK/Gas6/MerTK/SOCS3 signaling pathway to inhibit MMP9 activity and inflammatory response in RAW264.7 cells. Raw264.7 were primed with LPS (1 μg/ml, 6 h), and challenged with MSU suspension (200 μg/ml, 6 h) followed by treatment with CC (20 μM)/UNC2541(2.5 μM)/TP0903(100 nM). Ozone is given ten minutes after MSU treatment. A Western blot analysis and the expression of pAMPK, AMPK, SOCS3, MMP9 and β-actin. B Western blot analysis and the expression of pMerTK, Protein S, Gas6 and β-actin. C Western blot analysis showed that CC suppressed the expression of SOCS3, and Gas6 and Increase the expression of MMP9 in Raw264.7. D–F Expression of MMP9 and SOCS3 after UNC2541 or TP0903 treated in Raw264.7. G Phosphorylation of MerTK upon CC exposure in Raw264.7. H–I Expression of Gas6 and pMerTK after CC treated in the ankle joint. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. MSU-treated group; ^&p < 0.05, ^&&p < 0.01 and ^&&&p < 0.001 vs. MSU + ozone group

Fig. 4

Ozone inhibited MMP9 activity through the MerTK/SOCS3 signal pathway to relieve gout. A Mechanical allodynia was performed to evaluate the effect of ozone after UNC2541 was treated. B Time course of changes in MSU-induced ankle swelling after UNC2541 was treated. C, D Western blot analysis showed the expression levels of MMP9 and SOCS3 after being treated with UNC2541 in the ankle joint. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. MSU-treated group; ^&p < 0.05, ^&&p < 0.01 and ^&&&p < 0.001 vs. MSU + ozone group

Fig. 5

Gas6 played a key role in relieving gout pain with ozone. A Mechanical threshold of ipsilateral hind-paws between WT and Gas6-/- mice induced by MSU. B Ankle girth according to time after MSU crystal injection between WT and Gas6^−/− mice. C–I Ozone ameliorates the pain gait patterns of mice with MSU-treated. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs. control group; ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. MSU-treated group; ^&p < 0.05, ^&&p < 0.01 and ^&&&p < 0.001 vs. MSU + ozone group

Fig. 6

Decreased expression of plasma Gas6 and Protein S in patients. Gas6, Protein S and MMP9 levels in high UA patients and healthy controls. ^***p < 0.001 vs. control group

Fig. 7

Schematic indicating that ozone reduces inflammation and alleviates gout pain by activating AMPK to up-regulate Gas6/MerTK/SOCS3 signaling pathway