MaR1 has a strong effect on the expression of inflammatory factors induced by FAs + MSUc

To understand the anti-inflammatory activity of MaR1, we investigated its effect on NF-κB activation and inflammatory gene expression in BMDMs. BMDMs were incubated with or without MaR1 (10 nM) for 1 h and then stimulated with FAs + MSUc for 12 h. MaR1 significantly inhibited FAs + MSU crystal-induced P65 nuclear translocation (phosphorylation P65, p-P65) (Fig. 1A). Simultaneously, MaR1 decreased the mRNA levels of cytokines, chemokines and pro-inflammatory enzymes, many of which are responsive to NF-κB (Fig. 1B). Consistent with the RT‒qPCR data, MaR1 had similar effects on the secretion of proinflammatory cytokines (Additional file 1: Fig. S1). Moreover, MaR1 attenuated the INOS and COX2 protein levels induced by FAs + MSUc (Fig. 1C). MaR1 also decreased the mRNA and protein expression of the FA + MSU crystal-stimulated autophagic chaperone p62, which is encoded by the NF-κB-inducible Sqstm1 gene (Fig. 1D). These data indicate that MaR1 plays an important role in suppressing MSU crystal-induced inflammation in vitro.

Fig. 1

MaR1 inhibited nuclear localization of p-P65 and expression of inflammation-associated genes. A BMDMs were treated with or without MaR1 (10 nM) for 1 h, then stimulated with FAs + MSUc for 12 h. Western blot detection of p-P65 and P65 protein levels in the nucleus and cytoplasm. B Quantitative PCR for mRNA levels of IL-1β, IL-6, TNF-α, iNOS, COX-2 and MCP1. C Western blotting detection of INOS and COX-2 protein levels. D Quantitative PCR and Western blot to detect P62 protein level. *Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

NLRP3 inflammasome activation plays an essential role in the acute inflammatory response in the context of gout. We demonstrated the effects of MaR1 on pro-IL-1β processing and NLRP3 inflammasome activation. MaR1greatly decreased NLRP3 and NEK7 protein levels but had no effect on pro-IL-1β, ASC and pro-Casp1 protein levels (Fig. 2A). Importantly, MaR1 inhibited Casp1 activation and pro-IL-1β processing to mature IL-1β in BMDMs treated with FAs + MSUc. MaR1 also suppressed gasdermin D (GSDMD) cleavage (Fig. 2A). To validate NLRP3 inflammasome inhibition, we also evaluated MaR1’s effect on ASC oligomerization, an indicator of NLRP3 inflammasome assembly (Dick et al. 2016) (Fig. 2B). Consistent with its effect on Casp1, MaR1 decreased FA + MSU crystal-induced ASC speck formation (Fig. 2C).

Fig. 2

MaR1 inhibited NLRP3 inflammasome activation induced by FAs + MSUc. A Western blot for NLRP3, Pro-Casp-1, Pro-IL-1β, IL-1β (p17), Casp1(p20), NEK7 and GSDMD protein levels. B Western blotting was used to detect ASC oligomers formation. C Immunofluorescence detection of ASC speck formation. DAPI stains nucleus. Scale bar: 40 μm. *Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

MaR1 protected against mitochondrial dysfunction and mtDNA release induced by FAs and MSUc

Most NLRP3 inflammasome activators lead to mitochondrial dysfunction, mainly by stimulating mtROS production, inducing mitochondrial membrane depolarization and promoting mitochondrial permeability transition pore (mPTP) opening. FAs + MSUc stimulation induced a significant increase in mitochondrial ROS production, with increased MitoSOX fluorescence intensity (Fig. 3A). JC-1 staining revealed an important shift in fluorescence emission from JC-1 aggregates to JC-1 monomer in response to FAs + MSUc (Fig. 3B). According to the statistical analysis, the JC-1 monomer/JC-1 aggregate ratio associated with the MMP increased from 24% in BMDMs treated with vehicle to 46% in BMDMs treated with FAs + MSUc (Fig. 3B). FAs + MSUc challenge induced mPTP opening, as reflected by a significant decrease in Calcein fluorescence intensity (Fig. 3C). However, in the MaR1 intervention group, the mitochondrial functions of BMDMs were protected, as the level of mitochondrial ROS decreased, and the mPTP closure degree and mitochondrial membrane potential were improved (Fig. 3A–C). By performing live cell imaging, we observed the intracellular Calcein fluorescence intensity, and MaR1 ameliorated the decrease in fluorescence intensity caused by FAs + MSUc stimulation, further indicating that MaR1 inhibited the opening of the mPTP induced by FAs + MSUc (Fig. 3D). To further clarify the effects of MaR1 on mitochondrial function, we utilized Seahorse analysis to test the impact of MaR1 on mitochondrial respiration. As shown in Fig. 3E and F, MaR1 accelerated basal respiration, ATP production rate, maximal respiration rate, spare capacity and proton leak in BMDMs treated with FAs + MSUc.

Fig. 3

MaR1 protected against MSU crystal-induced mitochondrial dysfunction. A–C FCM was used to detect mitochondrial ROS (MitoSOX probe) generation, mitochondrial membrane potential (JC-1 probe) and mPTP (Calcein probe). D LCM imaging of mPTP. E OCR was measured by Seahorse analysis. F Quantification of OCR of basal respiration, ATP production rate, maximal respiration rate, spare capacity, proton leak and non mitochondrial respiration. G mitochondrial Ca2+ levels. Scale bar: 40 μm. DAPI stains nucleus. H Detection of cytoplasmic mitochondrial DNA (mtDNA) and oxidized mtDNA by qPCR and ELISA, respectively. I Western blotting was used to detect CMPK2 protein levels. *Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

NLRP3 inflammasome agonists drive mPTP opening caused by an elevated mitochondrial Calcium ion concentration ([Ca2+] m), which then drives mtDNA and Ox-mtDNArelease (Xian et al. 2022). We examined the effect of MaR1 on the [Ca2+] m. The fluorescence intensity of Rhod-2 (a dye that fluoresces in response to mitochondrial Ca2+ binding) in BMDMs stimulated with FAs + MSUc was strongly elevated (Fig. 3G). MaR1 treatment attenuated the increase in the [Ca2+] m (Fig. 3G). MaR1 strongly inhibited FAs + MSU crystal-stimulated mtDNA synthesis (Additional file 1: Fig. S2) and blocked the production of cytoplasmic mtDNA and Ox-mtDNA in BMDMs challenged with FAs + MSUc (Fig. 3H). Because CMPK2 controls mitochondrial DNA synthesis (Zhong et al. 2018), we further confirmed the effect of MaR1 on CMPK2 protein levels. MaR1 markedly blocked the upregulation of CMPK2 protein expression induced by FAs + MSUc (Fig. 3I).

MaR1 accelerated Prdx5 expression in BMDMs treated with FAs + MSUc

To investigate the molecular mechanisms of MaR1 in MSU crystal-induced inflammatory responses, we performed proteomic and Bioinformatics analyses of differentially expressed proteins (DEPs) in BMDMs treated with or without MaR1 and stimulated with FAs + MSUc. Hierarchal clustering partitioned the differentially expressed proteins (DEPs) into 2 distinct clusters separating BMDMs treated with or without MaR1 and stimulated with FAs + MSUc, implying that the proteomics data were highly reliable (Additional file 1: Fig. S3). A total of 53 differentially expressed proteins (DEPs) between the MaR1-treated and untreated groups were selectedin BMDMs treated with FAs + MSUc by significant difference protein screening, and the information about the genes encoded by the DEPs is provided in Additional file 1: Table S4. Among them, 28 DEPs were upregulated in the MaR1-treated group compared to the MaR1-untreated group, while 25 DEPs were downregulated. The results are illustrated by a volcano plot (Fig. 4A). Three of the 28 upregulated proteins were localized to mitochondria, including Prdx5, UCP2 and MICU2. Western blot assays were used to verify the effect of MaR1 on the expression of these three proteins. The data indicated that MaR1 could effectively alleviate the reduction in Prdx5, UCP2 and MICU2 protein expression induced by FAs + MSUc (Fig. 4B).

Fig. 4

MaR1 promotes Prdx5 expression by accelerating NRF2 activity. A DEPs between MaR1-treated and untreated FAs + MSU crystal-stimulated BMDMs are displayed by a volcano plot. The up-regulated proteins in the MaR1 treatment group are shown in red, down-regulated proteins are shown in blue, and those with no difference are shown in gray. B Western blot detection of Prdx5, UCP2 and MICU2 protein levels. C Western blot detection of Prdx3 protein level. D Detection of Prdx5 mRNA level by qPCR. E Western blot detection of p-NRF2 protein level. F Detection of nuclear localization of NRF2 by immunofluorescence staining. Scale bar: 40 μm. G BMDMs were treated with or without ML385 (5 μM) for 1 h, then treated with MaR1 and FAs + MSUc for 12 h. Quantitative PCR and Western blot to detect the effect of ML385 reversing MaR1 on the mRNA and protein levels of Prdx5. & Compared with no MaR1 and ML385 treatment, # compared with FAs + MSUc treatment. $ compared with MaR1 and FAs + MSUc treatment. &, # and $ means P < 0.05

Among these upregulated genes, we were particularly interested in Prdx5. Prdx5 belongs to a family of cysteine-dependent peroxidase enzymes with a remarkable ability to scavenge peroxides and peroxynitrite in mammalian cells. There are 6 members of the Prdx family (Prdx1-6), which are classified according to their subcellular localization (Wood et al. 2003). Compared to the other Prdxs, Prdx3 and Prdx5 are more strongly mitochondrial-targeted antioxidant enzymes because they are mainly localized in the mitochondria (Poynton and Hampton 2014). Prdx3 and Prdx5 can effectively scavenge cytosolic and mitochondrial ROS in several cell lines (Huh et al. 2012; Park et al. 2016). We confirmed the effects of FAs + MSUc on the protein levels of Prdx1-6. As shown in Additional file 1: Fig. S4A, there were almost no changes in the protein levels of PRDX1, PRDX2, PRDX4 and PRDX6 after FAs + MSUc treatment. In contrast, the protein level of PRDX5 decreased; however, the protein level of PRDX3 increased. These data suggest that Prdx5 and Prdx3 may play important roles in FAs + MSU crystal-induced inflammation. We further confirmed the effect of MaR1 on PRDX3 protein levels. MaR1 had little effect on the elevated PRDX3 protein expression induced by MSUc (Fig. 4C).

Next, we explored whether MaR1 affects Prdx5 expression at the mRNA level. Consistent with the Western blot data, MaR1 significantly inhibited FAs + MSU crystal-induced downregulation of Prdx5 at the mRNA level (Fig. 4D). It has been reported that MaR1 can accelerate Nrf2 activity, and the Keap1-Nrf2 signaling axis affects Prdx5 expression at the mRNA level (Graham et al. 2018; Chen et al. 2020). Western blot data showed that MaR1 greatly suppressed the downregulation of p-NRF2 (assessed by phosphorylation of NRF2 (S40)) induced by FAs + MSUc (Fig. 4E). Immunofluorescence data further showed that MaR1 significantly promoted Nrf2 nuclear translocation in BMDMs treated with FAs + MSUc (Fig. 4F). ML385, a specific inhibitor of Nrf2, significantly reversed the upregulation of Prdx5 mRNA and protein expression induced by MaR1 (Fig. 4G).

We wanted to know if MaR1 regulates Prdx5 expression via the Keap1-Nrf2 signaling axis. To further explore the interacting mode of Keap1 with MaR1, molecular docking study, based on the crystal structures of Keap1 (PDB code: 5CGJ), was performed by using the software. The results were shown in Fig. 5A. Finally, the calculated binding energy of MaR1 with Keap1 and Ki value were respectively was -6.85 kcal/mol and 9.55 nM. MaR1 repressed FAs + MSU crystal-induced Keap1 protein expression (Fig. 5B). Both Keap1 knockdown and the inhibitor of Keap1-Nrf2 axis (ML334) could accelerate p-NRF2 protein level in BMDMs treated with FAs + MSUc (Fig. 5C, D and Additional file 1: Fig. S4B). Consistent with this data, both Keap1 knockdown and the inhibitor of Keap1-Nrf2 axis obviously promoted Prdx5 mRNA level (Fig. 5E, )F). These data suggest that MaR1 may accelerate Prdx5 expression through the Keap1-Nrf2 axis in FAs + MSU crystal-induced inflammation.

Fig. 5

MaR1 regulates the Keap1-Nrf2signaling axis. A The docking mode of maresin1 with keap1 (PDB code: 5CGJ). Maresin1 and the amino acid residues that participate in the interactions were displayed as sticks and colored by the element, hydrogen bonds are showed as green dashed lines. B Western blot detection of KEAP1 protein level. C BMDMs were treated with or without ML334 (1 μM) for 1 h, then stimulated with FAs + MSUc for 12 h. Western blot detection of p-NRF2 protein level. D Effect of Keap1 knockdown on p-NRF2 protein level. E and F ML334 treatment or Keap1 knockdown increased Prdx5 mRNA expression. *Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

Prdx5 accelerated AMPK activity and improved mitochondrial fragmentation

A previous study showed that MSUc stimulation inhibits AMPK activity (as indicated by the phosphorylation of AMPKα and p-AMPK) (Wang et al. 2016) and that Prx5 overexpression induces the phosphorylation of AMPKα (Kim et al. 2020). We sought to investigate whether Prdx5 affects AMPKα phosphorylation during FAs + MSUc induced inflammation. The data indicated that Prdx5 overexpression reversed the reduction in AMPK phosphorylation induced by FAs + MSUc (Fig. 6A and Additional file 1: Fig. S5A). Since it has been shown that AMPK activation regulates the TXNIP/TRX balance (McWherter et al. 2018), we next explored the effects of Prdx5 overexpression on the expression of TRX isoforms and TXNIP. As shown in Fig. 6A, FAs + MSUc downregulated the expression of TRX1 and TRX2 isoforms while stimulating the expression of TXNIP. These effects were blocked by Prdx5 overexpression, indicating an ability of Prdx5 to maintain the balance between TRX isoforms and TXNIP. Immunofluorescence data all revealed that Prdx5 overexpression accelerated TRX2 expression in BMDMs treated with FAs + MSUc (Fig. 6B). Our data suggest that compound C, an AMPK inhibitor, hinders the effect of Prdx5 overexpression on TXNIP, TRX1 and TRX2 expression (Additional file 1: Fig. S5B).

Fig. 6

Prdx5 over-expression promotes AMPK activity. BMDMs were transfected with control (pcDNA3.1) or Prdx5 open reading frame (ORF) was cloned into pcDNA3.1 plasmids (Prdx5 OE) for 24 h and then stimulated with FAs + MSUc for 12 h. A Western blot analysis for p-AMPK, TXNIP, TRX1, TRX2 and p-DRP1 protein levels. B Immunofluorescence staining to detect the signal intensity of TRX2 protein. C BMDMs were stained with Mitotracker Red probe, then immunofluorescence staining for p-Drp1 (green). Scale bar: 40 μm. Areas outlined in white are shown enlarged in the right panels. Image is representative of 5 images/dish; n = 3 dishes/condition. D Representative TEM images of morphological changes in mitochondria (white box) in BMDMs. Scale bar: 1 μm. Image is representative of 10 images/sample; n = 3 samples/condition. E BMDMs were stained with Mitotracker Green probe and DAPI, and mitochondrial morphology was analyzed by LCM imaging. Image is representative of 5 images/dish; n = 3 dishes/condition. *Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

A previous study revealed that an AMPK activator blocked Drp1-mediated mitochondrial fission (Wang et al. 2017). We further determined the effect of Prdx5 overexpression on Drp1 activity and the mitochondrial ultrastructure. Prdx5 overexpression repressed Drp1 activity, as reflecting by reduced Drp1 (s616) phosphorylation (Fig. 6A) and Drp1 recruitment to mitochondria (Fig. 6C). This study showed that compound C could block the effect of Prdx5 overexpression on Drp1 activity (Additional file 1: Fig. S5B). Ultrastructure analysis performed by transmission electron microscopy (TEM) indicated that in FAs + MSUc induced BMDMS, the integrity of the mitochondrial cristae is severely disrupted (Fig. 6D). We analyzed the size of mitochondria by measuring the mitochondrial aspect ratio (AR). BMDMs treated with FAs + MSUc exhibited a significant decrease in the AR ratio, which was markedly reversed by Prdx5 overexpression (Fig. 6D), reflecting an improvement in mitochondrial morphology. Mitochondrial morphology in BMDMs stained with MitoTracker Green was further analyzed by laser confocal microscopy (LCM). In BMDMs that were not subjected to FAs + MSUc treatment, most mitochondria formed interconnected, tubular networks (Fig. 6E). In BMDMs treated with FAs + MSUc, the mitochondria were fragmented into small pieces in parallel with mitochondrial network breakdown (Fig. 6E). Prdx5 overexpression significantly inhibited mitochondrial fragmentation (Fig. 6E). However, in Prdx5-deficient BMDMs, MaR1 almost completely lost its ability to affect the expression of p-AMPK and its downstream targets (Additional file 1: Fig. S5C, D).

Prdx5 relieved impaired fatty acid oxidation induced by FAs + MSUc

It has been reported that Prdx5 regulates fatty acid oxidation (Kim et al. 2020). Fatty acid binding proteins (FABP) 3 and (FABP) 4 are responsible for intracellular shuttling of fatty acids. FABP3 is closely related to fatty acid oxidation (FAO) (Wu et al. 2022). CD36 is a scavenger receptor mediating long-chain fatty acid (FA) uptake (Guerrero et al. 2022). ACSL5 can activate saturated FAs (SFAs) and convert into fatty acyl-CoA esters, especially for palmitic acid (PA, C16:0). Therefore, we wondered whether FAs + MSUc would affect the expression of fatty acid transporters (FATs) and FAO-related enzymes. FAs or MSUc alone could upregulate the expression of FATs (including CD36, FABP3 and FABP4) and FAO-related enzymes (including CPT1A, CPT2 and CACT) to a certain extent, but the combination of FAs and MSUc synergistically increased the expression of these proteins (Fig. S6A). Next, we further investigated the effect of Prdx5 overexpression on the expression of FATs and FAO-related enzymes. Western blot and RT-qPCR analysis indicated that Prdx5 overexpression reduced the expression of FATs and FAO-related enzymes stimulated by FAs + MSUc (Fig. 7A and Additional file 1: Fig. S6B). Immunofluorescence data showed that FAs + MSUc also promoted CD36 expression, and Prdx5 overexpression significantly inhibited CD36 expression induced by FAs + MSUc (Fig. 7B). We further explored whether MaR1 inhibited FAs + MSU crystal-stimulated expression of FATs and FAO-related enzymes through upregulation of the Prdx5 protein. Similar to Prdx5 overexpression, MaR1 treatment attenuated the expression of FATs and FAO-related enzymes induced by FAs and MSUc (Additional file 1: Fig. S7). In Prdx5-deficient macrophages, MaR1 had almost no effect on the FAs + MSU crystal-induced protein expression of FATs and FAO-related enzymes (Additional file 1: Fig. S7). These data suggest that Prdx5 plays a key role in the regulation of fatty acid oxidation by MaR1.

Fig. 7

Prdx5 overexpression ameliorates impaired fatty acid oxidation (FAO). A BMDMs were transfected control (pcDNA3.1) or pcDNA3.1 inserted into the Prdx5 ORF for 24 h and then stimulated with FAs + MSUc for 12 h. Western blot detection of CPT1A, FABP3, CACT, FABP4, CPT2 and CD36 protein levels. B Immunofluorescence staining for CD36. Scale bar: 40 μm. C Non-targeted metabolomics (T500) detection and analysis of metabolite levels (Isovaleryl-carnitine, Isobutyryl-L-carnitine, 2-Methylbutyroylcarnitine, L-palmitoylcarnitine, L-carnitine, Acetyl-carnitine). D Levels of some metabolites (Ornithine, Citrulline, Argininosuccinate and Arginine) in the Urea cycle. E Levels of GABA. F Western blot for ASS1, p-JAK2, p-STAT1, JAK2 and STAT1 protein levels. * Compared with no FAs + MSUc treatment, # compared with FAs + MSUc treatment. * and # means P < 0.05

Since Prdx5 affects the expression of FATs and key enzymes for FAO, we further explored the effect of Prdx5 overexpression on metabolites in BMDMs treated with MSUc. A total of 269 metabolites were evaluated with the T500 untargeted metabolomics assay. There were 43 metabolites that were significantly different between pcDNA3.1 + Vehicle vs pcDNA3.1 + FAs MSU and pcDNA3.1 (FAs MSU) vs Prdx5 overexpression (FAs MSU), and the heat-maps are shown in Additional file 1: Figs. S8 and S9. Acylcarnitine levels are closely related to fatty acid oxidation. MaR1 reversed the decrease in short-chain acylcarnitine levels (including Isovaleryl-carnitine, Isobutyryl-L-carnitine, 2-Methylbutyroylcarnitine, L-carnitine, Acetyl-carnitine) and long-chain acylcarnitine (L-palmitoylcarnitine) (Fig. 7C). MaR1 reduced the elevated ratio of L-palmitoylcarnitine to L-carnitine induced by FAs + MSUc (Fig. 7C), suggesting that MaR1 inhibited the activity of CPT1A, which is the limiting enzyme of FAO. As shown in Additional file 1: Fig. S10A, FAs + MSUc stimulation resulted in a significant decrease in the levels of lysine and methionine which are the precursors for carnitine biosynthesis, but MaR1 treatment greatly upregulated the levels of Lysine and L-Methionine (Additional file 1: Fig. S10A). FAO is associated with ATP production, and we further explored the effect of MaR1 on ATP levels. MaR1 also blocked the decrease in ATP levels induced by FAs + MSUc (Additional file 1: Fig. S10B). In response to FAs + MSUc, there was a decrease in the levels of intermediate metabolites (including Ornithine, L-citrulline, Argininosuccinate and Arginine) in the urea cycle (Fig. 7D), while MaR1 treatment reversed this phenomenon (Fig. 7D). JAK2-STAT1 signaling and ASS1 expression levels have been reported to correlate with the urea cycle (Mao et al. 2022), and Prdx5 overexpression inhibited the JAK2-STAT1 signaling and ASS1 protein expression induced by FAs + MSUc (Fig. 7E). A recent study has reported that GABA inhibits NLRP3 inflammasome activation and IL-1β secretion in macrophages (Fu et al. 2022). Our metabolomics data showed that Prdx5 overexpression significantly upregulated GABA levels in BMDMs treated with FAs + MSUc (Fig. 7F).

MaR1 and Prdx5 are involved in MSU crystal-induced inflammation in vivo

Based on the anti-inflammatory function of MaR1 and Prdx5 in vitro, we investigated whether MaR1 treatment or Prdx5 deficiency influenced the severity of MSU crystal-induced gouty arthritis in vivo. A model of MSU crystal-induced swelling of mouse foot-pad was used to assess the severity of inflammation. After MSU crystal challenge, MaR1 treatment inhibited the increase in the swelling index in WT mice (Fig. 8A). Compared to WT mice injected with MSUc, Prdx5-deficient mice showed an increase in the swelling index, and MaR1 treatment did not improve the swelling index in Prdx5-deficient mice (Fig. 8A). As shown in Fig. 8B, MaR1 treatment inhibited immune cell infiltration, whereas Prdx5 deficiency exacerbated inflammatory cell infiltration (Fig. 8B). Western blot data indicated that MSUc injection into mouse foot-pad tissue resulted in elevated MPO, CPT1A and p-DRP1 protein expression, while PRDX5 protein expression was reduced. In Prdx5+/+ mice injected with MSUc, MaR1 treatment inhibited MPO, CPT1A and p-DRP1 protein expression while promoting PRDX5 protein expression (Fig. 8C). The myeloperoxidase (MPO) activity in a homogenate of foot-pad tissues was analyzed to investigate the recruitment of neutrophils induced by MSUc. MaR1 effectively decreased the MPO activity induced by MSUc (Fig. 8D). Immunofluorescence staining indicated that MaR1 blocked the distribution of CD68-, MPO-, Ly6G- and CPT1A-positive cells in foot-pad tissue sections (Additional file 1: Fig. S11).

Fig. 8

MaR1 inhibits MSUc induced joint inflammation in a mouse model. A Detection and analysis of swelling index of mouse foot-pad. B Detection of immune cell infiltration in mouse foot-pad tissue sections by hematoxylin–eosin (HE) staining. C Western blot detection of p-DRP1, MPO, CPT1A and PRDX5 protein levels in mouse foot-pad tissue. D Detection of MPO activity in mouse foot-pad tissue lysate. *Compared with Prdx5+/+  + Vehicle, # compared with Prdx5+/+  + MSUc, & compared with Prdx5+/+  + MSUc, $ compared with Prdx5+/+  + MSUc + MaR1. *, #, & and $ means P < 0.05. N = 5 each group

We further explored the function of MaR1 and Prdx5 in a model of MSU crystal-induced peritonitis. In comparison with that of WT mice injected with MSUc, the number of leukocytes in the peritoneal lavage fluid (PCLF) of mice treated with MaR1 was significantly reduced after MSU crystal challenge (Fig. 9A). MaR1 also reduced the infiltration of macrophages (CD11b+ F4/80+) and neutrophils (CD11b+ Gr1+) into the peritoneal cavity induced by MSUc (Fig. 9B and Fig. 9C). In WT mice injected with MaR1 + MSUc, MaR1 treatment also greatly attenuated IL-1β secretion in the peritoneal lavage fluid compared to WT mice injected with MSUc (Fig. 9D).

Fig. 9

MaR1 decreases MSUc induced peritoneal inflammation in a mouse model. A–C Detection of leukocytes (CD45), macrophages (F4/80), neutrophils (Gr-1) in peritoneal fluid by FCM. D ELISA for IL-1β levels in peritoneal fluid. N = 5 each group. *Compared with Prdx5+/+  + Vehicle, # compared with Prdx5+/+  + MSUc, & compared with Prdx5+/+  + MSUc, $ compared with Prdx5+/+  + MSUc + MaR1. *, #, & and $ means P < 0.05