Detail of materials are described in the online supplemental file.

Male C57BL/6 mice (No. SCXK (E) 2015-0018) were purchased from Hubei experimental animal research center (Wuhan, China), and Nlrp3 ^−/− mice were purchased from Shanghai Model Organisms Center, Inc. The animals were housed in a temperature-controlled, regular 12-h light/dark cycle and provided with standard chow and water ad libitum. All experimental procedures were performed in accordance with the regulations of the Animal Ethics Committee of South-Central University for Nationalities (SCUN) (approval number: 2020-scuec-025), all procedures involving animals were performed in accordance with the SCUN Animal Experimentation Guidelines.

Bone marrow-derived macrophages (BMDMs) were isolated from bone marrow of 6-8 weeks old mice and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% MCSF for 6–7 days. Human Lymphocyte Separation Medium (catalog no. P8610-200, Solarbio) was used to obtain human peripheral blood mononuclear cells (PBMCs). Prior to stimulation, PBMCs were cultured overnight in RPMI 1640 medium. To activate NLRP3 inflammasome, BMDMs or PBMCs were primed or un-primed with LPS (100 ng/ml, for 3 h) before being stimulated with nigericin (10 μM, for 1 h), or monosodium urate crystals (MSU) (200 μg/ml, for 6 h). To activate the other inflammasomes, salmonella typhimurium or poly A:T was added to the LPS-primed BMDMs.

Healthy adult male mice fed in clean-grade animal houses with free food and water. After 1 week of adaptive feeding, mice were randomly divided into five groups (n = 10 in each group). 1) control group, injected intraperitoneally or given orally with equal volumes of saline; 2) vehicle group, injected intraperitoneally with PO (300 mg/kg) and given orally equal amounts of saline, 3) and 4) methyl gallate groups, injected intraperitoneally with PO (300 mg/kg) and orally given methyl gallate 20 or 40 mg/kg, respectively; 5) allopurinol group, injected intraperitoneally with PO (300 mg/kg) and given orally allopurinol 5 mg/kg. Mice were given saline, methyl gallate or allopurinol by gavage 30 min before PO injection for 28 constitutive days.

Serum urate (Sur), urinary urate (Uur), serum creatinine (Scr), urinary creatinine (Ucr) and blood urea nitrogen (BUN) levels were measured using standard diagnostic kits. Protein concentrations were measured by the Bradford method using bovine serum albumin as a standard. Each test was performed in triplicate. The fraction excretion of uric acid (FEUA) was then calculated to assess the uric acid excretion-promoting effect of methyl gallate: FEUA = [(Uur) × (Scr)]/[(Ucr) × (Sur)] × 100, expressed as a percentage (Chen et al., 2011).

IL-6, IL-1β, TNF-a and IL-18 were detected by ELISA kits. The specific method and steps of the experiment were tested according to the experimental guidance given by R&D.

The liver tissue was removed from −80°C, 900 μL of pre-cooled physiological saline was added per 100 mg of tissue, homogenized in a grinder, and centrifuged at 8,000 r/min (4°C) for 10 min, the supernatant was obtained, and continue to centrifuge at 15,000 r/min for 30 min, remove the uppermost turbid material, and absorb the supernatant. The protein concentration was measured by BCA method, and the activity of XOD in liver and serum was detected by referring to the kit instructions.

BMDMs at 2 × 10⁵/ml were placed overnight on coverslips (Thermo Fisher Scientific) in 12-well plates. 12–18 h later, the medium was replaced with Opti-MEM containing 1% FBS. After that, methyl gallate was added for another 0.5 h as described, and then stimulated with MSU. Finally, BMDMs were stained with 2′,7′-dichlorofluorescin diacetate (DCFDA), and then washed with ice-cold PBS for three times and fixed with 4% PFA in PBS for 15 min.

BMDMs were cultured in 96-well plates (1 × 10⁶ cells/well) using phenol red-free RPMI medium (Gibco) for 24 h at 37°C in a humidified incubator with 5% CO₂. Cells were then loaded with the ROS-specific fluorescent probe H2DCFDA (20 μM final concentration; Sigma-Aldrich) for 30 min, washed twice with preheated medium, and exposed to MSU (200 μg/ml). In the treatment group, cells were incubated with 10 μM CP105,696 for 40 min prior to the addition of the MSU. Fluorescence was assessed at 10-min intervals over 1 h using a spectrofluorometer (Synergy 2; BioTek) with a fluorescein isothiocyanate filter (excitation 485 nm, emission 538 nm).

The left kidney of the mouse was fixed in 4% paraformaldehyde. After paraffin embedding, 4 μm serial sections were taken and H&E staining was performed by conventional methods.

The kidney tissue obtained from the experimental mice in each group was first rinsed gently with pre-chilled 1 × PBS buffer twice, then the kidney tissue was placed in 2 ml EP, 500 μL of protein lysis solution was added, and homogenized with a tissue homogenizer. It was then centrifuged at 4°C and 12,000 rpm for 10 min using a low-temperature high-speed centrifuge. After centrifugation, the supernatant was sucked out and placed in a 1.5 ml EP tube and marked on ice. Then the total protein content was detected by BCA kit.

The above protein samples were denatured in Laemmle buffer and then boiled at 95°C for 10 min, separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight with primary antibodies (caspase 1, IL-1β and GSDMD lysed forms) and proteins were detected by enhanced chemiluminescence with anti-rabbit secondary antibody or anti-mouse secondary antibody.

All experiments were performed at least three times. Values are presented as mean ± S.D. When means of two groups were compared, a 2-tailed Student’s t-test was used to determine significance. When multiple treatment groups were compared, one-way ANOVA and Tukey’s post hoc test were used to calculate significance between means. * p value < 0.05 was considered statistically significant. Data handling and statistical processing were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, United States).

We first investigated whether methyl gallate had the effect on improving renal function and suppressing high uric acid levels in PO-treated mice. According to our previous study, 4 weeks of intraperitoneal injection of PO can lead to HN in mice. Table 1 summarized the anti-hyperuricemia and renal protection effect of methyl gallate. Sur, Scr and BUN levels were significantly increased in the vehicle (PO-treated) mice compared to that of control mice. Methyl gallate at doses of 20 and 40 mg/kg and allopurinol at 5 mg/kg significantly reversed the levels of SUA, SCr and BUN in hyperuricemia mice to the normal value. Moreover, PO treatment could induce an obvious reduction in UUA and UCr levels compared with that of saline-treated mice. Methyl gallate at 20 and 40 mg/kg could significantly elevate UUA and UCr levels in hyperuricemia mice. As previously reported (Hu et al., 2009), FEUA, an important parameter of renal uric acid handling, was significantly reduced in hyperuricemia mice. In the present study, 20 and 40 mg/kg of methyl gallate and allopurinol significantly reversed FEUA in PO-treated HN mice.

HN is a form of chronic tubulointerstitial inflammation caused by the deposition of MSU in the distal collecting duct. Therefore, we further investigated whether methyl gallate could inhibit the chronic inflammation. The results showed that the levels of TNF-α, IL-6 and IL-1β were significantly increased in serum and renal tissues of PO-treated mice. However, correspondingly the production of TNF-α, IL-6 and IL-1β was effectively inhibited by methyl gallate and allopurinol (Figures 1A–C). It was speculated that this anti-inflammatory effect derived from their uric acid-lowering effect, especially allopurinol.

In the present study, the effect of methyl gallate on uric acid production was also investigated. XOD is a key enzyme that catalyzes uric acid production. XOD activity in the liver of HN mice was markedly attenuated by methyl gallate (Figure 1D), indicating that methyl gallate reduces uric acid production through inhibiting XOD activity in vivo.

HN is a chronic renal tubulointerstitial inflammation with histological changes including arteriosclerosis, glomerulosclerosis and tubulointerstitial fibrosis. As aforementioned results, compared to the control mice, the renal tubules of HN mice were significantly damaged, with severe dilatation of the proximal tubules, formation of casts, and massive detachment and necrosis of the tubular epithelium. All these tubulointerstitial lesions were improved to some extent after treatment with allopurinol and different doses of methyl gallate (Figure 1E). In addition, compared to the control mice, there were significant interstitial fibrous-like changes in the renal tissue of HN mice. However, methyl gallate were effective in alleviating these pathological damages, with only mild fibrous-like changes (Figure 1F).

NLRP3 has been reported to sense a variety of stimuli, ranging from bacterial toxins, ATP, crystalline structures such as MSU, silica, and alum (Ghaemi-Oskouie and Shi, 2011; Franklin et al., 2016). The up-regulated expression of NLRP3, ASC, caspase-1 and IL-1β proteins were observed in HN mice, while methyl gallate significantly reversed the levels of NLRP3, ASC, IL-1β and caspase-1 proteins to normal level (Figure 2). These results suggested that methyl gallate had a definite inhibitory effect on NLRP3 inflammasome activity in HN.

To confirm the mechanism of methyl gallate on inhibiting NLRP3 inflammasome activation, the present study was first investigated whether methyl gallate inhibited caspase-1 cleavage and IL-1β secretion in MSU-stimulated BMDMs. It was observed that methyl gallate treatment blocked MSU-induced caspase-1 cleavage, secretion of IL-1β and IL-18, and pyroptosis in BMDMs (Figures 3A–E). Moreover, methyl gallate was reported to inhibit cytokine-induced activation of NF-κB (Ding et al., 2021; Zhang et al., 2021). In consistent with these studies, methyl gallate significantly inhibits the production of TNF-a in MSU-treated BMDMs (Figure 3F). These results indicated that methyl gallate suppressed both NLRP3 inflammasome activation and related gene expression in MSU-treated BMDMs. It is known that ROS provides the upstream signaling for MSU activation of NLRP3 (Gross et al., 2011), it could be inferred that methyl gallate had an inhibitory effect on ROS overproduction in MSU-treated BMDMs. Indeed, methyl gallate decreased the production of ROS by MSU (Figures 3G,H). This result suggested that methyl gallate was a blocker of the upstream pathway of NLRP3 inflammasome activation.

To further confirm the inhibitory effect of methyl gallate on the activation of inflammasomes, whether methyl gallate could inhibit the secretion of IL-1β in LPS-primed BMDMs treated with nigericin was explored. It was found that methyl gallate treatment blocked nigericin-induced IL-1β secretion but had no effect on the production of TNF-α (Figures 4A,B). In contrast, when BMDMs were incubated with methyl gallate for 30 min before 3-h LPS treatment, methyl gallate inhibited the production of TNF-α (Figures 4C,D). These results further suggested that methyl gallate could suppress the two signaling events of NLRP3 inflammasome activation.

The noncanonical activation of NLRP3 inflammasome by intracellular LPS leads to an alternative inflammasome pathway. We tested the release of interleukin-1β in BMDMs stimulated with the TLR2/4 agonist Pam3CSK4, and then transfected wild type and NLRP3 effective cells with LPS. Treatment with methyl gallate reduced the release of IL-1β in BMDMs cells. Cells lacking NLRP3 showed low or no IL-1β production, confirming the requirement of NLRP3 for IL-1β release (Figure 4E).

In addition to nigericin, we also studied the effects of other NLRP3 agonists (including MSU, Alum, and ATP) to understand whether methyl gallate is a co-inhibitor of NLPR3 inflammasome (Figure 4F). Similar to nigericin, methyl gallate significantly blocked these agonists-induced IL-1β secretion. These results indicated that methyl gallate was a potent and broad inhibitor for NLRP3 inflammasome activation. Additionally, methyl gallate had no effect on the activation of NLRC4 or AIM2 inflammasomes, which were triggered by salmonella typhimurium infection or poly A:T transfection, respectively (Figure 4F). Taken together, these results demonstrated that methyl gallate could specifically inhibit the activation of NLRP3 inflammasomes and the subsequent production of IL-1β.

We next used immunoprecipitation (IP) of the NLRP3-ASC association to study the effect of methyl gallate on the formation of NLRP3 inflammasome in BMDMs stimulated by LPS/nigericin. Following the stimulation of LPS and nigericin, ASC was immunoprecipitated with NLRP3 confirming the oligomer formation (Figure 4G). Methyl gallate led to a reduction in the NLRP3-mediated ASC recruitment (Figure 4G, top and bottom). Using the full-length recombinant human NLRP3 protein, we next gauged the effect of methyl gallate on the nucleotide binding domain of NLRP3. Recombinant NLRP3 exhibited ATPase activity and was inhibited by methyl gallate at 10 and 50 μM (Figure 4H). In the same assay, MCC950, a known inhibitor of the NLRP3 inflammasome, was used as a positive control (Coll et al., 2019). In conclusion, these data suggested that methyl gallate reduced the release of mature IL-1β by blocking the oligomerization of NLRP3 inflammasome.

We next examined whether methyl gallate could inhibit NLRP3 inflammasome activation in vivo. As mentioned above, methyl gallate administration significantly improved PO-induced HN (including renal function, high uric acid levels and FEUA) in wild type C57BL/6 mice, but not in Nlrp3 ^−/− mice (Table.2). In addition, methyl gallate administration significantly improved renal histological changes and reduced renal levels of IL-6 and IL-1β in wild type mice but not in Nlrp3 ^−/− mice in PO-induced HN (Figures 5A–C). As expected, methyl gallate treatment significantly reduced TNF-a levels in PO-induced wild type mice. In particular, methyl gallate treatment further reduced TNF-a levels in Nlrp3 ^−/− mice (Figure 5D). Thus, these findings suggested that methyl gallate protected against inflammation and tissue damage by inhibition of NLRP3 inflammasome.

The recruitment of neutrophils and tissue infiltration are hallmarks of the inflammatory response to injury and infection. Thus, we evaluated the effect of methyl gallate on PBMCs after LPS and ATP treatment. As expected, methyl gallate prevented the release of IL-1β (Figure 6A) without affecting the production of TNF-a in PBMCs (Figure 6B). Methyl gallate (10 and 50 μM) also reduced caspase-1 activity in PBMCs (Figure 6C). Therefore, these results suggested that methyl gallate could prevent the activation of NLRP3 in human cells.

Finally, we assessed whether methyl gallate could affect the pre-activated NLRP3 inflammasomes on patient cells with abnormal NLRP3 activation. As expected, IL-1β secretion could be detected in the culture supernatant from synovial fluid cells (SFCs) isolated from two patients with gout arthritis were cultured in the absence of NLRP3 agonist stimulation (Figure 6D). However, when these cells were incubated with methyl gallate (50 μM), IL-1β production were reduced by 65 and 40%, respectively (Figure 6D). In reverse, TNF-α production was unaffected by methyl gallate treatment (Figure 6E). These results suggested that methyl gallate could inhibit the pre-activated NLRP3 inflammasome in patients’ SFCs, supporting the clinical use of methyl gallate for the control of NLRP3-driven disease.