CN114028376A

CN114028376A - Application of MG in preparation of NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis

Info

Publication number: CN114028376A
Application number: CN202111432521.0A
Authority: CN
Inventors: 陈旅翼; 刘鹏
Original assignee: Wuhan Yibo Jisheng Biotechnology Co ltd
Current assignee: Wuhan Yibo Jisheng Biotechnology Co ltd
Priority date: 2021-09-17
Filing date: 2021-11-29
Publication date: 2022-02-11

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of gallic acid methyl ester (MG) in preparation of an NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis. The gallic acid methyl ester can specifically inhibit activation of NLRP3 inflammatory bodies in BMDMs and PBMCs, and has no influence on activation of NLRC4 or AIM2 inflammatory bodies; and gallic acid methyl ester is a broad inhibitor of NLRP3 inflammasome activation: can inhibit the activation of NLRP3 inflammatory bodies stimulated by agonists such as LPS/MSU, alum, ATP and nigericin. In addition, the gallic acid methyl ester is widely distributed in various edible plants, and the source is wide. Therefore, the gallic acid methyl ester is expected to be developed into an NLRP3 pathway inhibitor for treating hyperuricemia nephropathy and/or gouty arthritis diseases by using the gallic acid methyl ester as an active ingredient.

Description

Application of MG in preparation of NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of gallic acid methyl ester (MG) in preparation of an NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis.

Background

Hyperuricemia is a metabolic disease caused by imbalance in uric acid synthesis or excretion in the body. Hyperuricemia is a state in which the blood uric acid level of adult male is not less than 420. mu. mol.L-1, and the blood uric acid level of adult female is not less than 324. mu. mol.L-1. Hyperuricemia is closely related to the occurrence of HN, and is an important stage of gout patients. Studies have shown that hyperuricemia is an important risk factor for gout, hypertension, diabetes, cardiovascular complications, metabolic syndrome, and kidney disease. There is increasing evidence that uric acid-induced inflammation is the central mechanism of hyperuricemia rodent kidney injury, and that the NLRP3 pathway may play a central role therein. At present, the marketed drugs for treating Hyperuricemia Nephropathy (HN) mainly comprise XOD inhibitors, uricosuric drugs, recombinant uricase supplements and the like, but the wide clinical application of the drugs is limited due to the great adverse reaction and hepatotoxicity of the drugs. In the treatment of HN, there is a need for a drug that both reduces circulating uric acid synthesis and increases renal uric acid excretion, while ameliorating renal inflammation, renal fibrosis, oxidative stress and other injuries.

Polyphenols have potent antioxidant, immunomodulating and anti-inflammatory effects, have a wide range of therapeutic effects, and have potential effects in different complications caused by oxidative stress, such as cardiovascular and neurodegenerative diseases. In recent years, polyphenols have received much attention for their potential in modulating immune signaling pathways, inhibiting inflammatory mediators, modulating ischemia reperfusion injury, neurodegenerative diseases, neuropathic pain and arthritis, among many pathological conditions.

Gallic acid methyl ester (MG), is a gallotannin, and is widely distributed in edible plants such as Rosa multiflora, Terminalia catappa, Smilax china, Wuren, Burdock, Ginkgo biloba, and peony. Several studies have shown that MG is associated with significant biological effects in these plants, such as their antioxidant, antitumor and antibacterial activities. In this study, we investigated the role of MG in HN and its molecular mechanism of anti-inflammatory activity in the context of NLRP3 inflammasome activation.

Disclosure of Invention

The invention aims to provide application of gallic acid methyl ester as an NLRP3 pathway inhibitor of hyperuricemia nephropathy and/or gouty arthritis.

Furthermore, the hyperuricemia nephropathy and the gouty arthritis are all caused by the induction of urate crystallization.

Preferably, the gallic acid methyl ester is used as an NLRP3 pathway inhibitor of hyperuricemia nephropathy.

Compared with the prior art, the invention has the advantages and beneficial effects as follows:

the invention provides a potential NLRP3 pathway inhibitor for treating hyperuricemia nephropathy and/or gouty arthritis diseases: gallic acid methyl ester. The gallic acid methyl ester can specifically inhibit activation of NLRP3 inflammatory bodies in BMDMs and PBMCs, and has no influence on activation of NLRC4 or AIM2 inflammatory bodies; and gallic acid methyl ester is a broad inhibitor of NLRP3 inflammasome activation: can inhibit the activation of NLRP3 inflammasome stimulated by agonists such as MSU, alum, silica, ATP, Pam3CSK4 and LPS/nigericin. In addition, the gallic acid methyl ester is widely distributed in various edible plants, and the source is wide. Therefore, the gallic acid methyl ester is expected to be developed into an NLRP3 pathway inhibitor for treating hyperuricemia nephropathy and/or gouty arthritis diseases by using the gallic acid methyl ester as an active ingredient.

Drawings

FIG. 1 is a graph showing the effect of MG on PO-induced HN. Wherein: (A-C) ELISA kit detects cytokines such as IL-6(A), IL-1 beta (B) and TNF-a (C) in kidney. (D) The activity of XOD in liver was determined with reference to kit instructions. (E and F) Kidney of different groups of miceHE staining (E) or Masson staining (F), scale bar, 100 μm. a: comparison; vehicle (PO treated); c: allopurinol (AL, 5 mg/kg); d: MG (20 MG/kg); e: MG (40 MG/kg). Values are expressed as mean ± SEM. Data were analyzed by one-way anova and Tukey HSD test. Compared to the control:^###P<0.001; compared with PO: p<0.05，**P<0.01，***P<0.001。

Fig. 2 is a graph of the effect of MG on the renal NLRP3 pathway. Wherein: (A) representative Western blot analysis of NLRP3, ASC, Caspase-1 and IL-1. beta. in the kidney of different groups of mice. (B-E) Western blot quantitative analysis of NLRP3, ASC, Caspase-1 and IL-1. beta. in the kidneys of different groups of mice. Values are expressed as mean ± SEM, n ═ 3. Data were analyzed by one-way anova and Tukey HSD test. Compared to the control:^#P<0.05，^##P<0.01; compared with PO: p<0.05，**P<0.01。

FIG. 3 is a graph showing the specific inhibition of activation of NLRP3 inflammasome in BMDMS by MG. Wherein: (A) western blot analysis of the inhibitory effect of MG (10 and 50. mu.M) on cleaved IL-1. beta., activated caspase-1(P20) and pro-IL-1. beta., pro-caspase-1 in culture Supernatant (SN), lysates of MSU-stimulated BMDMs. (B and C) inhibition of IL-1 β secretion and pro-IL-1 β release in MSU-stimulated BMDMs by MG (10 and 50. mu.M). Western blot analysis of the inhibitory effect of (D and E) MG (50. mu.M) on the expression of GSDMD (P30, P50 refer to the two C-and N-terminal domains of GSDMD, respectively) in MSU-stimulated BMDMs. (F) Inhibition of TNF- α secretion in BMDMs by MSU-stimulated LPS by MG (10 and 50 μ M). (G, H) inhibition of ROS production in MSU-stimulated BMDM by MG (50. mu.M) was determined by ROS (G) and ROS staining (H). MG: gallic acid methyl ester, Veh: vector, Con: and (6) comparison. Data are presented as mean ± SEM (n ═ 6) and represent two independent experiments. Statistical differences were calculated by one-way anova with Tukey HSD test (B-F) and unpaired student t test (G): p <0.01, P < 0.001.

FIG. 4 is a graph of MG-specific inhibition of activation of NLRP3 inflammasome in BMDMs. Wherein: (A and B) BMDMs were treated with different doses of MG for 0.5 hours and then stimulated with LPS for 3 hours. Thereafter, cells were stimulated with nigericin. The medium Supernatants (SN) were analyzed by ELISA for IL-1. beta. and TNF-. alpha.release. (C and D) BMDMs were stimulated with LPS for 3 hours, then treated with various doses of MG for 0.5 hours, and finally stimulated with nigericin. IL-1. beta. and TNF-. alpha.release in SN was analyzed by ELISA. (E) IL-1 β release from BMDMs was stimulated with the TLR2/4 agonist, Pam3CSK4, followed by transfection of WT and NLRP 3-deficient cells with LPS. Treatment with MG (10, 50. mu.M) reduced IL-1. beta. release in BMDMs cells. Cells lacking NLRP3 show low or no IL-1 β production. (F) After LPS-stimulated BMDMs were treated with MG (50. mu.M), BMDMs were stimulated with MSU, nigericin, ATP, alum, poly A: T and Salmonella, and IL-1. beta. expression in BMDMsSN was detected by ELISA. (G) Immunoprecipitation (IP) and analysis (Mean + -SEM) of NLRP3-ASC in LPS/nigericin-stimulated BMDMs cells. (H) Recombinant NLRP3 atpase activity (n-3) in the presence of MG (n-3) and MCC950(1 μ M). MG: gallic acid methyl ester, Veh: vector, Con: and (6) comparison. Data represent Mean ± SEM (n ═ 6). Statistical differences were determined by one-way anova with Tukey HSD test (a-D) and unpaired student t test (E and F): p <0.01, P < 0.001.

Fig. 5 is a graph of the effect of MG on the renal NLRP3 signaling pathway. Wherein: (A) HE staining of the kidneys of different groups of mice. (B-D) ELISA kit detects cytokines such as IL-6(B), IL-1 beta (C) and TNF-alpha (D) in kidney. Scale bar, 100 μm. a: WT; b: WT + MG (40 MG/kg); c: NLRP3^-/-；d：NLRP3^-/-+ MG (40 MG/kg). Values are expressed as Mean ± SEM. Data were analyzed by one-way anova and Tukey HSD test. Compared to the control:^###P<0.001; compared with PO: p<0.05，**P<0.01，***P<0.001。

FIG. 6 shows the effect of MG on cell inflammation in healthy or gout patients. Wherein: (A and B) IL-1. beta. (A) or TNF-. alpha. (B) levels in PBMCs supernatants stimulated with LPS and ATP following MG (10 and 50. mu.M) treatment. (C) Caspase-1 activity in human PBMCs after LPS + ATP stimulation in the presence of MG (10 and 50. mu.M). (D and E) MG (50. mu.M) for 20h, ELISA was performed to detect the expression of IL-1. beta. (D) and TNF-. alpha. (E) in supernatants of SFCs from gout patients. MG: gallic acid methyl ester, Veh: vector, Con: and (6) comparison. Data were analyzed as Mean ± SEM (n ═ 6) for PBMCs from 3 healthy donors and SFCs from 2 patients. Statistical differences were calculated by one-way analysis of variance using Tukey HSD test (a and B) or 2-way analysis of variance using post hoc Sidak test for multiple comparisons (C) or unpaired student t-test (D and E): p <0.05, P <0.01, P < 0.001.

FIG. 7 is a graph showing the effect of MG on gouty arthritis in mice. Wherein: effects of MG at doses (20 and 40MG/kg) on toe swelling (A) and hind limb weight bearing (B) in mice. Statistical differences were calculated by one-way analysis of variance using TukeyHSD test (a and B) or two-way analysis of variance using multiple comparisons using post hoc Sidak test or unpaired student t-test: compared to the control:^###P<0.001; compared with the MSU: p<0.05，**P<0.01，***P<0.001。

Fig. 8 is a graph of HN mechanization with MG improved PO induction.

Detailed Description

The following is a further description of the technical solution of the present invention by the applicant with reference to the specific embodiments and the drawings, but the scope of the claims of the present invention is not limited to these embodiments.

1 materials and methods

1.1 Experimental animals

Male C57 mice (accession number: SCXK (E)2015-0018) were purchased from the research center for experimental animals in Hubei (Wuhan, China), NLRP3^-/-Mice (NLRP3 group knockout mice) were purchased from the shanghai model organism center. A constant temperature environment, regular 12 hour light/dark cycles, and standard food and free drinking water are provided. All experimental procedures were performed according to the SCUN animal ethics Committee (approval No. 2020-scuec-025) and all animal-related procedures were performed according to the SCUN animal Experimental guidelines.

1.2 cell culture and stimulation

BMDMs (bone marrow macrophages) were isolated from bone marrow of 6-8 week old mice and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 1% MCSF for 6 to 7 days. Human lymphocyte isolation medium (catalog No. P8610-200, Solarbio) was used to obtain human PBMCs (human peripheral blood mononuclear cells). PBMCs were cultured overnight in RPMI1640 medium supplemented with 10% FBS, 100U/mL penicillin, 100. mu.g/mL streptomycin.

To activate the NLRP3 inflammasome, BMDMs or PBMCs were primed with LPS (100ng/mL, 3h) prior to stimulation with nigericin (10. mu.M, 1h) or MSU (200. mu.g/mL for 6 h). To activate another inflammasome, LPS-primed BMDMs were supplemented with Salmonella typhimurium or poly A: T.

1.3 reagents

Potassium Oxonate (PO), Colchicine (Colchicine), sodium urate crystals (MSU) and MG (methyl gallate) were all purchased from Sigma, where MG was used: animal experiments were performed using 0.5% CMCNa, cell experiments were performed using sterile PBS, and cytokine detection ELISA kits were purchased from R & D. The biochemical index detection kit is a product of Nanjing-built biological Limited company. The BCA protein detection kit is a product of Shanghai Biyuntian biology limited company. All antibodies were purchased from Cell Signaling Technology.

1.4 animals and models

Establishing an HN animal model: healthy adult male ICR mice were fed in SPF animal houses and were fed ad libitum. After 1 week of adaptive feeding, mice were randomly divided into control groups, PO, MG (divided into two groups of low and high doses: 20MG/kg, 40MG/kg) and allopurinol (5MG/kg) drug-treated groups, 10 per group. PO, MG and allopurinol drug treatment groups were injected intraperitoneally with PO (300MG/kg) to establish HN animal models, and control mice were injected with an equal volume of PBS solution (1 XPBS, 0.01mol/L, pH 7.2-7.4). MG (20MG/kg, 40MG/kg) and allopurinol (5MG/kg) were intragastrically administered 30min before drug therapy, and equal volume of PBS solution (1 XPBS, 0.01mol/L, pH 7.2-7.4) was administered to the other groups for 28 consecutive days. Taking the drug treatment group as an example: the administration of a certain amount of therapeutic drug and PO (300mg/kg) was performed once a day for 28 consecutive days.

Establishing a gouty arthritis animal model: healthy adult male C57 mice were fed in SPF animal houses and were freely fed. After 1 week of adaptive feeding, mice were randomized into 10 control, MSU, MG (low and high dose groups: 20MG/kg, 40MG/kg) and Colchicine (0.8MG/kg) drug-treated groups. The MSU, MG and Colchicine drug treatment groups injected 20 μ L200 μ g/mLMSU to one side of ankle joint of mice to establish the animal model of gouty arthritis, and the control group injected equal volume of PBS solution (1 XPBS, 0.01mol/L, pH 7.2-7.4). MG (20MG/kg, 40MG/kg) and Colchicine (0.8MG/kg) were gavaged 30min before drug treatment group modeling, and equal volume of PBS solution (1 XPBS, 0.01mol/L, pH 7.2-7.4) was given to the other groups for 3 consecutive days. Taking the drug treatment group as an example: one day for 3 consecutive days, gavage a certain amount of therapeutic drug and inject 20 μ L of 200 μ g/mL MSU, respectively, each day.

After the animal model is established, indexes such as uric acid, urea nitrogen, creatinine content and the like in a mouse are detected. The difference compared with the blank group has statistical significance (P <0.05), which indicates that the HN molding of the mice is successful. The difference between the ankle joint swelling of the mice and the blank group has statistical significance (P is less than 0.05), and the success of gouty arthritis modeling is shown.

1.5 measurement of uric acid, creatinine and Urea Nitrogen

Serum Uric Acid (SUA), Urinary Uric Acid (UUA), Serum Creatinine (SCR), Urinary Creatinine (UCR) and urea nitrogen (BUN) levels were measured using standard diagnostic kits. Protein concentration was determined according to the Bradford method using bovine serum albumin as a standard. Each assay was performed in triplicate. FEUA was then calculated as follows to assess uricosuric effect of MG: FEUA ═ ([ UUA ] × [ SCR ])/([ UCR ] × [ SUA ]) × 100, expressed in percentage.

1.6 enzyme-linked immunosorbent assay (ELISA)

Cytokines were detected by ELISA kit. The specific methods and procedures of the experiment were tested according to the reagent instructions.

1.7 measurement of Xanthine Oxidase (XOD) Activity

Liver tissue was removed at-80 ℃, 900. mu.L of pre-cooled physiological saline was added per 100mg of tissue, the homogenate was ground, centrifuged at 8000rpm (4 ℃) for 10min, the supernatant was taken, centrifuged at 15000rpm for 30min again, the uppermost turbid material was removed, and the supernatant was aspirated. The BCA method measures protein concentration and detects XOD activity in liver and serum with reference to kit instructions.

1.8 ROS staining

2 x 10 to⁵The/ml BMDMs were spread on a coverslip (Thermo Fisher Scientific) on a 12-well plateOvernight. After 12-18 hours, the medium was changed to Opti-MEM containing 1% FBS. Thereafter, 50. mu. mol/L MG was added and cultured for 0.5 hour. BMDMs were stimulated with LPS and stained with CD31 and GSDMD or 2',7' -dichlorofluorescein diacetate (DCFDA), then cells were washed 3 times with cold PBS solution (1 × PBS, 0.01mol/L, pH 7.2-7.4) and fixed with 4% PFA for 15 min. Thereafter, cells were washed 3 times with PBST (1 ×, pH 7.5) (PBS plus Tween-20).

1.9 active oxygen measurement

BMDMs in 96-well plates (1X 10)⁶Individual cells/well) were cultured in phenol red-free RPMI1640 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) in a humidified incubator at 37 ℃ for 24 hours, 5% carbon dioxide. Cells were then added with ROS-specific fluorescent probe H2DCFDA (2',7' -dichlorofluorescein diacetate, 20. mu.M final concentration; Sigma-Al drich) for 30 minutes, washed twice with pre-warmed medium, and exposed to MSU (200. mu.g/mL). In the treatment group, cells were incubated with 10 μ MCP105,696 for 40 minutes before adding MSU. Fluorescence was assessed every 10 minutes over 1 hour using a spectrofluorimeter (Synergy 2; BioTek) with fluorescein isothiocyanate filters (excitation 485nm, emission 538 nm).

1.10H & E staining, Masson staining

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

The left kidney of the mouse was fixed with 4% paraformaldehyde. After paraffin embedding, 4 μm serial sections were taken and Masson staining was performed by a conventional method.

1.11 protein extraction and Western blotting

The kidney tissue (right kidney) of each group of experimental mice was taken, gently rinsed 2 times with pre-cooled 1 × PBS buffer, then the kidney tissue was placed in a 2ml lep tube, 500 μ L of protein lysate was added, and homogenized with a tissue homogenizer. The mixture was then centrifuged using a low temperature high speed centrifuge at 2000rpm4 ℃ for 10 minutes. After centrifugation, the supernatant was aspirated, placed in a 1.5mL EP tube, and labeled. The total protein content was then measured with the BCA kit.

Cell lysates and culture supernatants were denatured in 2 × protein loading buffer (150mM Tris-HClpH 6.8, 20% glycerol, 10% SDS, 10% dithiothreitol, 4% β -mercaptoethanol, and bromophenol blue). The protein samples were then boiled at 95 ℃ for 10 minutes and separated by SDS-PAGE.

Protein samples were separated by SDS-PAGE. The separated proteins were transferred to PVDF membrane. Immunoblots were incubated overnight with primary antibodies and proteins were detected by enhanced chemiluminescence (GE Healthcare, RPN2236) using an anti-mouse secondary antibody.

1.12 measurement of swelling volume of molded lateral toes

Marking 0.5mm on the ankle joint of the left hind limb of the mouse by using a mark pen before molding, measuring the drainage volume from the left hind foot of the mouse to the marked position by using a toe volume measuring instrument, recording, and measuring twice and taking the average value as the toe volume measuring value of 0 h. After molding, toe volume values of 3h, 6h, 9h, 12h, 24h and 48h were measured by the same method and recorded.

Toe swelling rate was 100% × (toe volume measurement-0 h toe volume measurement) 0h toe volume measurement at the time point after molding

1.13 determination of differences in balance between the two feet of mice

The mouse is placed in a double-foot balance pain measuring instrument, after the mouse joint is modeled, the bearing of the left hind limb and the bearing of the right hind limb are changed immediately, and the joint pain of the mouse is measured by measuring the difference of the bearing of the hind limbs of the mouse. Before the experiment, the pedal is adjusted to zero in no-load mode, the instrument is calibrated, the measuring time is set to be 5s, the mouse is placed into the fixator, forelimbs of the mouse are erected on the fixator, the left foot and the right foot stand at the centers of the left pedal and the right pedal respectively, and the measurement is started after the animal is quiet. Three times were measured and the mean pressure values (R-L) were recorded three consecutive times in the resting state of the mice.

The rate of difference in balance between feet is 100% × (value on molding side-value on non-molding side)/value on non-molding side

1.14 statistical data

Values are mean ± Standard Error of Mean (SEM). In comparing the mean of the two groups, a two-tailed student's t-test was used to determine significance. In the case of comparing multiple treatment groups or genotypes, significance between the means was calculated using one-way analysis of variance and Tukey test to calculate P-value. P values <0.05 were considered statistically significant.

2 results

2.1 Effect of MG on hyperuricemia and renal insufficiency

We first investigated whether MG had the effect of improving renal function and inhibiting hyperauric acid levels in PO-treated mice. According to our previous study, intraperitoneal injections of PO for 4 weeks (once a day, 300mg/kg each) resulted in mice HN. Table 1 shows the anti-hyperuricemic and nephroprotective effects of MG. The levels of SUA, SCR and BUN were significantly increased in PO-treated (Vehicle) mice compared to control mice. MG doses of 20MG/kg and 40MG/kg significantly reduced the SUA, SCR and BUN levels in hyperuricemic mice to normal values, while allopurinol at 5MG/kg also significantly reduced the SUA, SCR and BUN levels. Furthermore, PO treatment can significantly reduce UUA and UCR levels compared to control (PBS treated) mice. The doses of MG 20MG/kg and 40MG/kg can obviously improve the UUA and UCR levels of mice with hyperuricemia. As previously reported, FEUA was significantly reduced in PO-treated mice as an important renal uric acid treatment parameter. In this study, MG and allopurinol at 20MG/kg and 40MG/kg doses significantly reversed FEUA.

HN is a chronic tubulointerstitial inflammation caused by the deposition of urate crystals in the distal collecting vessels. We therefore further investigated whether MG could inhibit this chronic inflammation. The results show that TNF-alpha, IL-6 and IL-1 beta levels are significantly elevated in serum and kidney tissues of PO-treated mice. However, the production of TNF- α, IL-6 and IL-1 β was correspondingly effectively inhibited by MG and allopurinol (FIGS. 1A-C). We speculate that this anti-inflammatory effect stems from their uric acid lowering effect, especially allopurinol.

In this study, we also investigated the effect of MG on uric acid production. We evaluated the inhibitory activity of MG on XOD (xanthine oxidase, a key enzyme catalyzing the production of uric acid). The results showed that MG significantly attenuated the increase in XOD activity in the liver of HN mice (fig. 1D). This confirms the mechanism by which MG reduces uric acid production by inhibiting XOD activity in vivo.

HN is a chronic tubulointerstitial inflammation of the kidney, with histological changes including arteriosclerosis, glomerulosclerosis and tubulointerstitial fibrosis. As described above, in HN mice, tubular injury was evident in the cortex, proximal tubules were severely dilated, casts were formed, and tubular epithelium was largely detached and necrosed, compared to control mice. These tubulointerstitial lesions were improved to some extent after treatment with allopurinol and various doses of MG (fig. 1E). Furthermore, PO-treated mice showed significant interstitial fiber-like changes in kidney tissue compared to control mice. However, different doses of MG were effective in reducing this pathological damage to the kidney with only slight fibrosis (fig. 1F).

Table 1 effect of MG on PO-induced HN mouse serum and uric acid, creatinine levels and metabolic rate, urea nitrogen.

Note: control: normal control group, Vehicle: HN control, AL: allopurinol. Values are expressed as mean ± SEM. Statistical differences were calculated by one-way anova and Tukey HSD test. Compared to the control:^#P<0.05，^##P<0.01，^###P<0.001; compared with PO: p<0.05，**P<0.01，***P<0.001。

2.2 Effect of MG on renal NLRP3 pathway

NLRP3 is reported to be able to perceive a variety of stimuli, including bacterial toxins, ATP, crystalline structures such as sodium urate (MSU), silica and alum. Upregulation of protein expression levels of NLRP3, ASC, Caspase-1 and IL-1 β was observed in PO-treated mice, whereas MG significantly reversed protein levels of NLRP3, ASC, IL-1 β, Caspase-1 to normal levels after MG drug treatment (FIG. 2). These results indicate that MG has a clear inhibitory effect on NLRP3 inflammatory body activity in HN.

2.3 MG-specific inhibition of NLRP3 inflammasome activation in BMDMs

To demonstrate the mechanism by which MG inhibits NLRP3 inflammasome activation, we first examined whether MG inhibits caspase-1 cleavage and IL-1 β secretion in MSU-stimulated BMDMs. The experimental steps of the activation of the NLRP3 inflammatory body by MSU stimulation are as follows: after incubation of BMDMs with MG (10. mu. mol/L, 50. mu. mol/L) for 30min, pre-induction was performed with LPS (100ng/mL) for 3h, followed by continuous stimulation with MSU (200. mu.g/mL) for 6 h. We did observe that treatment with MG blocks MSU-induced caspase-1 cleavage, secretion of IL-1. beta. and IL-18, and apoptosis of cells in BMDMs (FIGS. 3A-E). MG has been reported to inhibit cytokine-induced NF-. kappa.B activation. We then examined whether MG inhibited TNF-. alpha.production in MSU-treated BMDMs. As expected, MG significantly inhibited TNF- α production in this case (fig. 3F). These results indicate that MG can inhibit NLRP3 inflammatory body activation and related gene expression in MSU-treated BMDM. Since our work above suggests that ROS provide an upstream signal for MSU to activate NLRP3, we conclude that MG has an inhibitory effect on ROS levels in MSU-treated BMDMs. In fact, MG reduced ROS produced by MSU (fig. 3G and 3H). This result indicates that MG is a blocker of NLRP3 inflammasome activation of the upstream pathway.

2.4 MG-specific inhibition of activation of NLRP3 inflammatory bodies in macrophages

To further confirm the inhibitory effect of MG on inflammasome activation, we tested whether MG could inhibit IL-1 β secretion in BMDMs stimulated with nigericin. The experimental steps for stimulating the activation of NLRP3 inflammatory bodies by nigericin are as follows: (1) before LPS: MG (10. mu. mol/L, 50. mu. mol/L) was added to BMDMs and incubated for 30min, pre-induced with LPS (100ng/mL) for 3h, and then stimulated with nigericin (10. mu. mol/L) for 1 h. (2) After LPS: LPS (100ng/mL) was added to BMDMs for pre-induction for 3h, and after incubation for 30min with MG (10. mu. mol/L, 50. mu. mol/L), stimulation was continued with nigericin (10. mu. mol/L) for 1 h. We have found that: MG was added after LPS pre-induction of BMDMs, and MG treatment blocked Nigericin-induced IL-1 β secretion but had no effect on TNF- α production (FIGS. 4A and 4B). In contrast, when BMDMs were incubated with MG for 30 minutes prior to LPS treatment, MG inhibited LPS-induced TNF- α production (fig. 4C and 4D). These results further indicate that MG can inhibit two signaling events (IL-1. beta., TNF-. alpha.) for activation of NLRP3 inflammasome.

Intracellular LPS pairNon-classical activation of NLRP3 inflammasome leads to another inflammasome pathway. We tested WT and NLRP3 transfected with BMDMs and LPS stimulated with TLR2/4 agonist Pam3CSK4, lacking release of IL-1 β in cells, with specific experimental steps: adding MG (10. mu. mol/L, 50. mu. mol/L) into BMDMs/NLRP3^-/-After 30min incubation, pre-induction with LPS (100ng/mL) was performed for 3h, and additional Pam3CSK4 (1. mu.g/mL) was added for continuous stimulation for 3 h. MG therapy may reduce IL-1 β release in BMDMs cells. Cells lacking NLRP3 showed low or no secretion of IL-1 β, confirming that IL-1 β release requires NLRP3 in this model (FIG. 4E).

In addition to nigericin, we also investigated the effects of other NLRP3 agonists (including MSU, alum and ATP) to see if MG is a broad inhibitor of NLPR3 inflammasome (fig. 4F), and the experimental steps for agonist (MSU, alum or ATP) stimulation of activation of NLRP3 inflammasome were: MG (50. mu. mol/L) was added to BMDMs and incubated for 30min, pre-induced with LPS (100ng/mL) for 3h, and then stimulated with agonists (MSU: 200. mu.g/mL, alum: 300. mu.g/mL, ATP: 5mmol/L) for 1 h. Similar to nigericin, MG can significantly block IL-1 β secretion induced by these agonists. These results indicate that MG is a potent and broad inhibitor of NLRP3 inflammasome activation. Furthermore, MG (50. mu. mol/L) had no effect on the activation of NLRC4 or AIM2 inflammasome, which were triggered by Salmonella typhimurium (1. mu.g/mL) infection or poly A: T (5. mu.g/mL) transfection for 6h, respectively (FIG. 4F). Taken together, these results indicate that MG can specifically inhibit the activation of NLRP3 inflammasome and the subsequent production of IL-1 β.

We next investigated the effect of MG on NLRP3 inflammatory body formation in LPS/nigericin-stimulated BMDMs cells using NLRP 3-ASC-linked Immunoprecipitation (IP), with specific experimental steps: the cells were washed with pre-cooled PBS and then harvested and lysed by adding 500. mu.L of lysis buffer (50mM Tris-HCl, pH 7.6, 0.5% Triton X-100, 0.1mM PMSF). The lysate was centrifuged at 3500rpm for 10 minutes at 4 ℃. Following stimulation with LPS and nigericin, ASCs were immunoprecipitated with NLRP3, confirming oligomer formation following inflammatory body stimulation (fig. 4G). NLRP 3-mediated ASC recruitment was reduced following MG treatment (fig. 4G). Using the full-length recombinant human NLRP3 protein, we next measured the effect of MG on the NLRP3 nucleotide binding domain. Recombinant NLRP3 showed ATPase activity, inhibited at MG concentrations of 10. mu.M and 50. mu.M (FIG. 4H). In the same experiment, the known inhibitor MCC950 (1. mu. mol/L) of the NLRP3 inflammasome was used as a positive control. Taken together, these data indicate that MG reduces release of mature IL-1 β by preventing NLRP3 inflammasome oligomerization.

2.5 MG inhibition of NLRP3 dependent inflammation in vivo

We next examined whether MG could inhibit NLRP3 inflammatory body activation in vivo, with specific experimental steps: experimental groups: taking WT mice/NLRP 3^-/-Intragastric administration of MG (40MG/kg) to mice, 30min later, intraperitoneal injection of PO (300MG/kg), once a day for 28 consecutive days; control group: WT mouse/NLRP 3^-/-Mice were injected with an equal volume of PBS solution (1 XPBS, 0.01mol/L, pH 7.2-7.4) to MG/PO. As described above, administration of MG significantly improved PO-induced HN (including renal function, elevated uric acid levels, and FEUA) in WT mice, but at NLRP3^-/-There were no changes in the mice (table 2). Furthermore, in PO-induced HN, MG administration significantly improved renal histology and reduced renal levels of IL-6 and IL-1 β in WT mice, but at NLRP3^-/-Mice did not (FIGS. 5A-C). As expected, MG treatment significantly reduced the PO-induced TNF- α levels in WT mice. In particular, MG treatment further reduced NLRP3^-/-TNF- α levels in mice (FIG. 5D). Thus, these results indicate that MG prevents inflammation and tissue damage by inhibiting NLRP3 inflammasome.

TABLE 2MG vs PO-induced WT and NLRP3^-/-Effects of serum and uric acid, creatinine levels, and FEUA and BUN in HN mice.

Note: values are expressed as mean ± SEM. Statistical differences were calculated by one-way anova and Tukey HSD test. Compared with WT: p <0.05, P <0.01, P < 0.001. Compared to NLRP 3-/-: a, indicates insignificant.

2.6 in vivo Activity of MG on cells of healthy persons or gout patients

Recruitment of neutrophils and tissue infiltration are hallmarks of inflammatory responses to injury and infection. Therefore, we evaluated the effect of MG on LPS and ATP-treated PBMCs. The method comprises the following specific steps: MG (10. mu. mol/L, 50. mu. mol/L) was added to PBMCs and incubated for 30min, pre-induced with LPS (100ng/mL) for 3h, and stimulated with ATP (5mmol/L) for 45 min. As expected, MG prevented IL-1 β release (FIG. 6A) without affecting TNF- α production in HBN (FIG. 6B). MG (10 and 50. mu.M) also reduced caspase-1 activity in PBMCs (FIG. 6C). Thus, these results indicate that MG can prevent activation of NLRP3 in human cells.

Finally, we assessed whether MG would affect pre-activated NLRP3 inflammasome in patient cells with aberrant activation of NLRP 3. As expected, IL-1 β secretion could be detected in culture supernatants of freshly isolated synovial cells (SFCs) of two gout patients cultured without NLRP3 agonist stimulation (fig. 6D). However, when these cells were incubated with MG (50 μ M), IL-1 β production was reduced by 65% and 40%, respectively (fig. 6D). In contrast, TNF-. alpha.production was not affected by MG treatment (FIG. 6E). These results indicate that MG can inhibit pre-activated NLRP3 inflammasome in patient SFCs, and that MG is clinically useful in the control of NLRP3 driven diseases.

2.7 Effect of MG on gouty arthritis in mice

We investigated whether MG had an effect in ameliorating gouty arthritis in MSU-treated mice. We used the toe swelling rate to indicate the severity of swelling. Compared with the control group, the swelling degree of the joint at different time points after MSU molding is obviously increased (P <0.001), the swelling starts to be obvious 3h after the MSU molding, and then the increase amplitude is increased, and 9h reaches the peak value (figure 7A). Mice were shown to have an inhibitory effect on toe swelling at different time points after treatment with Colchicine and different doses of MG. The biped balance experiment can detect the body weight born by the MSU modeling side of the mouse and can reflect the anti-inflammatory and analgesic effects of the medicament. We used the rate of difference in bipedal balance to represent the change in weight bearing in rats. Hind limb weight bearing was significantly reduced in MSU-treated mice at different time points compared to control mice (P < 0.001). After treatment with the Colchicine and the MG, the bearing capacity of the model-making side joint of the mouse can be obviously increased, which shows that the Colchicine and the MG have anti-inflammatory and analgesic effects on the gouty arthritis mouse (fig. 7B).

Discussion of 3

In this experiment, we found that MG can significantly reduce uric acid levels by inhibiting hepatic XOD activity. Creatinine and urea nitrogen levels are common indicators for assessing kidney function. MG can reduce serum creatinine, urine creatinine, and urea nitrogen levels in a dose-dependent manner. Pathological section results show that the MG can obviously improve pathological changes of the kidney (including arteriosclerosis, glomerular sclerosis and tubulointerstitial fibrosis). These results indicate that MG can indeed improve HN.

Hyperuricemia patients activate NLRP3 through uric acid crystals (MSU) as an activation signal that is recognized and bound by the Leucine Rich Repeat (LRR) of NLRP 3. Activated NLRP3 recruits apoptosis-related sites (ASCs) and caspases (Caspase-1) to form NLRP3 inflammasome, which cleaves IL-1 β precursor protein and produces IL-1 β by modulating Caspase-1 activity. And simultaneously induces cell membrane perforation injury, leads to cell death, promotes IL-1 beta release, and causes inflammation. The research shows that MG can obviously reduce the activation of NLRP3 inflammasome (the protein expression level of NLRP 3), ASC, IL-1 beta and Caspase-1.

In this study, we demonstrated that MG has a dual inhibitory effect on NLRP3 inflammasome activation. First, MG can inhibit NF-. kappa.B activation and inhibit the production of inflammatory-corpuscle-independent cytokines, such as TNF-. alpha.. Second, MG also directly inhibits NLRP3 inflammatory body activation to reduce IL-1 β production. Importantly, we found MG pair NLRP3^-/-The deficient HN mice had no effect, indicating that the in vivo anti-inflammatory activity of MG is dependent on its inhibitory effect on the NLRP3 inflammasome. These results indicate that MG binds directly to NLRP3 and has significant anti-inflammatory activity in vitro and in vivo, suggesting that MG may have good therapeutic potential in NLRP3 driven diseases.While both NLRP3 inflammasome components and upstream signaling can be targeted, only targeting NLRP3 by itself specifically blocks its activation. Our results indicate that although upstream signals of NLRP3 activation, such as ROS, are affected by MG treatment, we also found that MG can block the interaction between NLRP3 and ASC, which is an important step in NLRP3 inflammasome assembly. MG failed to inhibit activation of NLRC4 or AIM2 inflammasome, suggesting that ASC interacts with NLRP3, but not with other inflammasome sensors, such as NLRC4 and AIM 2. Thus, our results indicate that MG is a classical NLRP3 inhibitor. In addition, MG has a protective effect on MSU-induced gouty arthritis in mice.

In summary, current studies indicate that MG has a protective effect on the kidney of PO-induced HN mice. This mechanism may be associated with blocking NLRP3 activation to exert its significant anti-inflammatory activity in NLRP3 driven diseases (figure 8).

Claims

1. Application of gallic acid methyl ester in preparing NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis.

2. Application of gallic acid methyl ester as sole active component in preparing NLRP3 pathway inhibitor for hyperuricemia nephropathy and/or gouty arthritis.

3. Application of gallic acid methyl ester in preparing NLRP3 pathway inhibitor of hyperuricemia nephropathy.

4. Application of gallic acid methyl ester as sole active component in preparing NLRP3 pathway inhibitor of hyperuricemia nephropathy.

5. The use according to any one of claims 1 to 4, wherein the methyl gallate is used to inhibit the activation of LPS and MSU stimulated NLRP3 inflammasome.

6. The use according to any one of claims 1 to 4, wherein the methyl gallate is used to inhibit the activation of LPS and alum stimulated NLRP3 inflammasome.

7. The use according to any one of claims 1 to 4, wherein the methyl gallate is used to inhibit the activation of LPS and ATP stimulated NLRP3 inflammasome.

8. The use according to any one of claims 1 to 4, wherein the methyl gallate is used to inhibit the activation of LPS and nigericin stimulated NLRP3 inflammasome.