A novel Chinese medicine formula against hyperuricemia-induced kidney inflammation involving pyroptosis inhibition via modulating AMPK-TLR4-NLRP3 pathway
Yuan-Yang Tian, Yu-Lin Wu, Koon Kit Lam, Hengzhou Zhu, Yan-Fang Xian, Zhi-Xiu Lin
TL;DR
A traditional Chinese medicine formula reduces kidney inflammation caused by high uric acid by inhibiting cell death pathways and modulating key proteins.
Contribution
The study demonstrates a novel Chinese medicine formula's efficacy in treating hyperuricemia-induced kidney inflammation via the AMPK-TLR4-NLRP3 pathway.
Findings
GF reduced pain sensitivity and improved grip strength in hyperuricemia-induced mice.
GF suppressed inflammatory markers and modulated uric acid transporters in mice and HK-2 cells.
GF inhibited pyroptosis and activated the AMPK pathway to reduce kidney injury.
Abstract
Hyperuricemia (HUA), characterized by elevated serum uric acid (SUA) level, serves as a precursor of gout. Gout formula (GF), an empirical formula, has been prescribed in Chinese medicine practice for many years and observed to be effective in relieving HUA and gout. This study aimed to investigate GF as an innovative therapeutic agent for HUA and to elucidate its underlying molecular mechanisms using in vivo and in vitro models of HUA. A HUA mouse model was established by orally administering potassium oxonate (PO) and injecting with hypoxanthine (HX) to mice. Behavioral tests and histopathological analysis were performed to measure the efficacy of GF. Biochemical indicators such as SUA, urine uric acid (UA), xanthine oxidase (XOD), blood urea nitrogen (BUN), and creatinine (Cre), inflammatory markers, signaling pathway markers, uric acid transporters (UAT), and renal injury were…
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Figure 6- —Peter Hung Pain Research Institute of The Chinese University of Hong Kong
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Taxonomy
TopicsGout, Hyperuricemia, Uric Acid · Inflammasome and immune disorders · Thyroid Disorders and Treatments
Introduction
The epidemiology of hyperuricemia (HUA) has become an important global public health issue, largely owing to its close association with multiple medical conditions such as gout, cardiovascular disease, and metabolic syndrome [1–3]. Recent studies have demonstrated an increasing prevalence of HUA in diverse populations. For instance, the prevalence of HUA in the United States has remained stable over the past decade, affecting approximately 20% of adults. The prevalence of HUA was 10–25% in Europe and 13–25% in Asian populations, with a prevalence of 10–20% reported in Latin America and the Middle East [4, 5]. Similarly, China has in recent years also witnessed a rapid rise in the prevalence of HUA. Based on the data from the national health surveys, a comprehensive analysis of HUA prevalence among Chinese adults revealed that the overall prevalence increased from 11.1% in 2015–2016 to 14.0% in 2018–2019, indicating a concerning upward trend in just a few years [6]. These epidemiological data indicate that HUA is a significant health problem of global scale, and underscore the need for effective public health interventions.
The treatment of HUA and its associated conditions primarily encompasses urate-lowering therapies (ULTs), which consist of xanthine oxidase inhibitors (XOIs) and uricosuric agents. However, various limitations are associated with their clinical use [4].
HUA, characterized by an elevated serum uric acid (SUA) level, can lead to various complications, including kidney damage and the activation of inflammatory response [7]. Research suggests that HUA can trigger pyroptosis, a form of programmed cell death associated with inflammation, particularly in renal tissues [8]. Pyroptosis is mediated by the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which is activated in response to various stressors, including elevated uric acid (UA) level. Existing research indicates that the activation of the NLRP3 inflammasome is a two-step process, comprising priming and activation [9]. The classical priming process is induced by the Toll-like receptors, leading to the activation of nuclear factor-kappa B (NF-κB) and subsequent gene transcription. In the activation phase, the common factor among the activators of NLRP3 is that they all induce cellular stress, including the efflux of potassium ions or chloride ions. The AMP-activated protein kinase (AMPK), as a core regulatory factor of cellular energy homeostasis, plays a crucial role in this process. The activation of AMPK is triggered by cellular stress, such as low ATP levels. In HUA, the activation of AMPK has been shown to alleviate renal tubular injury by reducing the lysosomal degradation of Na + -K + -ATPase (NKA), thereby maintaining the function of NKA, and attenuating downstream inflammatory signaling through the activation of Src and the NLRP3 inflammasome [10, 11]. This protective effect of AMPK is further supported by evidence indicating that AMPK activators, such as AICAR and metformin, mitigate the UA-induced renal injury in preclinical models, highlighting their therapeutic potential [10].
The Toll-like receptor 4 (TLR4) is a pattern recognition receptor that recognizes damage-associated molecular patterns (DAMPs) and intersects with the AMPK/NLRP3 axis in HUA. SUA or other DAMPs activate TLR4, initiating the NF-κB signaling, leading to the transcription of NLRP3 and pro-IL-1β, heralding the activation of the inflammasome. AMPK has been shown to inhibit TLR4 signaling, thereby reducing the transcriptional activation of NLRP3 and the subsequent production of IL-1β [12]. This interaction indicates that there exists a feedback loop where the activation of AMPK suppresses the initiation and activation phases of the NLRP3 inflammasome signaling pathway, providing a multifaceted approach to controlling inflammation in HUA.
The activation of the NLRP3 inflammasome leads to the release of pro-inflammatory cytokines such as interleukin-1 beta (IL-1β), exacerbating kidney injury and inflammation. Pyroptosis is not only a consequence of HUA but also a contributing factor to its pathological effects. The inflammatory environment created by pyroptosis can lead to tissue damage and exacerbate conditions such as chronic kidney disease and cardiovascular diseases [13, 14]. In the context of kidney injury, pyroptosis has been implicated in the progression of diabetic nephropathy and acute kidney injury, where the activation of caspase-1 (CASP1) and subsequent pyroptotic cell death contribute to renal inflammation and fibrosis [13]. In short, the AMPK/TLR4/NLRP3 pyroptosis pathway represents a critical molecular mechanism linking HUA to inflammatory diseases, with AMPK activation serving as a worthy therapeutic target to mitigate inflammation and restore metabolic balance.
Given the multi-component, multi-target properties and low toxicity characteristics, Chinese medicines (CM) has been used for the treatment of HUA in China for thousands of years. Gout formula (GF) is an empirical formula of the corresponding author Prof. Zhi-Xiu Lin, who is a Registered Chinese Medicine Practitioner in Hong Kong. GF has been prescribed in clinic for many years and observed to be effective in relieving HUA. GF contains 15 Chinese herbal medicines, including the dried fruit of Coix lacryma-jobi (Yiyiren in Chinese), the dried root of Smilax glabra (Tufuling in Chinese), the dried root of Dioscorea hypoglaua (Fenbixie in Chinese), the dried fruit of Vigna umbellata (Chixiaodou in Chinese), the dried fruit of Dolichos lablab (Baibiandou in Chinese), the dried root of Alisma orientable (Zexie in Chinese), the dried herb of Lycopus lucidus (Zelan in Chinese), the dried rhizome and root of Clematis chinensis (Weilingxian in Chinese), the dried root of Paeonia lactiflora (Chishaoyao in Chinese), the dried nut of Prunus persica (Taoren in Chinese), the dried body of Pheretima aspergillum (Dilong in Chinese), the dried tuber of Corydalis yanhusuo (processed with vinegar; Cuyanhusuo in Chinese), the dried rhizome of Curcuma Longa (Jianghuang in Chinese), the dried female insect body of Eupolyphaga sinensis (Tubiechong in Chinese), and the dried rhizome and root of Glycyrrhiza uralensis (Gancao in Chinese) at the ratio of 20:20:15:10:10:10:10:6:8:8:12:12:8:8:4. Among these, Coicis Semen, Smilacis Glabrae Rhizoma, and Glycyrrhizae Radix have been extensively validated for their therapeutic effects on HUA, demonstrating their ability to reduce UA level in HUA mice [15–19]. Dioscoreae Hypoglauae Rhizoma, Vignae Semen, Dolichoris Album Semen, Alismatis Rhizoma, and Lycopi Herba are commonly used for clearing away heat and removing dampness [15]. Some of these herbs also have the ability to invigorate the spleen. For example, Coicis Semen has the function of nourishing the spleen and stomach, as documented in the “Compendium of Materia Medica” written by Li Shizhen, a renowned CM physician in the Ming dynasty (AD 1368–1644) [20]. With the combination of the above herbs, detoxification of damp heat pathogens can be achieved. The herbs that promote blood circulation and remove blood stasis, such as Clematidis Radix, Paeoniae Rubra Radix, Persicae Semen, Pheretima, Eupolyphaga Steleophaga, Corydalis Rhizoma and Curcumae Longae Rhizoma are combined in the formula to achieve better pain-relieving effects [16, 17].
This study aimed to investigate the effect of GF on HUA using in vivo and in vitro experimental models, and to elucidate its underlying molecular mechanisms. We used ultra-performance liquid chromatography (UPLC)-Q Exactive-mass spectrometry (MS) methods to identify the chemical components and establish the quality control of GF. We then investigated that anti-HUA effects of GF using potassium oxonate (PO) plus hypoxanthine (HX)-induced HUA in mice. Finally, we used the monosodium urate (MSU)-induced HUA cell model with 5-aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR), an AMPK agonist, to confirm the anti-HUA effects of GF against HUA.
Materials and methods
Chinese herbal materials of GF
The crude decoction pieces of Coicis Semen, Smilacis Glabrae Rhizoma, Dioscoreae Hypoglauae Rhizoma, Vignae Semen*,* Dolichoris Album Semen, Alismatis Rhizoma, Lycopi Herba, Clematidis Radix, Paeoniae Rubra Radix, Persicae Semen, Pheretima, Corydalis Rhizoma (processed with vinegar), Curcumae Longae Rhizoma, Eupolyphaga Steleophaga, and Glycyrrhizae Radix were purchased from Zisun Medicine, a reputable GMP-certified Chinese medicines supplier. The identities of these herbal medicines were authenticated by Prof. Yanfang Xian, a seasoned pharmacognosist at the School of Chinese Medicine, CUHK. Authenticated voucher specimens have been deposited in the storeroom of the School of Chinese Medicine at CUHK with the voucher specimen no. 20230501–20230515.
Extraction of GF
The herbal mixture was macerated in a ten-fold volume of distilled water for 2 h, followed by boiling for 1 h (Mtops Heating Mantle E105). This extraction process was repeated twice. After filtration, the crude extract was centrifuged to remove undissolved particles. The pooled extract was then concentrated under reduced pressure (BUCHI Rotavapor R-200 and BUCHI Vacuum Pumps V-700) and freeze-dried (BenchTop Pro BTP-3ES00X). The yield of GF was 21.4%, and the dried aqueous extract was stored at − 20 °C. For the animal studies, the GF extract was dissolved in physiological saline, while for in vitro cellular studies, it was dissolved in the culture medium and filtered immediately prior to use.
Chemical compositions analysis
To identify the components of GF, we utilized an UPLC (Thermo, UltiMate 3000 RS) coupled with Q Exactive MS (UPLC-Q Exactive MS). GF was dissolved in 80% methanol, subjected to centrifugation at 20,000 × g for 10 min at 4 °C, and subsequently filtered through 0.22 μm Millipore filters. The resulting filtrate was injected into a reverse-phase C18 column (2.1 mm × 150 mm, 1.8 μm, Welch) maintained at 35 °C. The mobile phases consisted of 0.1% (v/v) formic acid in purified water (A) and 0.1% (v/v) formic acid in acetonitrile (B). The gradient elution protocol was as follows: 0–1 min, 2% B; 1–10 min, 2–50% B; 10–20 min, 50–95% B; 20–25 min, 95% B; 25–26 min, 95–2% B; and 26–30 min, 2% B. The injection volume was 5 μL, with a flow rate of 0.3 mL/min. The mass spectrometry full scan range was set to 150–2,000 m/z, employing high-purity nitrogen and argon as collision and desolvation gas, respectively. The results were compared against the mzCloud, mzVault, and ChemSpider databases.
Quality control of GF
HPLC fingerprinting was employed for the quality control of GF. GF, along with three standard references such as alisol B 23-acetate, paeoniflorin, and astilbin. GF and the three compound standards, purchased from Chengdu Must Bio-Technology Co., Ltd, were dissolved in methanol and subsequently filtered through a 0.20 μm filter prior to injection. The analysis was conducted using an ACQUITY UPLC system (Waters, USA) equipped with a Spherisorb ODS1 column (4 mm × 150 mm). The mobile phase consisted of acetonitrile (solvent A) and water (solvent B), utilizing a gradient from 5 to 90% solvent A over a duration of 30 min at a flow rate of 1 mL/min. Separations were carried out at room temperature, with the detection wavelength of 203 nm for alisol B 23-acetate, 230 nm for paeoniflorin, and 288 nm for astilbin. An injection volume of 10 μL was used. The compound identifications were achieved by comparing the relative retention times and UV characteristics of the standards with those of GF. The contents of the alisol B 23-acetate, paeoniflorin and astilbin were calculated based on pre-constructed standard curves.
Animals and drug treatments
The HUA mouse model was established using a combination of PO (Sigma, #156,124) and HX (Sigma #H9377), and this experimental model has been widely employed by many researchers for HUA investigations [21]]. The experimental procedure for model establishment and drug treatment was outlined as follows. Briefly, eight-week-old male C57BL/6 mice (readily available from LASEC, CUHK) were randomly divided into six groups of ten animals each: untreated normal control group, the HUA control group, the HUA plus allopurinol (AP, 7 mg/kg, positive control, Sigma, #A8003) group, and the HUA plus GF (1.5, 3 and 6 g/kg) groups. The doses of AP, PO, and HX were optimized and adjusted in accordance with the dosages reported in the published literature [22, 23], as well as our preliminary experimental results. In the clinical practice of Chinese herbal medicine, GF is usually prescribed at a daily dose of 202 g of raw herbal materials. We converted this human dose into an animal dose using following parameters: an extraction yield of 21.4%, a person of 60 kg in weight, and a conversion factor of 9 between human and mice, and came up to a dose equivalent to the high dose (6 g/kg) for this study.
HUA mice received intraperitoneal injections of PO at a dose of 300 mg/kg after oral administration with HX at a dose of 300 mg/kg for seven consecutive days, one hour prior to drug administration. Mice in each treatment group were administered intragastrically with GF and AP once daily for seven consecutive days. Mice in the normal control and HUA control groups were administered orally the same volume of physiological saline (0.9%). One hour after the final drug treatment, the animals were anesthetized, and blood and urine samples were collected. Subsequently, the mice were sacrificed by cervical dislocation under anesthesia, and the liver, kidney, and spleen tissues were harvested for further analysis. The procedures of all animal experiments in this study were conducted in accordance with the protocols approved by the Animal Experimentation Ethics Committee of CUHK (Ref. No.: 21–133-ITF).
The pain-related behavioral evaluation in the PO + HX-treated mice
To evaluate the pain-related behaviors in the PO + HX-treated mice, we employed two well-established assessment methods, including the hot plate test and the grip strength test [24, 25]. The hot plate test was performed using the XR1100 Hot/Cold Plate Analgesia Meter (Shanghai Xinruan Information Technology Co., Ltd., Shanghai, China), with the hot plate temperature maintained at 55 °C. Briefly, the mice were placed in a transparent plastic cylinder positioned on the hot plate. Their reflex actions, such as paw licking, jumping, or vocalizing discomfort, were measured and recorded at the intervals of 0, 30, 60, and 90 min after drug administration. To mitigate potential data errors arising from residual odors, the instrument was thoroughly cleaned with alcohol after each test.
The grip strength test was conducted using the BIO-GS4 (BX-ROD-M; Bioseb Instruments US, FL, USA). The instrument was positioned horizontally, and the mice were held by their tails and gently pulled backward, prompting them to grasp the metal grid with their forelimbs. The mice were then pulled horizontally until they released their grip, and the peak pulling force was recorded. Eight mice were used in each group for performing each test.
Histopathology analysis
The fresh kidney tissues were thoroughly rinsed with PBS to remove any debris, ensuring that the samples were clean and suitable for further processing. Following this, the tissues were fixed to preserve their structural integrity and prevent any degradation. After fixation, the samples were embedded in paraffin to create a solid medium that facilitates easier slicing for analysis. Once embedded, the tissues were sectioned into thin slices with a precise thickness of 5 μm, enabling detailed examination of the cellular architecture. The sections were then stained using hematoxylin and eosin (H & E) or periodic acid-Schiff (PAS) staining protocols, which are standard methods employed to highlight specific cellular components and histological features. Lastly, the histopathological alterations within the kidney tissues were analyzed under a microscope at a magnification of × 200. This level of magnification allowed for a thorough investigation of the tissue morphology and any pathological changes, thus providing valuable insights into the kidney’s health and function.
Serum and urine biochemical assays
The blood and urine samples were collected from the mice. The serum samples were harvested from the blood samples after incubation at room temperature for 2 h, then centrifuged at 3500 rpm for 10 min at 4 °C. The levels of SUA, urine UA, creatinine (CRE), and blood urea nitrogen (BUN) were subsequently measured using specific commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).
Cell culture and drug treatment
Human kidney 2 (HK-2, CRL-2190 ™) cells, sourced from healthy adult male kidneys, were purchased from the American Type Culture Collection (ATCC). These HK-2 cells were routinely maintained in the DMEM/F-12 medium with the addition of 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin, and were incubated in a humidified environment with 5% CO_2_ at 37 °C. Cells in the exponential growth phase were resuspended in DMEM and plated at a density of 10,000 cells per well across different types of well plates. Subsequently, the cell culture supernatant was discarded, and the cells were exposed to a culture medium enriched with exogenous UA (MSU, 0.15 mg/mL, Sigma, #U2875) for 24 h. The cells were treated with GF at the concentrations of 2.5, 5.0, or 10.0 mg/mL, or AP at a concentration of 0.1 mg/mL. After incubation with GF or AP for additional 24 h at 37 °C, the UA level in the cell culture supernatants and cell lysates were measured using the UA assay kit (Nanjing Jianchen Bioengineering Institute, Jiangsu, China). For AICAR experiment, AICAR (0.5 mM, MCE, HY-13417) and GF (10.0 mg/mL) were added into the MSU-stimulated HK-2 cells for additional 24 h, after stimulation with MSU for 24 h. The experiment was performed three times to confirm the reproducibility.
In vivo and in vitro xanthine oxidase (XOD) activity assay
The liver and cell tissues were homogenized and centrifuged at 3,000 rpm for 10 min at 4 °C. The supernatant was subsequently used to measure XOD activity using a commercial kit. For the in vitro assay, XOD activity was evaluated using a spectrophotometric method described previously [26]. To prepare the XOD enzyme working solution, 20 μL of XOD standard solution (100 U/mL, MCE, HY-P2755) was diluted with 980 μL of PBS at pH 7.4. Additionally, xanthine powder was dissolved in PBS to achieve a final concentration of 10 μg/mL. The reaction mixture was prepared by combining with 50 μL of the GF solution (at the concentrations of 0.25, 0.5, 1, 2, 4, 8, and 16 mg/mL), 127 μL of PBS, and 7 μL of the AXOD enzyme working solution. This mixture was incubated at 37 °C for 20 min. Following the incubation, 66 μL of the xanthine solution was added to initiate the reaction, bringing the final volume of the reaction system to 200 μL. The mixture was then incubated at 37 °C for an additional 30 min, and the UV absorbance was measured at 290 nm. ALLOP was employed as a positive control, and all steps were repeated three times. The rate of XOD inhibition was calculated using a specific formula and expressed as the mean ± standard deviation (n = 3). Furthermore, the IC_50_ value for the XOD inhibitory activity of both ALLOP and GF were determined using SPSS software.
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$XOD\,inhibitivity\left(\%\right)=\left[1-\frac{(S-{S}_{0})}{(B-{B}_{0})}\right]\times 100$$\end{document}In this context, let S and S_0_ represent the absorbance values of the reaction system with and without the presence of XOD, respectively. The variable B denotes the absorbance of the reaction system in the absence of any samples; B represents the enzyme activity without a sample, while B_0_ serves as the control for B in the absence of both the sample and XOD.
Immunofluorescence (IF) staining
Double immunofluorescence labeling method and conventional IF staining were conducted following the established protocols. Briefly, the paraffin-embedded kidney tissue sections were dewaxed, and antigen retrieval was performed. After this step, the slides were allowed to cool naturally and were washed three times with PBS (pH 7.4) on a shaker for 5 min each time. A hydrogen peroxide-sealed histochemistry pen was used to outline the tissue, after which the sections were immersed in a 3% hydrogen peroxide solution and incubated at room temperature in the dark for 30 min. Subsequently, the slides were washed three times in PBS (pH 7.4) for 5 min each. Following the PBS drying, 3% BSA was added dropwise to block nonspecific binding for 30 min. The primary antibody (NLRP3, Servicebio, GB114320, 1:2000) was then applied, and the slides were incubated in a humidified chamber at 4 °C overnight. After incubation, the slides were washed three times in PBS (pH 7.4) on a shaker for 5 min each. Once dried, the corresponding HRP-labeled secondary antibody was added to the outlined area and incubated at room temperature for 60 min. The washing step was repeated, and the slides were spun dry before adding TSA dropwise to the outlined area. This was incubated at room temperature for 10 min in the dark, followed by another washing step. The tissue sections were then placed in a repair box filled with antigen retrieval buffer and heated in a microwave oven: medium heat for 8 min, a pause for 8 min, and then medium–low heat for 7 min. The second primary antibody (Apoptosis-Associated Speck-like Protein Containing a CARD (ASC), Servicebio, GB113966, 1:4000) was applied, and the previous steps were repeated, including overnight incubation and the addition of the corresponding HRP-labeled secondary antibody. Finally, the washing step and TSA dyeing procedure were repeated, followed by a final wash. Finally, the sections were mounted using SlowFade Diamond Antifade Mountant with DAPI (Invitrogen, S36973). The images were observed with an image acquisition system (Nikon Instruments Inc. Melville, NY, USA) using a Nikon fluorescent inverted microscope.
The indicators of high mobility group box 1 (HMGB1) associated with cell pyroptosis were assessed using IF staining. Briefly, after the cells adhered to the slide, the culture medium was discarded, and the cells were washed three times with PBS. Following this, the cells were fixed with 4% formaldehyde dissolved in PBS for 30 min at room temperature, after which the washing steps were repeated. Subsequently, the cells were permeabilized with 0.2% Triton X-100 (prepared in PBS) for 10 min at room temperature. The washing steps were repeated, and then 5% BSA (also in PBS) was applied to block non-specific binding for 30 min at room temperature. After removing the blocking solution, the prepared HMGB1 antibody (Abcam, ab18256, 1:500) was added dropwise, and the slide was incubated at 4 °C overnight. The washing steps were repeated twice, and the cells were then treated with the secondary antibody, i.e., Alexa Fluor 488 Rabbit anti-Mouse IgG (Abcam, ab6728), for 1 h at room temperature. Finally, the sections were mounted using SlowFade Diamond Antifade Mountant with DAPI (Invitrogen, S36973). The images were observed with an image acquisition system (Nikon Instruments Inc. Melville, NY, USA) using a Nikon fluorescent inverted microscope.
Flow cytometry analysis
To identify and quantify the occurrence of pyroptosis in HK-2 cells, the Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) detection kit (One Step TUNEL Apoptosis Assay Kit Red Fluorescence, Beyotime, C1089) was used according to the manufacturer’s instructions. Briefly, cells were fixed with 4% formaldehyde in PBS at room temperature for 10 min, followed by three washes with PBS, each lasting 5 min. Subsequently, the cells were permeabilized with 0.2% Triton X-100 (prepared in PBS) for 10 min at room temperature, after which the washing steps were repeated. The TUNEL detection solution was prepared in accordance with the manufacturer’s instructions, and 50 μL of the solution was added to the samples, which were then incubated at 37 °C in the dark for 60 min, followed by another washing step. The cells were then resuspended in 300 μL PBS to obtain the cell suspension with concentration of 1 × 10^6^ cells/mL. After vortexing, the cells were subjected to the flow cytometry analysis on a flow cytometer (BD FACSCelesta™ Cell Analyzer), with FlowJo software being used for data analysis.
The enzyme-linked immunosorbent assays (ELISA)
The levels of tumor necrosis factor-α (TNF-α), IL-1β, interleukin-4 (IL-4), Interleukin-6 (IL-6), C–C chemokine receptor type 2 (CCR2), C–C Motif Chemokine Receptor 5 (CCR5), and urate transporter 1 (URAT1) in the kidney tissues of the mice were measured using ELISA kits (ABclonal Technology Co., Ltd. and ELK (Wuhan) Biotechnology Co., Ltd., Wuhan, China) as per the manufacturer’s instructions.
The reverse transcription-quantitative polymerase chain reaction (RT-PCR)
The total RNA was isolated from kidney tissues using Trizol reagent following the manufacturer’s instructions. The RNA purity was confirmed to be between 1.8 and 2.0. The extracted RNA was then reverse transcribed into cDNA. The mRNA expressions of IL-1β, IL-6, TNF-α, CCR2, CCR5, and C-X-C motif chemokine 10 (CXCL10) were determined using the HiScript^®^ II Q RT SuperMix (+ gDNA wiper) and ChamQ™ SYBR^®^ qPCR Master Mix Kit. The primer sequences were listed in Supplementary Table 1 (ST1) and were designed using online primer design software (Sangon Biotech Co., LTD, Shanghai, China). The gene expression levels were calculated using the 2^−ΔΔCt^ method, with β-actin serving as the normalization control.
The Western blot analysis
The total and nuclear proteins were extracted from mouse kidney tissues and HK-2 cells using the Total Protein and Nuclear Protein Extraction Kit (Abcam, ab113474). The protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23,225), and the samples were denatured. A total of 15 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies against AMPK (ABclonal, A12491), p-AMPK (ABclonal, AP0432), TLR4 (CST, #14,358; Servicebio, GB11519-100), MYD88 (Servicebio, GB115549-100), NF-κB p65 (Abcam, ab32536; CST, #8242; Servicebio, GB11997-100), p-p65 (Servicebio, GB113882-100), IκBα (GB151509-100; GB11212-100), p-IκBα (CST, #2859), NLRP3 (GB114320-100), ASC (Affinty, DF6304; Servicebio, GB115270-100; GB113966-100), TNF-α (Servicebio, GB11188-100), Pro-caspase-1(Abcam, ab179515), Caspase-1 (ABclonal, A21085; Servicebio, GB11383-100), GSDMD-NT (Affinty, #DF13758), OAT1 (ABclonal, A1814; Servicebio, GB113690-100), OAT3 (ABclonal, A14575), URAT1 (ABclonal, A5118; Servicebio, GB111847-100), GLUT9 (ABclonal, A14592), and β-actin (Santa Cruz, SC69879), and all of which were diluted to 1:1000 in 5% BSA-TBST. After washing, the membrane was incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature, followed by three washing with TBST. The chemiluminescent signals were detected using the SuperFemto ECL Chemiluminescence Kit (Vazyme, E423-02) and the Azure c300 Chemiluminescent Western Blot Imaging System. The band intensities were quantified using ImageJ software (NIH, USA).
Acute toxicity study of GF
An acute toxicity study was conducted using eight-week-old male and female C57BL/6 mice. The mice were randomly divided into three groups, with 10 animals in each group maintaining male to female at the ratio of 1:1. Prior to the administration of a single dose of GF (12 or 24 g/kg, dissolved in saline), the mice were fasted overnight for 12 h, with unrestricted access to water. Treatments were administered intragastrically at a volume of 10 mL/kg. The general behaviors were monitored continuously for 4 h following treatment and intermittently at 6 h intervals for a total of 24 h, and thereafter daily observation for 7 consecutive days.
Statistical analysis
The statistical analysis was performed using the GraphPad Prism 10.0 software (San Diego, CA, USA). The data were presented as mean ± standard error mean (SEM). Multiple groups comparisons were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc testing. The p value less than 0.05 was considered statistically significant.
Results
Quality control and composition identification of GF
Several compounds were identified in the GF extract, including betaine, L-isoleucine, astilbin, and ageratriol (Fig. 1A, Supplementary Table (ST2)) using UPLC/qExactive-MS analysis. The HPLC fingerprinting analysis was constructed to monitor the quality control of GF. GF was found to contain 0.51% alisol B 23-acetate, 4.09% paeoniflorin, and 1.43% astilbin (Table 1 and Fig. 1B–D).Fig. 1. Chemical compositions and quality control method and the therapeutic effects of GF on the severity of HUA in the HUA mice. A Chemical compositions of GF. B Chromatogram of GF and alisol B 23-acetate detected at 203 nm. C Chromatogram of GF and paeoniflorin detected at 230 nm. D Chromatogram of GF and astilbin detected at 288 nm. E Representative images of the kidneys of the HUA mice. F Pathological changes in the kidney tissues using H&E staining (scale bar represents 50 µm). G The kidney index; H SUA; I Urine UA; J BUN and K CRE in serum; L XOD activity in the liver; M URAT1 in the kidneys. N and O Representative western blotting images and quantitative analysis of the protein expressions of GLUT9, URAT1, OAT1, and OAT3 in the kidneys of the HUA mice. All data were expressed as mean ± SEM (G–M, n = 8–10; N–O, n = 3–4). ^#^p < 0.05 and ##p < 0.01 compared to the control group; *p < 0.05 and **p < 0.01 compared to the PO + HX vehicle control groupTable 1The content determination of the 3 compounds in GF by HPLC analysisCompoundsLinearityContent (%, w/w) in Range (μg/mL)EquationR^2^Alisol B 23-acetate7.5–200.0y = 250.45x–321.970.99630.51Paeoniflorin25–400.0y = 119.40x + 1004.200.99414.09Astilbin25–400.0y = 1040.6x–14,3360.99691.43
GF treatment reduces the UA level and improves the renal pathological changes in HUA mouse model
The HUA mouse model was successfully established through PO + HX treatment, leading to a marked increase in the renal edema, kidney index (F (5, 42) = 55.53, p < 0.001), and SUA level (F (5, 42) = 150.7, p < 0.001), which are critical pathological characteristics of HUA (Fig. 1E, G and H). In addition, PO + HX treatment could induce the kidney toxicity via increasing the levels of serum BUN (F (5, 42) = 96.84, p < 0.001) and CRE (F (5, 42) = 33.14, p < 0.001). However, treatment with GF greatly reduced the HUA-induced renal injury in mice, as evidenced by decreasing tubular dilatation, renal edema, cellular swelling, thickening of the basement membrane, and atrophy of the glomeruli (Fig. 1F).
The metabolism of purines involves the breakdown of nucleotides by the enzyme nucleotidase, generating free bases and ribose 1-phosphate, ultimately contributing to the UA formation. The XOD is crucial in this pathway. To evaluate the effects of GF on UA production, the XOD activity (F (5, 42) = 112.1, p < 0.001) and URAT1 level (F (5, 42) = 31.37, p < 0.001) were assessed in the liver tissues of mice (Fig. 1L). Additionally, the expression levels of the principal UAT including OAT1 (F (5, 12) = 29.13, p < 0.001), OAT3 (F (5, 12) = 34.15, p < 0.001), URAT1 (F (5, 12) = 17.15, p < 0.001), and GLUT9 (F (5, 12) = 15.43, p < 0.001) were analyzed to clarify the regulatory roles of GF on the UA metabolism (Fig. 1N, O). The results revealed a notable increase in XOD activity and the elevated expressions of URAT1 and GLUT9 in the HUA group, alongside the reduced expressions of OAT1 and OAT3, which were associated with a higher UA level. Treatment with AP resulted in a notable decrease in XOD activity, but did not affect UA transporters. Conversely, GF treatment not only resulted in significant reduction in the levels of SUA and urine UA, the expressions of XOD activity, URAT1, and GLUT9, but also led to increase in the expressions of OAT1 and OAT3 in a dose-dependent manner (Fig. 1H, I and L-O). These findings indicate that GF was able to modulate the UA level by inhibiting its synthesis and reabsorption while promoting its excretion. These results were also supported by the findings from the urine UA assay (F (5, 30) = 106.9, p < 0.001) (Fig. 1I).
GF treatment improves the renal inflammation and pain-related behavior by modulating inflammatory cytokines and chemokines in the HUA mice
The hot plate test and grip strength test were utilized to assess the improvement of the inflammation-related pain behavior in the HUA mice treated with GF. The experimental results indicated that GF treatment of varying doses significantly reduced the pain sensitivity, as evidenced by increasing the reaction times of the mice, as compared with the PO + HX-treated mice (Fig. 2A). Additionally, GF treatment markedly improved the tensile performance of the mice via increasing the strength (F (5, 42) = 172.0, p < 0.001) and strength/weight (F (5, 42) = 76.55, p < 0.001), as compared with the PO + HX-treated mice (Fig. 2B, C).Fig. 2. Anti-inflammatory effects of GF on the inflammation-related pain behavior and inflammatory cytokines and chemokines in HUA mice. A Reaction time of the hot plate test; B and C Relative and absolute Strength. D and E CCR2 mRNA and protein levels; F and G CCR5 mRNA and protein levels; H CXCL10 mRNA level; I and M IL-1β mRNA and protein levels; J and N TNF-α mRNA and protein levels; (K and O) IL-6 mRNA and protein levels; L IL-4 protein level. All data were expressed as mean ± SEM (n = 3–10). ^#^p < 0.05 and ^##^p < 0.01 compared to the control group; *p < 0.05 and **p < 0.01 compared to the PO + HX vehicle control group
As shown in Fig. 2D–H, a significant increase in the renal chemokines including CCR2 (F (5, 12) = 110.0*, p* < 0.001 for mRNA level; F (5, 48) = 29.81,* p* < 0.001 for protein level), CCR5 (F (5, 12) = 99.92, p < 0.001 for mRNA level; F (5, 48) = 96.09,* p* < 0.001 for protein level), and CXCL10 (F (5, 12) = 36.74,* p* < 0.001 for mRNA level) were observed in both the HUA and AP groups. GF treatment effectively reduced the levels of the chemokines such as CCR2, CCR5, and CXCL10 in the kidneys of HUA mice. The inflammatory cytokines such as TNF-α (F (5, 12) = 63.65, p < 0.001 for mRNA level; F (5, 42) = 149.3, p < 0.001 for protein level), IL-1β (F (5, 12) = 114.3, p < 0.001 for mRNA level; F (5, 42) = 213.8, p < 0.001 for protein level), IL-4 (F (5, 48) = 73.74, p < 0.001), and IL-6 (F (5, 12) = 58.87, p < 0.001 for mRNA level; F (5, 48) = 24.01, p < 0.001 for mRNA level) (Fig. 2I–O) were quantified using RT-PCR and ELISA kits. Notably, the HUA group elevated the levels of these pro-inflammatory cytokines, but reduced the concentrations of anti-inflammatory factors (Fig. 2D–O). In contrast, GF treatment significantly attenuated the levels of pro-inflammatory cytokines, while simultaneously accentuated the concentration of the anti-inflammatory factor such as IL-4. It is worth noting that these chemokines play vital roles in the recruitment and activation of immune cells, and their elevated levels indicate an exacerbated inflammatory response. These findings suggest that GF treatment was able to significantly mitigate the inflammatory response in the kidney tissues induced by HUA in mice.
GF inhibits the NLRP3 inflammasome-induced pyroptosis in the HUA mice via regulating the AMPK-TLR4-NLRP3 signaling pathway
Previous studies have demonstrated that the potential mechanism by which GF exerts its therapeutic effect on HUA may involve the regulation of AMPK-TLR4-NLRP3 signaling to provide renal protection and exert anti-inflammatory effects. One pathway leading to pyroptosis is inflammatory cell death induced by the NLRP3 inflammasome, characterized by cell swelling and the release of pro-inflammatory factors. This finding aligns with previous experimental observations of renal edema in the HUA mice, where changes in inflammatory factors and chemokines were consistent. Furthermore, the canonical pathway of pyroptosis depends on the activation of caspase-1 and the cleavage of GSDMD, which facilitates the release of inflammatory factors and the subsequent occurrence of pyroptosis. Additionally, IF staining results demonstrated significant activation of the NLRP3 inflammasome in the kidney tissues of HUA mice, as evidenced by the increased expression of NLRP3 and the formation of ASC specks (Fig. 3A). These findings indicate that HUA mice display marked pyroptotic NLRP3 inflammasome activation.Fig. 3GF inhibits pyroptosis by regulating the expression of the AMPK-TLR4-NLRP3 pathway in the PO + HX-treaded mice. A Representative images of NLRP3 and ASC co-localization in the kidney tissues of the HUA mice (scale bar represents 20 μm, ASC green, NLRP3 red, DIPI blue). B–O Representative western blotting images and quantitative analysis of the protein expression of p-AMPK/AMPK, TLR4, MYD88, p-IκBα/IκBα, p-NFκB-p65/NFκB-p65, NLRP3, Pro Caspase-1, GSDMD-NT, TNF-α, ASC, and Caspase-1 in the kidney tissues of the HUA mice. All data were expressed as mean ± SEM (n = 3–10). ^#^p < 0.05 and ^##^p < 0.01 compared to the control group; *p < 0.05 and **p < 0.01 compared to the PO + HX vehicle control group
To further substantiate the occurrence of pyroptosis, we detected the key proteins involving in the pyroptosis pathway using Western blotting analysis. Notably, the HUA group exhibited a decreased protein expression of p-AMPK/AMPK (F (5, 12) = 15.51, p < 0.001), while elevated expression levels of TLR4 (F (5, 12) = 88.01, p < 0.001), MyD88 (F (5, 12) = 16.66, p < 0.001), p-IκBα/IκBα (F (5, 12) = 16.66, p < 0.001), p-p65/p65 (F (5, 12) = 16.66, p < 0.001), NLRP3 (F (5, 12) = 24.88, p < 0.001), procaspase-1 (F (5, 12) = 16.66, p < 0.001), TNF-α (F (5, 12) = 16.66, p < 0.001) and ASC (F (5, 12) = 17.45, p < 0.001), and the activity of caspase-1 (F (5, 12) = 19.12, p < 0.001) and GSDMD-NT (F (5, 12) = 26.20, p < 0.001) (Fig. 3B–O). Following GF treatment, the AMPK phosphorylation was activated, and the AMPK-TLR4-NLRP3 pathway was regulated to improve the expression of the related proteins in HUA mice (Fig. 3B–O).
GF inhibits the NLRP3 inflammasome-induced pyroptosis in the MSU-stimulated HK-2 cells by regulating the AMPK-TLR4-NLRP3 signaling pathway
To further validate the results that GF treatment inhibited the NLRP3 inflammasome-induced pyroptosis in the HUA mice, a HUA cell model was established using HK-2 cells stimulated with MSU. The results of CCK8 assay confirmed that there was significantly cytotoxicity at the tested concentrations of MSU, AP, and GF (F (5, 30) = 47.38, p < 0.001) (Fig. 4A). Consequently, the dosage of 0.15 mg/mL of MSU was selected for 24 h exposure in the cell experiments. Microscopic observation revealed the distinct morphological characteristics of pyroptosis in the HUA cells, primarily characterized by osmotic swelling, an increase in cell size, and disintegration of some cells following swelling. Additionally, more pronounced yellow urate crystallization was observed (F (5, 12) = 89.38, p < 0.001) (Fig. 4B–D). The results of LDH assay indicated that the MSU-stimulated HK-2 cells exhibited trends similar to those observed in the kidney tissues of the PO + HX-treated mice, with an increase in LDH activity (F (5, 12) = 77.17, p < 0.001) (Fig. 4E). In addition, GF treatment could decrease the intracellular UA (F (5, 30) = 30.33, p < 0.001) but increase extracellular UA (F (5, 30) = 228.0, p < 0.001) in the MSU-stimulated HK-2 cells (Fig. 4F–G). Moreover, the in vitro XOD inhibition assay demonstrated that GF directly inhibited the XOD enzyme activity, with an IC_50_ of 4.45 mg/mL (Fig. 4H), consequently resulting in the reduced UA production. Furthermore, in the cell experiments, we selected HMGB1, a protein frequently highlighted in recent studies for its role in pyroptosis, to stain and observe the HUA cells. The IF staining results demonstrated that HMGB1 was distributed both within the nucleus and cytoplasm of HUA cells (Fig. 4I), indicating that HMGB1, whether actively secreted or passively released, plays a critical role in pyroptosis and the inflammation-related signaling pathways. Following GF treatment, the nuclear exocrine secretion of HMGB1 was significantly attenuated (Fig. 4I).Fig. 4GF reduces UA level in the MSU-stimulated HK-2 cells and inhibited pyroptosis by regulating the AMPK-TLR4-NLRP3 pathway. A Cell viability. B Images of the HUA HK-2 cell pyroptosis. C, D Flow cytometry analysis for TUNEL staining in HK-2 cells (n ≥ 3 per group, all experiments were performed in triplicate). E Level of LDH release in HK-2 cells. F–G Intracellular and extracellular UA levels. H the in vitro XOD inhibition. I Localization of HMGB1 detected by IF staining (scale bar represents 20 μm, HMGB1 green, DAPI blue). J–K Representative western blotting images and quantitative analysis of the protein expression of GLUT9, URAT1, OAT1, and OAT3 in the HUA HK-2 cells. L–X Representative western blotting images and quantitative analysis of the protein expression of AMPK-TLR4-NLRP3 pathway, including p-AMPK/AMPK, TLR4, MYD88, p-IκBα/IκBα, p-NFκB-p65/NFκB-p65, NLRP3, GSDMD-NT, TNF-α, ASC, Caspase-1 in the MSU-stimulated HK-2 cells. All data were expressed as mean ± SEM (B–H, n = 8–10; J–X, n = 3–5). ^#^p < 0.05 and ^##^p < 0.01 compared to the control group; *p < 0.05 and **p < 0.01 compared to the MSU vehicle control group
The expressions of the reabsorption-related proteins, specifically URAT1 (F (5, 18) = 38.85, p < 0.001) and GLUT9 (F (5, 18) = 15.44, p < 0.001), were reduced, leading to a diminished UA reabsorption (Fig. 4J–K). Conversely, the expressions of UA secretion-related proteins, including OAT1 (F (5, 24) = 39.67, p < 0.001) and OAT3 (F (5, 12) = 26.72, p < 0.001), were restored, thereby enhancing UA excretion (Fig. 4J–K). As shown in Fig. 4L–X, in the HUA cell model, the p-AMPK/AMPK was decreased (F (5, 12) = 16.98, p < 0.001), while the expression levels of TLR4 (F (5, 12) = 31.98, p < 0.001), MYD88 (F (5, 12) = 28.45, p < 0.001), p-IκBα/IκBα (F (5, 12) = 30.38, p < 0.001), p-NFκB p65/NFκB p65 (F (5, 12) = 39.51, p < 0.001), NLRP3 (F (5, 12) = 40.33, p < 0.001), GSDMD-NT (F (5, 12) = 22.22, p < 0.001), TNF-α (F (5, 12) = 32.29, p < 0.001), ASC (F (5, 12) = 24.13, p < 0.001), and Caspase-1 (F (5, 12) = 38.21, p < 0.001) were significantly elevated. This alteration activated the TLR4-NFκB-NLRP3 inflammatory pathway, resulting in the increased expression of downstream inflammatory molecules. However, GF treatment effectively activated AMPK phosphorylation, and modulated the expression of the proteins associated with the inflammatory pathways (Fig. 4L–X).
AICAR, an AMPK agonist, enhances GF to improve the MSU-induced cellular pyroptosis via inhibiting the activation of the AMPK-TLR4-NLRP3 pathway
Current in vivo and in vitro studies indicated that GF could alleviate HUA symptoms by inhibiting XOD activity and modulating the expression of UAT. Furthermore, GF exerted protective effects in renal function and anti-inflammatory effects and mitigated the MSU-induced pyroptosis through regulating the AMPK-TLR4-NLRP3 pathway via AMPK agonist-like effects. To ascertain whether AMPK is the primary target of the inhibitory effect of GF on the MSU-induced cell pyroptosis, we employed AICAR, an AMPK agonist, to further validate the effects of GF against HUA within the MSU-stimulated HK-2 cells. The CCK8 assay confirmed that the concentrations of GF used in the experiment remained within a safe range (F (4, 25) = 72.34, p < 0.001) (Fig. 5A). The results of the LDH and TUNEL assays demonstrated that GF exhibited a similar effect to that of AICAR, as both caused a reduction in the TUNEL level (F (4, 10) = 134.7, p < 0.001) and LDH (F (4, 10) = 125.3, p < 0.001) (Fig. 5B–D), while optimal results were achieved when GF and AICAR were administered concomitantly. Additionally, the IF staining of HMGB1 also corroborated these findings (Fig. 5E). The Western blot analysis of the key proteins within the AMPK-TLR4-NLRP3 pathway also revealed a consistent action pattern between GF and AICAR, as both activated AMPK phosphorylation and inhibited the expression of downstream inflammatory proteins, with the most favorable outcomes observed when GF and AICAR were used together (Fig. 5F–M). Additionally, the MSU treatment could reduce the protein expression of p-AMPK/AMPK (F (4, 10) = 54.79, p < 0.01), but increased the protein expression of TLR4 (F (4, 10) = 23.53, p < 0.01) and p-p65/65 (F (4, 10) = 22.47, p < 0.01) in HK-2 cells. Notably, similar results were also observed in key indicators associated with pyroptosis, including NLRP3 (F (4, 10) = 11.29, p < 0.01), GSDMD-NT (F (4, 10) = 14.68, p < 0.001), ASC (F (4, 10) = 21.04, p < 0.001), and Caspase-1 (F (4, 10) = 17.03, p < 0.001). These findings further confirmed that GF could inhibit the activation of the TLR4-NF-κB p65 and NLRP3 inflammatory pathways by promoting AMPK phosphorylation, leading to a reduction of the pyroptosis induced by NLRP3 inflammasome.Fig. 5AICAR enhances GF to activate the AMPK phosphorylation via inhibiting the activation of TLR4-NFκB-p65-NLRP3 pathway. A Cell viability. B, C Flow cytometry analysis for TUNEL staining in HK-2 cells (n ≥ 3 per group, all experiments were performed in triplicate). D Level of LDH release in HK-2 cells. E Localization of HMGB1 detected by IF staining (scale bar represents 20 μm, HMGB1 green, DAPI blue). F–M Representative western blotting images and quantitative analysis of the protein expression of AMPK-TLR4-NLRP3 pathway, including p-AMPK/AMPK, TLR4, p-NFκB-p65/NFκB-p65, NLRP3, GSDMD-NT, ASC, Caspase-1 in the MSU-stimulated HK-2 cells. All data were expressed as mean ± SEM (A–D, n = 8–10; E–M, n = 3–5). ^#^p < 0.05 and ^##^p < 0.01 compared to the control group; *p < 0.05 and **p < 0.01 compared to the MSU vehicle control group
Discussion
Growing evidence from both clinical and preclinical studies indicates a strong correlation between inflammation and pyroptosis [27], underscoring the crucial role of pyroptosis in the development and progression of various illnesses [28]. Pyroptosis, a form of programmed cell death driven by inflammasome activation, is intrinsically pro-inflammatory, creating a positive feedback loop between inflammation and cell death [29].
The canonical pyroptosis pathway is intricately linked to inflammation through several critical stages. A key step in this process is the activation of the NLRP3 inflammasome, one of the most extensively studied types of inflammasomes. This activation involves two distinct signals: a primary signal that upregulates inflammasome components via the Toll-like receptor (TLR) stimulation, and a secondary signal that facilitates inflammasome assembly [30]. Upon the formation of the inflammasome, caspase-1 is recruited and activated, leading to the cleavage of Gasdermin D (GSDMD). The N-terminal fragment of GSDMD forms pores in the plasma membrane, causing cellular swelling, lysis, and the release of inflammatory cytokines such as IL-1β and IL-18, resulting in the amplification of the inflammatory response [31, 32]. This cascade underscores the pivotal role of pyroptosis in sustaining and intensifying inflammation.
Our results demonstrated that GF treatment significantly attenuated the release of the key inflammatory mediators, including CCR2, CCR5, CXCL10, IL-1β, TNF-α, and IL-6, while simultaneously accentuating the anti-inflammatory cytokine IL-4 (Fig. 1). Furthermore, GF reduced the expression levels of XOD, URAT1, and GLUT9, while upregulating OAT1 and OAT3 **(**Fig. 2). These effects suggest that GF mitigates inflammation by disrupting the pyroptosis-induced inflammatory feedback loop, particularly through its inhibitory effect on inflammasome activation. The relationship between pyroptosis and inflammation has been observed in numerous pathological conditions, including arthritis and HUA [30, 33].
To date, numerous published studies have demonstrated the therapeutic potential of certain components in the AMPK/TLR4/NLRP3-mediated pyroptosis pathway in the treatment of the HUA and inflammation-related diseases. For instance, research has shown that regulating pyroptosis can prevent acute gouty arthritis [34], and the activation of AMPK can significantly reduce the activation of the NLRP3 inflammasome and subsequent pyroptosis [35]. Recent studies have elucidated the complex interactions between TLR4 and the NLRP3 inflammasome in mediating pyroptosis, a form of inflammatory cell death, which exacerbates the inflammatory cascade in HUA. These findings helped us to focus on the AMPK/TLR4/NLRP3-mediated pyroptosis pathway in the present study [36, 37].
A significant contribution of this study lies in its elucidation of the AMPK-TLR4-NLRP3 pathway as a mechanistic link between inflammation and pyroptosis in HUA. AMPK, a crucial regulator of cellular energy homeostasis, has been demonstrated to exert protective effects against various forms of cellular stress. Its activation inhibits the TLR4 signaling pathway, which is implicated in the inflammatory response associated with HUA [31, 32]. By suppressing TLR4 activation, AMPK reduces the recruitment of inflammatory cells and the activation of the NLRP3 inflammasome, thereby mitigating pyroptosis [38, 39]. These findings are consistent with previous research results related to inflammation. Although this study considers AMPK as an upstream regulatory factor of the TLR4-NLRP3 signaling axis based on the aforementioned literature and experimental results, there is increasing evidence suggesting a bidirectional interaction between AMPK and TLR4. AMPK activation can inhibit TLR4-mediated NF-κB signaling pathways and the activation of downstream inflammasomes, while excessive TLR4 stimulation and ROS production may inhibit the phosphorylation of AMPK. This mutual regulation may vary depending on cell types, experimental conditions, and disease stages. Therefore, in the context of HUA, it cannot be ruled out that there may exist a feedback regulatory loop, rather than a simple linear AMPK-TLR4-NLRP3 pathway, thus dynamically balancing cellular energy metabolism and inflammatory responses.
The involvement of AMPK in inhibiting the NLRP3 pathway is multifaceted, involving direct and indirect mechanisms that regulate NLRP3 activation, inflammasome assembly, and downstream inflammatory responses [37]. One of the primary mechanisms by which AMPK inhibits the NLRP3 pathway is through its ability to modulate mitochondrial function and reactive oxygen species (ROS) production. The AMPK activation enhances mitochondrial quality control by promoting mitophagy, the selective degradation of the damaged mitochondria, thereby reducing mitochondrial ROS, a key trigger for NLRP3 inflammasome activation [40]. AMPK also directly influences the expression and activity of NLRP3-related proteins. Activation of AMPK has been demonstrated to suppress the transcription and translation of NLRP3, caspase-1, and IL-1β, thereby attenuating inflammasome activation [37]. Our data indicate that GF promoted AMPK phosphorylation, and subsequently inhibits the TLR4-NF-κB p65-NLRP3 pathway, leading to a reduction in pyroptosis and inflammation.
The role of AMPK in renal health further underscores its therapeutic potential. In condition of HUA, the AMPK activation alleviates renal tubular injury by maintaining NKA function, thereby protecting against the HUA-induced damage [10]. Consistent with these findings, our study demonstrated that GF enhances the AMPK activation, leading to reduced histological damages in the kidneys of the HUA mice and improved inflammatory profiles. Our findings demonstrated that GF attenuated pyroptosis by targeting the TLR4-NF-κB p65-NLRP3 inflammatory pathway. Notably, the activation of AMPK was identified as a critical mechanism by which GF exerts its anti-inflammatory effects, an observation congruent with previous studies indicating that the AMPK activation can counteract the GSDMD-mediated pyroptosis [41, 42].
An important finding of this study is the observation of a synergistic effect between GF and the AMPK agonist AICAR. Compared to the use of either treatment alone, the combination therapy significantly reduced sepsis markers, such as LDH and TUNEL levels. Furthermore, changes in the AMPK phosphorylation and TLR4 protein levels support this observation, suggesting that GF functions similarly to an AMPK agonist and enhances its therapeutic effects when combined with pharmacological AMPK activators (Fig. 5). GF exhibits dual roles in regulating inflammasome activation and protecting kidney health, rendering it as a promising candidate for treating HUA and its complications. Our results demonstrated that GF enhanced AMPK phosphorylation and synergized with the AMPK agonist AICAR in inhibiting the downstream TLR4-NLRP3 signaling cascade in MSU-stimulated HK-2 cells, suggesting GF may serve as an AMPK agonist. However, more experiments such as specific AMPK inhibitors, siRNA-mediated knockdown, or molecular docking and binding assays are warranted to determine whether GF or its active components directly activate AMPK or act through upstream kinases. Importantly, in the acute toxicity test, we found no significant toxicity of GF, even at the highest dose of GF (24 g/kg).
Considering the advantages of CM in addressing complex physiological processes and targeting multiple pathways, we conducted an in-depth investigation of the herbal formula GF. Our current research findings confirm that GF ameliorated HUA by regulating UAT and inhibiting the activity of the XOD. Furthermore, GF markedly mitigated inflammation by inhibiting the activation of TLR4-NF-κB p65-NLRP3 pathway, with similar effects of AMPK agonists. GF also improved the pyroptosis induced by inflammation, thereby serving a renal protective and anti-inflammatory function.
According to the theory of CM, treatment principle for gout mainly involves invigorating the spleen and removing dampness, clearing away heat and detoxifying. GF consists of herbs that are commonly used for the treatment of gout [41, 42]. Coicis Semen, Smilacis Glabrae Rhizoma, Dioscoreae Hypoglauae Rhizoma, Vignae Semen, Dolichoris Album Semen, Alismatis Rhizoma, and Lycopi Herba are commonly used for clearing away heat and removing dampness. Some of these herbs also have the ability to invigorate the spleen. With the combination with herbs that promote blood circulation and remove blood stasis, such as Clematidis Radix, Paeoniae Rubra Radix, Persicae Semen, Pheretima, and Eupolyphaga Steleophaga, detoxification of damp heat pathogens could be achieved. Corydalis Rhizoma and Curcumae Longae Rhizoma are used to relieve pain as well as to promote blood circulation [41, 42]. Overall, the herbs in GF synergize for the treatment of gout through achieving the functions of invigorating the spleen and removing dampness, promoting blood circulation to stop pain, thereby ameliorating the symptoms of gout. Therefore, GF not only targets on the symptoms of HUA and gout, but may also tackle the root causes simultaneously to reduce the recurrence rate of HUA and gout. Unlike conventional monotherapy that targets a single enzyme or receptor, Chinese medicines have multiple-target, multi-pharmacology and low toxicity characteristics. Although the exact reasons underlying the effects of GF on gout are not well understood, the different components of GF may play an important role. Previous study demonstrated that astilbin isolated from Smilacis Glabrae Rhizoma significantly decreased the serum UA level by increasing urinary UA level and fractional excretion of urate, via suppressing GLUT9, URAT1 but up-regulating the ATP-binding cassette subfamily G member 2, organic anion transporter 1/3 and organic cation transporter 1 [43]. Coix seed oil extracted from Coicis Semen and smilacis glabrae Rhizoma extract have been reported to promote the UA homeostasis induced by PO + H-induced HUA mouse model in mice via enhancing the mitochondrial function and antioxidant capacity, as well as modulating the gut microbiota or inhibiting inflammation response [44, 45].
In conclusion, our study demonstrated that GF effectively ameliorated HUA by regulating the AMPK-TLR4-NLRP3 signaling pathway, thereby inhibiting pyroptosis and reducing inflammation. The results highlight the dual benefits of GF in mitigating the renal damage and restoring the inflammatory balance. The significance of the AMPK-TLR4-NLRP3 axis extends beyond HUA to a wide range of inflammation-driven diseases. Pyroptosis has been implicated in conditions such as arthritis, cardiovascular diseases, and neurodegenerative disorders [30, 33]. GF ameliorates HUA-induced renal inflammation through activation of AMPK phosphorylation and modulation of the TLR4–NF-κB–NLRP3 signaling axis. Although our findings indicate AMPK involvement, additional mechanistic studies are required to verify whether GF or its active constituents directly target AMPK or act via upstream regulatory pathways. Through demonstrating that GF could attenuate pyroptosis through AMPK activation, this study contributes to a growing body of evidence supporting the therapeutic targeting of this pathway. Furthermore, our findings also highlight the potential of integrating traditional herbal medicine with modern pharmacological approaches to address complex pathological processes such as HUA and gout.
Conclusion
Our experimental results have unequivocally demonstrated that GF inhibits the activation of the XOD in both in vivo and in vitro conditions, modulates the expression of UAT to reduce the UA level. Moreover, GF attenuates pyroptosis by downregulating the activation of the TLR4-NF-κB p65-NLRP3 signaling pathway, showing analogous effects to AMPK agonist (Fig. 6). This combined action mechanisms underscore the protective properties of GF against kidney injury and inflammatory response induced by HUA. The long-term exposure to GF causes no overt toxicity to the experimental animals. Our findings are strongly indicative that GF is a promising effective and safe long-term therapeutic option for managing gout and kidney complications associated with HUA.Fig. 6. The schematic diagram showing that GF mitigates renal inflammation and hyperuricemia (HUA) induced by potassium oxonate plus hypoxanthine (PO+HX) in mice and HUA induced by monosodium urate (MSU) in Human kidney 2 (HK‑2) cells via activating the phosphorylation of AMPK through regulating the activation of the TLR4–NF‑κB–NLRP3‑mediated pyroptosis pathway. GF inhibits the activation of xanthine oxidase (XOD) in both PO+HX‑treated mice and MSU‑stimulated HK‑2 cells, and modulates the expression of uric acid transporters (UAT) to reduce the uric acid (UA) level
Supplementary Information
Additional file 1. Supplementary Table 1. Primer sequences. Supplementary Table 2. Identification of Top25 chemical components in GF by UPLC/qExactive-MS analysis