Suppressive Effect of Huzhentongfeng on Experimental Gouty Arthritis: An In Vivo and In Vitro Study

Background Huzhentongfeng (HZTF) is an extract from four Chinese medical herbs for treating gout. This study aims to evaluate its antigout activity and preliminary explore its mechanism in vivo and in vitro. Methods The rats were intragastrically administered with HZTF for 5 days and then injected 0.1 ml (10 mg) of MSU crystals to their joints for generating a gout model to analyze the paw volume and histopathology of joint synovial tissues of rats with different doses. We also investigated the antioxidant capacity of HZTF in vitro using indication including lipid peroxidation, DPPH·, and ABTS+ radical-scavenging capacity; besides, we used qRT-PCR to measure the effect of HZTF on interleukin (IL)-1β, caspase-1, NLRP3, and NQO1 expression in hydrogen peroxide-stimulated RAW264.7 macrophages and IL-1β, IL-6, and tumor necrosis factor (TNF)-α in MSU crystal-induced THP-1 monocytes. Confocal microscopy analysis was used to observe the dimerization of ASC adapter proteins. In addition, we also established quality standard of HZTF by using the high-performance liquid chromatography (HPLC) method. Results HZTF could significantly suppress the paw swelling and neutrophil infiltration induced by MSU intra-articular injection in rats compared with the control group. HZTF also showed inhibition effects of inflammatory cytokines (IL-1β, IL-6, and TNF-α) secretion at 25.00 and 50.00 μg/ml in MSU-induced THP-1 cells but showed no effects of IL-1β, IL-6, and TNF-α mRNA expression in MSU-induced THP-1 cells. Furthermore, confocal microscopy analysis showed that HZTF could prevent the oligomerization of ASC. Moreover, HZTF also showed effects in cell-free and cell-base tests of antioxidant capacity. Conclusion The results prove that HZTF possessed the potential preventive effect against gout arthritis, and the effect may be attributed to its preventing effect on neutrophil infiltration and proinflammatory cytokines secretion such as IL-1β, IL-6, and TNF-α which were caused by the activation of inflammasome.


Introduction
Gout is a common type of arthritis caused by hyperuricaemia and subsequent accumulation of monosodium urate crystal deposition in the joints, tendons, and surrounding tissues. e signs and symptoms of gout include sudden and intense pain, redness, swelling, and stiffness in a joint of the big toe or in another joint, such as the ankle, knee, elbow, wrist, and finger [1]. At present, there are many medications in clinic, such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, or corticosteroids, used to treat the symptoms of gout attacks, prevent future flares, and reduce the risk of gout complications. But they all have some limitations. Over 80% of patients treated with colchicine have experienced abdominal pain before full clinical improvement. In addition, the side effects of NSAIDs are more common in the elderly. erefore, it is critical to develop more effective agents with less adverse reactions.
e dried root of Polygonum cuspidatum Sieb.et Zucc was known as "Huzhang" in China, as a traditional Chinese medicine, which was officially listed in the Chinese Pharmacopoeia. e compounds from Plantago depressa Willd have been demonstrated to have tremendous amount of pharmacological activities, including antioxidant, antibacterial, and anti-inflammatory activities [12,13]. Plantago depressa Willd contains large amounts of polyphenols such as resveratrol, and it is considered one of the best sources of resveratrol because it contains higher amounts of this compound than other plants or fruits [14]. Plantago depressa Willd or Cheqiancao in Chinese is widely distributed and utilised around world, which has no significant toxicities in a regular dosage and has been used as medicinal plants for centuries [15,16]. Currently, researches have shown that extracts from Plantain leaves exhibited antioxidant and antimicrobial activities. e above-mentioned bioactivities of Plantain leaves extracts are thought to be related to phenolic compounds and flavonoids [17]. Ligustrum lucidum Ait is a kind of evergreen shrub plant which can be found in Hu'nan, Jiangsu, Sichuan, and Zhejiang provinces in China. e mature fruits of this plant have a cardiac and roborant effect and known as a traditional herb medicine in China, which is also prescribed in Chinese Pharmacopoeia. Previous phytochemical investigations on Ligustrum lucidum Ait reported the isolation of secoiridoid glycosides, phenolic glycosides, and lignans [18,19]. Beehive is the hive of Parapolybia varia Fabricius, a widely used traditional Chinese medicine. It is widely distributed in China and be harvested in autumn and winter. After removing the dead wasps, the hive is manufactured into a kind of traditional Chinese medicine by openair drying. Because of its multiple pharmacological activities, including antivirus and anti-inflammatory beehive has been used in traditional Chinese medicine over thousands of years for treating various diseases, including malignant tumors, rheumatoid arthritis, lung diseases, skin disease, digestive and urinary disorders, and dental diseases [11,20].
Huzhentongfeng (HZTF) is an extract developed by GuangZhou (Jinan) Biomedical Research and Development Center for treating gout. In order to obtain a good antigout effect, we combined the traditional Chinese medicine theory system with modern medical theory to extract these four medicinal materials to produce HZTF. According to the theory of traditional Chinese Medicine, Polygonum Cuspidatum is a monarch drug, Plantain is a minister drug, Ligustrum is an assistant drug, and Nidus Vespae is the guide drug. And, its main function is to treat acute gouty arthritis.
Firstly, in order to ensure the stability of product quality of HZTF, we also established a standard for the content of medicinal materials by using the HPLC method.
Synovial cells, monocytes-macrophages, and neutrophils will produce multiple cytokines, such as IL-1β, IL-6, and TNF-α, when they were stimulated by MSU crystals in the joints. Our study demonstrated that HZTF showed prominent effect on neutrophil infiltration and paw swelling in rats induced by intra-articular MSU injection. However, the concrete mechanism of HZTF preventing gouty arthritis remains unclear. So, in this study, we investigated the inhibition effects on expression levels of IL-1β, caspase-1, NLRP3, and NQO1 in hydrogen peroxide (H 2 O 2 )-induced RAW264.7 macrophages and expression and secretion of IL-1β, IL-6, and TNF-α from MSU crystal-induced THP-1 cells to investigate the mechanism of HZTF in treating gouty arthritis.

Equipment.
Stainless steel tape gauge, produced by Southwest China Tool General Plant of Guizhou Aviation Industry Group, China. e model of UV-visible spectrophotometer used in this experiment is UV2600 (Shimadzu, Japan), the model of the centrifugal machine is HC-2518 (Anhui USTC Zonkia, China), the real-time thermal cycler is CFX96 Touch (BioRad, USA), and the microplate reader is ELx808 (BioTek, USA).

Quality Standard of HZTF by HPLC.
Quality standard of HZTF was performed as described in an earlier report with slight modifications [21]. Briefly, HZTF was dissolved in 70% ethanol, and HPLC analysis was conducted using Poroshell 120 SB-C18 (4.6 × 100 mm, 2.7 μm, Agilent, US). e chromatogram was monitored at wavelength 280 nm. e mobile phase consisted of A (0.1% formic acid) and B (acetonitrile) with an isometric elution as follows: A 16% and 2 Evidence-Based Complementary and Alternative Medicine B 84%. e flow rate of the mobile phase was 1.0 ml/min, and the injection volume was 10 μl.

Synthesis of MSU Crystals.
MSU crystals were synthesized as described [22]. Briefly, 1.68 g of uric acid in 0.01 M NaOH was in a water bath heated to 70°C. en, 0.5 M NaOH was added to maintain pH between 7.1 and 7.2, and the solution was incubated at room temperature with slow stirring and continuously for 24 h. e crystals were washed with absolute ethyl alcohol, freeze dried, and autoclaved.

Animals.
Male Sprague-Dawley rats (200 ± 20 g) were purchased from the Experimental Animal Center of Henan Province, China. e animals were allowed to adapt to their environment for at least one week before initiation of the experiments. e animals were housed five per cage under a normal 12 h/12 h light/dark schedule. e animals were housed at room temperature (20 ± 2°C) with relative humidity (60 ± 5%) and were free access to a standard diet in the duration of the study. All studies were operated in accordance with the Institutional Animal Care Committee at the Experimental Animal Center of Henan Province and the China Council on Animal Care at the Experimental Animal Center of Henan Province.

MSU Crystal-Induced Inflammation in Rats
2.6.1. Animal Gout Model and Drug Administration. e rats were randomly divided into six groups. Each group contained ten rats. Group I served as controls and were intra-articular injected in the right posterior foot with 0.1 ml physiological saline. In group II, gouty inflammation was induced by intra-articular injection of 0.1 ml (10 mg) of MSU crystal suspension into the right posterior foot. Groups III, IV, and V comprised MSU crystal-induced rats treated with HZTF (0.25, 0.50, and 1.00 g/kg body weight, respectively). Group VI comprised MSU crystal-injected rats treated with positive control Tongfengshu (a commercially available Chinese patent medicine for gout, 1.50 g/kg body weight). HZTF and Tongfengshu were fully dispersed in distilled water. All doses of the respective drugs were expressed as grams per kilogram body weight. Drugs were intragastrically administered for 5 d once daily and also administration at the 5th day 1 h before the MSU crystal injection. Isoflurane was used to anesthetize the rats, and then the MSU suspension injection was given.

Assessment of Paw Swelling.
e pathological degree was quantified by the swelling value of paw measured at 1, 2, 3, 4, 5, 6, and 7 h after MSU crystal injections. e degree of edema was expressed as paw swelling which was calculated by the following equation: Here, a is the foot volume before MSU crystal injection and b is the foot volume after MSU crystal injection.

Histopathological Analysis.
Rats were killed by cervical dislocation (7 h), and then the ankle joints of the right posterior foot of the rats were dissected and fixed in 10% neutral formaldehyde fixative for synovial tissue collection. After decalcification, the tissues were paraffin-embedded, sectioned, and stained with haematoxylin and eosin (H&E).

Cell-Free In Vitro Studies
e thiobarbituric acid (TBA) method was carried out according to the method described by Costa et al. [23][24][25], and modified according to our situation. Briefly, 1 mL of the different concentrations of HZTF or VC was mixed with 1 mL of 10% yolk homogenate in tris buffer (pH 8.0). en, 100 μl of 70 mM AAPH, 2.9 ml 20% acetic acid, and 3.0 ml 0.8% TBA solution were added. And, the mixture was boiled 60 min for full reaction; after cooling, adding 4.0 ml butanol, and fully extracting, the mixture was centrifuged at 2000 g for 10 min, extracting the organic layer. e final concentrations of HZTF or VC in the reaction system are 30.00, 60.00, 90.00, 120.00, and 150.00 μg/ml. e absorbance was measured at 532 nm by the ultraviolet-visible spectrophotometer. e decrease of absorbance indicates the increase of antioxidant activity of the samples. e antioxidant activity value of the samples was expressed as the percentage of lipid peroxidation inhibition rate which was calculated by the following equation: where A S is the absorbance of test sample and A C is the absorbance of blank control.

DPPH · Radical-Scavenging
Ability. DPPH · radicalscavenging capacity was determined according to the modified method by Luo et al. [26]. HZTF and VC samples were prepared in 70% ethanol at the difference concentrations, then 1 ml of each sample mixed with 1 ml DPPH solution (0.1 M in 50% ethanol) and 3 ml distilled water. e final concentrations of HZTF are 4.00, 12.00, 20.00, 28.00 and 36.00 μg/ml, and VC are 2.00, 4.00, 6.00, 8.00 and 10.00 μg/ml. e mixtures were vortexed and allowed to equilibrate for 20 min at room temperature in the dark, and then measured the absorbance at 517 nm. [27,28]. Scavenging activity was calculated using the formula: where A S is the absorbance of the samples at different concentration and A C is the absorbance of control.

ABTS + Radical-Scavenging
where A S is the absorbance of sample and A C is the absorbance of the black control. were cultured in Dulbecco's modified eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 1x antibiotic solution (streptomycin (100 U/ml) and penicillin (100 U/ml)) in a humidified atmosphere supplied with 5% CO 2 and maintained at a temperature of 37°C. Cells were allowed to grow till they reached a confluency of 80-90% and washed with phosphate buffered saline (PBS) with regular replacement of culture medium.
THP-1 cells were maintained in 1640 medium supplemented with 10% FBS in a humidified atmosphere supplied with 5% CO 2 and maintained at temperature of 37°C. Cells were allowed to grow till they reached a density of 2 × 10 6 and then added the same volume of culture medium.

Cell Viability.
Cell viability was performed by MTT assay. RAW264.7 macrophage cells were seeded in a 96-well culture plate. THP-1 cells were seeded in another 96-well culture plate for 3 hours with 30.82 μg/ml PMA, the day before drug treatment. After 24 h incubation, the cells were stimulated with varying concentrations of HZTF (3.12, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 μg/ml) in FBS-free medium for further 24 h. After incubation, 10.0 μL MTT DMSO solutions (5 mg/ml) were added to each well. en, the plates were incubated for 4 h at 37°C in dark. After incubation, 90 μL medium was removed from each well, 100 μL of DMSO was added to the wells, and then the plates were incubated for 15 min at 37°C under gentle shaking to dissolve the tetrazolium dye. e cell viability was calculated according to the results of absorbance at 570 nm and expressed as relative percentage viability. e relative cell percentage viability was calculated by the following equation: where A S is the absorbance of HZTF, A C is the absorbance of the control, and A b is the absorbance of cell blank.  25.00 and 50.00 μg/ml), stimulated by MSU (1 mg/ml) for 8 h, and collected the supernatant. Secretion of inflammatory cytokine (IL-1β, IL-6 and TNF-α) was measured by ELISA kits according to the assay procedure.
2.8.6. Western Blotting Analysis. Protein samples were obtained from the culture medium as same as in the ELISA assay and concentrated by the ultrafiltration device. Protein sample was denatured by mixing with 1/4 volume of 5x loading buffer, and metal bath at 100°C for 10 min. Samples were resolved by electrophoresis with 10% SDS-PAGE and transferred onto a PVDF membrane (Merk, Germany). After blocking nonspecific binding sites for 2 h with 5% dried skim milk dissolved in TBST, the membranes were individually incubated for overnight with anti-IL-1β, anti-IL-6, anti-caspase-1, anti-PGE2, and ASC. Develop the color of the blot rocking in 3, 3′-diaminobenzidine (DAB) substrate solution. Stop the reaction by pouring out the substrate after the expected band appears and then well rinsed with distilled water repeatedly. Dry the membrane, and store it in the dark place.

Confocal Microscopic
Analysis. THP-1 cells were pretreated with MCC950 (4.26 μg/ml) or HZTF (50.00 μg/ ml) and stimulated by MSU (1 mg/ml) for 8 h. Briefly, THP-1 cells were plated on coverslips and incubated with an anti-ASC antibody and then incubated with an anti-rabbit IgG-FITC antibody. Recording with a laser scanning confocal microscope (LSM710, Carl Zeiss, Oberkochen, Germany), the ASC speck formation was analyzed using Zen2010 software.
2.9. Statistical Analysis. All results were expressed as mean ± standard deviation (n � 3). Statistical analysis was carried out by SPSS 21 statistical software (IBM Inc., USA). A statistical difference was considered significant when P-values are less than 0.05. Figure 1, four substances in HZTF were selected as content indicators: (1) plantamajoside, (2) polygonum cuspidin, (3) specnuezhenide, and (4) resveratrol glucoside, and all of them had distinct peaks under 280 nm ultraviolet absorption.

Effect of HZTF and Tongfengshu in In Vivo Studies.
To determine the degree of edema, the swelling value of the right posterior foot ankle joints of rats were measured ( Table 2). e foot-swelling values of MSU crystal-induced rats were significantly higher than that of the control rats at 1 h to 7 h (P < 0.05). Paw edema decreased significantly in MSU crystal-induced rats treated with HZTF (0.50 and 1.00 g/kg body weight) at 1 h to 7 h and Tongfengshu (1.50 g/ kg body weight) at 1 h to 7 h (P < 0.05).
To compare the histological changes in the joints between different groups, joint tissues were sectioned and examined via H&E staining. e coverage of synovial cells on synovial cavity of the joints was fragmentary, and there was edema in loose connective tissue, as well as local infiltration of leukocytes ( Figure 2). However, rats treated with HZTF (0.50 and 1.00 g/kg body weight) and Tongfengshu (1.50 g/kg body weight) decreased edema and leukocyte infiltration after 7 h. is change correlated well with the decrease in paw swelling induced by HZTF and Tongfengshu treatment.

In Vitro Cell-Free Antioxidation Experiment of HZTF.
To investigate whether the antigout effect of HZTF is due to its antioxidant activity, we measured the antioxidant activity of HZTF using multiple methods. As shown in Figure 3(a), we can know that HZTF can significantly inhibit lipid peroxidation when the concentration was higher than 60.00 μg/ml, and the IC 50 values of VC and HZTF are 49.94 ± 3.67 and 114.62 ± 3.43 μg/ml, respectively, which mean the lipid peroxidation inhibition ability of HZTF is significantly weaker than VC (P < 0.05). It is observed that the DPPH · radical-scavenging activity of HZTF is shown in Figure 3(b). e IC 50 values of VC and HZTF are 2.81 ± 0.62 and 13.04 ± 2.80 μg/ml, respectively. ough it can inhibit the DPPH · radical, the IC 50 value of HZTF is significantly higher than VC (P < 0.05). As shown in Figure 3(c), the ABTS + radical-scavenging activity of both VC and HZTF are in dose-effect relationships. HZTF can scavenge the ABTS + radical significantly when the concentration was higher than 5.00 μg/ml and the IC 50 values of VC and HZTF are 1.48 ± 0.70 and 7.52 ± 0.50 μg/ml, respectively, which means the ABTS + radical-scavenging activity of HZTF is significantly weaker than VC (P < 0.05).

Confocal Microscopic Analysis.
As a symbol of inflammasome activation, ASC forms oligomers in response to NLRP3 activators. Confocal microscopy analysis intuitively showed that HZTF suppressed MSU crystal-induced formation of ASC speckles in THP-1 cells (Figure 8).

Discussion
Inflammatory response to MSU crystals triggered the acute symptoms of gout and mainly mediated by macrophages and neutrophils. Intra-articular injection of MSU crystal can simulate gouty arthritis in humans and produces a painful response which is similar to the acute gouty attacks occurrence spontaneously [32]. Significant swelling accompanied by an extensive inflammatory response after MSU injection in the ankle joint is an important symbol in determining the degree of inflammation and therapeutic efficacy. e primary pathological symbol of gout is the neutrophils accumulated and infiltrated in the joint tissues, which actively phagocytosed MSU crystals. en, the membrane lysed and released inflammatory cytokines and free radicals amplified the inflammatory response [33,34]. We observed similar changes in the joint MSU injection rat model, so this model is suitable for studying the pathological mechanism of gout arthritis.
Plants which give antioxidant activities should be highly potent in the management of gout because they often share xanthine oxidase (XO) inhibitory effects [35,36]. We measured the antioxidant activities to study whether the antigout effect of HZTF is related to its antioxidant capacity. Lipid peroxidation occurs in the process of reactive oxygen  Figure 1: e quality standard of HZTF was determined using an HPLC system, plantamajoside (1), polygonum cuspidin (2), specnuezhenide (3) and resveratrol glucoside (4).      species (ROS) oxidizing the biological membranes, which ROS reacts with membrane phospholipids, enzyme and membrane receptor-associated polyunsaturated side chain, and nucleic acid fatty acid and produces lipid peroxidation products such as malondialdehyde and 4-hydroxynonenal, and then the fluidity and permeability of cell membrane changed, resulting in the change of structure and function of cells [37,38]. e free radical-scavenging capacity of HZTF was measured using commercially available stable free radical DPPH · . HZTF presented DPPH · radical-scavenging capacity to some extent. It appears that antioxidant activity and reduction of DPPH · both require the presence of a free OH group on the ellipticine ring [39], and if HZTF can reduce DPPH · , it may suggest that HZTF contains compounds of similar structure. Based on the special chemical properties of formed free radicals, ABTS assay is frequently used to measure the radical-scavenging capacity of compounds [40]. e ABTS + radical elimination is a common antioxidant method as well as the DPPH · method to evaluate the antioxidant capacity of the chemical compound. Based on previous descriptions, Polygonum cuspidatum, Plantain, and Ligustrum, which were used to produce HZTF, can extract compounds with good antioxidant activity, and similar to HZTF, these compounds were also extracted from ethanol, which suggest that the main antioxidant compounds containing HZTF may be phenolic compounds too [41][42][43]. According to the results, the lipid peroxidation inhibition ability, DPPH · radical-scavenging ability and ABTS + radical-scavenging capacity of HZTF were significantly weaker than VC (P < 0.05). VC is a strong antioxidant [44], but seldom used in acute gout treatment. HZTF has weaker antioxidant capacity than vitamin C, but has a strong antigout capacity, so we believed that the antioxidant capacity of HZTF may not be the main mechanism of its antigout effect.
IL-1β is a proinflammatory cytokine that induces local and systemic inflammation aimed to eliminate foreign matters and microorganisms. However, exorbitant levels of inappropriate IL-1β production have been shown to be a key process in the etiology of the disease. In these conditions, blocking IL-1β has proven very effective in clinical studies [45]. Caspase-1 and NLRP3 are closely linked to the pathogenesis of various metabolic diseases including gouty arthritis [22,46]. At the same time, the expression increase of IL-1β, caspase-1, and NLRP3 is closely related to oxidative stress, and if we reduce the level of oxidative stress, we may suppress the expression of IL-1β, caspase-1, and NLRP3 [47]. NQO1 is an important cellular antioxidant enzyme, Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of MSU crystals, in joints and periarticular tissues. MSU engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active IL-1β [22]. Macrophages from mice deficient in components of the inflammasome such as ASC (RAW264.7 macrophages) are defective in crystal-induced IL-1β activation [48]. THP-1 cells were chosen for this experiment. According to the result, MSU crystals did not increase IL-1β mRNA expression, but increased IL-1β secretion, and HZTF also did not affect the mRNA expression of IL-1β, IL-6, and TNF-α, but significantly reduced the secretion of IL-1β, IL-6, and TNF-α in THP-1 cells stimulated by MSU. In the western blotting test, we can know that HZTF inhibited procaspase-1, cleaved-caspase-1, pro-IL-1, cleaved-IL-1β, and IL-6 at the same time, suggesting that HZTF may inhibit not only the inflammasomes but also the NF-κb signaling. Also, in the western blotting result, HZTF showed inhibition effect in PGE 2 secretion, which suggests that the mechanism of antigout effect of HZTF may be varied. At the same time, confocal microscopy analysis intuitively demonstrated the inhibitory effect of HZTF on ASC dimerization. ese results suggested that HZTF may prevent gout by inhibiting NALP3 inflammasome as well as blocking the NF-κb signaling pathway, which will increase the expression of IL-1β, IL-6, and TNF-α [49].

Conclusion
e present study confirmed the antigout effects of HZTF in in vivo experiments and showed its reduction effects on the secretion of proinflammatory cytokines IL-1β, IL-6, and TNF-α. At the same time, whether its antigout effect is related to its antioxidant capacity needs further study. is study provides a new promising therapeutic TCM for gout, and the precise mechanism of action is worth further to be investigated.