Folic Acid-modied Lysozyme-protected Gold Nanoclusters as Anti-inammatory Nanomedicine for Gout Flares

Gout normally occurs when excess urate crystals accumulate in the joints that induce inammation. The inammations further result in gout ares, causing serious pain. Some anti-inammatory drugs can relieve pain, but they have signicantly toxic and side effects. Nanomedicines offer opportunities to reduce various side effects but are mostly based on the delivery of additional organic drugs, which adds complexity and may cause other health problems including the bioaccumulation risk. We developed folic acid (FA)-modied lysozyme (Lys)-protected gold nanoclusters (AuNCs) (i. e., FLA) by a one-pot method, and FLA is developed as an anti-inammatory drug in the treatment of acute attacks of gout ares at high monosodium urate (MSU) levels. The morphological changes of the cells caused by were studied by Transmission electron microscopy (TEM) and uorescence microscopy. Western blotting was conducted to understand the inammation factors. AutoDock Vina was used for molecular docking of FLA to evaluate the binding mode of the ligand and the interaction in the active site. test in case of equal variance and Kruskal-Wallis test in case of uneven variance. Ankle swelling function disorder index and inammation index were analyzed using two-way ANOVA and Tukey post hoc test. p < 0.05 was considered to be statistically signicant.

Folic Acid-modi ed Lysozyme-protected Gold Nanoclusters as Anti-in ammatory Nanomedicine for Gout Flares Introduction Gout is a metabolic disease that occurs due to purine metabolism disorders and increased blood uric acid. The most common symptom of gout ares is a more serious pain, stiffness, and swelling in the joints than common arthritis [1] [2]. Current clinical management of gout ares relies on colchicine, nonsteroidal anti-in ammatory drugs, glucocorticoids, and adrenocorticotropic hormones [1,3]. Unfortunately, these treatments have caused various problems including irritability, mood disorders, increased blood glucose levels, immunosuppression, and uid retention [4]. On the other hand, due to the unsatisfactory therapeutic e ciency, the need for large doses and frequent administration causes di culties for long-term treatment [5]. There is an urgent need for safer and more effective drugs to treat gout ares.
In recent years, nanomedicines reduce the side effects for treating many diseases, and dosage is also reduced because of the larger active area. Several nano-drug deliver antiphlogistic durgs were used for treating arthritis [6][7][8], showing more satis ed biosafety and e ciency. Although signi cant improvement has been made, additional antiphlogistic durgs are still involved [9]. Several nano agents were directly applied for uric acid reduction without loading additional antiphlogistic durgs, but the in ammatory problems were hardly solved [10,11]. The limited e ciency of these nanomedicines might be attributed to their therapeutic strategies, which only focused on reducing MSU [12]. During the acute attack of gout ares, it is not suggested to use uric acid lowering drugs. Not only does it have insigni cant antiin ammatory and analgesic effects, but also causes the blood uric acid to drop too quickly, which promotes the dissolution of tophi in the joints, forming insoluble crystals and aggravating in ammation.
Uric acid-lowering treatment can be started only after the acute symptoms are relieved (≥2 weeks). Thus, We are trying to establish a method that can deal with acute attacks of gout ares, even in the case of high uric acid, which can still reduce in ammation.
AuNCs have been widely studied as antiphlogistic durg-free nanomedicines because of their biosafety, insigni cant bioaccumulation in the body, and antioxidant effects [13,14] [15]. AuNCs have been designed as a probe for uric acid, which is related to gout [16], indicating they may have various target effects for this disease. Herein, we developed FLA as antiphlogistic durg-free nanomedicine for the treatment of gout ares. This nanomedicine evokes immune function and shows excellent antiin ammatory effects on rat models with gout ares [17][18][19]. FLA also enables the joint swelling of gout rats to recover e ciently. To the best of our knowledge, this is the rst study to use AuNCs to successfully relieve the in ammations and other symptoms for rats with gout ares.

Results And Discussion
Characterization of FLA Based on a similar mechanism [22], the scheme for the synthesis of FLA (Fig. 1A) and the mechanism is proposed for the construction of FLA (Fig. 1B). Initially, Lys and Au(I), Au(I) binds to the 1-N of His15 of Lys. The free Au(I) in the mother liquor continuously diffuses into the Lys and then disproportionates into Au(0) and Au(III) in the crystal. Au(0) is further assembled as clusters, and Au(III) translocates and recombines to other sites in Lys. AutoDock Vina was used for molecular docking of FLA to evaluate the binding mode of the ligand and the interaction in the active site. PyMol has been used to generate a 3D pose of the recognized ligand that binds to the active site of LA, and its binding energy is 7.0 kcal/mol. Folate and amino acids form conventional hydrogen bonds (HB) GLU-35, ASN-59, TRP-62, ALA-107, ALA-110, and ASN-113. In addition, it forms π-anion interactions with ASP-52, and π-sigma and π-alkyl interactions with ALA-110. According to the molecular docking theory, folic acid molecules can be perfectly combined with AuNCs.
The AuNCs, FLA, and AuNPs were characterized with TEM (Fig. 1C). All of the AuNCs are well dispersed and the size was about 2.88 nm. Also, aggregation of the AuNCs cannot be found from the TEM images. Therefore, we assume that the use of Lys as a stabilizer can inhibit the formation of larger particles. The FA enabled AuNCs to grow to 4.5 nm in diameter. However, the AuNPs that mixed with Lys were 16 nm in diameter. Because of the small size and the biocompatible surface, these materials would be transferred into human cells. The alteration in the charge of the particles in the microenvironment and the attachment of FA on the surface of AuNCs may perturb the electron density into the metal nanoparticle and consequently change the inner interaction and the size.
Targeted Therapy for gout Normal RAW 264.7 cells were cultured with FLA (100 µM) containing medium and treated with LPS and MSU crystals for 24 h. Cells were labeled with FITC uorescent (green) clusters and the location of the cells was visualized by staining the nucleus with DAPI (blue) and the cytoskeleton with Tubulin (red). It was found that RAW 264.7 cells could effectively take up FLA ( Fig. 2A). the same treatment was followed by TEM observation of the cells and the results were the same as for confocal microscopy. RAW 264.7 cells could effectively take up FLA (Fig. 2B). The uorescent agent FITC was loaded into FLA and its distribution in vivo was studied (Fig. 2C). We could observe that the uorescent signal of FLA in the FLA group accumulated at the kidney before 8 h and was rapidly removed from the body after 8h, indicating that FLA was rapidly cleared in vivo. However, in the FLA+Model group, FLA aggregated at the kidney and ankle at 2 h. This suggests that FLA enhances the accumulation of uorescence in the ankle joint. It was noted that FLA aggregated in the kidney in both the FLA and FLA+Model groups, representing that FLA is excreted from the body mainly through the kidneys ( Fig. 2D-E). FLA can target the swollen and in amed ankle joints of rats and is phagocytosed by macrophages. Then, they are cleaned by the kidneys.
Therefore, FLA are promising to show therapeutic effects towards gout and biosafety.

Anti-in ammation in vitro
The FLA on MSU-induced in ammatory response in vitro was observed under confocal microscopy. We found that FLA inhibited the production of IL-1β, a key cytokine for gout in ammation that causes gout ares, and had a stronger effect than treatment with Lys and AuNCs alone ( Fig. 3A-B, D-E) Since gout is a dual-signal-driven disease. NLRP3 in ammatory vesicles are responsible for IL-1β maturation, and another signal upregulates IL-1β transcription and pro-IL-1β synthesis [1,25]. Therefore, the effects of FLA on NACHT, LRR, and PYD domain-containing protein 3(NLRP3) in ammatory vesicles, as well as the Toll-like receptors (TLR) signaling pathway, was examined. Western blot analysis revealed that FLA signi cantly reduced the protein levels of Caspase1 (Fig. 3F), ASC (Fig. 3G), NF-κB (Fig. 3K ) and was superior to Lys and AuNCs alone, suggesting that Lys acts as a shell ligand to enhance the e cacy of gold nanoclusters. At the same time, we found that FLA also decreased the protein levels of IL-6( Fig. 3H), TNF-α (Fig. 3I), and COX-2 (Fig. 3J). This may be related to the initiation of IL-1β signaling, leading to the production and secretion of pro-in ammatory mediators, and the up-regulation of pro-in ammatory cytokines through the binding of activated NF-κB to the promoter region of target genes [32][33][34]. As a result, it is concluded FLA can effectively inhibit the production of IL-1β and reduce the occurrence and expansion of gout ares. FLA also has promising therapeutic effects on common diseases that respond to IL-1β neutralization such as type 2 diabetes, heart failure, recurrent pericarditis, rheumatoid arthritis, and smoldering myeloma [35]. Thus, the administration of FLA can exhibit comprehensive therapeutic effects for gout patients.

Antioxidant effects
MSU crystals can activate NLRP3 in ammatory vesicles and thus increase IL-1β release via ROS [36]. To elucidate the mitigating effect of FLA on gout in ammation. We investigated the effect of FLA on oxidative stress using Raw 264.7 cells. We treated each group of RAW 264.7 cells with 100 µM FLA for 24 h and observed them with TEM, and found that the mitochondria in the sham group had normal morphology and clear mitochondrial ridges. The mitochondria in the FLA group showed signi cant improvement compared to the Model group (Fig. 4B). Also for exogenous ROS that was studied, Raw 264.7 was exposed to 20 µM H O for up to 30 minutes and incubated in the presence or absence of FLA, Lys, AuNPs for 4 h. The production of ROS was observed by confocal uorescence microscopy. A signi cant reduction of ROS in the FLA group could be found (Fig. 4C). The SOD-like enzyme activity, GSH level, and MDA level can re ect the antioxidant effects. The FLA group showed higher SOD-like activity than the Model, AuNPs, and Lys groups (Fig. 4D). The MDA content showed an opposite trend: the FLA group showed signi cantly lower MDA content than the Model, AuNPs, and Lys groups (Fig. 4E). Amount of GSH analysis showed that the FLA group had signi cantly higher GSH content than the Model, AuNPs, Lys group (Fig. 4F). These changes were attributed to the enhanced ability of FLA to resist oxidative stress after a gout attack. In addition, there are several other possibilities for FLA to inhibit ROS. The rst is to inhibit the NF-κB in ammatory factor leading to a decrease in the level of nitric oxide synthase (INOS) induced by downstream in ammatory factors, thereby reducing the production of free radicals such as NO, thereby reducing the level of ROS [37,38]. Secondly, the level of ROS may be reduced by regulating nuclear factor E2-related factor 2 transcription factor (Nrf2) [39,40], which may also be related to the buffering of oxygen radicals [41]. This suggests that FLA act as ROS scavengers in acute attacks of gout.

Anti-in ammatory effects in vivo
The injection of MSU crystals (0.5 mg/10 ml) into the ankle of SD rats can increase the protein level of the pro-in ammatory cytokine IL-1β in the tissues around the ankle [25,30]. Western blot analysis showed that FLA signi cantly reduced the protein levels of IL-1β (Fig. 5A), Caspase1 (Fig. 5D), ASC (Fig. 5E), NFκb (Fig. 5K), COX-2 (Fig. 5G), IL-6 ( Fig. 5F), and TNF-α (Fig. 5I). Its anti-in ammatory effect was consistent with the results of in vivo and in vitro experiments. COX-2 is a key enzyme that converts cell membrane arachidonic acid into in ammatory mediator prostaglandin 2 (PGE2), which is an important pain mediator. It plays an important role in the noxious stimulation of gout. Due to the down-regulation of COX-2, we assume that the protein level of PGE 2 will also down-regulate and have an analgesic effect on gout ares [42]. FLA effectively inhibits pro-in ammatory up-regulated cytokines, indicating the therapeutic effects on the acute attack of gout ares.

In vivo recovery of gout ares
The circumference of 1 cm above the thinnest point of the rat's ankle joint was measured to assess the degree of edema in the rat's ankle joint (Fig. 6A). By observing the injection of different concentrations of FLA with gout rats, we found that at 24h, the swelling reached its highest peak and was most effectively inhibited by FLA. According to the method to assess the in ammation and dysfunction index of rats, the in ammation and dysfunction index of the ankle of gout rats peaked at 24 with complete swelling and lameness on the swollen side. FLA (20 mg/kg) signi cantly improved their in ammation and dysfunction index (Fig. 6B-E). After 24 h, we selected the ankle of the rat for HE staining of the SD section and found it. In the gout model group, the joint cavity became smaller, with uid exudation and in ammatory cell in ltration. The cartilage layer becomes thinner, and the chondrocytes have vacuole-like changes and degenerative necrosis. There were fewer lesions in the treatment group. This is consistent with the hematoxylin and eosin-stained sections of the ankle joint (Fig. 6F) that FLA can reduce the ankle swelling caused by MSU. The administration of FLA also reduces tissue destruction caused by in ammation.
Biosafety plays an important role in determining whether nanomedicines can be used to treat diseases. Herein, Raw 264.7 cells were used to test cell viability by the MTT method. Cells were studied in the presence of FLA at 0 to 320 µM. No dose-response pattern was seen in Raw 264.7 at concentrations less than 320 µM. The cell viability of the cells was almost 100% (Fig. S1A). To study the toxicity of FLA in vivo. The organs were studied by histopathological analysis after injection of different concentrations of FLA (5, 10, 20 mg/kg) on the rst day and 14th day. Microscopically, myocardial bers were seen to be interwoven and arranged, with nuclear ellipses located centrally and capillaries abundant between myo bers. Hepatocytes were closely arranged, binucleated cells were common, and connective tissue content was low. The splenic white and red marrow structures were clear, and there was no signi cant increase in lymphoid tissue. The alveolar structure was intact and the interstitium was not brotic. There was no swelling of the renal nodules, the size of the renal capsule was normal, and the cubic renal tubule cells were not diseased. In addition, there was no signi cant structural difference between the material group and the control group (Fig. S1C-D). All these results indicate that FLA are safe for the treatment of gout ares in vivo and in vitro.

Conclusions
This study demonstrates a new anti-in ammation strategy for the fast therapy of gout ares using (FLA). FLA directly reduces the in ammation of the rat with gout ares without additional antiphlogistic durgs, which quickly repairs joints and other functions. This opens an avenue for relieving the serious pain for gout patients using antiphlogistic durg-free nanomedicines, which is promising for long-term use.

Materials and Instruments
HAuCl 4 , Lys, citrate sodium was obtained from Sigma Aldrich. The dialysis tube (MWCO 6000) was supplied by Spectrum. All other reagents were of analytical grades used as received without further puri cation. Ultrapure water (18.0 MΩ, Millipore) was used for all the experiments. Elemental analyses were performed by inductively coupled plasma mass spectrometry (ICP-MS) (Thermo). Microscopic images were investigated with a Tecnai G220 transmission electron microscope (TEM, 200 kV) (FEI, USA). Zeta potential was studied by Malvern sizer 3000. Fluorescence images were taken with Leica TCS SP5 II.

Preparation of FLA
Synthesis of AuNCs (LA) 2.5 mL of Lysozyme (Lys) solution (15 mg/mL) was mixed with 2.5 mL of aqueous HAuCl 4 (5 mM) solution in a 50 mL vial. The mixture was stirring for 5 minutes. Then, 100 μL of 100 mM NaOH solution was introduced with continuously stirring until transparent. Then, the vial was transferred to a 37 °C water bath and kept at this temperature for 24 h. The samples were puri ed by a dialysis tube (MWCO 6000) for more than 24 h before use.
Synthesis of FLA 10 ml of dimethyl sulfoxide (DMSO) was loaded in a 50 ml round bottom ask. FA (0.16 g, 0.36 mmol) was added into the ask and stirred until dissolved. 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-(3-Dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride (NHS) with a ratio of ( Folic acid: EDC: NHS=1: 1: 1) were added to the reaction and stirred for 20 h in the dark. Finally, AuNCs (2.5 mmol) were added to the mixture and further stirred for 16 h. The samples were puri ed by a dialysis tube (MWCO 6000) for more than 24 h before use.

Synthesis of AuNPs by the adsorption of Lys
The synthesis of the citrate stabilized AuNPs (Gold nanoparticles) was following a method described by the reference [20]. Next, 2.5 mL of Lys solution (15 mg/mL) was mixed with 2.5 mL of AuNPs (5 mM) (the concentration is corresponding to the Au element).

Molecular Dynamics
The molecular docking was performed using AutoDock Vina [21] to reveal the binding a nity and interactions of FA with the surface of AuNCs (PDB ID: 7BV2) [22]. FA was docked on the site using the optimized grid box and nine poses were generated. All of the docking results were ranked by energy and the lowest binding energy was selected. The docking interactions of FLA and FA were visualized and analyzed using PyMol [23].

Animals and Models
Adult SD rats (half male and half female, weight 180-220 g, 10-12 weeks) were purchased from Nanjing Institute of Biomedical Research, Nanjing University. The experimental procedures were approved by the Animal Protection and Use Committee of Jinzhou Medical University. ethical committee number:2021101.The animal data were reported following the ARRIVE 2.0 guidelines [24]. All rats were placed in standard cages in the SPF Laboratory Animal Center of Jinzhou Medical University. They had access to free food and water on a 12/12-hour day/night cycle and at the appropriate temperature. The animals were allowed to acclimatize to these conditions for at least 2 days before starting the experiments. For each group of experiments, animals were matched by age and body weight, and all procedures were performed at 1% sodium pentobarbital 50 mg/kg, with an intra-ankle injection of MSU crystal 1 suspension (1.25 mg/100μl) [25][26][27][28].
Cell preparation and stimulation Raw 264.7 was kept in moist 5% CO 2 at 37°C in Dulbecco supplemented with 10% (modi ed Eagle's medium v/v streptomycin (U/ml)) FBS, penicillin (100 U/ml) (Gibco, Grand Island, NY, USA) and. The cells were inoculated in cell dishes overnight and the medium was changed to serum-free medium the next morning, then the cells were treated with LPS (1 μg/ml) with or without Lys, AuNPs, and FLA. The treatment lasted for 12 h and was stimulated with MSU crystals (200 μg/ml) for 12 h. Analysis of cell extracts and precipitated supernatants by immunoblotting.

MSU crystal preparation
In 45 ml of 0.03 M NaOH solution (pH 7.5), 250 mg of uric acid (Solarbio, Beijing, China) was heated to boil. The solution was ltered and 1 ml of 5 M NaCl was added to accelerate crystal formation. The solution was gently stirred at room temperature for 24 h or until a milky white precipitate formed. The crystals were kept sterile, washed with ethanol and acetone, and dried at room temperature. The MSU crystals were resuspended in PBS at a concentration of 24 mg/ml, sonicated, and used under sterile conditions. MSU crystal preparations are evaluated with speci c Limulus reagent (Maclean's, Shanghai, China) and are endotoxin-free [29,30].

Cell viability
Cell viability was studied based on cell counting MTT assay analysis of Raw 264.7. First, cells were inoculated in 96-well plates at a density of 4 × 10 3 per well and incubated overnight. In addition, two types of cells were exposed to the medium for 24 h in the absence (control) and the presence of 5, 10, 20, 40, 80, 160, and 320 μM FLA, respectively. Next, the culture medium was discarded and washed carefully with PBS 2-3 times. Then 20 μL of MTT solution (5 mg/mL) was added to each well and the plates were incubated at 37°C, 5% CO 2 for 4 h until the purple methanogenic product appeared. After careful removal of the medium, the purple product is dissolved in 150 μL of dimethyl sulfoxide (DMSO). Cell viability was studied by recording the absorbance at 450 nm with an enzyme marker (n = 5/group).
Immuno uorescent dual-labeling staining RAW 264.7 cells in each group were incubated for a certain period and washed with PBS three times. Then, these cells were xed with 4% PFA for 30 minutes. Subsequently, the cells were washed 3 times with PBS, punched with Triton X-100 (0.3%) for 15 minutes, washed 3 times with PBS, and then blocked with 5% goat serum for 2 h. Then, these cells were incubated with primary IL-1β antibody and β-tubulin antibody (Invitrogen, Waltham, MA) in a culture medium at 4 °C overnight. Then the cells were washed 3 times with PBS. Subsequently, these cells were incubated with secondary antibodies (Alexa Fluor 546 labeled anti-rabbit IgG) and anti-mouse IgG (Alexa Fluor 488 labeled anti-mouse IgG) (Invitrogen, Waltham, MA) for 2 h and washed for 3 Secondly by PBS. Finally, the cells were stained with DAPI for 15 minutes. Similarly, RAW 264.7 cells were cultured with or without FLA (100 μM) medium, and FITC was added for 24 h. Then, the cells were xed with 4% PFA for 30 minutes. Subsequently, the cells were washed 3 times with PBS. The cells were incubated overnight at 4°C in a medium containing an anti-βtubulin antibody. Next, these cells were incubated with the corresponding secondary antibodies for 2 h, washed 3 times, and stained with DAPI for 15 minutes. The cells were then observed through a singlephoton confocal uorescence microscope for imaging experiments. The uorescence optical density was analyzed with ImageJ2x software (n = 3/group).

Antioxidant evaluation
Untreated RAW 264.7 cells were plated overnight in 24-well culture dishes without tissue culture treatment and treated with LPS (1 μg/ml) with or without Lys, AuNPs, and FLA. This was continued for 12 h and stimulated with MSU crystals (200 μg/ml) for 12 h. The glutathione (GSH), Superoxide dismutase-like enzyme (SOD-like enzyme), malondialdehyde (MAD), and ROS levels were assessed using GSH, SOD-like enzyme, MDA, and ROS assay kits (Solebro, Beijing, China), respectively, following the manufacturer's scheme (N≥3/group).

Hematoxylin-eosin staining
The ankle joints of anesthetized rat were xed in buffered 4% PFA for 24 h and decalci ed in 10% nitric acid + equal volume xative for 3 days. Finally, they were embedded in para n, sliced, and stained with hematoxylin and eosin (HE) (n=3/group). SD rats (180 g-220 g) were divided into four groups, and then different doses of FLA were injected intraperitoneally: control. After 30 days, the tissues (heart, kidney, liver, spleen, lung) were histologically analyzed by H&E staining (n = 3/group).

Target evaluation
Gout rat were randomly divided into the Sham group, model group, and FLA group. At the set time points (2, 4, 6, 12, and 24 h), the heart, liver, spleen, lung, kidney, and ankle of anesthetized rat were taken. And use IVIS Spectrum, PerkinElmer system to record the uorescence distribution (n = 3/group).
Swelling assessment 30 minutes after intraperitoneal injection of MSU into the ankle joint cavity, MSU crystals or FLA (5 mg/kg, 10 mg/kg, 20 mg/kg) were intraperitoneally injected into the ankle joint cavity. A soft ruler was used to measure the ankle edema. By subtracting the initial circumference from the ankle joint circumference measured at each time point, the ankle joint edema of each mouse was determined and expressed as Δmm/joint (n = 3/group).

In ammation and functional disorder evaluation
The classi cation criteria of in ammatory and dysfunctional indices are referred to the method according to the reference [31]. Grade 0 in ammatory index evaluation: normal; Grade 1: visible erythema, mild swelling, and bone signs; Grade 2: obvious redness and disappearance of bone signs, but the swelling is con ned to the joints; Grade 3: swelling of the lateral limbs of the left-hand joint. Grade 0 dysfunction index evaluation: normal gait, all four feet are evenly on the ground; grade 1: left foot relaxed, toes open, mild lameness; grade 2: left hind foot bent, toes touched on the ground, obviously lame; grade 3: left hind foot completely off the land.

Statistical Analysis
All values are expressed as mean ± SEM. In the case of multiple comparisons, we used one-way ANOVA followed by Bonferroni post hoc test in case of equal variance and Kruskal-Wallis test in case of uneven variance. Ankle swelling function disorder index and in ammation index were analyzed using two-way ANOVA and Tukey post hoc test. p < 0.05 was considered to be statistically signi cant. The experimental method was approved by the Animal Protection and Use Committee of Jinzhou Medical University. The animal data were reported following the ARRIVE 2.0 guidelines.ethical committee number:2021101.

Consent for publication
Not applicable.

Declaration of competing interest
The authors declare no con icts of interest.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.  Raw 264.7 cells treated with 100 μM of FLA after 24 h and observed by single-photon confocal microscopy (A). Raw 264.7 cells treated with 100 μM FLA after 24 h and observed by TEM; Scale bar = 5μM (B). Flow chart of joint imaging of live rat (C). Fluorescence images of the heart, liver, spleen, lungs, and kidneys of normal rat, FLA-injected normal rat, and FLA-injected gout rat from 2 to 24 h after injection. Scale bars are used for uorescence signal counting (D). Fluorescence images of the ankle of normal rat, FLA-injected normal rat, and FLA-injected gout rat from 2 to 24 h after injection. Scale bars are used for uorescence signal counting (E).