Potassium oxonate (PO, 98%) was obtained from Macleans Biotechnology Corporation (Shanghai, China), HX (98.5%) was provided by Yuanye Biotechnology Co., Ltd. (Shanghai, China), and allopurinol (99.5%) supplied by Hefei Jiulian Pharmaceutical Co., Ltd (Anhui, China). SUA, serum creatinine (SCr), blood urea nitrogen (BUN) kits were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse anti-OAT1 and OAT3 were purchased from Affinity (San Francisco, CA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Cell Signaling Technology Inc. (Beverly, MA, USA). All other reagents used in the experiment were analytically pure. Phenylmethanesulfonyl fluoride (PMSF) and radio immunoprecipitation assay were obtained from Beyotime Biotechnology (Shanghai, China). Armillaria mellea strains were high-quality Armillaria mellea (Vahl) P. Kumm. provided by Xingtianjian Pharmaceutical Group Company (Benxi, China).

Preparation of Armillaria mellea strains. The liquid shaker flask culture medium was transferred from the Armillaria mellea slant strain, placed in a constant temperature shaking incubator, 28 °C, 150 r/min culture for 15 days and subcultured 1–2 times for seven days each time to obtain stable liquid culture strains. The above liquid culture medium (Glucose 2%, Yeast powder 1%, KH${}_2$PO${}_4$ 0.15%, MgSO${}_4$ 0.075%, VB 10.01%) was incubated as a seed medium in 1 L culture flasks with 5% of the inoculum amount. The fermentation product (rhizomorphs and fermentation broth) was obtained after 40 days of incubation at constant temperature and away from light.

Considering the convenience of oral administration and avoiding secondary injury to mice caused by intraperitoneal injection, oral administration was chosen. Refer to the usage and dosage of Armillaria mellea in the Encyclopedia of Chinese Medicinal Materials, take 30–60 g of decoction or grind it orally, such that 60 g is the high dose and 30 g is the low dose. The dose for mice was converted according to a body weight of 70 kg and its equivalent dose ratio was 0.0026 (converted according to the body surface area). AFB: soaked 78.0 g fruiting body in water for 30 min and decocted twice for 60 min each time, combine the two filtrates and concentrate to 100 mL. AR: Filter the rhizomorphs, dry at 65 °C at a constant temperature, pulverized, soaked 78.0 g rhizomorphs in water for 30 min and decocted twice for 60 min each time, combine the two filtrates and concentrate to 100 mL. AFP: the fermentation product (after rhizomorphs removal) was dried at a constant temperature of 105 °C, pulverized, and 39.0 g of the fermentation product powder was taken and dissolved in 100 mL of normal saline.

Preparation of purine reference substance: Precisely weigh an amount of HX, xanthine and adenine reference substance, add water, and dissolve in a 10 mL volumetric flask to prepare HX (0.526 mg$\cdot$mL${}^{-1}$), xanthine (0.732 mg$\cdot$mL${}^{-1}$) and adenine (0.382 mg$\cdot$mL${}^{-1}$) reference solution (if HX is insoluble in water add a trace amount of 0.1 moL$\cdot$L${}^{-1}$ NaOH). Preparation of purine test sample: Weigh 1.0 g of fruiting body and rhizomorphs, respectively, add 10 mL of distilled water, ultrasonically extract for 90 min (80 kW), centrifuge at 5000 rpm for 10 minutes, take the supernatant and dilute to 10 mL.

Waters XSelect HSS T3 column (4.6 mm $\times$ 250 mm, 5 µm); Mobile phase: acetonitrile (A)-water (B), gradient elution (0–15 min, 99% B; 15–25 min, 99% B-99% B; 25–30 min, 99% B-90% B; 30–37 min, 90% B-5% B; 37–38 min, 5% B-5% B; 38–48 min, 5% B-99% B). Flow rate: 1.0 mL$\cdot$min${}^{-1}$; column temperature: 35 °C; injection volume: 10 µL. The detection wavelength of three purine components was 254 nm; the theoretical plate number should not be less than 1000 calculated according to the peaks of HX, xanthine and adenine.

90 healthy male C57 mice (SPF, weighing 18 $\pm$ 2 g) were obtained from Liaoning Changsheng Biotechnology Co., Ltd., animal experiments were conducted by the Animal Experiment Center of Changchun University of Traditional Chinese Medicine. The experimental animals freely ate and drank water (air temperature 23 $\pm$ 1 °C, relative humidity 55 $\pm$ 5%, 12 h light-dark cycle). The experimental program was approved by the Experimental Animal Ethics Committee of Changchun University of Traditional Chinese Medicine and strictly complied with the regulations on the use of experimental animals and the provisions of the China Animal Welfare Law.

The 90 mice were divided into nine each groups containing 10 mice. After one week of adaptation an animal model of hyperuricemia were established in the mice by intragastric administration of PO (0.9 g$\cdot$kg${}^{-1}$) and HX (0.5 g$\cdot$kg${}^{-1}$) for two weeks. The specific drug administration regimens were as follows. (a) Control group: Intragastric administration of an equal volume of normal saline. (b) Model group: Intragastric administration of an equal volume of normal saline. (c) Allopurinol group: Intragastric administration of 0.013 g$\cdot$kg${}^{-1}$ allopurinol. (d) AFB (3.9 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 3.9 g$\cdot$kg${}^{-1}$ AFB. (e) AFB (7.8 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 7.8 g$\cdot$kg${}^{-1}$ AFB. (f) AR (3.9 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 3.9 g$\cdot$kg${}^{-1}$ AR. (g) AR (7.8 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 7.8 g$\cdot$kg${}^{-1}$ AR. (h) AFP (1.95 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 1.95 g$\cdot$kg${}^{-1}$ AFP. (i) AFP (3.9 g$\cdot$kg${}^{-1}$) group: Intragastric administration of 3.9 g$\cdot$kg${}^{-1}$ AFP. The foregoing groups were administered daily for two weeks.

The serum obtained from mouse whole blood was centrifuged at 3000 rpm for 10 min at 4 °C. SUA, SCr, and BUN in mice serum were determined by commercially available kits.

Stomach tissue was fixed with 4% paraformaldehyde solution and embedded in paraffin, cut at a thickness of 5 µm, and stained with hematoxylin and eosin (H&E). Histopathological changes of the stomach were then observed by light microscope.

Following addition of 500 µL precooled protein lysate (containing PMSF) to 100 mg of kidney tissue, samples were homogenized and incubated on ice for 30 min. The protein concentration of the tissue supernatant was determined by Bicinchoninic Acid Assay (BCA) after centrifugation at 3500 rpm for 10 minutes at 4 °C. Electrophoresis was performed with equal amounts of total protein for sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to nitrocellulose membranes. Non-specific binding was prevented by incubating with 5% skimmed milk powder at room temperature for two hours. The blot was incubated with primary antibody (anti-OAT1, anti-OAT3, and anti-GAPDH) overnight at 4 °C, then incubated with the secondary antibody plus fluorescent markers for two hours at room temperature. A chemiluminescent detection system was used to detect bands and optical density analysis with ImageJ software (ImageJ 1.48v Jave1.6.0-20(64-bit), National Institutes of Health, USA) was used to measure band intensity. The ratio of OAT1 and OAT3 to the corresponding control GAPDH was calculated for each sample.

Part of the gastric tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned into 5 µm slices. Phosphate buffered saline with Tween (PBST) washed 3 times, 3% hydrogen peroxide room temperature was closed for 20 min, PBST washed 3 times, 5% bovine serum albumin was closed at room temperature for 1 hour, primary antibody incubation overnight at 4 °C. Samples were incubated with PBST and horse radish peroxidase-labeled secondary antibody at room temperature for 45 min, PBS washed three times, the diaminobenzidine was color developed, stained under the microscope, and washed with deionized water. Hematoxyfolk was counterstained, dehydrated after transparency, and sealed with neutral gum.

Data were processed with the aid of Graph Pad Prism 8.3 (Graph Pad Software Inc., San Diego, CA, USA), the difference between groups was analyzed by one-way ANOVA, and purine content was analyzed by paired t-test. Experimental data were expressed as mean $\pm$ SEM, and p 0.05 suggested significant statistical difference.

The chromatogram of sample and control products are given (Fig. 1) and show that HX, xanthine, and adenine in both AFB and AR exhibit a good chromatographic peak shape and that AFB contains more purine components. The content of three purines was too low and did not reach the detection limit in AFP. Analytic results show that the density of HX, xanthine, and adenine in AFB were 1001.87, 807.87, and 113.12 µg$\cdot$g${}^{-1}$, respectively, and that the sum of the three common purines was 2 mg$\cdot$g${}^{-1}$ (Fig. 2). The contents of HX, xanthine, and adenine in AR were 25.81, 59.09, 36.34 µg$\cdot$g${}^{-1}$, respectively, with the sum of the three common purines 120 µg$\cdot$g${}^{-1}$.

Fig. 1.

Liquid Chromatogram of Samples and Standards. 1: hypoxanthine; 2: xanthine; 3: adenine.

Fig. 2.

Comparative data of AFB and AR in hypoxanthine, xanthine, and adenine content. A: hypoxanthine content; B: xanthine content; C: adenine content. Data expressed as mean $\pm$ SD (n = 6). ${}^{****}$p 0.0001, versus the AR group. AFB, Armillaria mellea fruiting body; AR, Armillaria rhizomorph.

There was no significant difference in the kidney coefficient (bilateral kidney weight (mg)/body weight (g), Fig. 3) indicating that Armillaria mellea and their fermentation products had no effect on the kidney.

Fig. 3.

Effect of Armillaria mellea and its fermentation products on the kidney organ coefficient of mice. Data expressed as mean $\pm$ SD (n = 6). Con, control group; Mod, model group; All, allopurinol group; AFP, Armillaria mellea fermentation product.

Histopathological results showed (Fig. 4) that the surface envelope of kidney tissue in the control group was composed of dense connective tissue with uniform thickness. No obvious abnormalities were found in the kidney parenchyma, neither was there significant hyperplasia of the kidney interstitium, nor obvious inflammatory changes. Compared with the control group, punctate infiltration of lymphocytes (black arrow) and eosinophilic substances (yellow arrow) were seen in the kidney capsule in the model group. After administration, exception for a small amount of eosinophils in the AFB (7.8 g$\cdot$kg${}^{-1}$) and allopurinol groups, there were no obvious lesions in the renal tissue sections of any other group.

Fig. 4.

Effect of Armillaria mellea and its fermentation products on kidney tissue sections in mice kidney. Histopathological changes are shown following H&E staining (magnification, $\times$200).

Compared with the control group, the serum levels of SUA, SCr, and BUN in the model group were significantly improved (p 0.05, p 0.01 or p 0.0001, Fig. 5). The allopurinol group had only a significantly reducing effect on SUA (p 0.0001, Fig. 5). The AFB (3.9 g$\cdot$kg${}^{-1}$) group had a reduced effect on SUA and BUN (p 0.01, Fig. 5A,C), while the AFB (7.8 g$\cdot$kg${}^{-1}$) group had a reducing effect on SCr (p 0.05, Fig. 5B) when compared with the model group. The AR (3.9 and 7.8 g$\cdot$kg${}^{-1}$) groups and the AFP (1.95 and 3.9 g$\cdot$kg${}^{-1}$) groups significantly decreased the levels of serum SUA, SCr and BUN (p 0.05, p 0.01, p 0.001 or p 0.0001, Fig. 5). The findings showed that AR and AFP had a certain protective effect on kidney function.

Fig. 5.

Effect of Armillaria mellea and its fermentation products on SUA, BUN, and SCr. A: SUN content; B: SCr content; C: BUN content. Data are expressed as mean $\pm$ SD (n = 6). ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.0001, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}}$p 0.05 versus the model group. ${}^{****}$p 0.0001, ${}^{**}$p 0.01, ${}^{*}$p 0.05 versus the control group. SUA, serum uric acid; SCr, serum creatinine; BUN, blood urea nitrogen.

The OAT1/PAT3 protein results are shown in Fig. 6. The expression level of OAT1 and OAT3 proteins in the kidney tissue of the model group decreased significantly compared with the control group (p 0.05, Fig. 7A,B). Compared with the model group, the allopurinol group had no significant effect on the protein content of OAT1 and OAT3. The AFB (7.8 g$\cdot$kg${}^{-1}$) group had a significant increase in effect on OAT1 (p 0.05, Fig. 7A). Both AR (3.9 and 7.8 g$\cdot$kg${}^{-1}$) and AFP (1.95 and 3.9 g$\cdot$kg${}^{-1}$) groups significantly increased the protein content of OAT1 and OAT3 (p 0.05, p 0.01 or p 0.0001, Fig. 7).

Fig. 6.

OAT1/OAT3 protein expression results.

Fig. 7.

Armillaria mellea and its fermentation products increase the activation of OAT1 and OAT3 in kidney tissue. A: OAT1 protein content; B: OAT3 protein content. Data expressed as mean $\pm$ SD (n = 6). ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.0001, ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}}$p 0.05 versus the model group. ${}^{*}$p 0.05 versus the control group.

OAT1 protein levels were significantly reduced in the hyperuricemia model group (p 0.001, Figs. 8, 9) when compared to the control group. AFB (3.9 g$\cdot$kg${}^{-1}$) group significantly increased OAT1 protein content, the AR (3.9 and 7.8 g$\cdot$kg${}^{-1}$) and AFP (1.95 and 3.9 g$\cdot$kg${}^{-1}$) groups significantly increased OAT1 protein content in kidney tissue compared with the model group (p 0.0001, Figs. 8, 9). The experimental results were found to be identical to Western blot results.

Fig. 8.

OAT1 Immunohistochemical results.

Fig. 9.

Immunohistochemical analysis of Armillaria mellea and its fermentation products on mice OAT1 protein. Data expressed as mean $\pm$ SD (n = 3). ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.0001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 versus the model group. ${}^{***}$p 0.001 versus the control group.