S. apetala leaves and branches were obtained from Nansha Coast Wetland in Guangzhou, China (2017GDZJ011). Potassium oxonate (97%, 2207-75-2, PO) and HX (99%, 68-94-0) were purchased from Sigma-Aldrich (United States). FBX (98%, 144060-53-7) was from Shanghai Yuanye Bio-Technology Co. LTD. (Shanghai, China). The biochemical assay kits of UA (C012-2), creatinine (CRE, C011-2-1), blood urea nitrogen (BUN, C013-2-1), glutathione (GSH, A006-2), catalase (CAT, A007-1-1), superoxide dismutase (SOD, A001-3), malondialdehyde (MDA, A003-1), and XOD (A002-1-1) were the products of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The antibodies against JAK2 (ab108596), p-JAK2 (ab195055), STAT3 (ab68153), p-STAT3 (ab131103), and β-actin (ab8227) were obtained from Abcam Biosciences (Inc., United States). Glucose transporter 9 (GLUT9, 26486-1-AP) was purchased from Protein technology Co. LTD. (Inc., United States). Organic anion transporter (OAT1, DF6633), urate reabsorption transporter 1 (URAT1, DF12340), nuclear factor kappa-B P65 (NF-κB P65, AF5006), anti-histone H3 antibodies (AF0863), and secondary antibodies (110191) were obtained from Affinity Biosciences (Inc., United States). Other chemicals and reagents were obtained from local suppliers.

Fresh S. apetala leaves and branches were rinsed and extracted in boiling distilled water twice for 5 h. After filtration, the extracting solution was concentrated by reducing pressure and then freeze-dried to obtain SAL (Liu et al., 2019).

To determine the components of SAL, the ultra-performance liquid chromatography (UPLC)/qExactive-mass spectrum (MS) method was performed (Hossain S. J. et al., 2016). In brief, SAL was dissolved in 80% methanol, centrifuged (20,000 × g, 4°C, 10 min), and the filtered with millipore filters (0.22 μm). The filtrate was injected for UPLC analysis through an RP-C18 column (2.1 mm × 150 mm, 1.8 μm; Welch) at 35°C. The mobile phases were 0.1% (v/v) formic acid dissolved in purified water (A) and 0.1% (v/v) formic acid dissolved in acetonitrile (B). The elution program was set with a gradient procedure 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 of SAL was 5 μL and eluted at 0.3 ml/min. The MS full scan range was 150–2,000 m/z. The collision gas and desolvation gas were high-purity N₂ and Ar, respectively. Finally, the results were compared across the databases (mzCloud, mzVault, and ChemSpider).

Eight-week-old male Kunming mice were supplied by Guangdong Medical Laboratory Animal Center (Guangzhou, China, No.44007200077071). All the mice were acclimated for one week with a 12-h light/dark cycle, a controlled temperature (22 ± 2°C), and a relative humidity of 55 ± 5% before experiment. The mice were allowed to obtain food and water freely. The experiment was conducted under the supervision of the Laboratory Animal Ethics Committee of Guangzhou University of Chinese Medicine [SYXK (Yue) 2018-0085] and in accordance with Regulations for the Administration of Affairs Concerning Experimental Animals (Ethics NO.20200602006).

In brief, mice were randomly divided into seven groups (n = 10): intact, vehicle, BZM (10 mg/kg), FBX (10 mg/kg), and SAL groups (50, 100, and 200 mg/kg) (Liu et al., 2019). Except those in the intact group, all the mice were intragastrically given HX (300 mg/kg) and intraperitoneally injected with PO (300 mg/kg) (Lu et al., 2019). One h after administration with PO and HX, mice in SAL-treated groups, the BZM group, and the FBX group were given a corresponding dose of SAL, BZM, or FBX, while those in the intact and vehicle groups were fed with equal volumes of 0.5% of carboxymethylcellulose sodium (CMC-Na) solution by gastric gavage. All the operations were conducted once daily for consecutive 1 week. After the last treatment, all the mice were fasted for 4 h. Subsequently, the mice were anesthetized using 3% of pentobarbital sodium (Jiewei et al., 2018). Blood samples were obtained from the orbit and centrifuged for 10 min (1,000 rpm, 4°C) after clotting at room temperature for 120 min. Serum was obtained and stored at −80°C for the further analysis. Afterward, all the animals were sacrificed, and the kidney tissues were weighed and collected for following biochemistry analysis. The kidney index was calculated according to the following formula: the kidney index of the mice = (kidney weight of the mice/body weight of the mice) × 100%.

Fresh kidney tissues were rinsed thoroughly, fixed, and then embedded in paraffin. Subsequently, 5 -mμ tissues were cut and finally dyed with hematoxylin and eosin (H and E) or periodic acid-Schiff (PAS) routinely. Finally, kidney histopathological changes were examined using a microscope at × 200 magnification.

To detect the level of ROS in the kidney, dihydroethidium (DHE) staining was adopted according to the methods, as previously described (Semprun-Prieto et al., 2011). Fresh kidney tissues were prepared into 5-μm cryosections. The slices were then incubated with the DHE (100 μmol/L) in the dark for 30 min at 37°C. The results were quantified as fluorescence intensity.

The serum of the mice was obtained by centrifugation (3,500 rpm, 10 min, and 4°C) after being kept at 25°C for 2 h. Then the activities of serum UA, CRE, and BUN were measured by respective commercials kits.

The liver tissues were homogenized and centrifuged at 3,000 rpm for 10 min (4°C). Subsequently, the supernatant was used for the measurement of XOD activity by a commercial kit.

The supernatant of renal homogenate was obtained by the method of 2.8. Subsequently, under instruction of manufacture’s protocols, the renal GSH, MDA levels, and SOD, CAT activities were measured by using commercial kits.

Following the manufacturer’s instructions, the levels of interleukin-6 (IL-6), interleukin-18 (IL-18), IL-1β, and TNF-α in the kidney were detected by using ELISA kits obtained from MLBIO Biotechnology Co., Ltd. (Shanghai, China).

To isolate the total ribonucleic acid (RNA) from kidneys of the mice, Trizol reagent was used according to manufacturer’s instruction. The RNA purity was identified to be between 1.8 and 2.0. The obtained RNA was then reverse-transcribed into cDNA. The mRNA expressions of IL-1β, IL-6, IL-18, TNF-α, monocyte chemotactic protein 1 (MCP-1), transforming growth factor-β (TGF-β), suppressor of cytokine signaling 3 (SOCS3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by using HiScript^® II Q RT SuperMix (+ gDNA wiper) and ChamQ™ SYBR^® qPCR Master Mix Kit (Livak and Schmittgen, 2001). The sequences of primers, shown in Table 1, were designed by online primer design software (Sangon Biotech Co., LTD., Shanghai). The relative quantifications of genes expression were calculated using the 2^−ΔΔCq method with GAPDH served as a normalization control.

The kidney tissues were extracted with protein lysis buffer containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail to obtain the total protein. Besides, extraction of cytoplasmic and nucleus protein was performed using the extraction kit (Thermo). The protein concentration was measured by the Bicinchoninic Acid (BCA) Protein Assay Kit. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein samples, which were then transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk, the membranes were incubated with primary antibody and a horseradish peroxidase (HRP) goat anti-rabbit antibody (Abo-Youssef et al., 2020). The bands were detected by electrochemiluminescence (ECL) reagent. The density of each band was analyzed using ImageJ.

All the data were expressed as mean ± standard deviation (SD), and statistical analysis was performed by SPSS software 23.0. The data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s test. The figures were processed using GraphPad Prism 8.0.1. The value of p <0.05 was regarded as statistical significance.

SAL was obtained from S. apetala leaves by boiling water extraction, achieving a yield of 4.23%. The positive and negative ion chromatograms of SAL are shown in Figure 1, and the characterization of the compounds is presented in Table 2.

To investigate the effect of SAL on renal morphological changes in HUA mice, H and E and PAS staining were performed. As shown in results of H and E staining (Figure 2A), the morphology of the kidney cells in the intact group was in a healthy condition. The glomeruli, renal tubules, renal cortex, and medulla were clearly structured. In contrast, the HUA mice showed inflammatory cellular infiltration, along with obvious edema, necrosis, and balloon-like changes in the glomeruli, surrounding renal tubules and renal tubular epithelial cells. The appearance of kidneys in the vehicle group confirmed that the HUA model was established successfully. BZM and FBX treatment reduced the degree of edema and diminished the inflammatory cellular infiltration in the renal interstitium significantly. Moreover, all the renal pathological changes were also ameliorated by the SAL in a dose-dependent manner.

As presented in results of PAS staining (Figure 2B), the glomeruli basement membrane of HUA mice was significantly thickened compared with the intact group. The BZM and FBX treatment partially attenuated the thickened basement membrane. Moreover, the SAL treatment mitigated the thickening of glomeruli basement membrane dose-dependently.

To validate the effects anti-hyperuricemia and nephroprotective effects of SAL in HUA mice, UA, CRE, and BUN levels in serum were determined. As shown in Figures 3B–D, the levels of serum UA, CRE, and BUN in the vehicle group were significantly (p < 0.01, p < 0.01, and p < 0.01, respectively) increased compared to those in the intact group, which suggested that the HUA model was established successfully. The renal index in the vehicle group was also increased markedly (p < 0.01). BZM significantly decreased the levels of serum UA, CRE, and BUN (p < 0.01, p < 0.01, and p < 0.01, respectively). FBX also decreased the levels of UA, CRE, and BUN in serum (p < 0.01, p < 0.01, and p < 0.01, respectively). When compared with the vehicle group, SAL at 50, 100, and 200 mg/kg also reduced the levels of UA, CRE, and BUN dose-dependently.

To illuminate whether SAL could affect uric production in mice, XOD activity in liver was measured. As shown in Figure 4, hepatic XOD activity of the vehicle group was markedly elevated in mice (p < 0.01) compared to the intact group. On the contrary, FBX remarkedly inhibited the XOD activity in HUA mice (p < 0.01). However, BZM showed no inhibitory effect (p > 0.05) on XOD activity. Surprisingly, SAL attenuated the elevated XOD activity in the liver at 50, 100, and 200 mg/kg (p < 0.01 for all). Therefore, SAL might ameliorate HUA by suppressing UA production.

To evaluate ameliorative effect of SAL on renal oxidative stress in the HUA mice, activities of antioxidant enzymes were analyzed. As shown in Figure 5, compared with those of the intact group, the renal GSH level and SOD and CAT activity decreased significantly, while the MDA level increased significantly in the vehicle group. However, the BZM and FBX treatment could restore the renal SOD, CAT activity, MDA, and the GSH level in the HUA mice. Moreover, administration with SAL significantly increased the activities of SOD, GSH, and CAT (p < 0.01 for all) and reduced the level of MDA, especially at 100 and 200 mg/kg (p < 0.01 for both), suggesting that SAL could suppress oxidative stress in the HUA mice. In addition, the ROS level markedly elevated in the vehicle group compared with the intact group and decreased in both BZM- and FBX-treated groups (Figure 6). Meanwhile, SAL at 50, 100, and 200 mg/kg decreased the ROS intensity significantly (p < 0.01 for all), which showed obvious inhibitory effects on ROS production. Therefore, these results indicated that SAL might suppress renal oxidative stress in HUA mice.

To further investigate whether SAL could also suppress renal inflammation in vivo, levels of inflammatory cytokines involving IL-1β, IL-6, IL-18, and TNF-α were determined by ELISA and RT-PCR. Remarkable elevated levels of the renal inflammatory cytokines were observed in the vehicle group (p < 0.01 for all). However, treatment with SAL significantly decreased the levels of these inflammatory cytokines in the kidneys of the HUA mice. Of note, SAL at 200 mg/kg exhibited the same effect on reducing the levels of the inflammatory cytokines compared to the BZM and FBX treated groups (Figure 7).

The JAK/STAT signal pathway is closely related to inflammation that regulates the expression of inflammatory cytokines. As shown in Figure 8, the protein levels of p-JAK2 and p-STAT3 (p < 0.01 for both) were significantly increased in HUA mice compared to those in mice from the intact group. Compared to the vehicle group, SAL downregulated renal protein expression of p-JAK2 and p-STAT3 in a dose-dependent manner. Besides, the gene expression of MCP-1, TGF-β, and SOCS3 were increased significantly in the vehicle group. Contrarily, mRNA levels of all these downstream targets were reduced by treatment with BZM, FBX, and SAL (p < 0.01 for all). Moreover, after challenge with PO and HX, the protein level of nuclear NF-κB p65 was increased, whereas that of cytosol NF-κB p65 was decreased in the HUA mice (p < 0.01 for both). As shown in Figure 9, treatment with BZM, FBX, or SAL attenuated the translocation of NF-κB p65 from the cytoplasm to the nucleus. Furthermore, SAL at 200 mg/kg showed greater effects on reduction of nuclear NF-κB p65 and augmentation of cytosol NF-κB p65 (p < 0.01, both) relative to BZM and FBX (Figure 9).

To study whether SAL could directly promote the urate excretion, we measured the protein levels of renal urate transporters by Western blot, which was associated with uric acid reabsorption and excretion (Figure 10). After hyperuricemia induction, the protein levels of URAT1 and GLUT9 were significantly upregulated compared to the intact group (p < 0.01 for both), whereas those of OAT1 declined (p < 0.01). Treatment with SAL obviously inhibited the elevation of URAT1, GLUT9 expression, and dramatically reversed the decrease in OAT1 expression.