Pectolinarigenin (PEC) was obtained from Chengdu Chroma-Biotechnology Co., Ltd. (purity ≥99.0%). Antibodies against GAPDH, α-tubulin, fatty acid-binding protein (FABP4), IL-6, alpha-smooth muscle actin (α-SMA), janus kinase 2 (JAK2), p-JAK2, Smad3 and p-Smad3, and cleaved caspases 3 (C casp 3) were purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China). Antibodies against Collagen-1(Col I), Fibronectin (FN), signal transducer and activator of transcription 3 (STAT3), p-STAT3, BAX, and Bcl2 were bought from Abcam (Cambridge, MA, United States). Anti-TNF-α antibody was bought from Affinity Bioscience (Cincinnati, OH, United States).

The HN model was established in male C57BL/6J mice (8–10 weeks old; 20–25 g) provided by the Animal Laboratory Center of Sichuan University (Chengdu, China). Forty mice were randomly assigned to five groups: Control (n = 8), HN (n = 8), Allopurinol (n = 8), PEC 25 mg/kg (n = 8), PEC 50 mg/kg (n = 8). The HN model was established by feeding mice with a mixture of adenine (0.16 g/kg) and potassium oxonate (2.4 g/kg) every other day for 4 weeks, as previously described (Ren et al., 2021). Allopurinol (10 mg/kg) and PEC (25 and 50 mg/kg) were orally given daily during the experiment along with HN establishment (for 4 weeks). The mice were sacrificed, and the kidneys were collected at the end of study. Ethical approval was granted by the Animal Ethics Committee of West China Hospital of Sichuan University (No. 2020061A).

Tissue sections were fixed with 10% phosphate buffered formalin and embedded in paraffin after dehydrating. Kidney slides of 4-μm thickness were subject to PAS staining for morphologic analysis and Masson staining for fibrotic analysis (Ren et al., 2021). Six pictures (×400) per kidney were randomly captured by light microscopy for semiquantitative analysis. The tubular injury score was evaluated on the base of histopathology of injured/damaged renal tubules and was graded from 0 to 4 (0: 0%; 1: <25%; 2: 26–50%; 3: 51–75%; 4: ≥76% of injured/damaged renal tubules) (Liu et al., 2015). The collagen positive area was measured by the ImageJ software.

Total proteins were isolated from frozen kidney tissue or mouse kidney epithelial cells (TCMK-1, ATCC^® CCL-139™, Beijing bnbio Co., Ltd., Beijing, China) using radio immune precipitation (RIPA) lysis buffer (P0013B, Beyotime Biotechnology, China) and quantified using a Pierce™ BCA Protein Assay Kit (23225, Thermo Scientific, Billerica, MA, United States). Equal amounts of protein lysate were separated on 10–12% SDS-PAGE as previously described (Ren et al., 2021). Immunoblots were visualized by the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore Corporation, Billerica, MA, United States) with Bio-Rad Chemi Doc MP and densitometered by ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, United States).

Immunohistochemical staining was performed as previously described (Ren et al., 2021). The following primary antibodies were used: anti-α-SMA (1:100, Huabio), anti-STAT3 (1:200, Abcam), anti-p-STAT3 (1:100, Abcam), anti-FABP4 (1:100, Huabio). Images were examined and acquired with an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany).

Total RNA in kidney tissues of mice or TCMK-1 cells was isolated with a total RNA extraction Kit (TP-01121, Foregene, Chengdu, China) according to the manufacturer’s instructions. The concentration of mRNA was determined by a Scan Drop 100 (Analytik Jena, Thuringia, Germany) determiner. Quantitative real-time PCR assays were performed on a PCR system (CFX Connect; Bio-Rad, Hercules, CA, United States). The sequences of primers are shown in Supplementary Table S1. Statistical analysis was conducted using the comparative 2^−ΔΔCT method with GAPDH or β-actin as the internal standard.

Total RNA was extracted from kidney tissues with Trizol reagent (Invitrogen, Carlsbad, CA, United States). Total RNA quality was assessed using the RNA 6000 Nano LabChip Kit (Agilent, CA, United States) of the Agilent Bioanalyzer 2100 system. The RNA-seq were performed by LC-BIO Bio-tech Ltd. (Hangzhou, China). Differentially expressed genes were defined as those with fold changes ≥1.5 and p ≤ 0.05. Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the OmicStudio tools at https://www.omicstudio.cn/tool.

TCMK-1 cells were cultured in DMEM (Sigma-Aldrich) supplemented with 5% FBS (SH30084.03, Hyclone, Australia) in a humidified atmosphere (5% CO₂, 37°C). After incubating with DMEM containing 0.5% FBS for 24 h, cells were exposed to UA (800 μM) and treated with PEC of various concentrations (25, 50, 100, 150, and 300 μM) for 24 h.

A Cell Counting Kit-8 assay (CCK-8, Meilunbio, Dalian, China) was employed to assess cytotoxicity. Briefly, TCMK-1 cells were seeded into a 96-well plate at a density of 5,000–10,000 cells/well and incubated with various concentrations of PEC (25, 50, 100, 150, and 300 μM) with or without UA. Cells cultured in DMEM containing the same amount of DMSO were used as control. Twenty-four hours after incubation, the cells were incubated with 10% CCK-8 reagent for 1 hour (37°C, dark). Finally, the absorbance was detected by a microplate reader (Synergy Mx, Biotek, Winooski, VT, United States) at a wavelength of 450 nm.

Results are presented as the mean ± SD. Differences among multiple groups were compared using one-way analysis of variance (ANOVA) and a Tukey-Kramer post hoc test. Comparisons between two groups were performed using the two-tailed t test. All statistics were performed using Prism software (ver. 6.01; GraphPad, San Diego, CA, United States) and p < 0.05 was considered statistically significant.

Administration of adenine and potassium oxonate successfully induced HN experimental mice as evidenced by increased serum UA level and aggravated kidney function. According to Figure 1A, the serum levels of UA (253.4 ± 14.49 μM vs. 131.0 ± 5.631 μM, p < 0.05), blood urea nitrogen (BUN) (13.00 ± 0.7513 mM vs. 5.939 ± 0.2137 mM, p < 0.05), and creatinine (63.86 ± 2.183 μM vs. 20.33 ± 0.7468 μM, p < 0.05) were significantly higher than those of control mice. After allopurinol and PEC treatment, the serum levels of UA, urea nitrogen, and creatinine were significantly decreased, and PEC at a dose of 25 mg/kg seems more superior in reducing above indexes than PEC with a higher dose (50 mg/kg). Observation of kidney changes in mice by PAS staining also showed that pathological changes in HN mice were alleviated by allopurinol and PEC treatment (Figure 1B). However, tubular injury scores of mice in the PEC 25 mg/kg group were similar to those of the PEC 50 mg/kg group, indicating no superiority of low dose PEC in attenuating renal histopathology (Figure 1C).

To reveal the mechanism by which PEC improved kidney injury in HN mice, the RNA-seq analysis was applied. The results of volcano plot showed significantly different gene expression profile between control and HN mice (Figure 2A). Among these differentially expressed genes, 796 genes were up-regulated and 1,998 genes were down-regulated in kidneys of HN mice in comparison with control mice (p < 0.05). Remarkably, PEC 25 mg/kg significantly reversed the change of 1,421 down-regulated and 293 up-regulated genes (p < 0.05) (Figure 2B). The significant PEC-modulated genes were illustrated by heatmap in Figure 2C, and genes related to apoptosis (Bax), inflammation (il1b, Tnf), and fibrosis (Col-1a1, Fn1) were seen. Further GO and KEGG analysis also suggested that these differentially expressed genes were involved in processes of lipid metabolism, apoptosis, inflammatory response, and fibrogenesis (Figures 2D,E).

Consist with what transcriptome analysis found, the results from our western blot analysis showed that HN-induced kidney expression of apoptotic indicators was alleviated by PEC treatment (Figures 3A,B) (p < 0.05). In addition, the expression of proinflammatory cytokines (IL-6, TNF-α, MCP-1) was significantly increased in kidneys of HN mice and further decreased by PEC treatment (Figure 3C) (p < 0.05). Moreover, Masson’s staining (blue) revealed a remarkable increase of renal interstitial fibrosis in HN mice, which was ameliorated by PEC (Figure 4) (p < 0.05). Accordingly, the elevated accumulation of fibrotic markers of α-SMA, Col I, and FN was observed in kidneys of HN mice, and PEC significantly reduced the accumulation of these corresponding genes (Figure 5) (p < 0.05). The above results illustrated that PEC alleviated renal apoptosis, inflammation, and fibrosis in HN mice.

Our early study indicated that the lipid-binding chaperone FABP4 was increased in kidneys of HN mice and played crucial role in HUA-induced renal inflammation and fibrosis (Shi et al., 2020a). In line with our previous findings, the expression of FABP4 in kidneys of HN mice was significantly increased (p < 0.05). PEC treatment largely inhibited the expression of FABP4 both in the mRNA and protein level, further demonstrating the role of PEC in HUA-induced inflammation and fibrosis (Figure 6) (p < 0.05).

As the most potent fibrogenic factor, TGF-β was considered to contribute to HUA-mediated renal fibrosis via the activation of Smad3 (Liu et al., 2015). To investigate the effect of PEC on the activation of TGF-β/Smad3 signaling in mice of HN, we measured the expression of TGF-β by western blot analysis. It was shown that TGF-β expression was significantly increased in kidneys of HN mice and decreased by PEC treatment (Figure 7) (p < 0.05). Meanwhile, kidney injury resulted in the phosphorylation of Smad3, which was remarkedly suppressed by PEC (Figure 7) (p < 0.05). Altogether, these results suggested that PEC could inhibit activation of TGF-β/Smad3 signaling pathway in the kidneys of HN mice.

STAT3 is a cytoplasmic transcription factor that could elicit diverse biological outcomes. Considerable studies have elucidated the role of STAT3 in mediating HUA-induced renal inflammation, apoptosis, and fibrosis (Shi et al., 2020b; Pan et al., 2021). To examine whether PEC could abrogate the activation of STAT3 in HN, the immunochemical staining and western blot analysis was employed to measure the expression of phosphorylated STAT3 (p-STAT3). As shown by Figure 8, the phosphorylation level of STAT3 was significantly increased in kidneys of HN mice, which was restored by PEC (Figure 8) (p < 0.05). Additionally, immunochemical staining showed that HN-induced p-STAT3 was mainly located in renal tubules (Figures 8A,B).

To further investigate the role of PEC in HN, TCMK-1 cells were treated with soluble UA (800 μM) for 24 h. As shown in Figure 9A, PEC under 150 μM showed no cytotoxic effect for TCMK-cells and cells treated with PEC at 100 μM showed the highest cell viability. UA stimulation led to increased expression of IL-6, TNF-α, and FABP4 in TCMK-1 cells, and PEC (100 μM) significantly suppressed such expression (Figures 9B–D) (p < 0.05). Meanwhile, the fibrotic expression of α-SMA, FN, and Col I in UA-treated TCMK-1 cells was reduced by PEC (100 μM) (Figures 9B–D) (p < 0.05), thus confirming the anti-inflammatory and anti-fibrotic effects of PEC in vitro.

After UA treatment, the expression of TGF-β and phosphorylated Smad3 were significantly increased in TCMK-1 cells, indicating that HUA could directly activate the TGF-β/Smad3 signaling pathway (Figures 10A,B) (p < 0.05). PEC (100 μM) successfully suppressed the expression of TGF-β and the phosphorylation of Smad3 indued by UA (Figures 10A,B) (p < 0.05). Similarly, UA stimulation resulted in the phosphorylation of JAK2 and STAT3, which was abrogated by PEC treatment (100 μM) (Figures 10C,D) (p < 0.05). Hence, consistent with our in vivo findings, PEC (100 μM) could inhibit the TGF-β/Smad3 and JAK2/STAT3 activation in UA-treated TCMK-1 cells.