A total of thirty-six male C57BL/6 mice (8 weeks old) were randomly divided into four groups: control group (n = 6), HFD/STZ group (n = 6), HFD/STZ+20LCB group (n = 6), and HFD/STZ+40LCB group (n = 6). Mice (5-6 mice per cage) were acclimatized for 1 week under controlled environmental conditions (a 12 h light/dark cycle, temperature of 22 °C ± 1 °C, humidity of 45%–55%). Mice were reared with high-fat diet (HFD; 60% of kcal as fat; D12492 Research Diets, New Brunswick, NJ) for 4 weeks before being intraperitoneally injected with streptozotocin (STZ; dissolved in 0.1 M citrate buffer, pH 4.5) purchased from Macklin at a dosage of 50 mg/kg (Chen et al., 2022) for 5 days. The DN model was constructed and its performance was validated when the fasting blood glucose levels exceeded 16.7 mmol/L. Following STZ administration, mice in the treatment groups received LCB via gavage (20 and 40 mg/kg (Li et al., 2022); dissolved in 5% DMSO, 5% Tween-80, and 90% normal saline) purchased from Shanghai yuanye Bio-Technology Co., Ltd once every 2 days over an eight-week period. Body weight and blood glucose were monitored weekly. Finally, mice were housed in metabolic cages for 24 h to collect urine samples. Afterward, mice were euthanized utilizing isoflurane, and serum and kidney tissues were harvested for subsequent analysis.

The serum concentrations of blood urea nitrogen (BUN), total cholesterol (TC) and triglyceride (TG) were quantified utilizing corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. In addition, albumin and creatinine levels in 24-h urine were detected utilizing a mouse ALB ELISA kit (FineTest, Wuhan, China) along with a creatinine assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

In order to assess histopathologic alterations, renal fibrosis, and glycogen deposition, we carried out H&E, PAS and Masson staining. Briefly, kidney tissues were treated with a series of graded alcohol solutions (70%, 80%, 90% and 100%) to achieve dehydration. The kidney tissues were permeabilized with xylene (Aladdin, Shanghai, China) and embedded in paraffin. After freezing, tissues were sectioned into 5 μm slices. Subsequently, kidney tissue sections were dewaxed and stained with H&E (Solarbio, Beijing, China), Masson staining solution (Leagene, Beijing, China), and glycogen PAS staining solution (Leagene, Beijing, China), respectively. For H&E staining, sections were stained with hematoxylin for 5 min, rinsed, and then stained with eosin for 3 min. For PAS staining, sections were oxidized with a high-iodic acid solution for 10 min in a wet environment, followed by staining with Schiff’s reagent for 15 min. After rinsing for 5 min, hematoxylin is utilized for counterstaining for 2 min. For Masson staining, sections were stained with a Ponceau acid fuchsin staining buffer for 10 min, washed with phosphomolybdic acid solution for 2 min, and then stained with aniline blue solution for 1 min. Finally, a microscope (Olympus, Tokyo, Japan) was utilized for microscopic examination, followed by image acquisition and analysis.

TdT-mediated dUTP nick-end labeling (TUNEL) staining was employed to assess apoptosis levels. In brief, paraffin-embedded kidney tissues were made into 5-μm slices and then deparaffinized. Subsequently, the slices were stained utilizing an in situ cell detection kit (Roche, Basel, Switzerland) and observed under a microscope (Olympus, Tokyo, Japan). In addition, the activity of caspase-3 and caspase-9 was also determined according to the manufacturer’s instructions by corresponding kits (Solarbio, Beijing, China).

DHE staining was conducted to determine oxidative stress levels. Cell and tissue slices were incubated with DHE (Beyotime, Shanghai, China) under a dark environment at 37 °C for 30 min. Slices were observed and images were captured under a microscope (Olympus, Tokyo, Japan).

In accordance with manufacturer’s protocol, the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were employed to determine superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde (MDA) content in kidney tissues.

Extraction of total RNA was conducted from renal tissues in HFD/STZ-induced mice and HFD/STZ-induced mice administered with 40 mg/kg LCB. mRNA was captured from total RNA utilizing poly-T oligo-attached magnetic beads. Then, complementary DNA libraries were generated, and mRNA sequencing (mRNA-seq) was subjected to through the Illumina platform. The criteria for identifying upregulated differentially expressed genes (DEGs) are: fold change (FC) >1.5 and p < 0.05. The criteria for identifying downregulated DEGs are: FC <0.67 and p < 0.05. Enrichment analysis was carried out based on the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. GO enrichment analysis covering biological processes, molecular functions, and cellular components. The GO and KEGG terms with p < 0.05 were defined as being significantly enriched. The top 15 terms from GO and KEGG are presented.

HK-2 cells provided by iCell were cultured utilizing specialized medium (iCell, Shanghai, China). After 24 h of adherent growth, the medium was substituted with high glucose medium (HG, 35 mmol/L glucose) for 48 h to induce cell injury.

HK-2 cells were plated in 96-well plates at a density of 5 × 10³ cells/well. Various concentrations of LCB (1, 3, 6, 12, 25, and 50 μM) were added to treat cells. After 48 h, CCK-8 assay was performed utilizing a CCK-8 cell proliferation detection kit (KeyGEN, Nanjing, China). The OD values at 450 nm were determined. Additionally, cells were cultured with medium containing HG for 48 h and various concentrations of LCB (3, 6, 12, 25, and 50 μM) were utilized to intervene for 48 h, after which cell viability was evaluated via the CCK-8 assay.

Cells from each group were harvested through centrifugation at 150 g for 5 min after which the supernatant was removed. Cells were resuspended with 500 μL Binding Buffer (KeyGEN, Nanjing, China). Afterwards, cells were stained with 5 μL Annexin V–FITC and 5 μL Propidium iodide (KeyGEN, Jiangsu, China) for 15 min at room temperature in darkness. Eventually, flow cytometry was carried out to measure apoptosis levels.

In kidney tissues and HK-2 cells, total RNA was obtained through TRIpure reagent (BioTeke Bio., Beijing, China). cDNA was generated utilizing the all-in-one First-Strand SuperMix kit (Magen, Guangzhou, China). Real-time PCR was carried out on a fluorescent quantitative PCR instrument (Aperbio, Jiangsu, China) to detect the expression levels of heme oxygenase-1 (HO-1). PCR reaction conditions are as follows: pre-denaturation at 95 °C for 5 min, then performing 40 cycles (each cycle containing 10-s denaturation at 95 °C, 10-s annealing at 60 °C, and 15-s extension at 72 °C). The relative change in gene expression levels was quantified employing the 2^−ΔΔCt method. The primer sequences designed were as following: mus HO-1 forward: 5′-TGG​TGA​TGG​CTT​CCT​TGT-3′; reverse: 5′-ACC​TCG​TGG​AGA​CGC​TTT-3′; homo HO-1 forward: 5′-TTT​GAG​GAG​TTG​CAG​GAG​C-3′; reverse: 5′-AGG​ACC​CAT​CGG​AGA​AGC-3′.

Kidney tissue slices were treated with antigen retrieval solution for epitope recovery and incubated with 3% H₂O₂ for eliminating endogenous peroxidase activity. After blocking, the slices were incubated with HO-1 antibody (1:100; Proteintech Group, Inc., Rosemont, IL, United States) overnight at 4 °C, and then incubated with HRP-labeled goat anti-rabbit (1:500; Thermo Scientific, Pittsburgh, PA, United States) for 1 h at 37 °C. The slices were stained with DAB and observed under a microscope (Olympus, Tokyo, Japan).

IF staining was carried out as previously described (Cheng et al., 2016). For kidney tissues, samples were pretreated into 5-μm paraffin-embedded slices prior to staining. For cells, samples were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 30 min prior to staining. Tissue and cell samples were incubated with anti-Nrf2 antibody (1:100, Affinity, Changzhou, China) overnight at 4 °C and then incubated with CY3-conjugated goat anti-rabbit IgG (1:200; Abcam, Cambridge, United Kingdom) for 1 h. Finally, tissue and cell samples were counterstained with DAPI and observed through a microscope (Olympus, Tokyo, Japan).

HK-2 cells were lysed with RIPA lysis buffer (Solarbio, Beijing, China) and total protein was harvested by centrifugation at 4 °C (10,000 g) for 5 min. The BCA protein assay kit (Solarbio, Beijing, China) was applied to quantify the protein concentrations. Then, equal amounts of proteins (10–20 μg) were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene difluoride membranes. After blocking with western blocking buffer (Solarbio, Beijing, China) for 1 h, the membranes were incubated with HO-1 antibody (1:2000, Proteintech Group, Inc., Rosemont, IL, United States) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit IgG (1:3,000; Solarbio, Beijing, China) for 1 h at room temperature. Protein bands were visualized with ECL Western blotting substrate (Solarbio, Beijing, China) and optical density was analyzed via the Tanon Image software.

All data in this research were presented as mean ± SD. Statistical analysis was carried out through GraphPad Prism 9.0. Differences among multiple groups were assessed through one-way ANOVA with post hoc Tukey’s multiple-comparisons test. Two-way repeated-measures ANOVA with post hoc Tukey’s multiple-comparisons test was performed for analyzing mouse body weight and blood glucose results. Statistical significance was set at a P value <0.05. For in vivo animal experiments, “n” represents the number of independent animals. For in vitro cell experiments, “n” represents the number of independent experimental replicates.

We assessed the’drug-like’ the potential of LCB according to Lipinski’s rule. As summarized in Table 1, LCB (Figure 1) exhibits a molecular weight (MW) of 286.28 g/mol and an octanol/water partition coefficient (XLogP3) of 2.6. LCB contains 5 hydrogen-bond acceptors, 3 hydrogen-bond donors and 4 rotatable bonds. These data are obtained from the SwissADME database and comply with Lipinski’s rules. Furthermore, we found that human oral bioavailability (OB) of LCB is 76.76% and drug-likeness (DL) of LCB is 0.19. Given that common drug screening thresholds require OB more than 30% and DL more than 0.18. These results demonstrated that LCB is perceived as’drug-like’ compound.

To explore the therapeutic potential of LCB for DN mice (Figure 2A), we first assessed the typical symptoms of DN. As shown in Figures 2B,C, HFD/STZ-induced mice exhibited reduced body weight and elevated blood glucose (fasting blood sugar: 24.12 mmol/L) compared with control mice. After LCB administration, the weights of HFD/STZ-induced mice were enhanced, and the levels of blood glucose were declined. HFD/STZ induction resulted in an increase of the kidney size and weight, LCB treatment reduced the kidney size and weight (Figure 2D). Furthermore, LCB administration could decrease the levels of albumin and creatinine and the ratio of ACR in 24-h urine of HFD/STZ-induced mice (Figure 2E). The results showed that HFD/STZ induction increased the serum levels of TC, TG, BUN and Cr, while LCB administration could downregulate these levels, indicating that LCB can improve lipid metabolic disorder and renal function in DN mice (Figure 2F). Histologically, we further evaluated the impact of LCB on kidney injury by H&E, PAS and Masson staining. We observed increased glomerular area of the kidney in H&E staining in HFD/STZ-induced mice (Figure 2G; Supplementary Figure S2A). The results of PAS staining showed enhanced glycogen expression, thickened basement membrane and mesangial expansion (Figure 2H; Supplementary Figure S2B). Masson staining revealed more collagen deposition, renal tubular atrophy and interstitial fibrosis in HFD/STZ-induced mice (Figure 2I; Supplementary Figure S2C). However, LCB administration reduced kidney injury, glycogen deposition, and accumulation of collagen. Collectively, these findings demonstrated that LCB alleviated kidney injury and improved kidney function of DN mice.

We next investigated the impact of LCB on apoptosis and oxidative stress in renal tissues. TUNEL assay revealed that the apoptotic cells in HFD/STZ-induced mice were increased compared to control mice, while LCB treatment reduced excessive apoptosis (Figure 3A; Supplementary Figure S2D). Meanwhile, we measured the decreased levels of caspase-3 and caspase-9 in HFD/STZ-induced mice treated with LCB, further confirming anti-apoptotic role of LCB (Figure 3B). In addition, we also assessed the impact of LCB on oxidative stress via DHE staining. We observed stronger fluorescence in kidney tissues of HFD/STZ-induced mice, which was reversed by LCB administration (Figure 3C; Supplementary Figure S2E). Consistently, LCB could decrease the levels of MDA and increase the levels of SOD and CAT (Figure 3D). Together, our data suggested that LCB administration mitigated oxidative stress and suppressed apoptosis of kidney tissues in HFD/STZ-induced mice.

In order to investigate the underlying mechanisms, we carried out mRNA-seq to analyze the effects of LCB on the renal transcriptome in DN mice. Principal component analysis (PCA) showed clear distribution of samples in HFD/STZ-induced mice and HFD/STZ-induced mice treated with 40 mg/kg LCB, confirming that internal datasets had high repeatability (Figure 4A). The volcano plot exhibited a total of 423 DEGs including 147 downregulated genes and 276 upregulated genes (Figure 4C). Differences of DEGs levels were displayed in the heatmap (Figure 4D). As shown in Figure 4B, we performed the analysis of GO and KEGG enrichment. The pathways of GO enrichment included renal system development, kidney development and cellular response to oxidative stress, which were related to kidney function and oxidative stress. Protein-protein interaction (PPI) analysis revealed interactions among the factors related to renal system development, kidney development and cellular response to oxidative stress pathways (Supplementary Figure S5). Several genes, including HO-1, Arg2, and Hgf, were associated with these pathways. Herein, we focused on the levels of HO-1were upregulated (FC ≈ 1.51, p < 0.05).

We conducted a HG-induced HK-2 cell model to further investigate the cytoprotective effects of LCB on oxidative stress and apoptosis. Firstly, we measured the viability of LCB by CCK8 assay. We found that the LCB concentrations ranging from 1 to 6 μM had no significant effect on HK-2 cell viability (Supplementary Figure S1A). LCB at concentrations of 6, 12, and 25 μM enhanced the viability of HG-induced HK-2 cells (Supplementary Figure S1B). However, LCB concentrations of 12 and 25 μM markedly reduced HK-2 cell viability (Supplementary Figure S1A). Hence, 6 μM LCB was considered the safe and effectively therapeutic concentration for subsequent experiments. The results of DHE staining demonstrated that fluorescence intensity of HG-induced HK-2 cell was stronger than that of control cells, suggesting that HG induction led to the oxidative stress damage. However, LCB treatment markedly alleviated oxidative stress (Figure 5A; Supplementary Figure S3). Results of flow cytometry indicated that HG induction promoted apoptosis, while LCB treatment evidently repressed apoptosis (Figure 5B). This anti-apoptotic effect was further supported by decreased levels of caspase-3 and caspase-9 (Figure 5C). In summary, these findings indicated that LCB alleviated oxidative stress and suppressed apoptosis in HG-induced HK-2 cells.

We further investigated the impact of LCB on Nrf2/HO-1 pathway. Real-time PCR results indicated that the HO-1 mRNA level significantly increased after LCB intervention compared with HFD/STZ-induced mice (Figure 6A). As depicted in Figure 6B and Supplementary Figures S4A,B, the expression levels of HO-1 were remarkably downregulated in kidney tissues of HFD/STZ-induced mice compared to the control mice, which was reversed by LCB administration. IF staining results revealed that the expressions of Nrf2 were reduced in HFD/STZ-induced mice, while an increase was observed following LCB administration (Figure 6C). Consistently, we found that the expressions of HO-1 and Nrf2 were reduced in HG-induced HK-2 cells, while LCB treatment could enhanced their expression (Figures 6D–F; Supplementary Figures S4C,D; Supplementary Figure S6). Taken together, these results indicated that LCB could activate the Nrf2/HO-1 pathway.