The animals used for the experiment were 6-week-old male (n = 24) db/db mice and wild-type m/m mice obtained from Nanjing Institute of Biological Medicine (certificate number: SCXK2016-0010). All mice were housed in a facility maintained at a humidity of 60%, a constant temperature of 22-24°C, and a 12 h light/dark cycle. After 2 weeks of adaptive feeding, blood glucose levels were randomly measured in db/db mice, and mice were used in subsequent experiments when the blood glucose levels of mice exceeded 16.7 mmol/L on two occasions and the urinary albumin/creatinine ratio (UACR) was significantly elevated. The successfully generated db/db mice were randomly divided into a model group (db/db) and a BSTL group using the random number approach. The m/m mice were treated as the nondiabetic control group (con). Each group contained eight mice. The mice in the BSTL group received intragastric administration with 16.5 g/kg/d of crude BSTL based on the optimal concentration determined in previous studies by intragastric administration. BSTL was obtained from the Pharmacy of the Beijing Hospital of Traditional Chinese Medicine, Capital Medical University. The mice in the control and model groups were fed an equal volume of distilled water. Random blood glucose levels were tested every 2 weeks, and 8-h urine samples were collected from the mice every 4 weeks. After 12 weeks of treatment, all mice were anesthetized with sodium pentobarbital at 50 to 60 mg/kg. Meanwhile, we collected the serum of the mice, centrifuged at 4°C, 3000 rpm for 10 min, and stored at -80°C. After kidney extraction, the renal tissue was longitudinally sectioned into two halves: One half was fixed in 4% paraformaldehyde, followed by paraffin embedding for subsequent histopathological staining, immunohistochemistry, and immunofluorescence analyses. The other half was aseptically dissected to separate the cortex and medulla. The cortical portion was snap-frozen in liquid nitrogen and stored at −80°C for further protein extraction and western blotting. The mice were euthanized by decapitation. All animal experiments were performed in accordance with the protocol authorized by the Ethics Committee of the Beijing University of Chinese Medicine (BUCM-4-2020121804-4173).

Eight-week-old male Sprague-Dawley (SD) rats (n = 40) weighing 200 ± 30 g were obtained from Beijing Huafukang Biotechnology Co., Ltd. After 1 week of acclimatization, rats were randomly divided into a blank group and a BSTL group (n = 20). Rats were fed a normal diet. The rats in the BSTL group were gavaged with BSTL solution at a dose of 36.6 g/kg/d, while the rats in the blank group were gavaged with the same volume of distilled water for 7 days. Before the final gavage, all rats fasted for 12 h. Then, they were anesthetized with sodium pentobarbital at a dose of 30 to 50 mg/kg 1 h after administration, and blood was collected from the abdominal aorta. The rats were euthanized by decapitation. The serum was deactivated at 56°C for half an hour, filtered, aliquoted, and stored at -80°C. All animal experiments were performed in accordance with the protocol authorized by the Ethics Committee of the Beijing Institute of Chinese Medicine (BJTCM-R-2025-03-01).

Mouse podocyte clone-5 (MPC-5) cells, identified by the NCBI taxonomy number 10090 and catalogued as CVCL_AS87 (iCell-m081), were used. For the in vitro experiments, MPC-5 cells were cultured at 33°C and 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium (11879020/11875093, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (10099-141, Gibco), 100 µg/mL streptomycin, 100 U/mL penicillin G (V900929, Sigma, MO, USA), and 100 U/mL recombinant murine interferon (IFN)-γ (315-05-20, PeproTech, NJ, USA) for proliferation. After a culture period, podocytes were cultured at 37°C in RPMI 1640 medium without IFN-γ for 10–14 days to induce differentiation. The podocytes were used for subsequent experiments when the cell growth rate slowed, the cell volume significantly increased, and the foot processes expanded, indicating differentiation and maturation. Differentiated cells were cultured with normal glucose of 5.5 mmol/L (con), high glucose of 30 mmol/L (HG), and 30 mmol/L glucose with BSTL drug-containing serum (BSTL) for 24 h. The cells were collected for subsequent experiments.

PINK1 siRNA (44599, Santa Cruz Biotechnology) was transfected into cells with the Lipofectamine^® RNAiMAX transfection kit (13778150, Invitrogen) according to the manufacturer’s protocol. Briefly, podocytes were cultured in 6-well plates for 24 h. The RNAiMAX transfection reagent and PINK1 siRNA were added to the reaction mixture. The podocytes were cultured with serum-free medium and reaction mixture for 24 h, and the medium was changed for 6–8 h. After transfection was completed, the podocytes were treated for 24 h with various media. The PINK1 gene level was measured by PCR to confirm the success of the transfection. Podocytes were collected 24 h after transfection for the following experiments.

The level of serum creatinine (Scr) and urinary creatinine was measured with a creatinine assay kit (C011-2-1, Nanjing Jiancheng Biotechnology, JiangSu, China) following the manufacturer’s instructions. A mouse albumin ELISA kit (ab108792, Abcam, OR, USA) was used to measure the level of urinary albumin. Urinary albumin and urine creatinine levels were used to calculate UACR.

Renal tissues were immersed in 4% paraformaldehyde solution for 72 h, dehydrated, and embedded in paraffin. The kidney tissues were cut into 2-3 μm slices. Hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson staining were performed by the Department of Pathology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University. The stained sections were observed with a Leica microscope (Aperio CS2, Germany). Representative kidney tissue structures were selected and photographed.

Renal cortex tissues and MPC-5 cells were lysed with radioimmunoprecipitation (RIPA) lysis buffer (1:50, C1053, Applygen, Beijing, China) supplemented with protease inhibitors (1:100, P1260, Applygen). A bicinchoninic acid (BCA) protein quantification kit (P1511, Applygen) was used to quantify the protein in the samples. The protein extract was boiled at 95°C for 15 min. Samples from different groups (15 to 20 μg per well) were electrophoresed on polyacrylamide gels, which were electrotransferred to polyvinylidene difluoride (PVDF) membranes. After protein transfer, 5% skim milk was used to block the membranes for 1 h. The membranes were incubated at 4°C overnight with anti-Bcl-2 (1:2000, ab182858, Abcam), anti-Bax (1:5000, 50599-2-AP, Proteintech, Wuhan, China), anti-ATG5 (1:2000, 66744-1-Ig, Proteintech), anti-Beclin-1 (1:2000, ab207612, Abcam), anti-LC3B (1:2000, ab192890, Abcam), anti-PINK1 (1:500, ab23707, Abcam), anti-Parkin (1:1000, ab77924, Abcam) and protein loading control of anti-glyceraldehyde phosphate dehydrogenase (GAPDH, 1:5000, 10494-1-AP, Proteintech) primary antibodies and then incubated with secondary antibodies at room temperature for 1 h. Final detection was performed using enhanced chemiluminescence (ECL) hypersensitive substrate. Protein bands were primarily captured with a digital imaging system, while X-ray film was employed in Figure 5B .

Embedded kidney tissue was sectioned (2-3 μm). The sectioned tissues were baked at 60°C for 1 h, dewaxed with xylene three times for 45 min, hydrated with a gradient ethanol solution, and washed with deionized water. Subsequently, renal sections were immersed in the antigen retrieval solution, placed in a 95°C water bath for 20 min for antigen retrieval, and allowed to cool naturally to room temperature. Incubation with 3% H2O2 was used to quench the endogenous peroxidase activity. The renal sections were blocked with goat serum (ZLI-9056, ZSBIO company, Beijing, China) at 37°C for 30 min and incubated with anti-PINK1 (1:500, ab23707, Abcam) and anti-Parkin (1:400, 14060-1-AP, Proteintech) antibodies overnight at 4°C. The next day, the sections were washed with phosphate-buffered saline (PBS) three times, incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (PV-9001, ZSBIO business), and developed with diaminobenzidine (DAB, ZLI-9018, ZSBIO company). Finally, the nuclei were counterstained with hematoxylin. A Leica microscope (Aperio CS2, Germany) was used to observe the kidney sections.

The embedded renal sections were dewaxed, hydrated, and subjected to antigen retrieval as described for IHC. The renal sections were permeabilized with a 0.3% phosphate-buffered solution containing Tween-20 (PBST) and blocked with 3% donkey serum. Next, the renal sections were incubated with a mixture of rabbit anti-LC3B (1:200, ab192890, Abcam) antibody and mouse anti-TOM20 (1:50, sc17764, Santa Cruz Biotechnology) antibody overnight at 4°C. Renal sections were stained with Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:2000, A21206, Invitrogen, PA, USA) or a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:2000, A21206, Invitrogen) and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:2000, A32754, Invitrogen) as secondary antibodies at 37°C for 1 h after washing with PBS. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, ZL1-9557, ZSBIO company). Sections were observed by fluorescence microscopy (A1 HAL 100, ZEISS Scope, Germany) or laser scanning confocal microscopy (LSM 800, ZEISS, Germany).

In vitro, MPC-5 cells were cultured in 12-well plates with different media (con, HG, BSTL, and siPINK1) for 24 h after reaching 80% confluence. The podocytes were immersed in 4% paraformaldehyde. After permeabilization with 0.3% Triton X-100 and blocking with 3% donkey serum, podocytes were incubated with a mixture of rabbit anti-LC3B (1:200, ab192890, Abcam) antibody and mouse anti-TOM20 (1:50, sc17764, Santa Cruz Biotechnology) antibody overnight at 4°C. After washing with PBS, the podocytes were treated with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:2000) and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:2000) as secondary antibody at 37°C for 1 h, and the nuclei were counterstained with DAPI. A laser scanning confocal microscope (LSM 800, ZEISS, Germany) and a fluorescence microscope (A1 HAL 100, ZEISS Scope, Germany) were used to observe the cells.

In vivo, the Dead-End™ Colorimetric TUNEL System (G7130/G7160, Promega, WI, USA) was used to detect apoptotic glomerular cells. The embedded renal sections were dewaxed and hydrated as described for IHC. The renal sections were immersed in 4% paraformaldehyde, incubated with proteinase K, immersed in 4% paraformaldehyde again after being washed with PBS, and equilibrated for 10 min with an equilibration buffer. Renal sections were treated with an R-terminal deoxynucleotidyl transferase (rTdT) reaction mixture and immersed in 2X saline sodium citrate (SSC). The renal sections were incubated with 0.3% hydrogen peroxide after washing with PBS, treated with streptavidin horseradish peroxidase (HRP) solution, and developed with DAB. Finally, the renal sections were sealed with 100% glycerin and observed using a Leica microscope (Aperio CS2, Germany).

In vitro podocyte apoptosis was assessed with an in situ cell death detection kit (11684817910, Roche, BASEL, SWZ). MPC-5 cells were cultured in 6-well plates with different media according to the groups for 24 h. The cells were treated with the TUNEL reaction mixture at 37°C for 1 h, washed with PBS, and counterstained with DAPI. A fluorescence microscope (CKX41, OLYMPUS) was used to detect apoptotic podocytes.

Total RNA was extracted from cultured cells using TRIzol (15596-018, Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA. Real-time PCR was performed using SYBR Green reagent on a Bio-Rad CFX PCR System (Bio-Rad, Carlsbad, CA, USA).

Podocytes were cultured in 6-well plates and treated with different media for 24 h. The level of podocyte apoptosis was measured using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (556547, Becton Dickinson and Company, NY, USA). Briefly, podocyte density was adjusted to 1 × 106 cells/mL, and 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) were added to 100 μL cell suspensions. After incubation at room temperature for 30 min in the dark, podocytes were centrifuged and resuspended in a binding buffer. A flow cytometer (Calibur II, Becton, Dickinson, and Company, USA) was used to analyze the podocytes from the different groups. FlowJo software was used to analyze the Annexin V-FITC/PI levels.

All data were analyzed using IBM SPSS 26.0 software. The normality of distribution was assessed using the Shapiro-Wilk test, while homogeneity of variance was evaluated through both Bartlett’s and Levene’s tests. For data meeting both normality and homogeneity assumptions, one-way ANOVA was employed, followed by Dunn-Bonferroni post hoc tests for intergroup comparisons. When variances were unequal, Welch’s ANOVA was applied. Non-normally distributed data were analyzed using the nonparametric Kruskal-Wallis test. A p-value < 0.05 was considered statistically significant. Data visualization was performed using GraphPad Prism version 7.0.

By six weeks of age, db/db mice, an established model for type II diabetes mellitus, exhibited marked obesity and elevated fasting blood glucose levels, peaking between 8 and 12 weeks. To verify the efficacy of BSTL in db/db mice, at the end of 12 weeks of treatment, we collected blood and urine samples from mice and measured 8-hour urinary albumin, urinary creatinine, and serum creatinine levels. As shown in Figures 1A, B , db/db mice exhibited significantly higher UACR compared to control mice, along with elevated serum creatinine levels that did not reach statistical significance. In contrast, BSTL treatment markedly reduced UACR and partially improved Scr levels in db/db mice, indicating a protective effect on renal function.

To evaluate the impact of BSTL on renal pathology in db/db mice, we performed HE, PAS, and Masson staining on renal tissue sections from each group, examining pathological changes by optical microscopy. In the control group, the glomeruli exhibited a well-defined structure with a thin basement membrane and no increase in endothelial cells, mesangial cells, or mesangial matrix, characteristics typical of normal renal architecture. In contrast, db/db mice showed marked glomerular basement membrane thickening and increased mesangial matrix density. Notably, BSTL treatment significantly mitigated these pathological alterations ( Figure 1C ).

Expression levels of Bcl-2 and Bax serve as indicators of apoptosis, and the reduction of podocyte apoptosis is a key therapeutic approach for DKD. To evaluate the efficacy of BSTL, we analyzed Bcl-2 and Bax expression. Compared to the control group, db/db mice exhibited reduced Bcl-2, elevated Bax, and a lower Bcl-2/Bax ratio, reflecting increased apoptosis. BSTL treatment effectively reversed these changes ( Figures 2B, C ). TUNEL staining was also performed to assess glomerular cell apoptosis in renal tissues, producing consistent results ( Figures 2A, D ). To further explore anti-apoptotic effect of BSTL on podocytes, we conducted in vitro experiments with MPC-5 cells exposed to high glucose. TUNEL staining confirmed that high-glucose conditions significantly increased apoptosis in MPC-5 cells, while BSTL treatment markedly reduced the number of apoptotic cells ( Figures 2E, F ). These findings suggest that BSTL effectively mitigates podocyte damage, supports filtration barrier integrity, and reduces proteinuria.

A stable mitochondrial energy supply is essential to maintain the structural and functional integrity of podocytes. Mitophagy, a critical process for mitochondrial homeostasis, plays a complex role in the progression of DKD, acting as a double-edged sword. To further examine the impact of BSTL on mitophagy in DKD, we conducted immunofluorescence colocalization staining to assess LC3 and TOM20 expression. TOM20 was labeled with green fluorescence, LC3 with red fluorescence, and nuclei with DAPI (blue), with mitophagy indicated by yellow fluorescence resulting from the colocalization of LC3 and TOM20. As shown in Figures 3A, B , db/db mice exhibited greater colocalization of LC3 and TOM20 in the glomeruli compared to control mice, suggesting enhanced mitophagy in DKD. Notably, BSTL treatment significantly reduced this mitophagy level. We examined the expression of key mitophagy-related proteins to investigate the molecular mechanisms by which BSTL modulated mitophagy in podocytes. The results showed that the ATG5, Beclin-1, LC3 I, and LC3 II expression levels and the LC3 II/LC3 I ratio were elevated in db/db mice compared to controls. However, BSTL treatment reversed these alterations, indicating a regulatory effect on mitophagy ( Figures 3C–F ).

To further confirm the changes in mitophagy in podocytes, we performed immunofluorescence colocalization using MPC-5 cells from different experimental groups. LC3 was labeled with red fluorescence, TOM20 with green fluorescence, and nuclei with DAPI (blue). As shown in Figures 3G, H , MPC-5 cells cultured under high glucose conditions displayed an increased colocalization of LC3 and TOM20 compared to control cells, indicating an increase in mitophagy. However, BSTL-mediated serum treatment significantly reduced mitophagy levels, consistent with the findings observed in the animal model.

The PINK1/Parkin signaling pathway is a key regulator of mitophagy. To investigate whether BSTL modulates mitophagy to delay the progression of DKD through this pathway, we assessed the protein levels of PINK1 and Parkin in renal tissues by Western blotting. In db/db mice, both PINK1 and Parkin expression were significantly elevated, but these changes were markedly reversed after BSTL treatment ( Figures 4A–C ). To further examine the localization of these proteins in the glomeruli, we performed immunohistochemistry and observed consistent results ( Figures 4D–F ). In vitro, high-glucose treatment also led to increased expression of PINK1 and Parkin in MPC-5 cells, while BSTL-mediated serum treatment effectively downregulated the levels of both proteins ( Figures 4G–I ).

We silenced PINK1 in podocytes by transfecting them with a specific siRNA targeting PINK1, resulting in a marked decrease in PINK1 expression, as shown in Figure 5A . PINK1 and Parkin protein expression was significantly reduced in PINK1 siRNA-treated podocytes compared to the model group in a high-glucose environment ( Figures 5B–D ). Next, we performed immunofluorescence co-labelling of LC3 and TOM20 in each group of cells and showed that knockdown of PINK1 reduced the level of co-localization of LC3 and TOM20 in podocytes cultured in high glucose ( Figures 5E, H ). Additionally, inhibition of PINK1 expression attenuated high glucose-induced podocyte damage, as confirmed by the percentage of apoptotic cells ( Figures 5F, G ).