This study was conducted to examine the potential health benefits of thonningianin A (TA) on renal injury and interstitial fibrosis in diabetic nephropathy(DN) mice. In this study, a DN mice model was established using male C57BL/6 mice injected with streptozotocin(STZ, 50 mg/kg) intraperitoneally and treated with TA for 12 weeks. Firstly, the therapeutic and anti-fibrotic effects of TA on DN were evaluated. Secondly, the effect of TA on renal inflammation was evaluated and a western blot was used to detect the changes in NLRP3/ASC/Caspase-1 pathway-related protein expressions in the kidney. Furthermore, the effect of TA on impairments in the intestinal mucosa barrier was evaluated and the changes in lipopolysaccharide (LPS) levels in feces and serum were detected by ELISA. Finally, 16S rRNA sequencing was used to detect alteration of gut microbiota diversity and abundance in mice after TA treatment. The results showed that TA markedly mitigated blood glucose(Glu), decreased 24-hour urinary total protein(24hUTP), and improved renal dysfunction and kidney index(KI) in DN mice. Furthermore, TA significantly alleviated renal injury and interstitial fibrosis, repressing renal inflammation. Western blot results showed that the NLRP3/ASC/Caspase-1 signaling pathway-related proteins decreased after TA treatment. In addition, TA also ameliorated impairments in the intestinal mucosa barrier and restored the expressions of intestinal tight junction proteins(Claudin-1, Occludin, and ZO-1). Subsequently, it reduced LPS levels of DN mice in fecal and serum. Furthermore, 16S rRNA high-throughput sequencing showed that TA modulated gut microbiota dysbiosis and decreased the abundance of Gram-negative bacteria (Proteobacteria and Escherichia-Shigella). This study suggested that TA might exert a beneficial effect on renal interstitial fibrosis in DN mice by modulating gut microbiota dysbiosis, ameliorating impairments in the intestinal mucosa barrier, reducing the production and release of LPS, inhibiting the activation of NLRP3/ASC/Caspase-1 signaling pathway, and repressing renal inflammatory.

Animals and treatments

A total of 40 C57BL/6  male mice with an average body weight of 20g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK-2023-0011). The housing environment was maintained at 25°C ± 2°C, relative humidity was 50% ± 10%, the light-dark cycle was 12h/12h, and animals were given ad libitum access to food and water. The experimental design is shown in Figure 1B. Mice were randomly divided into 4 groups (10 mice for each group): negative control (NC) group, DN group, TA group, and Semaglutide (SE) group. After 1 week of adaptive feeding, the DN, TA, and SE groups were intraperitoneally injected with STZ (50 mg/kg, dissolved in 0.01M sodium citrate buffer, pH: 4.2) for 5 consecutive days to induce diabetic nephropathy. NC group was given equal citrate buffer as controls. The blood glucose(Glu) level of the mice was stable after a week. Subsequently, blood was taken from the mouse tail veins, and the level of fasting blood glucose (FBG) was measured. An FBG level > 11.1 mmol/L for 3 consecutive days was considered to indicate DN, and the mice were used for further research. The TA group was given 0.1mg/kg/d of TA, and the SE group was given 40ug/kg/3d of SE for 12 weeks, while the NC group and DN group were given equal normal saline during this time. The body weight and FBG of mice were measured every 2 weeks, and 24h urine was collected every 4 weeks. Serum creatinine(Scr) and blood urea nitrogen(BUN) were tested at 12 weeks after treatment. At the end of the study, mice were fasted overnight, and sacrificed by cervical dislocation. The blood samples were collected from the eyeballs and centrifuged (3000 r/min, 15 min) to obtain serum for biochemical analysis. Subsequently, the kidney tissues were rapidly harvested and weighed to calculate the kidney-to-body weight ratio (kidney index, KI). The renal and colonic tissues were used for western blot, immunohistochemical assay, histologic examinations, and other biochemical analyses. All serum and tissue samples were cryopreserved at -80 ℃. Colonic content was collected for the detection of LPS and microorganisms. All experimental procedures were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (Permission BUCM-2023042003-2110).

Biochemical Parameter Analysis

Levels of Scr and BUN were measured with commercial kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). For the 24-hour urinary protein (24hUTP) concentration, metabolic cages were used to record the 24-hour total urine volume and collect 24-hour urine samples from all mice. Then the supernatant of urine samples was collected to determine the 24hUTP levels using colorimetric methods as described previously. 

Pathology staining

Kidney and colon tissues were fixed in 4% paraformaldehyde for 48 hours, followed by a sequential process of dehydration through an ethanol gradient, clearance in xylene, immersion in wax, and embedding in paraffin, to prepare sections with a thickness of 3μm. After deparaffinization and rehydration, the tissue sections were stained according to the instructions provided in the staining kit using Hematoxylin and Eosin (H&E), Periodic Acid-Schiff (PAS), or Masson's Trichrome (MASSON) stains. Pathological alterations in the renal and colon tissues were examined under a light microscope, and representative images were captured for further analysis. 

Immunohistochemistry

Following deparaffinization, antigen retrieval, and inhibition of endogenous peroxidase activity, the 3 μm-thick paraffin-embedded kidney tissue sections were incubated with primary antibodies against IL-1β (1:2000, ab32362, Abcam, UK) and IL-6 (1:1000, ab216341, Abcam, UK), and Colon tissue sections were incubated with primary antibodies against ZO-1 (1:2,000, rabbit, 21773-1-AP, Proteintech, USA), occludin (1:5,000, rabbit, 27260-1-AP, Proteintech, USA) and claudin-1 (1:2,000, rabbit, ab211737, Abcam, UK), at 4℃ overnight.

Enzyme-linked immunosorbent assay

After 12 weeks of TA intervention, renal levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured in each group using ELISA kits(Elabscience, Wuhan, China), which was performed according to the manufacturer's instructions.

 Immunofluorescence staining

Kidney tissue sections were incubated with primary antibodies including NLRP3 (1:100, rabbit, # YT5382, ImmunoWay Biotechnology, USA), ASC (1:400, rabbit, 67824T, Cell Signaling Technology, USA), Caspase-1 (1:100, 22915-1-AP,  Proteintech, USA) at 4 ℃ overnight. The corresponding fluorescent secondary antibody was added and incubated at room temperature for 1 hour. Dihydrochloride (DAPI) was added and incubated for 5 minutes. All sections were imaged using a laser scanning confocal microscope (Olympus, Tokyo, Japan). Semi-quantitative statistical analysis was performed using Image J 1.48 version (National Institutes of Health, Bethesda, Maryland, United States) based on 6 fields of view.

Western Blot Analysis

Kidney and colon tissue samples were mechanically disrupted and lysed using RIPA lysis buffer (Applygen, Beijing, China) to facilitate protein extraction. Following centrifugation, the supernatant containing the proteins was harvested. The protein concentration was subsequently determined through a BCA protein assay, followed by heat denaturation in the presence of a loading buffer. Equal volumes of the protein samples (10μL per well) were loaded onto an SDS-PAGE gel for electrophoretic separation. After the separation process, the proteins were transferred onto a nitrocellulose membrane utilizing the wet transfer method. The membrane was then blocked with 5% skim milk and subsequently incubated overnight at 4°C with primary antibodies, including E-cadherin, α-SMA, NLRP3, ASC, Caspase-1, ZO-1, occludin, claudin-1 and β-actin. Post incubation, the membrane was washed with TBST to remove unbound antibodies, before incubation with HRP-conjugated secondary antibodies (either anti-rabbit or anti-mouse IgG) for 1 hour. The relative expression of target proteins was calculated using β-actin as an internal reference. Image J software was employed to analyze the densitometric values of the bands and to calculate the relative expression of the proteins of interest. 

Determination of LPS levels in serum and fecal samples

The collected whole blood samples were placed at room temperature for 2 hours and centrifuged at 1000 xg for 20 minutes. The supernatant was taken and the serum LPS content was determined using a LPS ELISA kit. The collected fecal samples (greater than 50 mg) were washed three times with phosphate-buffered saline (PBS), and the precipitate was collected by centrifugation and heavy. PBS buffer (9ml PBS buffer per gram of feces) was added, and they were crushed with a 4000 g ultrasound for 10 minutes. The supernatant was taken and the fecal LPS content was measured using the LPS ELISA kit.

16S rRNA high-throughput sequencing analysis of gut microbiota

DNA was extracted from mouse cecum feces using the HiPure Soil DNA Kit (Magen, Guangzhou, China), and the purity and concentration of the extracted DNA were determined with 2% agarose gel electrophoresis. The primer sequences 341F(5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) were used to amplify the V3-V4 hyper-variable region of the bacterial 16S rRNA gene (45). The samples were then sequenced in parallel utilizing the Illumina DNA Prep Kit (Illumina, CA, USA) according to the user manual. The resulting raw data files were manipulated and filtered with the QIIME (version 1.9.1) software package. Raw sequences were imported into FASTP (version 0.18.0), and FLASH software (version 1.2.11) was used for pair-end double-end sequence splicing and screening for sequence optimization. Sequences with >97% similarity were clustered and annotated to generate operational taxonomic units (OTUs) using UPARSE software (version 9.2.64). Alpha diversity and microbial taxon distribution analyses were performed with QIIME software.

Statistical Analysis

All data were reported as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by post hoc Fisher's least significant difference (LSD) and post hoc Tamhane’s test was used in assessing the differences between the two groups. All statistical analyses were performed using SPSS 20 software (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.