Specnuezhenide Ameliorates Diabetic Nephropathy in db/db Mice via Nrf2, A20/NF-κB, and Gut Microbiota Regulation

Summary

Specnuezhenide alleviates diabetic nephropathy in db/db mice, associated with improved renal function, intestinal barrier integrity, and reduced inflammation and oxidative stress.

Abstract

The pathogenesis of diabetic nephropathy (DN) is multifactorial, involving inflammation, oxidative stress, and the gut–kidney axis. Specnuezhenide is the main active component of Ligustri Lucidi Fructus and exhibits antineoplastic, antioxidant, and anti-inflammatory activities. However, the molecular mechanisms underlying its effects on DN need to be further studied. This study, therefore, aims to investigate the molecular mechanisms of Specnuezhenide on DN. A DN mouse model was established using db/db mice, and they were administered Specnuezhenide for 8 weeks. The effects of Specnuezhenide on these mice were assessed through serum glucose and renal function evaluations. Histopathological alterations were evaluated using hematoxylin and eosin staining. An enzyme-linked immunosorbent assay was employed to quantify inflammatory cytokine levels. The Nrf2 pathway and A20/NF-κB pathways were analyzed via western blotting (WB), while the expression of intestinal tight junction proteins was assessed using WB and immunofluorescence. Alterations in the gut microbiota were examined through high-throughput 16S rRNA sequencing. Compared with the model control, Specnuezhenide improved renal function in db/db mice, reducing blood glucose (from 37.13 mmol/L to 28.96 mmol/L), blood urea nitrogen (from 18.27 mmol/L to 15.15 mmol/L), and urinary protein (from 28.5 mg to 20.41 mg) in the high-dose group. Meanwhile, gut microbiota analysis showed increased microbial abundance and decreased lipopolysaccharide levels following Specnuezhenide administration. Meanwhile, Specnuezhenide can upregulate the expression of intestinal tight junction proteins, thereby enhancing the integrity of the intestinal barrier. Specnuezhenide can upregulate the expression of Nrf2 and A20, and downregulate the expression of NF-κB. Furthermore, Specnuezhenide can ameliorate renal functional injury while regulating the Nrf2 and A20/NF-κB pathways.

Introduction

Diabetes is a major cause of kidney disease, with the increase in the number of diabetes patients, its prevalence is rising¹. Traditional treatments for diabetic nephropathy (DN) have been shown to delay the progression of chronic kidney disease². However, there remains a need to identify more effective drugs for DN prevention and treatment. The pathogenesis of DN is multifaceted and involves inflammation, oxidative stress, and the gut-kidney axis¹. With its multi-target approach, Traditional Chinese Medicine (TCM) has demonstrated remarkable efficacy in treating chronic metabolic diseases. Furthermore, the growing research on TCM in the prevention and treatment of DN has shown promising results³.

Diabetes mellitus is a complex metabolic disorder, with the gut playing a central role in its pathophysiology through interactions with metabolic organs⁴. The interplay between the host and gut microbiota is crucial for maintaining host homeostasis. Disruption of a healthy microbiota structure can produce toxins that damage the kidneys², whereas impaired renal clearance can alter microbial metabolism and composition^5,6. The intestinal barrier functions as both a chemical and physical defense and is maintained by epithelial tight junctions and a mucus layer composed of mucin⁵. Evidence suggests a correlation between DN and impaired intestinal barrier function⁷, which permits the translocation of toxins and bacterial byproducts, such as lipopolysaccharide (LPS), into the bloodstream^8,9. LPS translocation across the compromised barrier can trigger dysregulation of adaptive and innate immune responses and stimulate the production of pro-inflammatory cytokines^7,10, ultimately leading to a chronic inflammatory state characteristic of DN^11,12.

Ligustri Lucidi Fructus is clinically used for treating diabetic nephropathy (DN)¹³, with Specnuezhenide identified as a key active component¹⁴. Specnuezhenide, a unique secoiridoid glycoside¹⁵, exhibits anti-inflammatory, antioxidant, and immunomodulatory properties^16,17,18,19. The db/db mouse is a well-established spontaneous type 2 DN model that recapitulates key human DN features, including persistent hyperglycemia, albuminuria, and mesangial matrix expansion²⁰. A previous study reported that Specnuezhenide exerted renoprotective effects in DN rats²¹; however, the molecular mechanisms remain largely unexplored. Notably, Specnuezhenide has a low oral bioavailability (approximately 1.93%)²², which makes it difficult to explain its pharmacological effects solely through direct absorption. Emerging evidence suggests that Specnuezhenide can be rapidly metabolized by the gut microbiota, and its pharmacological activity may be mediated through gut-microbiota-dependent pathways¹⁹. Based on these considerations, it was hypothesized that Specnuezhenide alleviates DN by modulating the gut microbiota and downstream inflammatory and oxidative stress pathways. To test this hypothesis, we evaluated the dose-dependent effects of Specnuezhenide in db/db mice, selecting doses based on preliminary experiments and prior pharmacological studies.

Therefore, this study aimed to evaluate the potential impact and mechanism of Specnuezhenide in the context of DN, with a focus on its ability to mitigate renal inflammation and oxidative stress via the gut–kidney axis. This study contributes to elucidating the mechanisms by which Specnuezhenide contributes to the prevention and treatment of DN.

Protocol

The Ethics Committee of Shanxi University of Chinese Medicine approved this study (2022DW167). The reagents and the equipment used are listed in the Table of Materials.

Animal experiments

Male db/db mice, aged 8–9 weeks, were obtained from a commercial source. The mice were housed in the SPF-level animal room of the Experimental Management Center at Shanxi University of Chinese Medicine and maintained at a stable humidity of 55% ± 5% and a comfortable environment of 25 °C ± 1 °C with a 12/12-h light/dark cycle and ad libitum access to food and water. Before the experiment, all mice were acclimated for 7 days. Humane endpoints: Mice showing >20% body weight loss, moribund state, or inability to reach food/water were humanely euthanized prior to the scheduled endpoint. Euthanasia: All mice were euthanized by intraperitoneal injection of pentobarbital sodium, followed by cervical dislocation, following institutionally approved protocols.

The db/db mouse is a genetically spontaneous diabetic model (not an induced model). Model success was confirmed at 9 weeks of age by measuring fasting blood glucose (≥16 mmol/L) and urine microalbumin (positive). All db/db mice met the criteria, and no animals were excluded. Once the DN model was successfully established in db/db mice, the mice were randomly assigned to groups using a computer-generated random sequence. The investigator performing the experiments was blinded to group allocation. Each cage housed only one treatment group to avoid cross-contamination. The mice were randomly divided into four groups (n = 10 per group): the model control group (MC), irbesartan group (Irb), low-dose Specnuezhenide group (Spe-L), and high-dose Specnuezhenide group (Spe-H), with db/m mice (n = 10) used as the normal control (NC) group. The NC and MC groups were administered distilled water. In contrast, the Irb, Spe-L, and Spe-H groups received daily oral gavage of 43.75 mg/kg Irbesartan, 57 mg/kg Specnuezhenide, and 114 mg/kg Specnuezhenide, respectively, for 8 weeks. The gavage dosage was increased in proportion to body weight gain. After 8 weeks of administration, all mice were fasted overnight (12 h) with free access to water before euthanasia and the serum (centrifuged at ~1500 x g for 15 min and the supernatant was collected), urine (collected 24-h urine in the metabolic cage), feces, and intestinal and kidney tissues were collected immediately after euthanasia, snap-frozen in liquid nitrogen, and stored at -80 °C until analysis. After data collection, blinding was broken to perform statistical analysis.

Blood and urine indicators

The serum levels of LPS, SCr, and BUN were measured according to the manufacturer’s instructions. The 24-h urine albumin concentration was detected according to the kit instructions. Serum glucose levels were measured using an automatic biochemical analyzer.

ELISA detection

The concentrations of TNF-α, IL-6, and IL-1β in the mouse serum were measured using a 352 model microplate reader according to the manufacturer’s instructions.

Pathological observation of mouse kidney tissues

The intestinal and kidney tissues were fixed in 4% paraformaldehyde solution for 48 h before washing, dehydration, and paraffin embedding. Sections (4 µm-thick) were obtained using a paraffin microtome. Following deparaffinization, the sections were stained with hematoxylin and eosin (HE) and scanned using a digital pathology slide scanner.

16S rRNA sequencing

Bacterial genomic DNA was extracted from cecal contents using a magnetic bead-based soil DNA extraction kit following the manufacturer’s protocol. DNA concentration was measured with a fluorometer.

The V3–V4 hypervariable region of the 16S rRNA gene was amplified using the primers 338F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′). Amplification was carried out with a PCR Master Mix under the following cycling conditions: an initial denaturation at 95 °C for 3 min; 5 cycles of 95 °C for 30 s, 45 °C for 30 s, and 72 °C for 30 s; followed by 20 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. The PCR amplicons were subsequently purified using magnetic bead-based DNA purification and size-selection beads.

Libraries were constructed using Illumina adapters and indexes, and sequencing was performed on a benchtop next-generation sequencing (NGS) platform (2 × 250 bp paired-end). Raw reads were assembled using paired-end sequencing read-merging software, quality-filtered, and clustered into operational taxonomic units (OTUs) at 97% similarity using amplicon sequence analysis software. Chimeric sequences and singleton OTUs were removed. Taxonomy was assigned by BLAST against the RDP database (for bacteria) and UNITE (for fungi).

Alpha diversity was calculated using Mothur v3.8.31. Beta diversity was evaluated by principal coordinate analysis (PCoA) based on Bray-Curtis distances using the R vegan package. Differential abundance analysis was performed with STAMP v2.1.3 and LEfSe v1.1.0. Functional prediction was conducted using PICRUSt v1.1.4.

Immunofluorescence staining

Following dewaxing, samples underwent antigen retrieval using sodium citrate at high temperatures for 15 min, followed by a 60-min block with 5% BSA. Primary antibodies for occludin and claudin-1 (1:500) were then applied to ileal and colonic tissues and incubated overnight. Subsequently, secondary antibody staining was performed for 60 min. After a 10-min DAPI stain, samples were preserved with an anti-fluorescence quenching reagent, and immunofluorescence images were acquired using an automated fluorescence imaging system.

Western blotting

Total protein was extracted using RIPA buffer and quantified with a BCA protein assay kit. Samples (15 µL/well) were loaded onto an 8%–12% separating gel for approximately 2 h of electrophoresis. Proteins were then transferred to a PVDF membrane under cooling conditions at 100 V for 2 h using a wet transfer system. After blocking with 5% skim milk, the membrane was incubated with the primary antibody overnight at 4 °C. Following washes, a suitable secondary antibody was applied, and the membrane was incubated for 1 h at room temperature. Finally, membranes were developed in a darkroom using ECL chemiluminescence and analyzed with ImageJ software. For normalization, the mean value of the NC group was set as the reference. The raw data of each individual sample from all experimental groups were then divided by this mean value, so that the average of the NC group became 1, and all other values were expressed as fold-change relative to NC.

Statistical analysis

Statistical analysis was conducted using SPSS 26.0 software, and the experimental data are presented as the means ± SD. Biological replicates (n) represent individual mice per group (n = 10 for physiological parameters; n = 3 for Western blot, and n = 6 for 16S sequencing). No animals or samples were excluded from the analysis. If the sample met the assumptions of a normal distribution and homogeneity of variance, one-way analysis of variance was used to compare multiple groups. When homogeneity of variance was confirmed, the least significant difference (LSD) test was applied for pairwise comparisons; otherwise, Dunnett’s T3 test was used. Given the exploratory nature of the study, no additional correction for multiple comparisons was applied across different endpoints. A statistically significant difference was indicated when P < 0.05.

Results

Specnuezhenide administration decreased blood glucose levels, improved renal function, and alleviated renal injury in mice with DN

DN model mice were treated with Specnuezhenide for 8 weeks after successful modeling. The mice in the MC group displayed marked increases in serum glucose levels, whereas those in the Specnuezhenide group were significantly decreased (Figure 1B). The MC group exhibited significantly elevated levels of urinary albumin, ...

Discussion

DN, a persistent microvascular complication of diabetes, is characterized by elevated urinary albumin excretion and impaired renal function²⁴. Albuminuria is a frequently used early clinical indicator of DN progression²⁵. Patients with DN may exhibit abnormal renal function, as evidenced by significantly increased SCr and BUN levels²⁶. In this study, the Specnuezhenide group demonstrated notably reduced urinary albumin and significantly decreased ser...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albustix	Guangzhou Huadu Gaoerbo Biotechnology Co., Ltd.	20211203
Analytical balance	Beijing Sartorius Instrument System Co., Ltd.	CPA
antibodie A20	Cell Signalling Technology	5630s	(1:1000)
antibodie Bax	Cell Signalling Technology	2772s	(1:1000)
antibodie Bcl-2	Cell Signalling Technology	3498s	(1:1000)
antibodie claudin 1	Abcam	EPR25359-48	(1:1000)
antibodie COX2	Proteintech Group	12375-1-AP	(1:2000)
antibodie GAPDH	Abcam	EPR16891	(1:10000)
antibodie GCLC	Proteintech Group	12601-1-AP	(1:10000)
antibodie HO-1	Proteintech Group	10701-1-AP	(1:10000)
antibodie IκBα	Cell Signalling Technology	4812s	(1:1000)
antibodie Myd88	Abcam	EPR21824	(1:1000)
antibodie NF-κB p65	Cell Signalling Technology	8242s	(1:1000)
antibodie NLRP3	Proteintech Group	68102-1-Ig	(1:5000)
antibodie NQO1	Proteintech Group	11451-1-AP	(1:5000)
antibodie Nrf2	Proteintech Group	16396-1-AP	(1:5000)
antibodie occludin	Cell Signalling Technology	91131S	(1:1000)
antibodie p-IκBα	Cell Signalling Technology	2859s	(1:1000)
antibodie p-NF-κB p65	Cell Signalling Technology	3033s	(1:1000)
antibodie TAX1BP1	Proteintech Group	14424-1-AP	(1:2000)
antibodie TLR4	GeneTex	GTX75742	(1:1000)
antibodie TRAF6	Santa Cruz Biotechnology	sc-8409	(1:1000)
antibodie ZO-1	Proteintech Group	21773-1-AP	(1:10000)
Automatic biochemical analyzer for blood glucose measurement	Biobase Biodusty (Shandong) Co., Ltd.	BK-280
blood urea nitrogen	Nanjing Jiancheng Bioengineering Institute	C03-2-1
Centrifugal machine	Hunan Xiangyi Laboratory Instrument Development Co., Ltd	HI650
db/db mice (8-9 week-old male)	Changzhou Kavens Laboratory Animal Co., Ltd.	SCXK (Su) 2021-0013
IL-1β ELISA Kit	Wuhan Fine Biotech Co., Ltd.	EM0109
IL-6 ELISA Kit	Wuhan Fine Biotech Co., Ltd.	EM1158
Irbesartan	Zhejiang Huahai Pharmaceutical Co., Ltd.	0000019353
LPS	Jiangsu Meimian Industrial Co., Ltd.	MM-0634M1
microscope	Olympus Corporation	BX53
microtome	Leica Biosystems Nussloch GmbH	RM 2016
Organized pizza slicing machine	Wuhan Junjie Electronic Co., Ltd	JK-6
Paraffin slicing machine	RWD Life Science Co., Ltd.	S710
Pathological slide scanner	Ningbo Konfoong Bioinformation Tech Co., Ltd.	KFBIO PRO-005
serum creatinine	Nanjing Jiancheng Bioengineering Institute	C011-2-1
Specnuezhenide	National Institutes for Food and Drug Control	39011-92-2	purity >98%
SPSS statistics 26.0
Tissue Embedding Center	Wuhan Junjie Electronic Co., Ltd	JB-P5
Tissue Processor	Wuhan Junjie Electronic Co., Ltd	JT-12J
TNF-α ELISA Kit	Wuhan Fine Biotech Co., Ltd.	ER1393
urinary protein	Wuhan Fine Biotech Co., Ltd.	EM0632