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10.1080/0886022X.2024.2398710
2398710
Version of Record
Research Article
Chronic Kidney Disease and Progression
Chrysophanol-mediated trx-1 activation attenuates renal fibrosis through inhibition of the JNK/Cx43 signaling pathway
N. Bao et al.
https://orcid.org/0000-0002-6841-1311
Bao Neng a
Wang Jin b
Yue Qiyu c
Cao Fang d
Gu Xuejing d
Wen Kejian d
Kong Wei a
https://orcid.org/0000-0001-9677-8279
Gu Mingjia d
a Department of Nephrology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing city, Jiangsu, China
b Department of Gastroenterology, Affiliated Hospital of Jiangnan University, Jiangsu, China
c School of Chinese Medicine & School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Jiangsu, China
d Department of Nephrology, Changshu Hospital affiliated to Nanjing University of Chinese Medicine, Changshu city, Jiangsu, China
Supplemental data for this article can be accessed online at https://doi.org/10.1080/0886022X.2024.2398710.

CONTACT Mingjia Gu fsyy00893@njucm.edu.cn Department of Nephrology, Changshu Hospital Affiliated to Nanjing University of Chinese Medicine, 6 Huanghe Road, Changshu city, Jiangsu 215500, China
Wei Kong kw-9009@163.com Department of Nephrology, Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, 157 Daming Road, Nanjing city, Jiangsu 210000, China
5 9 2024
2024
5 9 2024
46 2 239871030 3 2024
7 8 2024
26 8 2024
KnowledgeWorks Global Ltd.5 9 2024
published online in a building issue5 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Purpose

This study aimed to investigate the inhibitory effect of chrysophanol on renal fibrosis and its molecular mechanism.

Methods

Initially, potential targets of chrysophanol were predicted through network pharmacology analysis, and a protein-protein interaction network of these targets was constructed using Venn diagrams and the STRING database. GO enrichment analysis predicted the biological process of chrysophanol in treating renal fibrosis. Subsequently, both in vivo and in vitro experiments were conducted using unilateral ureteral obstruction (UUO) induced CKD mouse model and HK-2 cell model, respectively. In the mouse model, different doses of chrysophanol were administered to assess its renal protective effects through biochemical indicators, histological examination, and immunofluorescence staining. In the cell model, the regulatory effect of chrysophanol on the Trx-1/JNK/Cx43 pathway was evaluated using western blotting and flow cytometry.

Results

Chrysophanol treatment significantly ameliorated renal dysfunction and histopathological damage in the UUO mouse model, accompanied by a reduction in serum oxidative stress markers. Furthermore, chrysophanol markedly upregulated the expression of Trx-1 in renal tissues and inhibited the activation of the JNK/Cx43 signaling pathway. At the cellular level, chrysophanol enhanced the activity of Trx-1 and downregulated the JNK/Cx43 signaling pathway, thereby inhibiting TGF-β induced oxidative stress and cell apoptosis.

Conclusion

This study demonstrated a significant inhibitory effect of chrysophanol on renal fibrosis, mediated by the activation of Trx-1 to inhibit the JNK/Cx43 pathway. These findings provide experimental support for the potential use of chrysophanol as a therapeutic agent for renal fibrosis.

Keywords

Chronic kidney disease
chrysophanol
renal fibrosis
Trx-1
JNK
Cx43
Jiangsu Province Traditional Chinese Medicine Technology Development Plan Project No. MS2022085 Natural Science Foundation Project of Nanjing University of Traditional Chinese Medicine XZR2020063 and XZR2021092 Suzhou Gusu Health Talent Program Research Project No. GSWS2022099 Suzhou Youth Science and Technology Project No. KJXW2021070 Changshu Science and Technology Development Plan Project No. CS202231 Wuxi Municipal Health Commission’s research project plan No. Q202312 A joint support was given to this study by Jiangsu Province Traditional Chinese Medicine Technology Development Plan Project (No. MS2022085), Natural Science Foundation Project of Nanjing University of Traditional Chinese Medicine (Nos. XZR2020063 and XZR2021092), Suzhou Gusu Health Talent Program Research Project (No. GSWS2022099), Suzhou Youth Science and Technology Project (No. KJXW2021070), and Changshu Science and Technology Development Plan Project (No. CS202231), and the Wuxi Municipal Health Commission’s research project plan (No. Q202312).
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pmc1. Introduction

The increasing incidence of chronic kidney disease (CKD) is a worldwide health issue that impacts more than 800 million individuals, that is, more than 10% of the global population [1]. Renal fibrosis is the characteristic pathological basis of CKD, and oxidative stress is a significant contributor to this condition. Oxidative stress-induced hypoxia plays a significant role in the pathogenesis of kidney injury [2,3]. Consequently, the importance of redox balance in the pathogenesis of CKD is increasingly being recognized. However, the detailed mechanisms underlying the development of renal fibrosis due to redox imbalance remain incompletely understood.

Thioredoxin 1 (Trx-1) plays a crucial role in maintaining redox balance. Trx-1 has a molecular weight of 12 kilodaltons and contains a primary active site that is characterized by the consensus sequence ‘-Cys-Gly-Pro-Cys-’. The Trx-1 sequence is composed of redox-active dithiol/disulfide bonds, which predominantly govern its binding to specific substrate proteins, thereby influencing their functions [4]. Trx-1 plays a role in controlling different cell signaling pathways by reacting to oxidative stress, thereby exerting anti-inflammatory and anti-apoptotic effects [5]. Studies have revealed a notable increase in Trx-1 levels in the urine of individuals suffering from acute kidney injury (AKI). Conversely, Trx-1 in renal tubular epithelial cells was notably decreased in a rat model of AKI [6]. Importantly, the use of inhibitors to decrease Trx-1 levels in rat renal tubular epithelial cells (NRK-52E cells) has been proven to markedly increased the severity of fibrosis [7]. These data highlight the critical need to explore the link between Trx-1 and CKD, given the substantial impact of Trx-1 on the development of CKD.

Gap junctions (Gaps) are specialized membrane structures that facilitate communication between two adjacent cells [8]. These factors are vital for many physiological functions, including cellular metabolism, equilibrium, and growth. The structure of a gap junction consists of a hexagonal connectome that is formed by six subunit connexins that surround a hydrophilic channel in the middle; this structure creates a channel that spans the gap between two neighboring cells. Connexins are a family of proteins with several subgroups, including Cx26, Cx32, Cx36, and Cx43 [9]. Among these, Cx43 is the most highly expressed connexin in the kidney. Cx43 not only is involved in the intercellular transmission of information but also plays regulatory roles in gene transcription, cytoskeletal dynamics, energy metabolism, and the stress response [10]. Research indicates that in a mouse model of CKD caused by unilateral ureteral obstruction (UUO), the expression of Cx43 in the kidney is significantly increased. This upregulation is positively correlated with inflammatory responses and the degree of kidney fibrosis. Moreover, knockout of the Cx43 gene or the use of Cx43 inhibitors significantly increases the degree of fibrosis in mouse kidneys [11]. These results indicate that Cx43 is involved in regulating kidney function and that it potentially contributes to the onset of CKD.

Chrysophanol, a key ingredient in rhubarb, which is a traditional Chinese medicinal herb, has widespread applications in the food processing and pharmaceutical sectors [12]. Recent studies have highlighted the important role of chrysophanol in protecting kidney function [13]. Earlier studies confirmed that chrysophanol decreases fibrosis in the kidneys of mice with UUO-induced CKD by suppressing inflammation [14]. Despite these findings, few studies have examined the precise mechanism by which chrysophanol ameliorates renal fibrosis, and its pharmacological mechanisms warrant further exploration. Network pharmacology and molecular docking, which are emerging technologies that integrate bioinformatics and systems biology, have emerged as essential tools for investigating renal fibrosis treatments that involve monomers from traditional Chinese medicine [15–17]. These methodologies enable systematic analysis of the interaction network between traditional Chinese medicine monomers and molecular targets, providing a scientific foundation for evaluating efficacy, screening drug molecules, and designing optimal drugs. Consequently, the objective of this research was to investigate the antifibrotic effects of chrysophanol and to determine whether it alleviates TGF-β induced oxidative stress and apoptosis by regulating the Trx-1/JNK/Cx43 signaling pathway.

2. Materials and methods

2.1. Network pharmacology

Data on the 3D SDF structure and standard SMILES configuration of chrysophanol were obtained from the PubChem database (https://pubchem.ncbi. nlm.nih.gov/). Next, the molecular configurations were transferred into the Swisstarget database (http://www.swisstargetprediction.ch/) and the TargetNet database (http://targetnet.scbdd.com/calcnet/index/) to predict proteins that are associated with chrysophanol. Subsequently, the term ‘renal fibrosis’ was used to search the GeneCards database (https://www.genecards.org/) to identify relevant targets associated with renal fibrosis. After target identification, a Venn diagram was generated using a digital instrument (http://www.bioinformatics.com.cn/login/) to illustrate the intersecting targets, which represent potential chrysophanol targets associated with renal fibrosis. Then, the targets were uploaded to the STRING database (https://string-db.org/) to create a protein–protein interaction (PPI) network analysis schematic. To gain insights into the biological functions of the identified targets, Gene Ontology (GO) functional analysis was performed. The GO functional analysis results were graphically presented and arranged through a web portal (https://www.bioinformatics.com.cn) (last retrieved on 10 Nov 2023).

2.2. Drugs and reagents

Chrysophanol was purchased from McLean Reagents Co., Ltd. (Shanghai, China). and positive drug Losartan was obtained from McLean Reagent Co., Ltd (Shanghai, China). Px-12 was obtained from Selleckchem (S7947, Houston, USA). TGF-β1 was acquired from PeproTech (#100-21, Cranbury, USA). Antibodies against thioredoxin 1 (#2429, diluted 1:400 for immunofluorescence analysis; 1:1000 for Western blotting), connexin 43 (#83649, diluted 1:400 for immunofluorescence analysis), p-JNK (#4668, diluted 1:1000), JNK (#9252, diluted 1:1000), α-SMA (#19245, diluted 1:1000), TGF-β (#3711, diluted 1:1000), p-Smad2/3 (#8828, diluted 1:1000), Smad2/3 (#8685, diluted 1:1000), SOD (#37385, diluted 1:1000), HO-1 (#43966, diluted 1:1000), GAPDH (#2118, diluted 1:1000), and anti-rabbit IgG (H + L) (#14708, diluted 1:5000) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). NRF2 (#16396-1-AP, diluted 1:1000) was purchased from Proteintech (Chicago, Illinois, USA). α-SMA (#AF1032, diluted 1:1000) was purchased from Affinity (Elgin, Illinois, USA). Goat anti-rabbit IgG H&L (Alexa Fluor® 594) (ab150080, diluted 1:1000), goat anti-rabbit IgG H&L (Alexa Fluor®488) (ab150077, diluted 1:1000), anti-Connexin43 (ab230537, diluted 1:1000 for Western blotting), and anti-NQO1 (ab80588, diluted 1:1000) antibodies were obtained from Abcam (Cambridge, UK). BCA protein assay kit (# P0010S), Cell Counting Kit-8 (#C0037) and RIPA lysis buffer (#P0013C) were obtained from Beyotime Biotechnology (Shanghai, China). An ELISA kit (β2-MG) was purchased from Sangon Biotech (Shanghai, China). The DCFH-DA reactive oxygen species (ROS) fluorescent probe (#D6470), malondialdehyde (MDA) assay kit (#BC0025), superoxide dismutase (SOD) activity assay kit (#BC0175), catalase (CAT) assay kit (#BC0205), serum creatinine (SCr) assay kit (#BC4910), and blood urea nitrogen (BUN) assay kit (#BC1535) were purchased from Solarbio Co., Ltd. (Beijing, China). The alanine aminotransferase assay kit (# C009-2-1) and aspartate aminotransferase assay kit (# C010-2-1) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). An Annexin V-FITC/PI apoptosis detection kit was acquired from Vazyme Co., Ltd. (#A211-01, Nanjing, China).

2.3. Animal model and drug treatments

Under license SCXK (Beijing) 2019-0010, 60 male C57/Bl6 mice aged 7 weeks and weighing between 22 and 25 grams were acquired from Spefford (Beijing) Biotechnology Co., Ltd. Prior to the experiments, the mice were allowed to acclimate for one week in the Experimental Animal Center at Nanjing University of Chinese Medicine. The mice were housed in a controlled environment with a light/dark cycle of 12:12 h, a room temperature of 21.0 ± 2.0 °C, a humidity of 55.0 ± 5.0%, and free access to food and fresh water. This animal study was approved by the Ethical Review Committee of Nanjing University of Chinese Medicine. According to the experimental protocol from our previous study [14], the twelve mice were randomly divided into five groups: the control group, the UUO group, the UUO group treated with a low dose (20 mg/kg) of chrysophanol, the UUO group treated with a high dose (40 mg/kg) of chrysophanol and the UUO group treated with losartan (10 mg/kg). The mice in the UUO groups, but not the control group, underwent UUO surgery in accordance with published methods [18]. The mice in the UUO and control groups received 0.5% sodium carboxymethylcellulose orally. For two weeks, the mice in the chrysophanol treatment groups received 20 mg/kg/d or 40 mg/kg/d of the drug and the mice in the losartan treatment groups received 10 mg/kg/d of the drug. The mice were put to sleep at the end of the experiment, and kidney tissue and serum samples were collected for further examination.

2.4. Cell culture and treatments

The HK-2 cell line was obtained from the China Type Culture Collection in Shanghai, China. The cells were cultivated at 37 °C In a moisture-controlled incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Sangon; Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Sangon). The cells were plated at a density of 1 × 106 cells per well in 6-well culture plates once they reached 80% confluence. After being serum-starved in medium supplemented with 0.5% FBS overnight, the cells were subdivided into the following experimental groups: the CHR-H, Px-12, Mod, CHR-H, and CHR-H + Px-12 groups. The optimal drug concentration was determined using the Cell Counting Kit-8 assay. According to the literature, after being serum-starved for 24 h, HK-2 cells in the Mod group were treated with 20 ng/mL TGF-β1 [14]. Then, the CHR-H group was treated with 100 μM chrysophanol and 20 ng/mL TGF-β1 for 24 h. In the Px-12 group, serum-starved HK-2 cells were treated with 20 ng/mL TGF-β1 for 8 h, and then 10 μM Px-12 was added for a total of 24 h. In the CHR-H + Px-12 group, HK-2 cells that were serum-starved underwent an 8-h treatment with 20 ng/mL TGF-β1 and 100 μM chrysophanol, followed by the addition of 10 μM Px-12; then, the cells were incubated for an entire 24 h.

Every cell culture experiment was conducted three times, and the cells were collected for subsequent analysis via flow cytometry and western blotting.

2.5. Biochemical analysis

The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (SCr) and the serum levels of β2-microglobulin (β2-MG), superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were measured using specialized kits according to the manufacturer’s instructions. Kidney tissues were fixed in 4% paraformaldehyde for a minimum of 24 h and then embedded in paraffin wax. Subsequently, the sections were processed and stained with hematoxylin and eosin (HE) or Masson’s trichrome staining. Within every group, three mice were chosen at random, and three specific areas on each slide were examined and imaged using a microscope. The assessment of cellular damage and pathological characteristics in kidney tissues was conducted through a semiquantitative approach, and the results were categorized as follows: 0: absence of pathological alterations; 1: negligible pathological alterations (<25% tissue impacted); 2: moderate pathological alterations (25%∼50% tissue impacted); 3: intense pathological alterations (51%∼75% tissue impacted); and 4: extremely critical pathological alterations (>75% tissue impacted) [19]. The quantification of the area of renal fibrosis were performed in Masson’s staining [20].

2.6. Determination of intracellular ROS levels

The levels of reactive oxygen species (ROS) in cells were measured using a dichlorofluorescein assay. In brief, the production of ROS was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), a marker of oxidative damage. Following predefined experimental protocols, the cells were collected and treated. Subsequently, the cells were incubated for 20 min with 100 μM DCFH-DA in a 5% CO2 atmosphere. Subsequently, the cells were washed three times with phosphate-buffered saline (PBS, pH 7.4) to remove excess DCFH-DA. Next, the fluorescence emitted by the stained cells was measured using flow cytometry.

2.7. Apoptosis analysis

Apoptosis was analyzed by flow cytometry. As per the experimental procedure, the cells were subjected to treatment and were then separated using EDTA-free trypsin. Subsequently, the cells were subjected to centrifugation, and the supernatants were removed. To generate cell suspensions, 0.5 mL of buffer was added, and the cells were subsequently resuspended using a pipette. The Annexin V-PI dual-staining technique and flow cytometry agents were performed. Following the manufacturer’s instructions, 5 μL of Annexin V-FITC was added, mixed, and left to react in the dark for five minutes. Next, a mixture of 10 μL of propidium iodide solution was prepared, and the reaction proceeded in the dark for an additional 15 min. Subsequently, the proportion of HK2 cells undergoing apoptosis was quantified through flow cytometry.

2.8. Western blotting analysis

Lysis of kidney tissues and cell cultures was performed on ice using RIPA buffer enriched with 1% PMSF and 2% phosphatase inhibitor (Thermo Fisher, Rockford, IL, USA). A BCA assay kit was used to measure the levels of the proteins that were extracted. Subsequently, the proteins were mixed with loading buffer and heated. Proteins from each specimen were subsequently subjected to 12% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were incubated first with primary antibodies and then with secondary antibodies. Protein bands were visualized with an advanced chemiluminescence agent (Bio-Rad, Bio-Rad Hood Universal II, California, United States), and the bands were detected via chemiluminescence using Tanon devices (Tanon, Shanghai, China). Image J software was used to measure the gray intensity of the protein bands.

2.9. Immunofluorescence analysis

For immunofluorescence staining, kidney tissues were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% BSA. Next, the tissue slices were incubated overnight at 4 °C with primary antibodies. Subsequently, the samples were incubated for 1 h with secondary antibodies at ambient temperature in the dark. Subsequently, the specimens were subjected to 10 min of DAPI staining to visualize the cell nuclei. Using a fluorescence microscope, immunofluorescence images were obtained and examined to determine the expression of particular proteins and their cellular position in kidney tissue.

2.10. CCK-8 assay

The viability of HK-2 cells was assessed using the CCK-8 assay. Briefly, after stimulation, 10 ul of CCK-8 solution was added to each well and incubated for 1 h at 37 °C. The absorbance at 450 nm was then measured using a microplate instrument.

2.11. Molecular docking

Initially, the protein structure associated with Trx-1 was obtained from the Protein Data Bank (PDB) repository (https://www.rcsb.org/). Pymol 2.4.0 was used for protein alteration activities, including removing water molecules and ligands. A search of the chrysophanol structure was conducted through PubChem, followed by downloading the SDF files and transforming them into PDB format using Open Babel 3.1.1. The standard handling of protein receptors and small-molecule ligands utilized AutoDock 4.2.6, with the resulting structures stored in the ‘pdbqt’ format. Next, calculations of molecular docking and evaluations of binding energy were conducted utilizing AutoDock Vina 1.1.2, accompanied by a script. Ultimately, PyMOL software facilitated the visualization and comparison of docking outcomes, facilitating the creation of demonstrative representations.

2.12. Statistical analysis

The experimental results are expressed as the mean ± standard deviation (SD). Statistical analyses involving intergroup comparisons were performed using GraphPad Prism 8.0 software and one-way variance analysis. A value of *p < 0.05 was considered to indicate a significant difference.

3. Results

3.1. Potential targets and GO functional enrichment analysis of chrysophanol in the treatment of renal fibrosis

The 2D structure of chrysophanol is shown in Figure 1A. Comprehensive analysis identified 515 potential targets associated with chrysophanol that intersected with 882 disease targets related to renal fibrosis (RF). A Venn diagram was constructed to visualize the overlap, revealing 52 targets that were shared between chrysophanol and RF (Figure 1B). Subsequently, these 52 common targets were uploaded to the STRING database, and interactions with a minimum confidence score of ‘high confidence (0.700)’ were considered. The resulting protein interaction network diagram is shown in Figure 1C. Key target genes were screened using Cytoscape 3.7.0 software based on centrality analysis of the interaction network. Within the protein–protein interaction (PPI) framework, every node symbolizes a protein, while each edge indicates the interplay among proteins. Proteins located in the network’s core likely play pivotal roles in chrysophanol-mediated treatment of RF. CytoNCA analysis identified the top 20 core targets based on degree centrality values, including MMP9, SRC, HSP90AA1, EGFR, PIK3CA, BCL2, MMP2, ESR1, CASP3, MTOR, PTGS2, MET, KDR, MDM2, BRAF, NOS3, RAF1, MAP2K1, PTPRC, and PLAU. Functional analysis revealed that chrysophanol was involved in the regulation of biological processes (BPs) associated with RF, including responses to reactive oxygen species, the regulation of MAP kinase activity, and the modulation of phosphatidylinositol 3-kinase signaling. In terms of cellular components (CCs), chrysophanol primarily impacted membrane rafts, the basal plasma membrane, and the collagen-containing extracellular matrix in the treatment of RF. Additionally, the molecular functions (MFs) in RF that were influenced by chrysophanol in RF included scaffold protein binding, transmembrane receptor protein tyrosine kinase activity, and serine-type peptidase activity (Figure 1D–F).

Figure 1. Identification of therapeutic targets for chrysophanol against renal fibrosis. (A) The molecular structural formula of chrysophanol; (B) the Venn diagram illustrates the intersection of targets between chrysophanol and renal fibrosis; (C) protein-protein interaction analysis of the common target proteins in the intersection; (D–F) gene ontology enrichment analysis of biological processes, cellular components and molecular functions for the common target proteins in the intersection.

3.2. Chrysophanol reverses renal function damage caused by UUO

Our research focused on the kidney-protective effects of chrysophanol in a UUO-induced mouse model of CKD. The animal experimental procedure is shown in Figure 2A. Mice in the normal group exhibited normal food and water intake, were responsive, and had glossy fur. In contrast, mice in the model group showed decreased food and water intake compared to those in the normal group, were less responsive, and had disheveled and dull fur. Kidney morphology in the control group was dense and intact, with a smooth red surface. However, mice in the UUO group exhibited significantly increased kidney swelling, extensive urine retention, and an elevated kidney index. Administering chrysophanol decreased these morphological alterations, resulting in notable amelioration of the symptoms of mice that were treated with a substantial dose of chrysophanol (p < 0.05) (Figure 2B,C). The histological results of HE and Masson staining are shown in Figure 2E. In the control group, there was a clear glomerular structure, no swelling of tubular epithelial cells, and no inflammatory cell infiltration in the interstitium. In the model group, most glomeruli were markedly reduced in size, which was accompanied by epithelial cell atrophy and detachment. In the treatment group, a small number of shrunken glomeruli and some cell swelling was observed, along with a small amount of interstitial connective tissue hyperplasia and mild edema. With increasing drug concentrations, kidney tissue damage significantly decreased, as indicated by the pathological scores and the area of renal fibrosis (p < 0.05) (Figure 2K,L). SCr, BUN and β2-MG levels are common indicators of renal function damage. Consistent with the changes in the kidney index, the model group showed a notable increase in the SCr, BUN, and β2-MG levels compared with those of the control group. Nonetheless, after treatment with chrysophanol, there was a notable increase in these metrics (p < 0.05) (Figure 2D–G). Furthermore, the western blot data showed that UUO caused a significant increase in α-SMA, TGF-β and p-Samd2/3 protein expression in CKD mice (Figure 2H). As expected, UUO-induced increases in α-SMA, TGF-β and p-Samd2/3 level was significantly attenuated by chrysophanol treatment (Figure 2I–M). In terms of safety, no liver function abnormalities were observed in mice from the CHR-H group (Figure S1).

Figure 2. Chrysophanol mitigated the functional damage to the kidneys induced by unilateral ureteral obstruction (UUO). (A) Summary of animal experiment process; (B) the morphological characteristics of mice kidneys exhibit variations among different groups; (C) the ratio of kidney weight to body weight was measured; (D) comparison for SCr levels of mice in each group; (E) representative micrographs of kidney tissues from different groups were obtained through HE & Masson staining; (F–G) comparison for BUN and β2-MG levels of mice in each group; (H–K) western blotting was performed to detect the levels of α-SMA, TGF-β and p-Smad expression; (L) tubular injury score; (M) collagen volume fraction.

The data for figure (C,D, F,G) was expressed as the means ± SD (n = 12); The data for figure (H–M) was expressed as the means ± SD (n = 3); ##p < 0.01 vs. Con group; *p < 0.05 vs. Mod group; **p < 0.01 vs. Mod group.

3.3. Chrysophanol inhibits oxidative stress caused by UUO

Based on the network pharmacology GO enrichment analysis results, we focused on oxidative stress. The results showed a marked reduction in serum MDA and SOD concentrations and an increase in CAT levels in mice in the UUO induced CKD model group. Treatment with chrysophanol reversed these effects (p < 0.05) (Figure 3A–C). The results of flow cytometry revealed a significant increase in DCFH-DA-positive cells in the UUO group, indicating elevated ROS levels in these cells. The administration of chrysophanol significantly decreased ROS levels in the UUO group (Figure 3D,F). Western blotting analysis of SOD, NQO1 and HO-1 expression further confirmed the antioxidative effects of chrysophanol. Treatment with chrysophanol successfully counteracted the oxidative stress damage caused by UUO, as demonstrated by changes in protein expression levels (Figure 3E,G–I).

Figure 3. Chrysophanol inhibits oxidative stress induced by unilateral ureteral obstruction (UUO). (A–C) Comparison of SOD, MDA and CAT levels of mice in each group; (D) Percentage of DCF-positive cells; (E) Western blotting was performed to detect the expression levels of NQO1, SOD and HO-1proteins; (F) Flow cytometric analysis of DCF-positive cells by DCFH-DA staining; (G–I) Bar graphs showing the expression levels of NQO1, SOD and HO-1 proteins;

The data for figure (A–C) was expressed as the means ± SD (n = 12); The data for figure (D–I) was expressed as the means ± SD (n = 3). ##p < 0.01 vs. Con group; *p < 0.05 vs. Mod group. **p < 0.01 vs. Mod group.

3.4. Chrysophanol regulates the Trx-1/JNK/Cx43 pathway in UUO-treated mice

Trx-1 plays a crucial role in the redox signaling pathway and can scavenge intracellular ROS to mitigate apoptosis. It is regulated by Nrf2 through two antioxidant response elements in its promoter [21]. Western blotting analysis of Nrf2 and Trx-1 expression revealed a notable reduction in the UUO group. Nonetheless, chrysophanol markedly elevated the Nrf2 and Trx-1 levels. Molecular docking was conducted to mimic the interaction between chrysophanol and the thioredoxin fusion protein (PDB ID: 6LUR). The three-dimensional (3D) binding site analysis reveals that chrysophanol is deeply embedded within the active site pocket of the thioredoxin fusion protein. Key protein residues, including LEU-529, GLU-483, VAL-480, and PHE-534, engage in hydrophobic interactions with chrysophanol (Figure 4C). The deduced binding energy was −6.5 kcal/mol, which indicated a tight interaction among these proteins (Figure S2). The immunofluorescence results (Figure 4A) further confirmed this observation. To elucidate the molecular processes underlying the suppressive effect of chrysophanol on oxidative stress damage caused by UUO, we assessed MAPK pathway-associated protein levels via network pharmacology. Our findings indicate notable activation of p-JNK and its downstream protein Cx43 in the UUO group. Nonetheless, chrysophanol administration markedly reduced the protein levels of Cx43 and p-JNK, as shown in Figure 4F,G. Additionally, immunofluorescence analysis of Cx43 confirmed these results (Figure 4B).

Figure 4. Chrysophanol regulates the Trx-1/JNK/Cx43 pathway. (A,B) Comparison of immunofluorescence results for Trx-1 and Cx43 in the kidneys of different mice groups. (C) The 3-dimensional map of the binding sites between chrysophanol and thioredoxin fusion (PDB ID: 6LUR). Chrysophanol is shown in yellow, target protein is displayedas cyan; (D) expression of Nrf2, Trx-1, p-JNK and Cx43 through western blotting; (E–H) Bar graphs showing the expression levels of Nrf2, Trx-1, p-JNK and Cx43 proteins.

The data are expressed as the means ± SD (n = 3). ##p < 0.01 vs. Con group; *p < 0.05 vs. Mod group. **p < 0.01 vs. Mod group.

3.5. Inhibiting Trx-1 weakens the inhibitory effect of chrysophanol on TGF-β1-induced oxidative stress and apoptosis in HK2 cells

Based on the results of the CCK-8 assay, the concentration of chrysophanol was set to 100 μM for subsequent cell experiments (Figure S3). Investigating the regulatory effect of chrysophanol on Trx-1 involves Trx-1 inhibition via Px-12. The findings indicated that compared with those in the model group, the cells in the Px-12 group exhibited increased apoptosis and oxidative stress. Furthermore, the cells in the CHR-H + Px-12 group exhibited highers levels of apoptosis and oxidative stress than did the cells in the CHR-H group (Figure 5A,B). Western blotting analysis revealed a notable increase in p-JNK and CX43 protein levels in the CHR-H + Px-12 group compared with those in the CHR-H group, while Trx-1 protein expression was markedly reduced (Figure 6A–D).

Figure 5. Chrysophanol inhibits TGF-β1-induced apoptosis and oxidative stress in HK2 cells. (A) Comparison of apoptotic cells from different groups detected by flow cytometry and (B) Flow cytometric analysis of DCF-positive HK2 cells by DCFH-DA staining.

The data are expressed as the means ± SD (n = 3). **p < 0.01 vs. CHR-H group.

Figure 6. Chrysophanol inhibits the JNK/Cx43 pathway by regulating Trx-1 protein. (A–D) Expressions of Trx-1, p-JNK and Cx43 through western blotting.

The data are expressed as the means ± SD (n = 3). **p < 0.01 vs. CHR-H group.

4. Discussion

Many studies have confirmed a strong link between the progression of CKD and oxidative stress. The accumulation of ROS within the body disrupts redox balance, resulting in kidney damage [22,23]. Oxidative stress, which is a defense mechanism, is characterized by an imbalance between the generation of oxidative free radicals and the degradative functions of the antioxidant system, and ROS are the principal oxidative compounds [24]. The kidneys, which are metabolically active organs that consume high energy levels, are particularly susceptible to oxidative stress and the resulting functional impairment [25]. In response to oxidative stress, enzymatic antioxidants, which includes SOD, NQO1, HO-1 and CAT that ameliorate tissue and cellular damage, is activated [26]. Network pharmacology predictions have indicated that chrysophanol might alleviate renal fibrosis by regulating oxidative stress. Subsequent experiments confirmed that the levels of SOD, HO-1, NQO-1, and CAT in the kidneys of CKD model mice were significantly decreased. Nonetheless, chrysophanol administration mitigated the impairment of kidney function in mice, elevating the levels of these indicators. Notably, the involvement of Trx-1 in the progression of CKD has recently attracted increasing attention.

Trx-1 plays a crucial role in the thioredoxin mechanism, which is an essential thiol antioxidant system that reduces oxidative stress [27]. Several studies have confirmed that Trx-1 is regulated by nuclear factor erythroid 2-related factor 2 (Nrf2) in oxidation reactions [28,29]. Nrf2 is located in the cytoplasm and combines with Keap1 to form a stable compound. When subjected to reactions such as oxidative stress, Nrf2 detaches from Keap1 and enters the nucleus to combine with AREs, causing an increase in the secretion of Trx1 by cells [30]. Trx-1 participates in the pathogenesis of various diseases such as chronic pancreatitis [31], pulmonary fibrosis [32] and systemic sclerosis [33] by influencing tissue fibrosis. A report revealed that renal tubular epithelial cells secrete Trx-1 into the renal tubule and that it is subsequently excreted in the urine when kidneys are damaged due to ischemia and hypoxia, leading to a decrease in Trx-1 levels in renal tissues [34]. Our study is the first to show that Trx-1 expression is substantially reduced in a UUO-induced CKD mouse model. It has been reported that chrysophanol binds to Nrf2 to dissociate it from Keap1 [35], and inhibits oxidative reactions and tissue fibrosis by activating Nrf2 [36]. Therefore, we detected the levels of Nrf2 and Trx1, the results showed chrysophanol increased Nrf2 and Trx-1 expression in the kidney in a concentration- dependent manner and ameliorated renal dysfunction in mice with CKD. To further substantiate the therapeutic effect of chrysophanol on improving renal fibrosis, we used the Trx-1 inhibitor Px-12 in cell experiments. These results confirmed that chrysophanol exerted a therapeutic effect by enhancing Trx-1 activity.

Gap junctions, which are composed of specific proteins, play a crucial role in maintaining tissue structural integrity and are ubiquitously present in nearly all cell types. These junctions consist of two intercellular hemichannels known as connexons [37]. Each connexon is a hexamer composed of six connexins, and Cx43 is the most widely expressed connexin. In the context of kidney disease, Cx43 is involved in the structure and function of the renal cortex, and inhibiting its activity has been shown to inhibit the progression of kidney disease. Studies using various CKD models demonstrated that decreased expression of Cx43 significantly delayed the progression of renal dysfunction and ameliorated renal fibrosis [11,38]. Recent investigations have revealed increased activity of Cx43 in the damaged renal tubules of mice with CKD, which exacerbates inflammatory responses and induces renal fibrosis [39]. Our study confirmed a positive correlation between Cx43 levels in the kidneys of mice with UUO-induced CKD and the severity of renal fibrosis. Chrysophanol reduced the expression of Cx43 in a concentration-dependent manner both in vivo and in vitro.

Zhou et al. showed that reduced Trx-1 levels led to enhanced JNK phosphorylation, which in turn activated Cx43 and damaged rat renal tubular epithelial cells in vitro [7]. This is the first study to demonstrate that, in a UUO-induced CKD mouse model, phosphorylated JNK and Cx43 were expressed at higher levels and that Trx-1 levels were reduced. Chrysophanol significantly increased Trx-1 levels while reduced p-JNK activity and Cx43 expression. JNK, which is a key player in the MAPK pathway, affects various cellular processes, including proliferation, differentiation, migration, and apoptosis [40]. Animal experiments have confirmed the rapid activation of the JNK pathway following kidney damage caused by ischemia–reperfusion [30]. Studies on HepG2 cells have shown that oxidative stress activates JNK, leading to cell apoptosis [41]. Additionally, research on hPDL cells has revealed an interaction between JNK and Cx43. Consequently, we posit that oxidative stress after kidney damage decreases Trx-1 levels, concurrently increasing JNK and Cx43 levels. Renal tubular epithelial cells undergo apoptosis as a result of the interaction of these variables, which exacerbates the development of renal fibrosis. Chrysophanol increases Trx-1 activity and reduces the expression of JNK and Cx43, thus protecting renal function.

5. Conclusion

This study revealed the significant antifibrotic effects of chrysophanol on kidney fibrosis. Furthermore, it elucidated the mechanism underlying the effects of chrysophanol, highlighting its role in activating Trx-1 to inhibit the JNK/Cx43 signaling pathway both in vivo and in vitro. These results provide empirical support for the potential use of chrysophanol as to treat renal fibrosis.

Supplementary Material

Supplementary.docx

Authors’ contributions

Mingjia Gu and Wei Kong conceived and designed research. Neng Bao, Qiyu Yue, Fang Cao and Xuejing Gu conducted experiments. Jin Wang, Xuejing Gu and Kejian Wen analyzed data. Neng Bao, Jin Wang and Mingjia Gu wrote the manuscript. All authors read and approved the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Disclosure statement

There are no conflicts to declare.
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