1. INTRODUCTION
The prevalence of diabetes has surged with lifestyle changes. According to the International Diabetes Federation (IDF), there were more than 537 million people with diabetes in 2021 [1] (https://diabetesatlas.org). Patients with diabetes often have multiple complications, such as retinopathy, nephropathy, neuropathy, and cardiovascular diseases, which reduce the quality of life and life expectancy [2]. Moreover, it has been estimated that approximately 40% of patients with diabetes have diabetic kidney disease (DKD) [3].
DKD is one of the principal causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD) [4]; however, the mechanisms underlying DKD are complex and incompletely investigated. DKD is a multifactorial disease, the pathogenesis of which includes glomerular hypertension, changes in renal hemodynamics, renal ischemia and hypoxia, and upregulation of the renin-aldosterone system [5]. Therefore, treatment of DKD also relies on multiple interventions, including renin-angiotensin system (RAS) blockade, hypoglycemic drugs, and other methods achieving metabolic control [6, 7]. In addition, large clinical studies have identified drugs that alleviate the progression of DKD. Specifically, the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical studies of Evaluation (CREDENCE) trial reported that SGLT2 inhibitors improve the prognosis of DKD and reduce the risk of kidney failure [8]. The Finerenone in Reducing Kidney Failure and Disease Progression in Diabetic Kidney Disease (FIDELIO-DKD) trial showed that finerenone lowers the risks of CKD progression and cardiovascular events compared with placebo [9]. Unfortunately, even though these interventions can delay the progression of DKD, ESRD is inevitable in some patients. Therefore, promising and novel treatments preventing DKD are still needed.
Verbascoside or acteoside (PubChem CID: 5281800) is a ubiquitous phenylethanoid glycoside that is distributed among > 200 plant species from 23 plant families in different concentrations [10]. Verbascoside has been shown to have wound-healing, neuroprotective, anti-inflammatory, antioxidant, and anti-tumor properties [10, 11]. Moreover, these properties have potential nephroprotective effects. Previous studies have also indicated that drugs containing verbascoside reduce the blood glucose level and improve renal function in an experimental model of diabetic mice [12, 13]. The versatile role of verbascoside against DKD is not well-characterized, thus the present study determined the therapeutic effect of verbascoside in DKD and the underlying mechanism.
2. MATERIAL AND METHODS
2.1 Chemicals and reagents
Verbascoside was purchased from MedChem Express (HY-N0021; Monmouth Junction, NJ, USA). Antibodies to NR4A1 (25851-1-AP) and caspase-3 (66470-2-Ig) were purchased from Proteintech (Rosemont, IL, USA). Antibodies to LKB1 (sc-32245), podocin (sc-518088), and nephrin (sc-376522) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies to AMPK (5831), pAMPK (50081), cleaved caspase-3 (AF0120), caspase-3, Beclin-1 (3495), p62 (8025S), and Atg5 (2630S) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-CXCL10 (ab9807) was purchased from Abcam (Cambridge, Cambridgeshire, UK). Anti-LC3B (AF4650), Bcl-2 (AF6139), Bcl-xL (AF6414), Bax (AF0120), and anti-MCP-1 (DF7577) were purchased from Affinity Bioscience (Melbourne, Victoria, Australia). Antibodies to β-actin (ET1701-80) and IL-6 (EM170414) were purchased from Hangzhou HuaAn Biotechnology (Hangzhou, Zhejiang, China).
2.2 Animal experiments
The animal experimental protocols were approved by the Ethics Committee for Experimental Research and Animal Care and Use Ethics Committee of Sichuan University (2020061A). The animal care and experimental procedures were performed following The Guide for the Care and Use of Laboratory Animals from the National Research Council (USA) Committee. Male C57BLKS/J db/db and db/+ mice (6 weeks old) were purchased from GemPharmatech Biotechnology Company (Nanjing, China). The mice were housed in a temperature-controlled room (23 ± 2°C) under a 12-h light/dark cycle with free access to food and water. After a 2-week of the adaptation period, the C57BLKS/J db/db mice were randomly divided into 3 groups, as follows: diabetic group (n=6); low-dose treatment group (75 mg/kg; n=6); and high-dose treatment group (150 mg/kg; n=6). The db/+ mice were divided into the control group (n=12). Verbascoside was dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% normal saline, further diluted in normal saline, then fed by gavage to db/db mice. Equal volumes of normal saline were administered once daily to the db/+ control and db/db model groups. All mice were sacrificed after 4 weeks of treatment.
2.3 Biochemical analyses
The 12-h fasting blood glucose levels in mice were collected from the tail vein every week and the urine albumin level was determined using an automatic biochemical analyzer (BS-240; Mindray, Shenzhen, China).
2.4 Cell culture and treatments
Mouse podocyte clone 5 [MPC5] (ATCC® CCL-171™; Beijing Bnbio Co., Ltd., Beijing, China) cells were propagated at 33°C and treated with 10 U/mL of interferon (IFN-γ). Next, cells were differentiated without IFN-γ at 37°C for 14 days. For further study, MPC5 cells were cultured in RPMI-1640 (SH30809.01B; HyClone, Beijing, China) with 10% fetal bovine serum [FBS] (SH30084.03; Hyclone) and 0.5% penicillin and streptomycin (SV30010; HyClone, Beijing, China) at 37°C in 5% CO2. The cells were serum-starved in medium containing 0.5% serum for 24 h, then treated with high glucose [HG] (30 mM glucose) or mannitol [MA] (24.5 mM MA + 5.5 mM glucose) and incubated with or without verbascoside for 24 h.
2.5 Cell viability assay
To determine the potential effects of verbascoside on cell viability, a Cell Counting Kit-8 assay (CCK-8, APExBIO, Houston, TX, USA) was used. In brief, MPC5 cells (5000-10,000 cells/well) were seeded into 96-well plates for 24 h and later incubated with verbascoside at various concentrations (25, 50, 75, 100, 200, 300, or 400 μM). Then, the culture media from each well was replaced with 10 μL of CCK-8 solution, and the cells were incubated in the dark at 37°C for 1 h. The absorbance of the solution in each well was detected at a wavelength of 450 nm using a microplate reader (Synergy Mx; Biotek, Winooski, VT, USA).
2.6 Renal histologic examination
Kidney tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm. After deparaffinization and rehydration, kidney sections were stained with periodic acid-Schiff (PAS), Weigert’s iron hematoxylin (Masson), or hematoxylin and eosin (HE). The sections were viewed by light microscopy at ×200 or ×400 magnification. Finally, the mesangial expansion index of renal tissue was evaluated from 10 randomly-selected fields.
2.7 Transmission electron microscopy
Kidney tissues were fixed in cold 2.5% glutaraldehyde for 2 h at 4°C, then treated with standard procedures, including dehydration, osmosis, embedding, sectioning, and staining, and finally visualized on a Hitachi microscope (H-7650; Akihabara, Tokyo, Japan) at ×8000, ×12,000, and ×20,000 magnification.
2.8 Quantitative real-time PCR analysis
Renal tissue and total cellular RNA were extracted using a total RNA isolation kit (Foregene, Chengdu, China); the concentration and purity were determined by ScanDrop 100 (Analytik Jena, Thuringia, Germany). Then, reverse transcription was performed with HiScript III SuperMix (Vazyme, Nanjing, China) and iTaqTM Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) was used for quantitative real-time PCR in a PCR system (CFX Connect; Bio-Rad). Primer sequences were listed in Supplementary Table 1. The mRNA levels were normalized to β-actin and calculated using the comparative cycle threshold (2− ΔΔCt) method.
2.9 Immunohistochemistry
After fixation in 10% phosphate-buffered formalin overnight, the fixed kidneys were dehydrated through a graded series of ethanol, embedded in paraffin, sectioned (4 μm), and mounted on glass slides. The slides were blocked with 2.5 % normal goat serum and incubated with primary antibodies at 4°C. The slides were thrice-washed in PBS, and a Vectastain ABC Kit (Vector, Burlingame, CA, USA) was used for staining following the manufacturer’s instructions. The sections were counterstained with hematoxylin. Images were captured using an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany).
2.10 Immunofluorescence staining
OCT-embedded kidney sections (4 μm) were incubated with PBS containing 5% horse serum for 1 h at room temperature to block non-specific binding sites. Then, the specimens were incubated with primary antibody (anti-nephin [1:200]) or (anti-podocin [1:200]) in a humidified chamber overnight at 4°C. After washing, the secondary antibody (1:500) was used for 1 h and the samples were washed again. Then, the samples were stained with DAPI (1:500). Finally, the samples were sealed with coverslips. Images were acquired using an AxioCamHRc digital camera (Carl Zeiss) at magnifications of ×400 with ZEN 2012 microscopy software (blue edition).
2.11 Western blot analysis
Western blot analysis was performed as described earlier [14]. Densitometry analysis was performed using Image J 6.0 software (National Institutes of Health, Bethesda, MD, USA).
2.12 RNA-Seq transcriptomic assay
Total RNA from the kidneys of each treatment group (n = 3) was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Library constriction and sequencing were performed by LC-BIO Biotech Ltd. (Hangzhou, China). The libraries were sequenced on an Illumina NovaSeqTM 6000 platform and 2 × 150-bp paired-end reads were generated. Further bioinformatic analysis was performed using the OmicStudio tools at https://www.omicstudio.cn/tool.
2.13 Cell siRNA transfection
Transient transfections of the MPC5-1 cells with siRNAs were performed with transfection reagent (riboFECTTM CP transfection kit, 166T; RiboBio, Guangzhou, China) according to the manufacturer’s instructions [15, 16]. The NR4A1 siRNA and negative control (NC) siRNA sequences were as follows: NR4A1 siRNA, sense 5’-GCCCUGUAUUCAAGCUCAATT-3’ and antisense 5’- UUGAGCUUGAAUACAGGGCTT-3’; and NC siRNA, sense 5’-UUCUCCGAACGUGUCACGUTT-3’ and antisense 5’-ACGUGACACGUUCGGAGAATT-3’ (GenePharma, Shanghai, China). MPC5 cells were seeded in 6-well plates and transfected with a final concentration of 100 nM siRNA using riboFECTTM CP reagent (RiboBio) in RPMI-1640 medium containing 0.5% fetal bovine serum (SH30084.03; HyClone) without penicillin-streptomycin for 24 h.
2.14 Molecular docking
The three-dimensional structure of verbascoside was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The crystal structure of NR4A1 was downloaded from the Protein Data Bank [PDB] (https://www.rcsb.org/). The AutoDockTools (version 1.5.6; http://autodock.scripps.edu/) were used to convert the downloaded verbascoside and NR4A1 to the pdbqt format. Molecular docking was performed with the AutoDock Vina (version 1.12; http://vina.scripps.edu/). The docking pose of verbascoside and NR4A1 was visualized with the PyMOL software (version 2.2; https://pymol.org/2/).
2.15 Statistical analysis
All experiments were performed in triplicate unless otherwise stated. Data are presented as the mean ± SD. Statistical differences between the two groups were performed by two-tailed Student’s t-test for parametric data or the Mann-Whitney U test for non-parametric data. Comparisons between multiple groups were analyzed with one-way ANOVA for one experimental parameter or two-way ANOVA for two experimental parameters, followed by Tukey’s multiple comparisons test using GraphPad Prism 9.0.0 (GraphPad Software, San Diego, CA, USA). A P < 0.05 was considered statistically significant.
3. RESULTS
3.1 Verbascoside improved the blood glucose level, albuminuria, and renal histopathologic injury in diabetic mice
The chemical structure of verbascoside is shown in Figure 1a . To determine whether verbascoside is protective against DKD, we treated 8-week-old db/db mice with verbascoside at a dose of 75 or 150 mg/kg for 4 weeks. As presented in Figure 1b , verbascoside at a dose of 150 mg/kg/d (db-150VB) significantly lowered the fasting blood glucose (FBG) level and albumin-to-creatinine ratio (ACR) compared to db/db mice (P < 0.05). In addition, the 150 mg/kg group (db-150VB) alleviated kidney pathologic damage, which was characterized by mesangial matrix expansion and glomerulosclerosis compared to diabetic mice ( Figure 1c and e ). In contrast, the 75 mg/kg group (db-75VB) had no apparent therapeutic effect.
Verbascoside ameliorated kidney injury in db/db mice.
(a) Chemical structure of verbascoside. (b) Fasting blood glucose (FBG) and albumin-to-creatinine ratio (ACR). Vs. db/db group (n = 6). (c) Representative pathologic staining micrographs (PAS, Masson, and HE staining, ×1000, scale bar = 10 μm). (d) Representative immunofluorescence staining micrographs of nephrin (red) and podocin (green) in the renal cortex (×400, scale bar = 20 μm). (e) Quantification of the fraction of mesangial area. (f) Measurements of glomerular basement membrane (GBM) thickness. (g) Transmission electron microscopy showed thickening of the basement membrane, fusion of podocyte foot processes (arrows), and hyperplasia of mesangial cells (stars) in db/db mice, while the pathologic lesions were alleviated after verbascoside treatment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Furthermore, as shown by the results of immunofluorescence staining and electron microscopy, verbascoside ameliorated podocyte injury ( Figure 1d and g ). Nephrin and podocin are transmembrane podocyte proteins, the expression of which indicate the degree of podocyte injury. Immunofluorescence staining showed decreased expression of nephrin and podocin in db/db mice. At the same time, verbascoside administration restored the decreased expression of nephrin and podocin ( Figure 1d ). Moreover, transmission electron microscopy analysis revealed that 150 mg/kg of verabascoside (db-150VB) alleviated foot process fusion, glomerular basement membrane thickness, and mesangial cell proliferation ( Figure 1f and g ). Taken together, these results indicated that verbascoside protects against DKD.
3.2 Verbascoside suppressed cell stress responses in kidneys of diabetic mice and high-glucose triggered MPC5 podocytes
Collective evidence indicates that dysregulated inflammation, apoptosis, and autophagy are vital mechanisms underlying DKD [17–21]. First, we used q-PCR and Western blot to detect the expression of pro-inflammatory cytokines and chemokines in kidneys. As expected, both mRNA and protein levels of IL-6, CXCL10, and MCP-1 in kidney tissues were increased in db/db mice compared with db/+ mice. At the same time, verbascoside effectively reduced the corresponding expression, especially in the 150 mg/kg/d dose group ( Figure 2a and b ). Next, Western blot results implicated apoptosis increased in db/db mice compared with db/+ mice, which showed a decreasing trend of Bcl-2 and Bcl-xL and an increasing trend of Bax and cleaved caspase-3 (P < 0.05). Verbascoside treatment increased the levels of Bcl-2 and Bcl-xL protein, and decreased the levels of cleaved caspase-3 and Bax protein, especially at the 150 mg/kg dose ( Figure 2d ). Verbascoside reduced the level of Bax mRNA, and increased the levels of Bcl-2 and Bcl-xL mRNA ( Figure 2c ). Additionally, verbascoside promoted the levels of autophagy in db/db mice. Western blot analysis demonstrated that verbascoside increased the levels of Atg5, LC3-II, and Beclin-1, which were downregulated in the kidneys of db/db mice. Furthermore, the level of p62/SQSTM1 protein in the kidney was also decreased by verbascoside treatment ( Figure 2i ).
Verbascoside ameliorated cell stress responses in vitro and in vivo.
The levels of IL-6, CXCL10, and MCP-1 mRNA in renal tissues (a) and MPC5 cells (e) were analyzed by RT-PCR. The levels of IL-6, CXCL10, and MCP-1 protein in renal tissues (b) and MPC5 cells (f) were detected by Western blot. The levels of Bcl-2, Bax, and Bcl-xL mRNA in renal tissues (c) and MPC5 cells (g) were analyzed by RT-PCR. The levels of Bcl-2, cleaved caspase-3/caspase-3, Bax, and Bcl-xL protein in renal tissues (d) and MPC5 cells (h) were detected by Western blot. Western blot showed the levels of Beclin-1, LC3-II, Atg5, and p62 protein expression of renal tissues (i) and MPC5 cells (j). All data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Then, we used the CCK-8 assay to determine the cytotoxic effect of verbascoside (0-400 μM) in murine podocytes (MPC5 cells), and showed that cell viability was inhibited at the 200 μM dose ( Supplementary Figure 1 ). Accordingly, 3 therapeutic concentrations (25, 50, and 100 μM) were selected for the follow-up experiments, and MPC5 cells were cultured in HG media (30 mM) for 24 h. HG stimulation increased the pre- and post-transcriptional IL-6, CXCL10, and MCP-1 levels compared to the MA control group. Verbascoside reversed the elevated expression of these pro-inflammatory cytokines and chemokines; the 100 μM dose (100VB) was the most significant ( Figure 2e and f ). Consistent with the in vivo results, apoptosis was raised by HG stimulation and alleviated by verbascoside. The expression of pro-apoptotic markers (cleaved caspase-3 and Bax; Figure 2g and h ), as well as the apoptotic rate by flow cytometry ( Supplementary Figure 2 ) was significantly increased by HG stimulation and further decreased by verbascoside treatment (P < 0.05). In addition, verbascoside increased the expression of anti-apoptotic markers (Bcl-2 and Bcl-xL) that were reduced by HG ( Figure 2g and h ). Furthermore, Western blot results showed verbascoside increased levels of Atg5, LC3-II, and Beclin-1 protein, and decreased levels of p62 protein compared with the HG group ( Figure 2j ).
3.3 Predicting the verbascoside target in the kidneys of diabetic mice
To reveal the mechanism underlying verbascoside treatment in db/db mice, RNA-seq transcriptomic analysis was used. Significant verbascoside-regulated genes are illustrated by the heatmap in Figure 3a . Synaptopodin, a podocyte injury marker, was downregulated in the kidneys of db/db mice and upregulated in the VB-150 group. This finding is consistent with our previous findings that verbascoside protected against podocyte injury. Genes related to inflammation (IL-1β and CXCL10), metabolism (Pparg), and the cell cycle (Ccnb1, Mcm5, Bub1b, and Espl1) were also shown to change among groups. Additionally, we found that the transcription factor, NR4A1, the expression of which was markedly upregulated in the kidneys of db/db mice compared to db/+ mice, while verbascoside reversed the upregulation.
Verbascoside ameliorated DKD by regulating NR4A1 expression.
(a) Representative heatmap of differentially-expressed genes in kidneys. (b) KEGG pathway analysis of genes regulated by verbascoside. (c) Molecular docking of verbascoside binding to NR4A1. The mRNA (d) and protein (e) levels of NR4A1 in murine renal tissues were shown. The mRNA (f) and protein (g) levels of NR4A1 in MPC5 cells were detected. (h) Representative images of immunohistochemistry staining for NR4A1 in kidneys of diabetic mice. All data are represented as the mean ± SD (n = 3).*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the AMPK and PPAR signaling pathways, fatty acid degradation, and other metabolic pathways were involved ( Figure 3b ). This finding suggested that verbascoside may protect injured kidneys by affecting metabolic pathways. Notably, AMPK pathways, which are responsible for regulating cell metabolism, have vital roles in DKD.
To further elucidate the underlying mechanism, molecular docking was performed. Verbascoside exhibited good affinity with NR4A1 protein, and verbascoside had a compact binding pattern with the NR4A1 protein active pocket by forming hydrogen bonds with the following amino acid residues: GLN240; TYR244; ARG241; GLY65; SER222; HLS63; RPO62; LEU142; ASP153; ALA145; ARG241; and TYR244 ( Figure 3c ). Therefore, we hypothesized that verbascoside has a protective role in DKD by targeting NR4A1.
Previous studies have suggested that NR4A1 regulates the phosphorylation of AMPKα [22, 23]. Given that NR4A1 has been reported to bind LKB1 and inhibit the activation of AMPKα [22, 23], we speculated that verbascoside alleviates DKD via the NR4A1/LKB1/AMPK pathway. Further, we showed that NR4A1 expression is increased in the kidneys of db/db mice compared to db/+ mice. In addition, NR4A1 expression was reduced after verbascoside administration ( Figure 3d, e, and h ). HG stimulation increased NR4A1 expression in podocytes, while verbascoside decreased NR4A1 expression ( Figure 3f and g ).
3.4 Verbascoside regulated the NR4A1/LKB1/AMPK pathway in kidneys of diabetic mice and HG triggered podocytes
To verify the hypothesis, we analyzed the expression of LKB1 and phospho-AMPKα in the kidneys of mice. We found that both LKB1 and phospho-AMPKα of db/db mice were downregulated compared with db/+ mice. Verbascoside reversed the downregulation ( Figure 4a and b ) and increased the expression of LKB1 and phospho-AMPKα in HG-stimulated MPC5 cells ( Figure 4c and d ). Next, NR4A1 knockdown by siRNA technology ( Supplementary Figure 3a and b ) promoted the expression of LKB1 and phospho-AMPKα to a similar extent as verbascoside treatment ( Figure 4e and f ).
Effects of verbascoside on NR4A1/LKB1/AMPK signaling pathway in vitro and in vivo.
The levels of LKB1 mRNA in renal tissues (a) and HG-stimulated MPC5 cells (c) were detected by RT-PCR. The levels of LKB1, pAMPKα and AMPKα protein in renal tissues (b) and HG-stimulated MPC5 cells (d) were detected by Western blot. MPC5 cells were transfected with negative control (NC) siRNA or NR4A1 siRNA for 24 h with or without HG or verbascoside treatment. Then, the level of LKB1 mRNA (e) and levels of LKB1, pAMPKα, and AMPKα protein (f) was evaluated. All data are represented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with the HG group. ## P < 0.01, #### P < 0.0001 compared with the control group.
3.5 Inhibition of NR4A1 improved cell stress in HG-triggered podocytes
As delineated in Figure 5 , NR4A1 knockdown attenuated HG-stimulated inflammation, as evidenced by reduced pro-inflammatory cytokines and chemokines (IL-6, MCP-1, and CXCL10; Figure 5a, d, e, and f ). In addition, NR4A1 knockdown reduced apoptosis stimulated by HG. Silencing NR4A1 triggered a decrease in pro-apoptotic markers (cleaved caspase-3 and Bax; Figure 5b and i ) and apoptotic rate by flow cytometry ( Supplementary Figure 4 ). Silencing NR4A1 caused an increase in anti-apoptotic markers (Bcl-2 and Bcl-xL; Figure 5b, g, and h ). In addition, NR4A1 knockdown promoted autophagy, as evidenced by decreased expression of p62 and increased autophagy-related protein (Atg5, LC3-II, and Beclin-1; Figure 5c ). All in all, the results indicated that NR4A1 knockdown protected podocyte injury against HG stimulation by inhibiting inflammation and apoptosis, and improving autophagy.
NR4A1 knockdown alleviated cell stress responses in high glucose-triggered podocytes.
MPC5 cells were transfected with negative control (NC) siRNA or NR4A1 siRNA for 24 h with or without HG or verbascoside treatment. Then, the protein (a) and mRNA (d–f) levels of IL-6, CXCL10, and MCP-1 were analyzed. (b) The levels of Bcl-2, cleaved caspase-3/caspase-3, Bax, and Bcl-xL protein were detected by Western blot. (g–i) The levels of Bcl-2, Bax, and Bcl-xL mRNA were analyzed by RT-PCR. (c) Western blot showed the levels of Beclin-1, LC3-II, Atg5, and p62 protein expression. All data are represented as the mean ± SD (n =3). ****P < 0.0001 compared with the HG group. ## P < 0.01, ### P < 0.001, #### P < 0.0001 compared with the control group.
4. DISCUSSION
In the present study we determined the nephroprotective role of verbascoside in DKD. By treating db/db mice with verbascoside for 4 consecutive weeks, proteinuria and pathologic injury were significantly relieved. Moreover, the disordered cell stresses (increased inflammatory response and apoptosis, as well as dysfunctional autophagy) were alleviated after verbascoside administration in a dose-dependent manner. Verbascoside downregulated the expression of NR4A1, and further upregulated the expression of LKB1 and phospho-AMPKα. Notably, NR4A1 knockdown in podocytes also promoted LKB1 and phospho-AMPKα ex[ressopm. These observations support the therapeutic effect of verbascoside against DKD.
Previous studies reported that verbascoside or drugs containing verbascoside reduce the blood glucose level, which consistent with the results of our study. Specifically, Cistanche tubulosa, the main components of which include verbascoside, was shown to significantly inhibit the increase in fasting blood glucose and improve insulin resistance in db/db mice and STZ-induced diabetic rats [12, 24]. In addition, verbascoside alleviate endoplasmic reticulum stress in islet β cells [25]. Verbascoside improves the anti-cytotoxicity of HG in gingival cells [26]. Verbascoside also promotes bone formation in osteocytes of STZ-induced diabetic rats [26]. Taken together, these results indicate the potential nephroprotective efficiency of verbascoside in diabetic complications.
NR4A1 was thought to be a possible target of verbascoside in DKD based on the combined results of transcriptomics and molecular docking. NR4A1, a member of the nuclear orphan receptor superfamily (NR4A), regulates various cellular activities, widely participates in cell metabolism, apoptosis, inflammation, and other biological events, and is closely related to a variety of metabolic diseases [27]. NR4A1 is an early response gene, changes in which are rapidly induced by a variety of substances, such as cytokines, glucose, fatty acids, lipopolysaccharides, and other small molecular compounds [28–31]. Hyper- or dys-function of NR4A1 affects metabolic processes in primary metabolic tissues, such as influencing glucose and lipid metabolism in the liver, skeletal muscle, pancreatic tissue, and adipose tissue [32–34]. Notably, NR4A1 is also an essential regulator for diabetes and diabetic complications. Previous studies have shown that NR4A1 expression increased in type 1 and 2 diabetic animal models [34, 35], which is consistent with our results. Overexpression of NR4A1 by adenovirus induced the expression of gluconeogenesis-related genes [34]. Then, glucose production was promoted and the blood glucose level increased [34]. NR4A1 agonists, such as octaketide cytosporone B (CSN-B), have a similar effect on elevating hepatic glucose production and the blood glucose level [36]. In addition, the cAMP axis induces hepatic NR4A1 expression in response to glucagon and fasting [34]; however, the biological function of NR4A1 is complex and poorly understood. For example, knockout of NR4A1 leads to systemic inflammation in aged mice [37], aggravates colitis in mice [38], and promotes the differentiation of macrophages into the pro-inflammatory M1 phenotype [39]. These results are contrary to our results, which showed that NR4A1 knockout mitigated podocyte inflammation. There are also results in agreement with our finding that NR4A1 upregulation promotes inflammation, such as the NF-κB pathway [31], and knockdown of NR4A1 inhibited inflammation-related molecules, such as TNF-α and IL-6 [22]. It appears that the results can be explained in specific experimental conditions, like different diseases and stimulation conditions because of the sophisticated function of NR4A1. Additional work will need to be conducted to determine the role of NR4A1.
In conclusion, our study illustrated the therapeutic effect of the phenylethanoid glycoside, verbascoside, in DKD. Verbascoside downregulated NR4A1 and further influenced the LKB1/AMPK pathway. Our results point to verbascoside as a potential therapeutic approach for DKD.
