Puerarin alleviates the high glucose-induced oxidative stress via the RAGE/PKC/NOX4 axis in renal mesangial cells

Abstract

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes, of which progression is related to high glucose (HG)-induced oxidative stress in renal mesangial cells. Our study aims to explore the antioxidant activity and the underlying mechanism of Puerarin (Pu) in renal mesangial cells exposed to HG. After the cells finished different treatments, DCFH-DA was used to detect the generation of ROS while the expression of AGE, MDA, SOD, and GSH-PX was measured by the ELISA and corresponding kits. The cell morphology was captured by optical microscopy. The mRNA expressions of RAGE, PKCα, PKCβ, PKCγ, and NOX4 were calculated by RT-PCR assays, while the protein expressions of RAGE, NOX4, and PKCβ were quantified via western blotting. Compared with the normal glucose (NG) group, the ROS level, SOD activity, and GSH-PX expression were markedly reduced in the HG group while the MDA expression was increased in the HG group. Then, Pu treatment was proved to significantly prevent the HG-induced up-regulation of ROS level, MDA expression, and down-regulation of SOD activity and GSH-PX expression. Besides, Pu treatment can notably inhibit the AGE expression and reverse the increased RAGE, PKCβ, and NOX4 expressions by HG environment at both RNA and protein levels. Moreover, the antioxidant effect of Pu against access glucose could not be observed in PKCβ knockdown cells. Pu can alleviate the HG-induced oxidative stress via the RAGE/PKC/NOX4 axis in renal mesangial cells, which innovatively suggests the therapeutic potential of Pu for DN treatment.

INTRODUCTION

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes and accounts for approximately 50% of cases with end-stage renal disease (ESRD) (Li et al., 2022). It was reported that nearly 15 ~ 40% of patients suffering from diabetes mellitus may develop DN during their lifespan (Tayek, 2008). The longstanding hyperglycemic injury will usually lead to a relentlessly progressive decline in renal function, such as hyperfiltration, albuminuria, interstitial fibrosis, and even ultimately renal failure, thus making a great burden for successful therapy as well as the quality of the patient’s life (Sagoo and Gnudi, 2020; Oshima et al., 2021; Calle and Hotter, 2020). Currently, considering the principal factor of hyperglycemia acting in the pathological process of DN, intensive blood glucose control remains the general approach for DN treatment. However, many therapeutic strategies, such as oral hypoglycemic agents or angiotensin receptor blockades, fail to prevent the progression to ESRD in many cases with DN. Therefore, the development of novel agents is still urgent for DN treatment.

A deep understanding of the pathological mechanism will undoubtedly contribute to discovering novel therapeutic targets for DN treatment. However, to date, the thorough mechanism of DN remains quite complex and partly unknown. Nearly all types of renal cells are affected by choric hyperglycemia, no matter resident or nonresident renal cells. Renal mesangial cells are one of the resident renal cells, that is especially sensitive to glucose concentration by the presence of GLUT1, a surface receptor for extracellular glucose. Besides, several events, such as oxidative stress and inflammation, induced in the mesangial cells will also aggravate DN, such as glomerulosclerosis. Therefore, protecting mesangial cells from high glucose (HG)-induced damage will be a promising approach for DN treatment.

Advanced glycation end products (AGE) are the final substances by a nonenzymatic reaction, named Maillard reaction, between reducing sugars and amino acids. Several studies have revealed the accumulation of AGE as a dangerous factor in diabetes (Pathomthongtaweechai and Chutipongtanate, 2020; Chen et al., 2022). When the level of AGE is abnormally evaluated by the hyperglycemic environment, the receptor for AGE (RAGE) is activated and promotes a cascade of inflammation and oxidative damage in renal cells. Preimmunized AGE-modified albumin, a kind of anti-AGE therapy, has been proven to be effective against proteinuria, glomerular hypertrophy, and mesangial expansion in DN rats (Mashitah et al., 2015). Shu et al. also found that catalpol ameliorates renal dysfunction in DN by blocking the RAGE pathway (Shu et al., 2021). Consequently, it is reasonable to assume the therapeutic potential for DN treatment by blocking the HG-induced overexpression of AGE/RAGE systems.

The activation of protein kinase C (PKC) is a centerpiece in the pathogenesis of DN (Kanwar et al., 2011). PKC activation always occurs under hyperglycemic ambient and following AGE/RAGE interaction. Among a crowd of downstream events of AGE/RAGE systems, such as transforming growth factor β, the cross-talk between PKC and reactive oxygen species (ROS) is considered to accentuate the hyperglycemia-induced injury. Many experimental models of diabetes mellitus represent increased renal ROS generation, triggered greatly by hyperglycemia (Alshehri, 2023; Li et al., 2021). Additive ROS could exasperate the DN process via various signaling pathways associated with inflammation, apoptosis, and fibrosis (Wu et al., 2021; Huang et al., 2020). For example, Tong et al. have revealed the renoprotective effect of ROS-scavenging multifunctional nanoparticles in streptozotocin-induced renal injury rat model (Tong et al., 2020). Of multifarious ROS-generating enzymatic systems, such as xanthine oxidase, uncouples NOS, and mitochondrial oxidases, activation of NADPH oxidase (NOX) is particularly important in diabetes. Hitherto, there are seven proteins, including NOX1-7, that have been identified in this prototype oxidase family. Wherein, NOX4 is the most abundantly expressed in the kidney, hence originally termed renal oxidase (“Renox”). Wang et al. found that Smad3 promotes acute kidney injury in diabetic mice via NOX4-dependent ROS production (Wang et al., 2020a). However, more details underlying the relations between NOX4 and AGE/RAGE systems in DN remain for further exploration.

Puerarin (Pu) is one of the major isoflavone glycosides extracted from the root of Pueraria lobata. According to previous research, Pu has been extensively studied for its multiple pharmacological activities, such as cardioprotection, anti-inflammation, anti-oxidant, and neuroprotection (Yen et al., 2023; Zhang et al., 2019; Ou et al., 2021). Recently, the therapeutic effect of Pu against diabetes mellitus as well as relative microvascular complications has been reported. For example, Pu has been proven effective in treating STZ-induced diabetic rats via oxidative stress (She et al., 2014). Li et al. also found that Pu attenuates DN by promoting autophagy in podocytes (Li et al., 2020). Previous studies reveal the prospect of Pu as a therapeutic drug for DN treatment, but more details of the AGE/RAGE pathways on the Pu treatment remain largely unknown. Thus, in this study, we aim to determine the therapeutic effect of Pu against DN and further explore the underlying mechanism.

MATERIALS AND METHODS

Reagents

Pu preparation (purity ˃ 99%) was purchased from NANJING CHIA TAI TIANQING (06060521).

Cell culture and drug treatment

Mouse mesangial cell line, SV40-MES-13, was obtained from Procell Life Science & Technology Co., Ltd. (Pricella, Wuhan, China). All cells were cultured in Dulbecco’s modified eagle medium supplemented with 1% penicillin/streptomycin at 37°C in a 5% CO₂ humidified environment. All SV40-MES-13 cells were preliminarily divided into six groups: (1) Normal glucose (NG) group: cells treated with 5.5 mM glucose; (2) Mannitol (MN) group: cells treated with 5.5 mM glucose and 24.5 mM mannitol; (3) HG group: cells treated with 30 mM glucose; (4-6) Pu administration group: cells treated with 30 mM glucose and 2.5/5/10 μM Pu accordingly (HG+PU 2.5/5/10 μM).

Cell viability assay

The SV40-MES-13 cells taken in the logarithmic phase were trypsinized and counted. Then all cells were seeded into 96-well plates (5*10³ cells per well) in a medium with 10% FBS. To determine the safety of Pu in glomerular membrane epithelial cells, the concentration gradients (0/1/2.5/5/10/20 μM Pu) were added into the corresponding well for 24 hr. After different treatments, the medium was replaced with 100 μL fresh one containing 10 μL Cell Counting Kit-8 (CCK-8) agent. After incubation for 2 hr in a humidified incubator at 37°C with 5% CO₂, the absorbance was measured via a microplate reader at 450 nm to quantify the cell viability. The appropriate concentration gradients of Pu for the subsequent research were 2.5, 5, and 10 μM.

Detection of intracellular ROS level

DCFH-DA was applied to identify the ROS level. SV40-MES-13 cells were cultured with different conditions for 48 hr. After being loaded with 10 μmol/L DCFH-DA for 30 min at 37°C, DCF fluorescence was quantified at 488 nm excitation and 520 nm emissions via a microplate reader. Each experiment was performed in triplicate.

Evaluation of AGEs, MDA, SOD, GSH-PX levels

AGE protein was measured via a commercial ELISA kit according to manufacturer’s instructions (Cell Biolabs Inc., San Diego, CA, USA). For the detection related to oxidative stress, all cells (1 × 10⁵ cells/well) were pretreated with Pu (2.5/5/10 μM) for 1 hr and then stimulated with NG/MN/HG for 24 hr. The superoxide dismutase (SOD) value (U/mg) was evaluated via the xanthine oxidase activity assay kit; the malondialdehyde (MDA) level (nmol/mg) was measured by using the thiobarbituric acid method (MDA colorimetric assay kit); and the glutathione peroxidase (GSH-PX) value (U/mg) was quantified by a colorimetric assay kit (Elabscience Biotechnology Co., China).

RNA interference

In order to knock down PKCβ expression, SV40-MES-13 cells were transfected with PKCβ-specific siRNAs (Qiagen, Valencia, CA) by using Lipofectamine RNAi MAX (Invitrogen). The Stars negative control (NC) siRNAs (Qiagen, Valencia, CA) were applied as controls. Then the expression of PKCβ in transfected SV40-MES-13 cells was confirmed by RT-PCR and Western blotting assays after 48 hr transfection.

RT-PCR

The total RNA of SV40-MES-13 cells was extracted via Trizol reagent to measure the effects of Pu on the mRNA expression of relative genes (RAGE, NOX4, PKCα, PKCβ, and PKCγ) under HG condition. GAPDH was used as the internal control. Reverse transcription was performed by the Go Script™ Reverse Transcription kit (Promega Corporation, WI, USA). The primer sequences of RAGE, NOX4, PKCα, PKCβ, PKCγ, siRNA#1, siRNA#2, and GAPDH were listed in Supplementary Table S2. The gene expression level was analyzed via the SYBR Green kit (Promega Corporation, WI, USA). Quantitation analysis was conducted based on the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

Western blot (WB)

Briefly speaking, all samples were separated by SDS-PAGE (10% gel) and then transferred to the PVDF membrane. All membranes were blocked with 10% skim milk for 1.5 hr and incubated with corresponding primary antibodies overnight. The membranes were incubated with secondary antibodies for 1 hr the next day. Finally, the ECL Kit was utilized to detect the protein bands. The relative antibodies used in the present research were purchased by the following companies: RAGE (BosterBio, USA, M03438), NOX4 (Gene Tex, USA, GTX121929), PKC-β (Biorbyt, UK, orb650430), SOD (Abcam, UK, ab80946), GPX4 (Abcam, UK, ab125066), and GAPDH (Abcam, UK, ab8245). More details about antibodies used in this study are listed in Supplementary Table S1.

Statistical analysis

GraphPad Prism (8.0.0 version; GraphPad Software, CA, USA) was applied in this study. The data were exhibited as mean ± SEM and analyzed using a one-way analysis of variance followed by Tukey’s post hoc test. P < 0.05 represents the statistically significant difference.

RESULTS

HG induces oxidative stress in renal mesangial cells

Since the osmotic pressure is a non-ignorable variable affecting renal mesangial cells under high-glucose conditions, we first aim to figure out the leading role of HG itself, not the attendant hypertonic environment, in promoting the oxidative stress in SV40-MES-13 cells. Thus, the MN group was designed to be isotonic as the HG group and applied to remove interference (Fig. 1A). As shown in Fig. 1B, compared with the NG group, the ROS level was notably elevated in the HG group, while the difference between the NG and MN groups was not statistically significant. MDA, a product of intracellular lipid peroxidation, was also markedly increased by HG condition (Fig. 1C). Besides, some important antioxidant enzymes, such as SOD and GSH-PX, were decreased by the HG treatment (Fig. 1D and E). Taking together, we preliminarily clarified that HG, not hyperosmotic surroundings, make a difference in stimulating oxidative stress in renal mesangial cells.

Fig. 1

HG induces oxidative stress in renal mesangial cells. The information on group assignment is shown (A). The intracellular ROS level in each group was detected by the DCFH-DA method (B), and each measuring scale equals 100 μm. The expressions of MDA (C), SOD (D), and GSH-PX (E) were measured by the corresponding kit. (*P < 0.05, **P < 0.01, and ***P < 0.001 versus the MN group.)

Pu alleviates the HG-induced oxidative stress

Pu is the active ingredient of traditional Chinese medicine with promising potential for DN treatment, whose chemical structure is shown in Fig. 2A (Li et al., 2020). However, the safety of Pu, at least under experimental parameters, should be confirmed before conducting the research. According to the results of CCK8 assays, Pu has no distinct cytotoxicity in SV40-MES-13 cells when the concentration reaches 20 μM (Fig. 2B). The optimal concentration gradient for the following experiment was 2.5, 5, and 10 μM. Besides, compared with the normal group (0 μM Pu), the cellular morphology of the Pu treatment group (2.5, 5, and 10 μM Pu) was similar with a star-shaped morphology and multiple protrusions (Fig. 2C). No obvious nuclear fragmentation or decreased cell quantity can be seen even by the maximum drug concentration (10 μM Pu).

Fig. 2

Pu alleviates the HG-induced oxidative stress. The chemical structure of Pu is displayed (A). The cell viability after treatment of gradient concentration was quantified by CCK-8 assays (B). The cell morphology was captured by optical microscopy, and each measuring scale equals 100 μm (C). Besides, the effect of Pu treatment on the ROS level was quantified by the DCFH-DA detection (D), and each measuring scale equals 100 μm. The expressions of MDA (E), SOD (F), and GSH-PX (G) were measured by the corresponding kit. (No significance (NS) was considered when P > 0.05; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus the HG+DMSO group; ***P < 0.001 versus the NG+DMSO group.)

Considering the essential role of oxidative stress in DN, we wonder if Pu could exert a therapeutic effect by suppressing HG-stimulated oxidative stress. As shown in Fig. 2D, the ROS level was markedly down-regulated by Pu treatment in a dose-dependent manner. HG-induced up-regulation of MDA was notably reversed by Pu treatment (Fig. 2E), while the activity of SOD and GSH-PX were also inhibited by Pu administration (Fig. 2F and G). These outcomes proved that Pu could alleviate the HG-induced oxidative stress in renal mesangial cells.

Pu inhibits the HG-induced up-regulation of RAGE/PKC/NOX4 axis

After the antioxidant effect of Pu under HG conditions was determined in Fig. 2, we further investigated the underlying mechanisms. It is reported that AGE/RAGE systems can be abnormally activated in DN and thus stimulate the sequence of downstream events, including oxidative stress (Cho et al., 2023). Similarly, compared with the NG group, the expressions of AGEs and RAGE were notably increased in the HG, which was reversed by Pu treatment (Fig. 3A and B). Activation of PKC is one of the key elements that affect oxidative stress following the AGE/RAGE systems (Qin et al., 2019). The classical PKC subtypes include PKCα, PKCβ (I and II), and PKCγ. According to the results of PCR assays, it is PKCβ that significantly increased by access glucose and was down-regulated after Pu treatment, not PKCα or PKCγ (Fig. 3C-E). Besides, the mRNA expression of NOX4 was also suppressed by Pu treatment under HG conditions (Fig. 3F). The above-mentioned outcomes about the RAGE, NOX4, and PKCβ expressions were reconfirmed by western blotting (Fig. 3G and H). To sum up, the RAGE/PKC/NOX4 axis may be the molecular mechanism by which Pu exerts an antioxidant effect under HG conditions.

Fig. 3

Puerarin inhibits the HG-induced up-regulation of the RAGE/PKC/NOX4 axis. The influence of Pu treatment on the AGE expression was measured by ELISA kits (A). The mRNA expressions of RAGE (B), PKCα (C), PKCβ (D), PKCγ (E), and NOX4 (F) were determined by RT-PCR assays. The protein expressions of RAGE, NOX4 (G), and PKCβ (H) were quantified by western blotting. (*P < 0.01, ***P < 0.001 versus the NG+DMSO group; ^#P < 0.05, ^##P <0.01, and ^###P < 0.001 versus the HG+DMSO group.)

Knocking down PKCβ reverses the suppression effect of Pu on HG-induced oxidative stress

Finally, the knockdown of PKCβ was used to further validate the suppression effect of Pu on HG-induced oxidative stress. Knocking efficiency of PKCβ was determined at both RNA and protein levels (Fig. 4A and B), and PKC-siRNA#2 was selected for the following experiments based on the results. As expected, Pu could dramatically decrease the ROS level under HG conditions, but no measurable difference could be observed between DMSO and Pu groups in PKCβ knockdown cells, indicating that Pu exerts antioxidant effects mainly through the PKCβ pathways (Fig. 4C). The same outcomes also occur in the expression of MDA, SOD, and GSH-PX (Fig. 4D-G).

Fig. 4

Knocking down PKCβ reverses the suppression effect of Pu on HG-induced oxidative stress. The knockdown efficiency of PKCβ and the downstream event was ensured at both RNA (A) and protein (B) levels. The intracellular ROS level in each group was detected by the DCFH-DA method (C), and each measuring scale equals 100 μm. The expressions of MDA (D), SOD (E), and GSH-PX (F) were measured by the corresponding kit. The effects of PKCβ on the expression of SOD and GPX4 proteins are explored via western blotting assays (G). (No significance (NS) was considered when P > 0.05; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the groups indicated by horizontal lines.)

Collectively, our findings revealed that under HG conditions, the AGE/RAGE was upregulated, which further activated the PKCβ. Increased PKCβ induced excessive ROS production and redox imbalance in renal mesangial cells, such as increased MDA and decreased GPX4 and SOD. Pu treatment successfully alleviates the HG mediated-oxidative stress by suppressing the RAGE/PKC/NOX4 axis in vitro, which might be a promising therapeutic strategy for DN (Fig. 5).

Fig. 5

The graphical summary of the research.

DISCUSSION

Pu, an active compound of radix pueraria, is a major compound used in Chinese herbal medicines for DN treatment. According to the research of Li et al., Pu exerts renoprotective effects in STZ-induced DN mice by activating sirtuin 1 and anti-oxidative stress (Li et al., 2017). Similarly, in the present study, Pu treatment also successfully reduced the HG-induced increased ROS level and MDA expression. These findings reveal the therapeutic effect of Pu as an antioxidant agent for DN treatment, which urges further exploration of the underlying mechanisms.

DN is a well-known microvascular complication of diabetes mellitus, a metabolic malady in which chronic hyperglycemia causes dysfunction in multiple renal cells, such as glomerular podocytes as well as endothelial and mesangial cells (Kanwar et al., 2011). AGE is a product synthesized by nonenzymatic condensation of sugar and a free amino group. Although AGE is produced under normal physiological conditions, chronic hyperglycemia-induced abnormally increased AGE will lead to a series of hazardous events by interaction with RAGE. Lycopene was proved to be effective in glycation-induced DN by suppressing oxidative stress via the AGE/RAGE axis (Tabrez et al., 2015). In our study, Pu treatment also reverses the HG-induced activation of AGE/RAGE systems in a dose-dependent manner, thus suggesting that the AGE/RAGE axis may be the approach by which Pu exerts antioxidation activity.

Extracellular matrix (ECM) accumulation is a typical clinicopathological manifestation of DN, whose deterioration may lead to glomerulosclerosis, fibrosis, and even renal failure (Flyvbjerg, 2017; Yang and Liu, 2022). It was reported that the ability of AGE to cross-link with ECM exacerbates glomerulosclerosis and further promotes DN progression (Wang et al., 2020b). Mayura M Apte et al. found that syzygium cumini skeels could alleviate ECM accumulation by blocking the RAGE pathway (Apte et al., 2024). Many ECM structural proteins, such as fibronectin and laminin, are involved in the cross-linking of glucose to form extracellular AGE. These proteins may desensitize the enzymatic hydrolysis via matrix metalloproteinases, thus allowing them to accumulate in the extracellular space (Ringström et al., 2023). Interestingly, Hou et al. also found that Pu can notably alleviate the excessive ECM accumulation in DN rats (Hou et al., 2024). Considering the inhibition of AGE/RAGE by Pu treatment in this study, it is reasonable to assume that the AGE/RAGE system is also important in the renoprotective effect of Pu in vivo. However, the precise role of AGE/RAGE interaction in Pu treatment remains for further investigation.

In addition to aggravating ECM biology, extracellular AGE can stimulate multiple signaling kinases by interaction with its receptor, RAGE. Among numerous signaling kinases, PKC seems a centerpiece in the pathogenesis of DN (Ghaiad et al., 2023). PKC is a group of serine/threonine protein kinases. The AGE: RAGE interaction raises the cytosolic Ca²⁺ and DAG via membrane-bound phospholipase C, thus activating PKC cellular events. To date, there are 15 isozymes identified in the PKC family (Kawano et al., 2021). Wherein, PKC-α, -β, -γ, -δ, and -ε, are expressed in the nephridium and thus activated in diabetic patients (Pan et al., 2022). Vivian Soetikno et al. revealed that curcumin attenuates DN in diabetic rats by inhibiting PKC-α and PKC-β (Soetikno et al., 2011). Jiang et al. also found that PKC-βII knockdown alleviated HK-2 cell apoptosis and autophagy induced by AGE treatment (Jiang et al., 2020). In light of the present study, Pu treatment significantly reduced the PKC-β under HG conditions, not PKC-α or -γ. Moreover, knockdown PKC-β reverses the suppression effect of Pu on HG-induced oxidative stress, suggesting that PKC-β may be the main PKC isoform affected by Pu treatment. However, more information on the PKC isoform and the precise PKC-β subtypes (I or II) in the Pu treatment should be discussed in future research. Transcriptional regulation mainly contains two interconnected sections: the first involves transcription factors and apparatus, and the second refers to chromatin and specific regulators (Lee and Young, 2013). Wherein, the promoter DNA methylation has been reported to regulate the transcriptional regulation of PKCβ. Gao et al. found that the DNA methylation status of CpG sites within the PKCβ gene promoter notably reduced the PKCβ transcription activity in pre-eclampsia, while the application of 5-Aza, an inhibitor of DNA methylation, increased the transcription of PKCβ (Gao et al., 2019). Considering the Pu mediated-downregulation of PKCβ transcription, we wonder if DNA methylation is involved in the regulation of PKCβ by Pu treatment. However, the precise role of DNA methylation and the specific methylation sites by Pu treatment remains further exploration.

PKC activation can influence a number of downstream events, such as decreased endothelial nitric oxide synthase, upregulation of vascular endothelial growth factor, and oxidative stress (Sekar et al., 2020; Tanase et al., 2022). Oxidative stress is a complex event and can exacerbate DN progression via various pathways, such as podocyte apoptosis and radical damage. For example, Ma et al. revealed that Baicalin exerts a renoprotective effect partly by increasing GSH-PX, SOD, and catalase, and reducing MDA levels in DN rats (Ma et al., 2021). In this study, Pu treatment also markedly suppressed the HG-induced up-regulation of ROS and MDA levels. Enzymes that can scavenge ROS directly or indirectly, such as SOD and GSH-PX, maybe the approach that is conducive to attenuating HG-induced oxidative stress in Pu treatment.

Given the prominent antioxidant effects of Pu against additive glucose in renal SV40-MES-13 cells, we wonder if Pu treatment can inhibit the generation of ROS, not only elimination. There are mainly two systems generating ROS, mitochondrial oxidative phosphorylation, and NOX system, among which the latter are proved to be essential in DN progression (Gorin and Block, 2013; Urner et al., 2020). NOX4, also named “Renox”, is a member of the NOX family and is widely distributed in the kidney cells, thus playing a vital role in ROS-induced renal disease. Wang et al. found that Smad3 bound with the promoter region of NOX4 and induced ROS production and inflammation (Wang et al., 2020c). Likewise, Pu treatment also decreased NOX4 levels in HG groups, indicating that inhibition of NOX4 may also be momentous in the antioxidant effect of Pu. Besides, there is sophisticated crosstalk between PKC and NOX4. PKC has been shown to activate NOX4-dependent ROS generation in renal mesangial cells. This event may be supported by PKC-induced phosphorylation of NOX4 subunits, and up-regulation of inducible NOX4 gene expressions (Cosentino-Gomes et al., 2012). On the other hand, NOX4-induced ROS generation also regulates PKC activity in various cells. For example, the oxidants can demolish the zinc finger conformation of PKC via relieved autoinhibition (Lin et al., 1998), resulting in a PKC form that is catalytically active without Ca²⁺ or phospholipids. Donghee Kim et al. found that cudrania tricuspidata root extract can decrease oxidative stress via the PKC/NOX4 pathway in renal cells against AGE stimulation (Kim et al., 2021). In the present study, NOX4 was also significantly down-regulated by PKCβ knockdown, indicating that NOX4 may be the essential downstream protein of the RAGE/PKC pathway by which Pu exerts antioxidant activity against DN.

In summary, our research revealed the treatment potential of Pu as an antioxidant agent for DN treatment and further identified the RAGE/PKC/NOX4 as the underlying mechanism. However, in the present study, the therapeutic effects of Pu treatment should also be verified in the appropriate animal models. More details for the underlying mechanism of Pu treatment against DN remain for further exploration.

Our study identified the antioxidant activity of Pu against HG-induced oxidative stress. Moreover, the molecular mechanism underlying the antioxidant effect of Pu may be via inhibiting the RAGE/PKC/NOX4 axis. In conclusion, our study suggested the therapeutic potential of Pu as an antioxidant drug for DN treatment.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES

•  Alshehri,  A.S. (2023): Kaempferol attenuates diabetic nephropathy in streptozotocin-induced diabetic rats by a hypoglycaemic effect and concomitant activation of the Nrf-2/Ho-1/antioxidants axis. Arch. Physiol. Biochem., 129, 984-997.
•  Apte,  M.M.,  Khattar,  E. and  Tupe,  R.S. (2024): Mechanistic role of Syzygium cumini (L.) Skeels in glycation induced diabetic nephropathy via RAGE-NF-κB pathway and extracellular proteins modifications: A molecular approach. J. Ethnopharmacol., 322, 117573.
•  Calle,  P. and  Hotter,  G. (2020): Macrophage Phenotype and Fibrosis in Diabetic Nephropathy. Int. J. Mol. Sci., 21, 2806.
• Chen, J., Peng, H., Chen, C., Wang, Y., Sang, T., Cai, Z., Zhao, Q., Chen, S., Lin, X., Eling, T. and Wang, X. (2022): NAG-1/GDF15 inhibits diabetic nephropathy via inhibiting AGE/RAGE-mediated inflammation signaling pathways in C57BL/6 mice and HK-2 cells. Life Sci., 311 (Pt A), 121142.
•  Cho,  C.H.,  Yoo,  G.,  Kim,  M.,  Kurniawati,  U.D.,  Choi,  I.W. and  Lee,  S.H. (2023): Dieckol, Derived from the Edible Brown Algae Ecklonia cava, Attenuates Methylglyoxal-Associated Diabetic Nephropathy by Suppressing AGE-RAGE Interaction. Antioxidants, 12, 593.
•  Cosentino-Gomes,  D.,  Rocco-Machado,  N. and  Meyer-Fernandes,  J.R. (2012): Cell signaling through protein kinase C oxidation and activation. Int. J. Mol. Sci., 13, 10697-10721.
•  Flyvbjerg,  A. (2017): The role of the complement system in diabetic nephropathy. Nat. Rev. Nephrol., 13, 311-318.
•  Gao,  Q.,  Fan,  X.,  Xu,  T.,  Li,  H.,  He,  Y.,  Yang,  Y.,  Chen,  J.,  Ding,  H.,  Tao,  J. and  Xu,  Z. (2019): Promoter methylation changes and vascular dysfunction in pre-eclamptic umbilical vein. Clin. Epigenetics, 11, 84.
•  Ghaiad,  H.R.,  Ali,  S.O.,  Al-Mokaddem,  A.K. and  Abdelmonem,  M. (2023): Regulation of PKC/TLR-4/NF-kB signaling by sulbutiamine improves diabetic nephropathy in rats. Chem. Biol. Interact., 381, 110544.
•  Gorin,  Y. and  Block,  K. (2013): Nox4 and diabetic nephropathy: with a friend like this, who needs enemies? Free Radic. Biol. Med., 61, 130-142.
•  Hou,  B.,  Ma,  P.,  Yang,  X.,  Zhao,  X.,  Zhang,  L.,  Zhao,  Y.,  He,  P.,  Zhang,  L.,  Du,  G. and  Qiang,  G. (2024): In silico prediction and experimental validation to reveal the protective mechanism of Puerarin against excessive extracellular matrix accumulation through inhibiting ferroptosis in diabetic nephropathy. J. Ethnopharmacol., 319, 117281.
•  Huang,  S.,  Tan,  M.,  Guo,  F.,  Dong,  L.,  Liu,  Z.,  Yuan,  R.,  Dongzhi,  Z.,  Lee,  D.S.,  Wang,  Y. and  Li,  B. (2020): Nepeta angustifolia C. Y. Wu improves renal injury in HFD/STZ-induced diabetic nephropathy and inhibits oxidative stress-induced apoptosis of mesangial cells. J. Ethnopharmacol., 255, 112771.
•  Jiang,  W.,  Zhao,  W.,  Ye,  F.,  Huang,  S.,  Wu,  Y.,  Chen,  H.,  Zhou,  R. and  Fu,  G. (2020): Inhibiting PKCβ2 protects HK-2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy. Ann. Transl. Med., 8, 293.
•  Kanwar,  Y.S.,  Sun,  L.,  Xie,  P.,  Liu,  F.Y. and  Chen,  S. (2011): A glimpse of various pathogenetic mechanisms of diabetic nephropathy. Annu. Rev. Pathol., 6, 395-423.
•  Kawano,  T.,  Inokuchi,  J.,  Eto,  M.,  Murata,  M. and  Kang,  J.H. (2021): Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials. Pharmaceutics, 13, 1748.
•  Kim,  D.,  Cheon,  J.,  Yoon,  H. and  Jun,  H.S. (2021): Cudrania tricuspidata Root Extract Prevents Methylglyoxal-Induced Inflammation and Oxidative Stress via Regulation of the PKC-NOX4 Pathway in Human Kidney Cells. Oxid. Med. Cell. Longev., 2021, 5511881.
•  Lee,  T.I. and  Young,  R.A. (2013): Transcriptional regulation and its misregulation in disease. Cell, 152, 1237-1251.
•  Li,  X.,  Cai,  W.,  Lee,  K.,  Liu,  B.,  Deng,  Y.,  Chen,  Y.,  Zhang,  X.,  He,  J.C. and  Zhong,  Y. (2017): Puerarin attenuates diabetic kidney injury through the suppression of NOX4 expression in podocytes. Sci. Rep., 7, 14603.
•  Li,  X.,  Lu,  L.,  Hou,  W.,  Huang,  T.,  Chen,  X.,  Qi,  J.,  Zhao,  Y. and  Zhu,  M. (2022): Epigenetics in the pathogenesis of diabetic nephropathy. Acta Biochim. Biophys. Sin. (Shanghai), 54, 163-172.
•  Li,  X.,  Zhu,  Q.,  Zheng,  R.,  Yan,  J.,  Wei,  M.,  Fan,  Y.,  Deng,  Y. and  Zhong,  Y. (2020): Puerarin Attenuates Diabetic Nephropathy by Promoting Autophagy in Podocytes. Front. Physiol., 11, 73.
•  Li,  Y.,  Hou,  J.G.,  Liu,  Z.,  Gong,  X.J.,  Hu,  J.N.,  Wang,  Y.P.,  Liu,  W.C.,  Lin,  X.H.,  Wang,  Z. and  Li,  W. (2021): Alleviative effects of 20(R)-Rg3 on HFD/STZ-induced diabetic nephropathy via MAPK/NF-κB signaling pathways in C57BL/6 mice. J. Ethnopharmacol., 267, 113500.
•  Lin,  Y.L.,  Shivji,  M.K.,  Chen,  C.,  Kolodner,  R.,  Wood,  R.D. and  Dutta,  A. (1998): The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair. J. Biol. Chem., 273, 1453-1461.
•  Livak,  K.J. and  Schmittgen,  T.D. (2001): Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method. Methods, 25, 402-408.
•  Ma,  L.,  Wu,  F.,  Shao,  Q.,  Chen,  G.,  Xu,  L. and  Lu,  F. (2021): Baicalin Alleviates Oxidative Stress and Inflammation in Diabetic Nephropathy via Nrf2 and MAPK Signaling Pathway. Drug Des. Devel. Ther., 15, 3207-3221.
•  Mashitah,  M.W.,  Azizah,  N.,  Samsu,  N.,  Indra,  M.R.,  Bilal,  M.,  Yunisa,  M.V. and  Arisanti,  A.D. (2015): Immunization of AGE-modified albumin inhibits diabetic nephropathy progression in diabetic mice. Diabetes Metab. Syndr. Obes., 8, 347-355.
•  Oshima,  M.,  Shimizu,  M.,  Yamanouchi,  M.,  Toyama,  T.,  Hara,  A.,  Furuichi,  K. and  Wada,  T. (2021): Trajectories of kidney function in diabetes: a clinicopathological update. Nat. Rev. Nephrol., 17, 740-750.
•  Ou,  Q.,  Zhang,  S.,  Fu,  C.,  Yu,  L.,  Xin,  P.,  Gu,  Z.,  Cao,  Z.,  Wu,  J. and  Wang,  Y. (2021): More natural more better: triple natural anti-oxidant puerarin/ferulic acid/polydopamine incorporated hydrogel for wound healing. J. Nanobiotechnology, 19, 237.
•  Pan,  D.,  Xu,  L. and  Guo,  M. (2022): The role of protein kinase C in diabetic microvascular complications. Front. Endocrinol. (Lausanne), 13, 973058.
•  Pathomthongtaweechai,  N. and  Chutipongtanate,  S. (2020): AGE/RAGE signaling-mediated endoplasmic reticulum stress and future prospects in non-coding RNA therapeutics for diabetic nephropathy. Biomed. Pharmacother., 131, 110655.
•  Qin,  J.,  Peng,  Z.,  Yuan,  Q.,  Li,  Q.,  Peng,  Y.,  Wen,  R.,  Hu,  Z.,  Liu,  J.,  Xia,  X.,  Deng,  H.,  Xiong,  X.,  Hu,  J. and  Tao,  L. (2019): AKF-PD alleviates diabetic nephropathy via blocking the RAGE/AGEs/NOX and PKC/NOX Pathways. Sci. Rep., 9, 4407.
•  Ringström,  N.,  Edling,  C.,  Nalesso,  G. and  Jeevaratnam,  K. (2023): Framing Heartaches: The Cardiac ECM and the Effects of Age. Int. J. Mol. Sci., 24, 4713.
•  Sagoo,  M.K. and  Gnudi,  L. (2020): Diabetic Nephropathy: an Overview. Methods Mol. Biol., 2067, 3-7.
•  Sekar,  V.,  Mani,  S.,  Malarvizhi,  R.,  Barathidasan,  R. and  Vasanthi,  H.R. (2020): Positive interaction of mangiferin with selected oral hypoglycemic drugs: a therapeutic strategy to alleviate diabetic nephropathy in experimental rats. Mol. Biol. Rep., 47, 4465-4475.
•  She,  S.,  Liu,  W.,  Li,  T. and  Hong,  Y. (2014): Effects of puerarin in STZ-induced diabetic rats by oxidative stress and the TGF-β1/Smad2 pathway. Food Funct., 5, 944-950.
•  Shu,  A.,  Du,  Q.,  Chen,  J.,  Gao,  Y.,  Zhu,  Y.,  Lv,  G.,  Lu,  J.,  Chen,  Y. and  Xu,  H. (2021): Catalpol ameliorates endothelial dysfunction and inflammation in diabetic nephropathy via suppression of RAGE/RhoA/ROCK signaling pathway. Chem. Biol. Interact., 348, 109625.
•  Soetikno,  V.,  Watanabe,  K.,  Sari,  F.R.,  Harima,  M.,  Thandavarayan,  R.A.,  Veeraveedu,  P.T.,  Arozal,  W.,  Sukumaran,  V.,  Lakshmanan,  A.P.,  Arumugam,  S. and  Suzuki,  K. (2011): Curcumin attenuates diabetic nephropathy by inhibiting PKC-α and PKC-β1 activity in streptozotocin-induced type I diabetic rats. Mol. Nutr. Food Res., 55, 1655-1665.
•  Tabrez,  S.,  Al-Shali,  K.Z. and  Ahmad,  S. (2015): Lycopene powers the inhibition of glycation-induced diabetic nephropathy: a novel approach to halt the AGE-RAGE axis menace. Biofactors, 41, 372-381.
•  Tanase,  D.M.,  Gosav,  E.M.,  Anton,  M.I.,  Floria,  M.,  Seritean Isac,  P.N.,  Hurjui,  L.L.,  Tarniceriu,  C.C.,  Costea,  C.F.,  Ciocoiu,  M. and  Rezus,  C. (2022): Oxidative Stress and NRF2/KEAP1/ARE Pathway in Diabetic Kidney Disease (DKD): new Perspectives. Biomolecules, 12, 1227.
•  Tayek,  J.A. (2008): Multifactorial intervention and mortality in type 2 diabetes. N. Engl. J. Med., 358, 2292, author reply, 2292-2293.
•  Tong,  Y.,  Zhang,  L.,  Gong,  R.,  Shi,  J.,  Zhong,  L.,  Duan,  X. and  Zhu,  Y. (2020): A ROS-scavenging multifunctional nanoparticle for combinational therapy of diabetic nephropathy. Nanoscale, 12, 23607-23619.
•  Urner,  S.,  Ho,  F.,  Jha,  J.C.,  Ziegler,  D. and  Jandeleit-Dahm,  K. (2020): NADPH Oxidase Inhibition: Preclinical and Clinical Studies in Diabetic Complications. Antioxid. Redox Signal., 33, 415-434.
•  Wang,  J.-N.,  Yang,  Q.,  Yang,  C.,  Cai,  Y.-T.,  Xing,  T.,  Gao,  L.,  Wang,  F.,  Chen,  X.,  Liu,  X.-Q.,  He,  X.-Y.,  Wei,  B.,  Jiang,  L.,  Li,  C.,  Jin,  J.,  Wen,  J.-G.,  Ma,  T.-T.,  Chen,  H.-Y.,  Li,  J. and  Meng,  X.-M. (2020a): Smad3 promotes AKI sensitivity in diabetic mice via interaction with p53 and induction of NOX4-dependent ROS production. Redox Biol., 32, 101479.
•  Wang,  J.,  Zhang,  Q.,  Li,  S.,  Chen,  Z.,  Tan,  J.,  Yao,  J. and  Duan,  D. (2020b): Low molecular weight fucoidan alleviates diabetic nephropathy by binding fibronectin and inhibiting ECM-receptor interaction in human renal mesangial cells. Int. J. Biol. Macromol., 150, 304-314.
•  Wang,  J.N.,  Yang,  Q.,  Yang,  C.,  Cai,  Y.T.,  Xing,  T.,  Gao,  L.,  Wang,  F.,  Chen,  X.,  Liu,  X.Q.,  He,  X.Y.,  Wei,  B.,  Jiang,  L.,  Li,  C.,  Jin,  J.,  Wen,  J.G.,  Ma,  T.T.,  Chen,  H.Y.,  Li,  J. and  Meng,  X.M. (2020c): Smad3 promotes AKI sensitivity in diabetic mice via interaction with p53 and induction of NOX4-dependent ROS production. Redox Biol., 32, 101479.
•  Wu,  X.,  Li,  H.,  Wan,  Z.,  Wang,  R.,  Liu,  J.,  Liu,  Q.,  Zhao,  H.,  Wang,  Z.,  Zhang,  H.,  Guo,  H.,  Qi,  C.,  Jiao,  X. and  Li,  X. (2021): The combination of ursolic acid and empagliflozin relieves diabetic nephropathy by reducing inflammation, oxidative stress and renal fibrosis. Biomed. Pharmacother., 144, 112267.
•  Yang,  J. and  Liu,  Z. (2022): Mechanistic Pathogenesis of Endothelial Dysfunction in Diabetic Nephropathy and Retinopathy. Front. Endocrinol. (Lausanne), 13, 816400.
•  Yen,  P.T.,  Huang,  S.E.,  Hsu,  J.H.,  Kuo,  C.H.,  Chao,  Y.Y.,  Wang,  L.S. and  Yeh,  J.L. (2023): Anti-Inflammatory and Anti-oxidative Effects of Puerarin in Postmenopausal Cardioprotection: Roles of Akt and Heme Oxygenase-1. Am. J. Chin. Med., 51, 149-168.
•  Zhang,  Y.,  Yang,  X.,  Ge,  X. and  Zhang,  F. (2019): Puerarin attenuates neurological deficits via Bcl-2/Bax/cleaved caspase-3 and Sirt3/SOD2 apoptotic pathways in subarachnoid hemorrhage mice. Biomed. Pharmacother., 109, 726-733.