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. 2025 Jan 6;20(1):e0316615. doi: 10.1371/journal.pone.0316615

Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathy

Liping Bao 1,*, Yuhan Gong 1, Wenji Xu 2, Jun Dao 1, Jinjin Rao 1, Haihui Yang 1,*
Editor: Jordan Robin Yaron3
PMCID: PMC11703029  PMID: 39761239

Abstract

Diabetic nephropathy (DN) is the single largest cause of end-stage renal disease (ESRD). Inflammation reaction mediated by NLRP3 inflammasome and Nrf2-related oxidative stress have been considered to play a very important role in the progress of diabetic nephropathy (DN). Effective drugs for the treatment of diabetic nephropathy still need to be explored. Chlorogenic acid (CGA) is a kind of polyphenol with a Nrf2 activation property widely existed in nature. The aims of this study were to evaluate the renoprotective effect of CGA and to elucidate the anti-inflammation mechanisms involved. In the present study, we established a diabetic rat model to investigate the renoprotective effect of CGA in vivo. The results show that the level of serum creatinine (Scr), blood urea nitrogen (BUN), and urinary protein excretion in diabetic rats were significantly decreased after CGA intervention. CGA administration can active the Nrf2 pathway and inhibit NLRP3 inflammasome activation. Notably, Nrf2 siRNA transfection nullified the inhibitory effects of CGA on NLRP3 inflammasome activation in vitro. To summarize, our present study provided evidence that chlorogenic acid can slow the progression of diabetic nephropathy progression, and the effect is associated with suppression of NLRP3 inflammasome activation via through modulation of the Nrf2 pathway, suggesting its therapeutic implications for diabetic nephropathy.

1. Introduction

Diabetic nephropathy (DN), one of the most threatening complications of patients with diabetes, has become the leading cause of end-stage renal disease (ESRD) worldwide [1]. DN is characterized by proteinuria and the progressive decline in renal function. Numerous studies have shown that inflammation reaction plays a certain part in the development of DN [2,3].

Reports found that inflammation reaction mediated by NOD-like receptor protein 3 (NLRP3) inflammasome was responsible for the DN progression. NLRP3 inflammasome is composed of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) [4]. In the diabetic state, NLRP3 inflammasome is activated, then NLRP3 recruits ASC and cleaves caspase-1. Next, cleaved caspase-1 promotes the conversion of inflammatory factors such as pro-IL-1β and pro-IL-18 into IL-1β and IL-18 and released outside the cell, which leading to pro-inflammatory responses [5]. Excessive activation of NLRP3 inflammasome may lead to uncontrolled inflammation and cell death. Hence, therapies targeting activation of NLRP3 inflammasome may play an effective role in protecting renal function and delay the progression of DN. Oxidative stress is one of the fundamental causes of chronic complications of diabetes. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) takes on a critical part in the defense system against oxidative stress. In response to cellular oxidative stress injury, Nrf2 evades repression by Kelch-like epichlorohydrin-associated protein 1 (Keap1) and accumulates within the nucleus, and then activates the transcription of phase II detoxifying anti-oxidant enzymes, including heme oxygenase-1 (HO-1) [6]. Recent studies have shown that activating the Nrf2 pathway could inhibit the activation of NLRP3 inflammasome and significantly reduced the expression of downstream factors caspase-1, IL-18, and IL-1β in acute liver injury, cerebral artery occlusion-reperfusion injury and severe lupus nephritis [7–9]. We hypothesized that Nrf2 may negatively regulated the activation of NLRP3 inflammasome in DN.

Chlorogenic acid (CGA), a phenolic compound, is widely found in Chinese herbal medicines, such as Flos lonicerae and Eucommia ulmoides [10,11]. Recent studies have showed that CGA possesses various pharmacological activities, including anti-inflammatory [12], antioxidant [13], antibacterial activities [14], and anticarcinogenic [15]. Moreover, several recent experiments indicated that CGA treatment attenuated acute liver and lung impairments by hindering NLRP3 inflammasome activation [16,17]. However, the effect of CGA on NLRP3 inflammasome and the underlying mechanisms in DN are still unclear. Our previous study has shown that pre-treatment with CGA increased the renal expression of Nrf2 and the downstream target heme oxygenase-1 (HO-1) [18]. The aim of the present study was to explore the effect of CGA on NLRP3 inflammasome in the high-fat diet (HFD)/streptozotocin (STZ)-induced diabetic rat kidney and human proximal tubule epithelial cell line HK-2 and to further explore the potential connection between the Nrf2 pathway and NLRP3 inflammasome.

2. Materials and methods

2.1. Reagents

Chlorogenic acid (CGA) was purchased from MACKLIN Company (Shanghai, China). Biochemical kits for serum creatinine (Scr), blood urea nitrogen (BUN) and urinary protein were purchased from Changchun Huili Company (Changchun, China). The anti-NLRP3(DF7438), anti- cleaved caspase-1(AF5418), anti- IL-1β(AF5103), anti- IL-18(DF6252), and anti- HO-1(AF5393) antibodies were purchased from Affinity (Melbourne, Australia). Anti- Nrf2(16396-1-AP) and anti-H3(17168-1-AP) antibodies were purchased from Wuhan Sanying Biotechnology Co., Ltd (Wuhan, China).

Fetal bovine serum (FBS) and dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) medium were obtained from HyClone (Logan, UT, USA).

2.2. Animals and drug treatments

A total of 18 male specific pathogen-free (SPF) grade Wistar rats (aged 6–8 weeks, 180–200 g of body weight) were obtained from the Disease Prevention and Control Centers of Hubei Province. All animal experiments were conducted according to a protocol approved by Animal Care and Use Committee of the Center for Disease Prevention and Control in Hubei Province (No. 202220148). After 1 week of adaptive feeding, the rats were randomly selected and divided into three groups (6 rats in each group): a normal control group (NC), type 2 diabetes mellitus model group (DM), and DM+CGA group. The DM and DM + CGA groups were fed a high-fat diet (D12492, Hua Fu Kang Biotechnology Co., Ltd. Beijing, China) for 4 weeks to cause insulin resistance. And then all of the insulin resistance rats were injected with low-dose STZ (35 mg/kg, St. Louis, MO, USA) dissolved in citrate buffer solution once. The DM and DM + CGA groups were continued to be fed a high-fat diet until the end of the experiment. The successful establishment of the diabetes model was confirmed on the 3rd day after STZ injection by measuring the blood glucose level greater than 16.7 mmol/L. The rats in DM + CGA group were intraperitoneally injected with CGA for 10 weeks at a dose of 10 mg/kg/d. Rats in the NC and DM groups were intraperitoneally injected with equivoluminal distilled water.

2.3. Blood and urine chemistries analysis

After 10 weeks, animals were sacrificed with intraperitoneal injection of 1% pentobarbital solution, and if animals show signs of pain, increase the dosage appropriately. Serum creatinine, BUN and urinary protein excretion were measured using biochemical kits according to the manufacturer’s instructions.

2.4. Histopathology

The kidney tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The embedded kidney tissues were cut into 3-μm sections. The tissue sections were subsequently stained with hematoxylin and eosin (HE) and periodic acid- Schiff (PAS).

2.5. Immunohistochemistry

After deparaffinization and hydration, the slices were subjected to antigen heat repair using an electric pottery stove for 15 minutes. To block endogenous peroxidase, the sections were incubated with 3% H2O2 for 15 minutes. Then the sections were blocked with goat serum for 30 minutes. Subsequently, the sections were incubated with primary antibodies (1:100) overnight at 4°C. Washed with phosphate-buffered saline (PBS) for three times, the sections were incubated with horseradish peroxidase (HRP)-labelled secondary antibodies for 30 minutes, then stained with 3,3’-diaminobenzidine (DAB) and re-stained with haematoxylin. The sections were observed by a microscope and analyzed Image J software. The protein expression levels were reflected with the average optical density (AOD) values.

2.6. Cell culture and stimulation

Human proximal tubule epithelial (HK-2) cells were obtained from Nanjing KeyGen Company(Nanjing, China).The cells were cultured in low concentration of glucose (5.6 mmol/l) DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin at 37°C in an incubator with 5% CO2. Cultured cells were divided into five groups: the normal control group (NC, 5.6 mmol/L glucose); the high concentration of glucose group(HG,30 mmol/L glucose); the different concentrations of CGA(20, 50 and 100 μM) +30 mmol/L glucose group(CGA + HG). After 48 h of CGA and HG treatment, HK-2 cells were collected for subsequent experiments.

2.7. siRNA transfection

Predesigned small interfering RNA (siRNA) targeting Nrf2 and negative controls siRNA were obtained from Guangzhou Ribo Company. (Guangzhou, China). HK-2 cells were transfected with control or Nrf2 sense siRNA using LipofectamineTM 2000 transfection reagent. HK-2 cells were seeded onto 6-well culture plates in serum-free OPTI-MEM medium (Invitrogen, Carlsbad, CA, USA). When the cells at 60–80% confluence, they were incubated with 50 nM control or Nrf2 siRNA. After 6 h incubation, the cells were incubated with high concentration glucose medium alone or administrated with CGA (100 μM) for an additional 48 hours before being harvested for the further experiments.

2.8. Western blot analysis

Total proteins were extracted from kidney tissues and cultured cells, and RIPA buffer was used for the sample suspension. Protein concentration of cells was determined with bicinchoninic acid (BCA) protein assay kit (ASPEN, USA). 30 μg proteins in mixture solution were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then electrophoretically transferred to PVDF membranes of 0.45 μm. After being blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for one hour, the blocked membranes were incubated separately with diferent kinds of primary antibodies at 4°C overnight. The PVDF membranes were washed 3 times by TBST for 10 minutes each time and subsequently incubated with horseradish peroxidase-conjugated secondary antibody for two hours at room temperature. The protein bands were detected by the chemiluminescence method. The relative protein level of each group was analyzed by the IPP analysis software.

2.9. Statistical analysis

All experimental data are based on three biological replicates. The data were analyzed using GraphPad Prism 12 software. All data are presented as the means ± SD. Differences between two groups were conducted by t-test. It was considered statistically significant when the value of P < 0.05.

3. Results

3.1. CGA alleviated albuminuria and improved renal function in high-fat diet (HFD)/ streptozotocin (STZ) -induced diabetic rats

As shown in Fig 1 kidney weight of diabetic rats was significantly higher than normal rats, diabetic rats treated with CGA had lower kidney weight than untreated diabetic rats. However, body weight of diabetic rats was significantly lower than normal rats, diabetic rats treated with CGA had higher body weight than untreated diabetic rats. In addition, the DM group had significantly increased Scr and BUN concentrations and urinary protein excretion compared with the control group. CGA treatment reduced Scr and BUN concentration and urinary protein excretion. These data showed that CGA protects the kidneys in diabetic rats. Specific values are in S1 Table.

Fig 1. Effects of CGA on renal biochemical markers.

Fig 1

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A. Kidney weight. B.Body weight. C. Urinary protein. D. serum creatinine (Scr). E. Blood urea nitrogen (BUN). *P < 0.05 vs. NC group, **P < 0.01 vs. NC group, #P < 0.05 vs. NC group, ##P < 0.01 vs. DM group.

3.2.CGA attenuated renal histopathological injury in high-fat diet (HFD)/ streptozotocin (STZ) -induced diabetic rats

The changes in renal histopathology are shown in Fig 2. HE and PAS staining staining showed that diabetic rats had notable glomerular hypertrophy, mesangial matrix deposition (red arrow), renal tubular vacuolar degeneration (black arrow) and renal tubular dilatation (green arrow). 10 weeks of treatment with CGA significantly improved these pathological changes. The results indicated that CGA attenuated renal morphological abnormalities in diabetic rats.

Fig 2. The effects of CGA on renal histopathological injury.

Fig 2

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Renal tissues were stained with haematoxylin and eosin (HE ×200) and periodic acid-Schiff (PAS ×200). The glomerular area and mesangial area were quantitatively analyzed. A total of 10 glomeruli were analyzed from each rat to calculate the mesangial area using ImageJ software.

3.3. CGA inhibited NLRP3 inflammasome and activated the Nrf2 pathway in high-fat diet (HFD)/ streptozotocin (STZ) -induced diabetic kidneys

In vivo results showed that expression of NLRP3 protein was remarkably increased in the DM group, and similar changes in cleaved caspase-1(c-caspase-1), IL-1β and IL-18 were consistently observed. However, these increases were significantly inhibited by CGA treatment in diabetic kidneys, as shown in Fig 3A. In immunohistochemistry, the expression of NLRP3 and ASC were significantly increased in the DM group, which further validated the activation of the NLRP3 inflammasome. Chlorogenic acid treatment can reduce the expression of NLRP3 and ASC, as show in Fig 3B. We also evaluated the effect of CGA on the Nrf2 pathway using Western blotting. The results demonstrated that nuclear Nrf2 and the Nrf2 downstream target heme oxygenase-1(HO-1) expression were decreased in the diabetic group. The expression of nuclear Nrf2 and HO-1 were enhanced in response to CGA treatment, as shown in Fig 3C. The membrane images of the immunoprotein blotting are in S1 File.

Fig 3. The effects of CGA on NLRP3 inflammasome and the Nrf2 pathway in high-fat diet (HFD)/ streptozotocin (STZ) -induced diabetic kidneys.

Fig 3

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A. The protein levels of NLRP3, c-caspase-1, IL-1β and IL-18 were detected by Western blotting in renal tissues. *P < 0.05, **P < 0.01 vs. NC. #P < 0.05, ##P < 0.01 vs. DM. B. The protein levels of NLRP3 and ASC were detected by immunohistochemical staining. C. The protein levels of nuclear Nrf2 and HO-1 were detected by Western blotting in renal tissues. *P < 0.05, **P < 0.01 vs. NC. #P < 0.05, ##P < 0.01 vs. DM.

3.4. CGA inhibited NLRP3 inflammasome and activated the Nrf2 pathway in HK-2 cells

Cell experiment results showed that high glucose (HG) induced increases in the levels of NLRP3, cleaved caspase-1, IL-1β and IL-18, whereas treatment with CGA markedly decreased the levels of these NLRP3 inflammasome activation-related biomarkers, as shown in Fig 4A. Furthermore, CGA inhibited NLRP3 inflammasome activation in a concentration-dependent manner. We assessed the activation effect of CGA in vitro. As shown in Fig 4B, the expression of nuclear Nrf2 and HO-1 were decreased in the HG group. CGA increased the expression of nuclear Nrf2 and HO-1 compared with the HG group in a concentration-dependent manner.

Fig 4. The effects of CGA on NLRP3 inflammasome and the Nrf2 pathway in HK-2 cells.

Fig 4

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A. The protein levels of NLRP3, c-caspase-1, IL-1β and IL-18 were analysed using Western blotting, and GAPDH served as a control. B. The protein levels of nuclear Nrf2 and HO-1 were detected by Western blotting. *P < 0.05, **P < 0.01 vs. NC. #P < 0.05, ##P < 0.01 vs. HG.

3.5.CGA inhibited NLRP3 inflammasome activation through modulation of the Nrf2 pathway in HK-2 cells

To verify the effect of Nrf2 in CGA-mediated protection against HG-induced NLRP3 inflammasome activation, we knocked down Nrf2 expression using siRNA, as shown in Fig 5. The results showed that HG treatment significantly increased NLRP3 inflammasome activation in HK-2 cells, which could be down-regulated by the intervention of CGA. However, silencing of Nrf2 inhibited the effect of CGA on NLRP3 inflammasome activation induced by HG. These results demonstrated that the NLRP3 inflammasome inhibitory effect of CGA was mediated by the Nrf2 activation at least partly. Nrf2 plays pivotal role in CGA’s attenuation of DN progression through inhibiting NLRP3 inflammasome activation.

Fig 5. Intervention in the CGA-induced activation of the Nrf2 pathway and its inhibition of NLRP3 inflammasome in HK-2 cells.

Fig 5

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The protein expression of HO-1, NLRP3, c-caspase-1, IL-1β and IL-18 protein expression was analysed using Western blotting. *P < 0.05, **P < 0.01 vs. NC. #P < 0.05, ##P < 0.01 vs. HG. ^P < 0.05, ^^P < 0.01 vs. HG. + Control siRNA. +P < 0.05, ++P < 0.01 vs. HG. + Control siRNA + CGA.

4. Discussion

Diabetic nephropathy (DN) is one of fatal microvascular complications of diabetes patients in morbidity and mortality. Although several available therapeutic interventions have been reported to retard the progression of DN, the high morbidity and high mortality rates of DN have not declined significantly [19]. Therefore, the novel and effective therapeutic strategies have become a hot topic around world. Growing evidence has demonstrated that some natural plant compounds or herbal products have therapeutic potential via regulation of inflammation to prevent and potentially treat DN [20–22], such as abelmoschus manihot ameliorates podocyte pyroptosis and injury in high glucose conditions by targeting NLRP3 inflammasome activation [22]. Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory properties. Little information concerning CGA is available on DN, and the underlying mechanism still remains to be further revealed. In the current study, CGA obviously improved the renal function of diabetic rats. And we verified that CGA treatment ameliorated the pathological injury in the kidney tissues of diabetic rats through the histological study.

The activation of NLRP3 inflammasome plays a pivotal role in the occurrence and progression of DN. Extensive evidence has shown that circulating inflammasome and pro-inflammatory cytokines secretion were increased in DN patients and animal models, while decreasing the activation of the inflammasome mitigates diabetic kidney damage and delays progression [23]. Hence, therapies targeting NLRP3 inflammasome activation may effectively protect renal function renal function and prevent the progression of DN. Huang X et al. [24] reported that Nrf2 and NLRP3 participated in the CGA-mediated anti-inflammatory reaction. Shi A et al. [16]suggested that CGA protected against CCl4-induced acute liver injury through inhibiting NLRP3 inflammasome activation. Zeng J et al. [25] indicated that CGA prevented colitis by inactivating the NF-κB/NLRP3 inflammasome pathway in macrophages. In this study, we observed that the NLRP3 inflammasome was activated, indicated by the increased expression of NLRP3, cleaved caspase-1(c-caspase-1), IL-1β and IL-18 in diabetic rats and HG-induced HK-2 cells. Increased expression of NLPR3 and ASC in immunohistochemistry further confirmed the activation of the NLRP3 inflammasome. While CGA obviously suppressed the NLRP3 inflammasome activation by decreasing the expression of NLRP3, c-caspase-1, IL-1β and IL-18 in diabetic rats and HG-induced HK-2 cells. Our cell experiments also showed that CGA could suppress NLRP3 inflammasome activation. These findings support that treatment with CGA may be a potential therapeutic drug for DN by inhibiting NLRP3 inflammasome activation.

In recent years, many studies have explored the possible mechanisms of NLRP3 inflammasome activation, such as flux of ions, rupture of lysosomes, generation of ROS, release of mitochondrial DNA [26]. Although the mechanisms participated in NLRP3 inflammasome activation are still not been fully elucidated, oxidative stress has been reported to play a significant role in activating NLRP3 inflammasome [27]. Nrf2 plays a crucial role in endogenous oxidative stress. Numerous studies have suggested that CGA play important roles in activating of Nrf2 and stimulating antioxidant enzymatic activities to alleviate oxidative damage. Resent studies have shown that the treatment with CGA ameliorated the expressions of Nrf2, HO-1, NLRP3, ASC and inhibited the contents of IL-1β, IL-18 [24]. Our data displayed that CGA promoted Nrf2 and its target gene HO-1 expression in vitro. CGA also promoted Nrf2 activation, and promoted the expression of downstream antioxidant enzyme HO-1in HG- induced HK-2 cells. These findings show that treatment with CGA significantly improved kidney function and reduced urinary protein excretion and the effects were associated with the Nrf2 signalling pathway activation. Zhang C et al. [28] identified that Nrf2 is the upstream regulator of NLRP3 in parkinson’s disease by using three mice models of genetic defects (Nrf2-KO, NLRP3-KO and Caspase-1-KO). Another study also showed that the expression of NLRP3 was upregulated after Nrf2 silencing, while knocking down NLRP3 did not affect the expression of Nrf2 [29]. Hurtado-Navarro L et al. [30] also highlighted the potential application of Nrf2 inducers in the prevention of NLRP3 inflammasome activation. The further mechanisms are still elusive. Several studies attempted to explain the further mechanisms between the Nrf2 and the NLRP3 inflammasome [8,31]. Hou Y et al. [8]suggested that Nrf2 is a protective regulator against NLRP3 inflammasome activation by regulating the regulating thioredoxin1 (Trx1) / thioredoxin interacting protein (TXNIP) complex in cerebral ischemia reperfusion injury. Moreover, generation of ROS can promote NLRP3 inflammasome activation [26]. Nrf2 may suppress NLRP3 inflammasome activation by reducing ROS generation. In our study, silencing the Nrf2 using small interfering RNA (siRNA) increased the expression of NLRP3, c-caspase-1, IL-1β and IL-18 in HG-induced HK-2 cells. The inhibitory effect of chlorogenic acid on NLRP3 inflammasome was abolished after the intervention of Nrf2 siRNA. The results suggesting that the Nrf2 pathway play a vital role in regulating NLRP3 inflammasome activation. And CGA inhibited NLRP3 inflammasome activation through modulation of the Nrf2 pathway in DN. However, the role of Trx1/ TXNIP complex in the regulation of NLRP3 inflammasome by Nrf2 remains to be further investigated.

In conclusion, CGA protects against diabetic kidney injury in vitro and in vivo through activation of the Nrf2 pathway and inhibition of the activation of NLRP3 inflammasome. Moreover, the CGA induced inhibitory effect on NLRP3 inflammasome activation through modulation of the Nrf2 pathway. These findings suggest that CGA is a novel therapeutic choice for the treatment of DN.

Supporting information

S1 Table. The biochemical data and protein expression levels.

(XLSX)

pone.0316615.s001.xlsx (17.6KB, xlsx)
S1 File. The membrane images of the immunoprotein blotting.

(PDF)

pone.0316615.s002.pdf (554.3KB, pdf)

Data Availability

All relevant data are within the paper and Supporting Information files.

Funding Statement

The present study was supported by Yunnan Provincial Science and Technology Department (202101AY070001-304) and Yunnan Provincial Health Commission(D-2018021 to HY).

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Decision Letter 0

Jordan Robin Yaron

28 Aug 2024

PONE-D-24-12451Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathyPLOS ONE

Dear Dr. Bao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

 Specifically, please address the following points:

  • Inclusion of complete western blot membranes with molecular weight markers important for evaluating pro- and cleaved forms of respective enzymes and cytokines. This may be included as supplementary information.

  • Review and address concerns regarding the different dosing concentrations described throughout the manuscript.

  • Review and address concerns regarding language some of the reviewers found confusing. For example, the statement regarding Nrf2 and its negative regulation of the NLRP3 inflammasome. You may consider addressing this by expanding the introduction and/or discussion.

  • Address concerns regarding lack of detail in the methodology including, but not limited to, catalog numbers, dilution factors, etc.

  • Address concerns regarding the presentation of figures including, but not limited to, inclusion of arrows to emphasize respective areas of figures, normalization of figure presentation, etc.

  • Address recommendations regarding inclusion of NLRP3 inhibitors or NLRP3 KO mice, and potential explanations for mechanisms of NLRP3 activation of DN.

Please submit your revised manuscript by Oct 12 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Jordan Robin Yaron, Ph.D.

Academic Editor

PLOS ONE

Journal requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.  When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: [The present study was supported by Kunming Medical University applications of Yunnan (grant number 202101AY070001-304 to Liping Bao]We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:  [The author(s) received no specific funding for this work.] Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Please provide a complete Data Availability Statement in the submission form, ensuring you include all necessary access information or a reason for why you are unable to make your data freely accessible. If your research concerns only data provided within your submission, please write "All data are in the manuscript and/or supporting information files" as your Data Availability Statement. 5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.   In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper was mainly explored through two dimensions in vivo and in vitro. In vivo, the diabetic model rats were experimentally verified by ELISA, HE staining, PAS staining and Western blot. In vitro, HK-2 cells were used to explore the protein expression changes of inflammasome and Nrf2 pathway by Western blot, and the expression changes of inflammasome and Nrf2 pathway proteins were explored by Western blot after siRNA knockdown of Nrf2 expression, so as to verify that chlorogenic acid inhibits the activation of NLRP3 inflammasome in diabetic nephropathy by activating Nrf2.

The completion of this paper is good, the basic indicators have been verified, and the main problems are that the experimental grouping is thin, and the experimental design and the use of experimental methods are relatively simple and not novel enough. The content is relatively routine, and the verified mechanism has become universal. The main issues of the manuscript are as follows:

1. The weight of the Chinese drug group in Fig1 B was higher than that of the model, which was inconsistent with the description of the results.

2. It is recommended that the WB section provide the whole membrane；

3. Only one dose was used in the treatment group, and the angle was relatively simple. There was no experimental attempt to discuss the correlation between the drug dose and the treatment effect.

4. The content of the experimental design is relatively modeled, not novel enough, and there are some bright spots lacking in technology, so it is recommended to enrich the experimental design.

Reviewer #2: Notes on Review for PLOS One

“Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathy”

Accept with Minor Revisions

Review Comments to Author:

This manuscript is technically sound, and the data supports all conclusions presented in the final document. Statistical Analysis has been performed and indicates that the results presented are statistically significant. The authors have also made it clear that all data underlying the findings in the manuscript are fully available.

Summary:

Diabetic neuropathy is a serious complication of both type 1 and type 2 diabetes, and it often affects the kidneys’ ability to remove waste products and extra fluid from the body. Inflammation caused by the NLRP3 inflammasome and further Nrf2 activation is a large contributing factor to the progression of diabetic nephropathy (DN). Chlorogenic acid (CGA) could activate Nrf2, which is found in nature. This study evaluated the ability of CGA to reduce inflammation. Findings showed that serum creatinine (Scr), blood urea nitrogen (BUN), and urinary protein excretions decreased after applying CGA. This occurs because CGA can activate the function of Nrf2 and halt the activity of NLRP3. Thus, this study demonstrated that chlorogenic acid could decrease DN progression and showed further that this impact is largely due to suppression of the NLRP3 inflammation and is modulated through the Nrf2 pathway. This research represents potential therapeutic implications for DN.

Abstract:

Comments: Overall, the abstract explains the background and premise of the article concisely and fully.

Major Revisions:

Minor Revisions:

1. In the abstract, you wrote: “CGA administration can active the Nrf2 pathway and inhibited NLRP3 inflammasome activation.” However, it should be noted that “CGA administration can activate the Nrf2 pathway and inhibit NLRP3 inflammasome activation.”

Introduction:

Comments:

The introduction concisely explains DN and how NLRP3 plays a role in developing the disease. It provides an adequate basis for the overall study.

Major Revisions:

1. The hypothesis should be rephrased; the way it is currently explained it does not explain how the Nrf2 negatively regulates NLRP3 inflammasome, particularly in this segment “negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome.”

Minor Revisions:

None

Methods:

The methods described here were sufficient

Major Revisions: None

Minor Revisions:

I see that there are statistics performed on each of these experiments, but I could not find where there was a mention of how many times these experiments were completed. If they were done in duplicate or triplicate, please state this in the methods, particularly for the western blot analysis results and subsequent relative protein quantification.

Results:

Major Revisions: None

Minor Revisions: None

Though this is minor, I would make sure that all figures are uniform, there are a few that do not have the same borders which may make interpreting where certain bars are on the y axis confusing.

I think it was wise to quantify the relative levels of protein in western blot analysis.

Discussion:

Major Revisions: None

Minor Revisions:

1. The discussion mentions that “Growing evidence has demonstrated that some natural plant compounds or herbal products have therapeutic potential via regulation of inflammation to prevent and potentially treat DN[20-22]” but does not provide further details about where these compounds are found. This section would be improved in examples of where these compounds are found were also included.

2. Some small grammar repairs, consider changing “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory property. Little information concerning CGA is available on DN, and the underlying mechanism still remain to be further revealed.”

To “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory properties. Little information concerning CGA is available on DN, and the underlying mechanism still remains to be further revealed.”

Reviewer #3: The primary claim made by the authors of this submission is that the administration of Chlorogenic acid (CGA) demonstrates a renoprotective effect in the context of Diabetic Neuropathy (DN). Further the authors posit this effect is partially mediated through down-regulation of the NLRP3 inflammasome activation via the Nrf2 pathway. Reduction in the progression of kidney damage after treatment with CGA is significant due high rates of mortality despite treatment.

The authors cite multiple studies demonstrating the role inflammation reactions play in the development of DN as well as their own previous work demonstrating increased renal Nrf2 expression following CGA treatment.

Based on previous literature reports and their own previous studies on CGA treatment, the authors hypothesize that “Nrf2 negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome in DN.” This statement is not written clearly and adds confusion to the mechanism the authors are targeting. The hypothesis of any research study should be clear and unambiguous as to the authors intended meaning. This statement should be re-written to explain their reasoning and scientific justification more clearly.

The authors used both an in-vitro model of cells supplemented with high levels of glucose as well as a high-fat/STZ induced diabetic rat model to replicate the Diabetes Mellitus condition. They clearly demonstrated their intended targets of study associated with NLRP3 inflammasome mechanism of action such as caspase-1, IL-1b, and IL-18. Additionally, they have identified a downstream protein of interest, heme oxgenase-1 (HO-1), which is associated with the Nrf2 pathway activation. The significance of these targets to clinical disease and oxidative stress were supported by previous literature. The methods associated with the experimental assays – histology, western blot, and blood and urine chemistries - were detailed and informative to allow for experiments to be reproduced.

Most of the results and data figures were clearly labeled and supported the conclusions stated by the authors. However, in section 3.1 the authors state that diabetic rats treated with CGA [DM + CGA group] had a lower body weight compared to untreated diabetic rats [DM group]. This is result is not what is represented in figure 1B – the DM + CGA treatment group are shown to have a higher body weight compared to the DM group. This result should be re-written to match the corresponding figure.

Overall, I felt this was a strong manuscript with a clearly defined objective and orthogonally designed experiments. The conclusions drawn by the authors are supported by the results and delve into the possible mechanisms associated with the benefits of CGA treatment in Diabetes Mellitus and inflammatory reactions. The hypothesis should be revised to clearly state their goal as well as address the conclusion drawn from figure 1B.

Reviewer #4: In this manuscript, Bao et al. explore the effects of chlorogenic acid (CGA) on diabetic nephropathy (DN). Using a diabetic rat model, they find that CGA has potential therapeutic implications for slowing the progression of DN by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. However, the data presented in the manuscript are sometimes not convincing and lack proper description. Addressing the following comments could help improve the quality of this study:

1. It would be better if providing histological quantification and using arrowheads to indicate pathological damage in Figure 2.

2. The catalog numbers of the antibodies used in the manuscript are not provided.

In all Western blot images, it is unclear whether IL-1β, IL-18, and caspase-1 proteins are in their pro or cleaved forms, since cleaved caspase-1 and cytokines (IL-1β, IL-18) are indicators of NLRP3 inflammasome activation. Providing additional data, such as ASC oligomerization or immunohistochemistry for NLRP3 and ASC proteins in the kidney or cells, could offer more solid evidence for NLRP3 activation. Alternatively, it would be best to use NLRP3-deficient mice to determine if NLRP3 is truly involved in the progression of DN would strengthen the findings. If knockout mice are unavailable, NLRP3 inhibitors could be used.

3. Please include molecular weight markers in all Western blot images.

4. The NLRP3 inflammasome activates the downstream effector caspase-1, leading to caspase-1-mediated pyroptosis. Since the authors have identified NLRP3 activation under their experimental conditions, it would be interesting to investigate whether CGA could prevent DN by inhibiting NLRP3 inflammasome activation and subsequently reducing pyroptosis,

5. The discussion section could be improved by exploring possible mechanisms of NLRP3 activation in DN and how Nrf2 is induced as part of the defense system against oxidative stress, as well as how NLRP3 inhibition occurs under these conditions.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Nicole Grigaitis-Esman

Reviewer #4: No

**********

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Attachment

Submitted filename: Notes on Review for PLOS One.docx

pone.0316615.s003.docx (17.5KB, docx)
PLoS One. 2025 Jan 6;20(1):e0316615. doi: 10.1371/journal.pone.0316615.r002

Author response to Decision Letter 0

9 Nov 2024

Reviewer #1:

1. The weight of the drug group in Fig1 B was higher than that of the model, which was inconsistent with the description of the results.

Response: Thank you very much for your careful review and pointing out this issue. Kidney weight of diabetic rats was significantly higher than normal rats, diabetic rats treated with CGA had lower kidney weight than untreated diabetic rats. However, body weight of diabetic rats was significantly lower than normal rats, diabetic rats treated with CGA had higher body weight than untreated diabetic rats. We have revised this description in the resubmitted manuscript.

2. It is recommended that the WB section provide the whole membrane；

Response: Thank you very much for your suggestion, we have submitted the membranes for three biological replicates of WB.

3. Only one dose was used in the treatment group, and the angle was relatively simple. There was no experimental attempt to discuss the correlation between the drug dose and the treatment effect.

Response: Thank you very much for your suggestion. Because of the difficulties in establishing the diabetic rat model, the long duration of drug intervention, and the high cost, we only used one dose. To remedy this limitation, we set up different drug doses(20uM,50uM,100uM) in the cell experiment to discuss the relationship between drug dose and treatment effect. The result showed CGA inhibi NLRP3 inflammasome activation and increased the expression of nuclear Nrf2 and HO-1in a concentration-dependent manner. These mean that the treatment effect of CGA is closely related to its dose.

4. The content of the experimental design is relatively modeled, not novel enough, and there are some bright spots lacking in technology, so it is recommended to enrich the experimental design.

Response: Thank you very much for your suggestion. NLRP3 inflammasome is composed of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). In the diabetic state, NLRP3 inflammasome is activated, then NLRP3 recruits ASC and cleaves caspase-1. We demonstrated the effect of chlorogenic acid on NLRP3 inflammasome by immunohistochemistry for NLRP3 and ASC proteins.

Reviewer #2:

Abstract:

In the abstract, you wrote: “CGA administration can active the Nrf2 pathway and inhibited NLRP3 inflammasome activation.” However, it should be noted that “CGA administration can activate the Nrf2 pathway and inhibit NLRP3 inflammasome activation.”

Response: Thank you very much for your careful review and pointing out this issue. We have revised this description in the resubmitted manuscript.

Introduction:

The hypothesis should be rephrased; the way it is currently explained it does not explain how the Nrf2 negatively regulates NLRP3 inflammasome, particularly in this segment “negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome.”

Response: Thank you very much for your careful review and pointing out this issue. We have revised this description in the resubmitted manuscript.

Methods:

I see that there are statistics performed on each of these experiments, but I could not find where there was a mention of how many times these experiments were completed. If they were done in duplicate or triplicate, please state this in the methods, particularly for the western blot analysis results and subsequent relative protein quantification.

Response: Thank you very much for your suggestion, all experimental data are based on three biological replicates, we have further clarified this issue in Statistical analysis.

Results:

Though this is minor, I would make sure that all figures are uniform, there are a few that do not have the same borders which may make interpreting where certain bars are on the y axis confusing.

I think it was wise to quantify the relative levels of protein in western blot analysis.

Response: Thank you very much for your suggestion. We have modified the figures in the resubmitted manuscript.

Discussion:

1. The discussion mentions that “Growing evidence has demonstrated that some natural plant compounds or herbal products have therapeutic potential via regulation of inflammation to prevent and potentially treat DN[20-22]” but does not provide further details about where these compounds are found. This section would be improved in examples of where these compounds are found were also included.

Response: Thank you very much for your suggestion. In the discussion section, we have added the example of abelmoschus manihot. Abelmoschus manihot ameliorates podocyte pyroptosis and injury in high glucose conditions by targeting NLRP3 inflammasome activation.

2. Some small grammar repairs, consider changing “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory property. Little information concerning CGA is available on DN, and the underlying mechanism still remain to be further revealed.” to “Chlorogenic acid (CGA) with a good Nrf2 activation effect possessed anti-inflammatory properties. Little information concerning CGA is available on DN, and the underlying mechanism still remains to be further revealed.”

Response: Thank you very much for your careful review and pointing out this issue. We have revised this description in the resubmitted manuscript.

Reviewer #3: The primary claim made by the authors of this submission is that the administration of Chlorogenic acid (CGA) demonstrates a renoprotective effect in the context of Diabetic Neuropathy (DN). Further the authors posit this effect is partially mediated through down-regulation of the NLRP3 inflammasome activation via the Nrf2 pathway. Reduction in the progression of kidney damage after treatment with CGA is significant due high rates of mortality despite treatment.

The authors cite multiple studies demonstrating the role inflammation reactions play in the development of DN as well as their own previous work demonstrating increased renal Nrf2 expression following CGA treatment.

Based on previous literature reports and their own previous studies on CGA treatment, the authors hypothesize that “Nrf2 negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome in DN.” This statement is not written clearly and adds confusion to the mechanism the authors are targeting. The hypothesis of any research study should be clear and unambiguous as to the authors intended meaning. This statement should be re-written to explain their reasoning and scientific justification more clearly.

The authors used both an in-vitro model of cells supplemented with high levels of glucose as well as a high-fat/STZ induced diabetic rat model to replicate the Diabetes Mellitus condition. They clearly demonstrated their intended targets of study associated with NLRP3 inflammasome mechanism of action such as caspase-1, IL-1b, and IL-18. Additionally, they have identified a downstream protein of interest, heme oxgenase-1 (HO-1), which is associated with the Nrf2 pathway activation. The significance of these targets to clinical disease and oxidative stress were supported by previous literature. The methods associated with the experimental assays – histology, western blot, and blood and urine chemistries - were detailed and informative to allow for experiments to be reproduced.

Most of the results and data figures were clearly labeled and supported the conclusions stated by the authors. However, in section 3.1 the authors state that diabetic rats treated with CGA [DM + CGA group] had a lower body weight compared to untreated diabetic rats [DM group]. This is result is not what is represented in figure 1B – the DM + CGA treatment group are shown to have a higher body weight compared to the DM group. This result should be re-written to match the corresponding figure.

Overall, I felt this was a strong manuscript with a clearly defined objective and orthogonally designed experiments. The conclusions drawn by the authors are supported by the results and delve into the possible mechanisms associated with the benefits of CGA treatment in Diabetes Mellitus and inflammatory reactions. The hypothesis should be revised to clearly state their goal as well as address the conclusion drawn from figure 1B.

Response: Thank you very much for your careful review and positive comments on this study. “Nrf2 negatively regulated the activation of NLRP3 inflammasome via regulation of the NLRP3 inflammasome in DN.”- this statement is not written clearly. We have modified it to “Nrf2 negatively regulated the activation of NLRP3 inflammasome in DN.”. Our explanation in section 3.1 contains errors. Kidney weight of diabetic rats was significantly higher than normal rats, diabetic rats treated with CGA had lower kidney weight than untreated diabetic rats. However, body weight of diabetic rats was significantly lower than normal rats, diabetic rats treated with CGA had higher body weight than untreated diabetic rats. We have made revisions in the resubmitted manuscript. Thank you very much for pointing out the errors in our manuscript.

Reviewer #4: In this manuscript, Bao et al. explore the effects of chlorogenic acid (CGA) on diabetic nephropathy (DN). Using a diabetic rat model, they find that CGA has potential therapeutic implications for slowing the progression of DN by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation. However, the data presented in the manuscript are sometimes not convincing and lack proper description. Addressing the following comments could help improve the quality of this study:

1. It would be better if providing histological quantification and using arrowheads to indicate pathological damage in Figure 2.

Response: Thank you very much for your careful review and suggestion. We have marked the pathological damages with arrows and quantified the glomerular area and mesangial area.

2. The catalog numbers of the antibodies used in the manuscript are not provided.

Response: Thank you very much for your careful review and suggestion. We have provided the catalog numbers of the antibodies in 2.1. Reagents.

In all Western blot images, it is unclear whether IL-1β, IL-18, and caspase-1 proteins are in their pro or cleaved forms, since cleaved caspase-1 and cytokines (IL-1β, IL-18) are indicators of NLRP3 inflammasome activation. Providing additional data, such as ASC oligomerization or immunohistochemistry for NLRP3 and ASC proteins in the kidney or cells, could offer more solid evidence for NLRP3 activation. Alternatively, it would be best to use NLRP3-deficient mice to determine if NLRP3 is truly involved in the progression of DN would strengthen the findings. If knockout mice are unavailable, NLRP3 inhibitors could be used.

Response: Thank you very much for your careful review and pointing out this issue. In the introduction, we mentioned “cleaved caspase-1 promotes the conversion of inflammatory factors such as pro-IL-1β and pro-IL-18 into IL-1β and IL-18 and released outside the cell, which leading to pro-inflammatory responses”. So, IL-1β and IL-18 are their cleaved forms in Western blot images. Capase-1 is its cleaved form in WB images. And we have modified capase-1 to c-capase-1 (cleaved capase-1) in all Western blot images. According to your suggestion, we performed immunohistochemistry for NLRP3 and ASC proteins in the kidney to offer more solid evidence for NLRP3 activation.

3. Please include molecular weight markers in all Western blot images.

Response: Thank you very much for your careful review and suggestion. We have included molecular weight markers in all Western blot images in revised manuscript.

4. The NLRP3 inflammasome activates the downstream effector caspase-1, leading to caspase-1-mediated pyroptosis. Since the authors have identified NLRP3 activation under their experimental conditions, it would be interesting to investigate whether CGA could prevent DN by inhibiting NLRP3 inflammasome activation and subsequently reducing pyroptosis.

Response: Thank you very much for your careful review and suggestion. As you said, it's interesting to investigate whether CGA could prevent DN by inhibiting NLRP3 inflammasome activation and subsequently reducing pyroptosis. The manuscript only describes caspase-1 dependent pyroptosis. In our next step of research, we will explore the effect of chlorogenic acid on pyroptosis and other possible mechanisms.

5. The discussion section could be improved by exploring possible mechanisms of NLRP3 activation in DN and how Nrf2 is induced as part of the defense system against oxidative stress, as well as how NLRP3 inhibition occurs under these conditions.

________________________________________

Response: Thank you for your suggestion. In recent years, many studies have explored the possible mechanisms of NLRP3 inflammasome activation, such as flux of ions, rupture of lysosomes, generation of ROS, release of mitochondrial DNA. Our previous study has shown that pre-treatment with CGA increased the renal expression of Nrf2 and the downstream target heme oxygenase-1 (HO-1). Hou Y et al. suggested that Nrf2 is a protective regulator against NLRP3 inflammasome activation by regulating the regulating thioredoxin1 (Trx1) / thioredoxin interacting protein (TXNIP) complex in cerebral ischemia reperfusion injury. Moreover, generation of ROS can promote NLRP3 inflammasome activation. Nrf2 may suppress NLRP3 inflammasome activation by reducing ROS generation. We also conducted relevant discussion in the discussion section.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0316615.s004.docx (22.1KB, docx)

Decision Letter 1

Jordan Robin Yaron

15 Dec 2024

Chlorogenic acid inhibits NLRP3 inflammasome activation through Nrf2 activation in diabetic nephropathy

PONE-D-24-12451R1

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Acceptance letter

Jordan Robin Yaron

26 Dec 2024

PONE-D-24-12451R1

PLOS ONE

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    S1 Table. The biochemical data and protein expression levels.

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