Protocatechuic acid reduces H2O2-induced migration and oxidative stress of fibroblast-like synoviocytes in rheumatoid arthritis by activating Nrf2-Keap1 signaling pathway

Yan Liu; Yucheng Zhang; Keke Zhang; Yue Wang

doi:10.4103/cjop.CJOP-D-22-00087

ORIGINAL ARTICLE

Year : 2023 | Volume : 66 | Issue : 1 | Page : 28-35

Protocatechuic acid reduces H₂O₂-induced migration and oxidative stress of fibroblast-like synoviocytes in rheumatoid arthritis by activating Nrf2-Keap1 signaling pathway

Yan Liu¹, Yucheng Zhang², Keke Zhang³, Yue Wang¹
¹ Department of Rheumatology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
² Department of Critical Care Medicine, Changshu Hospital Affiliated to Nanjing University of Chinese Medicine, Changshu, Jiangsu, China
³ School of Integrated Traditional Chinese and Western Medicine, Jiangsu Health Vocational College, Nanjing, Jiangsu, China

Date of Submission	07-Sep-2022
Date of Decision	17-Oct-2022
Date of Acceptance	03-Nov-2022
Date of Web Publication	20-Feb-2023

Correspondence Address:
Dr. Yue Wang
Department of Rheumatology, Affiliated Hospital of Nanjing University of Chinese Medicine, No. 155, Hanzhong Road, Nanjing, 210029, Jiangsu
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjop.CJOP-D-22-00087

Abstract

Honeycomb (Nidus vespae) is traditional Chinese medicine and can treat rheumatoid arthritis (RA), and protocatechuic acid (PCA) is a bioactive component of honeycomb. This study aimed to investigate whether PCA could reduce the H₂O₂-induced migration and oxidative stress of RA fibroblast-like synoviocytes (RA-FLSs). H₂O₂-induced RA-FLSs were used to simulate the in vitro model of RA. The viability, apoptosis, migration, invasion, and oxidative stress of RA-FLSs were detected by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, wound healing, transwell assays, DCFDA staining, and malonaldehyde and superoxide dismutase enzyme-linked immunosorbent assay kits. The expression of migration and invasion-related proteins and Nrf2/Keap1 signaling pathway-related proteins was analyzed by western blotting. As a result, PCA suppressed the viability, migration, invasion, and oxidative and promoted apoptosis of H₂O₂-induced RA-FLSs by activating the Nrf2/Keap1 signaling pathway. ML-385, an Nrf2 inhibitor, could enhance the viability, migration, invasion, and oxidative and inhibited apoptosis of H₂O₂-induced RA-FLSs. In conclusion, PCA reduced H₂O₂-induced migration and oxidative stress of RA-FLSs by activating the Nrf2-Keap1 signaling pathway.

Keywords: Fibroblast-like synoviocytes, Nrf2-Keap1 signaling pathway, protocatechuic acid, rheumatoid arthritis

How to cite this article:
Liu Y, Zhang Y, Zhang K, Wang Y. Protocatechuic acid reduces H₂O₂-induced migration and oxidative stress of fibroblast-like synoviocytes in rheumatoid arthritis by activating Nrf2-Keap1 signaling pathway. Chin J Physiol 2023;66:28-35

How to cite this URL:
Liu Y, Zhang Y, Zhang K, Wang Y. Protocatechuic acid reduces H₂O₂-induced migration and oxidative stress of fibroblast-like synoviocytes in rheumatoid arthritis by activating Nrf2-Keap1 signaling pathway. Chin J Physiol [serial online] 2023 [cited 2023 Sep 28];66:28-35. Available from: https://www.cjphysiology.org/text.asp?2023/66/1/28/370015

Yan Liu, Yucheng Zhang: These authors contributed equally.

Introduction

Rheumatoid arthritis (RA) is a common chronic and systemic autoimmune disease characterized by chronic synovitis and cartilage destruction. Patients are often accompanied by persistent joint pain, swelling, stiffness, and severe cases will lead to cardiovascular, pulmonary, skeletal, and other complications.^[1],[2] The incidence of RA is reported to be high in the world and in our country, and it can occur at any age.^[3] Since the pathogenesis of RA is still unclear and existing drugs cannot completely cure RA,^[4] it is of great significance to develop new anti-RA drugs.

Honeycomb (Nidus vespae) is a traditional Chinese medicine and can treat RA.^[5] Protocatechuic acid (PCA) is a bioactive component of honeycomb.^[6] Through HERB database and literature review, it was found that PCA is one of the important anti-inflammatory and antioxidant compounds.^[7],[8],[9] One study has shown that PCA can inhibit the proliferation, migration, and inflammatory response of RA fibroblast-like synoviocytes (RA-FLSs).^[10] In osteoclasts, PCA can reduce the levels of reactive oxygen species (ROS) and oxidative stress, and promote the nuclear translocation of Nrf2.^[11] The activation of the Nrf2-Keap1 signaling pathway plays an important regulatory role against oxidative stress.^[12] Reducing oxidative stress is crucial for inhibiting the development of RA.^[13] It has been reported that activation of the Nrf2-Keap1 signaling pathway can reduce oxidative stress and apoptosis of RA-FLSs induced by H₂O₂,^[14] which indicates that the Nrf2-Keap1 signaling pathway functions in RA-FLSs induced by H₂O₂.

However, whether PCA regulates the H₂O₂-induced migration and oxidative stress of RA-FLSs through the Nrf2-Keap1 signaling pathway remained unknown. Nrf2 inhibitor, ML-385, was used to verify the protective effect of PCA on RA-FLSs induced by H₂O₂ through the Nrf2-Keap1 signaling pathway.

Materials and Methods

Cell culture

Human RA-FLSs (MH7A cells) were obtained from BeNa Culture Collection (BNCC; China) and cultured in DMEM (Gibco-BRL, Paisley, UK) containing 10% fetal bovine serum, 0.1% penicillin/streptomycin, and 2 mM L-glutamine at 37°C with 5% CO₂. PCA (CAS No. 99-50-3; purity 97%) was provided from Sigma-Aldrich and ML-385 (CAS No. 846557-71-9) was obtained from MedChemExpress. DMSO was the solvent for PCA and ML-385. For the PCA group, RA-FLSs were treated with different concentrations of PCA (10, 20, and 40 μM) for 48 h.^[10] For the H₂O₂+ PCA group, RA-FLSs were exposed to 5 μM H₂O₂ for 12 h^[15] following 48 h of treatment with 10, 20, and 40 μM PCA. For the H₂O₂+ PCA + ML-385 group, RA-FLSs were pretreated with 5 μM H₂O₂ for 12 h and 1.25 μM ML-385 for 1 h,^[16] and then treated with 40 μM PCA for 48 h.

Cell counting kit-8 assay

After the above-indicated treatment, MH7A cells were seeded into 96-well plates and incubated for 24 h at 37°C. After incubation, MH7A cells were treated with 10 μl Cell Counting Kit-8 (CCK-8) solution for continuous incubation for 2 h at 37°C. The optical density values reflecting cell viability were recorded at 450 nm using a microplate reader.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

After the above-indicated treatment, MH7A cells were fixed with 4% paraformaldehyde solution in phosphate buffer solution (PBS) for 1 h and washed with PBS three times. After the permeabilization of cells, MH7A cells were stained with DAPI or TUNEL. TUNEL-positive cells were counted in a randomly chosen field using a fluorescence microscope (Olympus).

Wound healing assay

After the above-indicated treatment, MH7A cells were seeded in six-well plates and cultured until the cell confluence reached to 90%. The scratch was made by a pipette tip, followed by washing to remove the floating cells. The remaining cells were incubated with serum-free medium for 24 h. The width of the scratch was photographed at 0 and 24 h using an inverted light microscope.

Transwell assay

The upper chambers were coated with Matrigel matrix (Corning, USA) at 37°C for 7 h. After the above-indicated treatment, MH7A cells were added into the upper chambers with 200 μl medium and 600 μl medium containing 10% FBS was added to the lower chambers, which were incubated at 37°C for 48 h. A cotton swab was used to gently wipe the cells on the upper surface of the chamber and the invaded cells were fixed with 4% paraformaldehyde and stained with 0.4% crystal violet, respectively, for 10 min at room temperature. Images of the invaded cells were photographed using an inverted light microscope.

Western blot

After the above-indicated treatment, MH7A cells were treated with radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime) and centrifuged to obtain the total proteins. Nuclear protein was extracted with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The concentration of total protein was determined by the BCA method. Then, the proteins (30 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). After blocking with 5% nonfat milk, the membranes were incubated overnight at 4°C with primary antibodies against MMP2, MMP9, Keap1, HO-1, GAPDH, N-Nrf2, and Lamin B. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Beyotime) and the intensity of the protein bands was quantified by ImageJ 1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

DCFDA staining

After the above-indicated treatment, MH7A cells were incubated with 10 μM dichlorofluorescein diacetate (DCFH-DA; Beyotime) for 20 min at 37°C and washed with serum-free DMEM for three times. The level of ROS in cells was detected by spectrofluorophotometer (Shimadzu Corporation) at the wavelength of 488/525 nm.

Enzyme-linked immunosorbent assay

After the above-indicated treatment, the culture medium of MH7A cells was centrifuged to collect the supernatants. The levels of oxidative stress factors malonaldehyde (MDA) and superoxide dismutase (SOD) were determined by MDA and SOD enzyme-linked immunosorbent assay (ELISA) kits (Jianglai Bio).

Statistical analysis

Data analysis was conducted using GraphPad Prism software (version 8.0.1; GraphPad Software, Inc., San Diego CA, USA). Multiple comparisons were calculated using a one-way analysis of variance with Tukey's post hoc test. P < 0.05 were considered statistically significant.

Results

Protocatechuic acid reduces H₂O₂-induced proliferation of rheumatoid arthritis fibroblast-like synoviocytes

To investigate the effect of PCA on the viability and apoptosis of MH7A cells with or without H₂O₂ induction, CCK-8 assay and TUNEL assay were used. The chemical structure of PCA was shown in [Figure 1]a. The viability of MH7A cells was decreased gradually by PCA [Figure 1]b. H₂O₂ induction promoted the viability of MH7A cells, which was suppressed by PCA [Figure 1]c. The apoptosis of MH7A cells was inhibited by H₂O₂ induction and PCA aggravated the apoptosis of H₂O₂-induced MH7A cells [Figure 1]d, [Figure 1]e.

Figure 1: PCA reduces H₂O₂-induced proliferation of RA-FLSs. (a) The chemical structure of PCA. (b and c) The viability of MH7A cells with or without H₂O₂ induction was detected by CCK-8 assay. (d and e) The apoptosis of H₂O₂-induced MH7A cells treated by PCA was analyzed by TUNEL assay. **P < 0.01 and ***P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 versus H₂O₂ (5 μM) group. PCA: Protocatechuic acid, RA-FLSs: Rheumatoid arthritis fibroblast-like synoviocytes, CCK-8: Cell counting kit-8, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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Protocatechuic acid inhibits H₂O₂-induced migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes

To estimate whether PCA affected the migration and invasion of MH7A cells with H₂O₂ induction, wound healing assay and transwell assay were performed. To further uncover the influence of PCA on the migration and invasion of MH7A cells with H₂O₂ induction, proteins associated with migration and invasion were measured by western blot. The migration and invasion of MH7A cells were induced by H₂O₂, which could be suppressed by PCA [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d. Accordingly, H₂O₂ promoted the expression of MMP2 and MMP9 in MH7A cells and PCA could also suppress the expression of the above proteins in H₂O₂-induced MH7A cells [Figure 2]e.

Figure 2: PCA inhibits H₂O₂-induced migration and invasion of RA-FLSs. (a and b) The migration of H₂O₂-induced MH7A cells treated by PCA was detected by wound healing assay. (c and d) The invasion of H₂O₂-induced MH7A cells treated by PCA was detected by transwell assay. (e) The expression of proteins associated with migration in H₂O₂-induced MH7A cells treated by PCA was detected by western blot. ***P < 0.001 versus control group. ^##P < 0.01 and ^###P < 0.001 versus H₂O₂ (5 μM) group. PCA: Protocatechuic acid, RA-FLSs: Rheumatoid arthritis fibroblast-like synoviocytes.

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Protocatechuic acid inhibits H₂O₂-induced oxidative stress levels in rheumatoid arthritis fibroblast-like synoviocytes

To evaluate whether PCA affected the oxidative stress of MH7A cells with H₂O₂ induction, the levels of ROS, MDA, and SOD were measured by DCFDA staining and respective ELISA assay kits. The levels of ROS and MDA were increased, whereas SOD level was decreased in MH7A cells after H₂O₂ induction. PCA treatment could reverse the effect of H₂O₂ induction on the oxidative stress factors levels in MH7A cells [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d.

Figure 3: PCA inhibits H₂O₂-induced oxidative stress levels in RA-FLSs. (a and b) The ROS level in H₂O₂-induced MH7A cells treated by PCA was detected by DCFDA staining. (c and d) The levels of MDA and SOD in H₂O₂-induced MH7A cells treated by PCA were determined by respective ELISA assay kits. ***P < 0.001 versus control group. ^#P < 0.05 and ^###P < 0.001 versus H₂O₂ (5 μM) group. PCA: Protocatechuic acid, RA-FLSs: Rheumatoid arthritis fibroblast-like synoviocytes, MDA: Malonaldehyde, SOD: Superoxide dismutase, ELISA: Enzyme-linked immunosorbent assay.

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Protocatechuic acid activates Nrf2/Keap1 signaling pathway in H₂O₂-induced rheumatoid arthritis fibroblast-like synoviocytes

For further investigation into the regulatory mechanism of PCA in protecting against H₂O₂-induced MH7A cell injury, the involvement of the Nrf2/Keap1 signaling pathway was explored. Western blot was to detect Nrf2/Keap1 signaling pathway-associated proteins N-Nrf2, Keap1, and HO-1 of cells. As shown in [Figure 4], H₂O₂ induction declined the expression of N-Nrf2 and HO-1, whereas upregulated the expression of Keap1 in MH7A cells, which was reversed by PCA treatment.

Figure 4: PCA activates Nrf2/Keap1 signaling pathway in H₂O₂-induced RA-FLSs. The expression of proteins associated with Nrf2/Keap1 signaling pathway in H₂O₂-induced MH7A cells treated by PCA was detected by western blot. ***P < 0.001 versus control group. ^#P < 0.05 and ^###P < 0.001 versus H₂O₂ (5 μM) group. PCA: Protocatechuic acid, RA-FLSs: Rheumatoid arthritis fibroblast-like synoviocytes.

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Protocatechuic acid reduces H₂O₂-induced proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes by activating Nrf2/Keap1 signaling pathway

To verify whether PCA exerted its protective effect on the proliferation, migration, and invasion of H₂O₂-induced MH7A cells through activation of the Nrf2 signaling pathway, ML-385 (1.25 μM) was used in the following experiments. CCK-8, TUNEL, wound healing, and transwell assays were applied to investigate the effect of ML-385 on the viability, apoptosis, migration, and invasion of H₂O₂-induced MH7A cells. The expression of proteins associated with cell migration affected by ML-385 was detected by western blot. ML-385 increased the viability, inhibited the apoptosis, and improved the migration and invasion of H₂O₂-induced MH7A cells after PCA treatment [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]e, [Figure 5]f, [Figure 5]g. The expression of MMP2 and MMP9 in H₂O₂-induced MH7A cells treated by PCA was upregulated by ML-385 [Figure 5]h.

Figure 5: PCA reduces H₂O₂-induced proliferation and migration of RA-FLSs by activating Nrf2/Keap1 signaling pathway. (a) The viability of H₂O₂-induced MH7A cells treated by PCA and ML-385 was detected by CCK-8 assay. (b and c) The apoptosis of H₂O₂-induced MH7A cells treated by PCA and ML-385 was analyzed by TUNEL assay. (d and e) The migration of H₂O₂-induced MH7A cells treated by PCA and ML-385 was detected by wound healing assay. (f and g) The invasion of H₂O₂-induced MH7A cells treated by PCA and ML-385 was detected by transwell assay. (h) The expression of proteins associated with migration in H₂O₂-induced MH7A cells treated by PCA and ML-385 was detected by western blot. **P < 0.01 and ***P < 0.001 versus Control group. ^###P < 0.001 versus H₂O₂ group. ^&P < 0.05, ^&&P < 0.01 and ^&&&P < 0.001 versus H₂O₂ + PCA group. PCA: protocatechuic acid, CCK-8: Cell counting kit-8, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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Protocatechuic acid inhibits H₂O₂-induced oxidative stress levels in rheumatoid arthritis fibroblast-like synoviocytes by activating Nrf2/Keap1 signaling pathway

To verify whether PCA exerted its protective effect on the oxidative stress of H₂O₂-induced MH7A cells through activation of the Nrf2 signaling pathway, ML-385 (1.25 μM) was used. The levels of oxidative stress factors in H₂O₂-induced MH7A cells after treatment of PCA and ML-385 were determined by DCFDA staining and ELISA assay kits. ML-385 elevated the levels of ROS and MDA while declining the SOD level in H₂O₂-induced MH7A cells treated by PCA [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d.

Figure 6: PCA inhibits H₂O₂-induced oxidative stress levels in RA-FLSs by activating Nrf2/Keap1 signaling pathway. (a and b) The ROS level in H₂O₂-induced MH7A cells treated by PCA and ML-385 was detected by DCFDA staining. (c and d) The levels of MDA and SOD in H₂O₂-induced MH7A cells treated by PCA and ML-385 were determined by respective ELISA assay kits. ***P < 0.001 versus control group. ^###P < 0.001 versus H₂O₂ group. ^&&&P < 0.001 versus H₂O₂ + PCA group. PCA: Protocatechuic acid, RA-FLSs: Rheumatoid arthritis fibroblast-like synoviocytes, MDA: Malonaldehyde, SOD: Superoxide dismutase, ELISA: Enzyme-linked immunosorbent assay.

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Discussion

RA-FLSs are important cells to maintain the normal structure and function of joints and are also the main target cells of RA. Inhibiting their lesions is an important method to alleviate RA.^[17] RA-FLSs are proliferative cells similar to tumor cells, and the expression of antiapoptosis-related genes is upregulated. The enhanced activity and decreased apoptosis of RA-FLs are the key causes of synovial dysplasia in RA patients.^[18] A large number of RA-FLSs will promote the secretion of inflammatory cytokines and promote the development of RA. RA-FLSs can also invade into cartilage and even bone tissue, leading to enlarged joints and reduced mobility.^[19],[20] This study also indicated that the viability, migration, and invasion of MH7A cells were enhanced and apoptosis of MH7A cells declined after H₂O₂ induction, which proved that the in vitro RA model was successfully constructed.

PCA alleviated brain edema and blood–brain barrier disruption in intracerebral hemorrhage by activating the Nrf2/HO-1 signaling pathway.^[21] PCA improved the macrophage endogenous antioxidant potential by JNK-mediated Nrf2 activation.^[22] PCA-activated JNK/Nrf2 survival signals to inhibit ROS overproduction in macrophages. Here, we also found that PCA could activate the Nrf2/Keap1 signaling pathway to suppress the viability, migration, invasion, and oxidative stress and promote the apoptosis of H₂O₂-induced MH7A cells.

Oxidative stress is one of the important known risk factors in the pathogenesis of RA,^[23] which runs through the whole process of RA pathogenesis and promotes the progression of the disease.^[13] Oxidative stress refers to a state in which the balance of ROS produced in the process of self-resistance to oxidation is destroyed.^[24] Nrf2, a key transcription factor of oxidative stress, belongs to the transcriptional activator family of leucine zippers.^[25] Keap1 is a precursor of the adaptor protein of the Cullin3 (CUL3)-E3 ubiquitin ligase complex.^[26] Studies have shown that activation of the Nrf2-KEAP1 pathway can effectively restore redox homeostasis.^[27],[28] Resveratrol can reduce inflammatory arthritis and prevent osteoarthritis in rats by NF-κB and HO-1/Nrf2 signaling pathways.^[29] To verify the role of the Nrf2/Keap1 signaling pathway in the protective effect of PCA on H₂O₂-induced MH7A cells and ML-385, a Nrf2 inhibitor, was applied and the results presented that ML-385 could weaken the protective effect of PCA on H₂O₂-induced MH7A cells, thereby promoting the viability, migration, invasion, and oxidative stress and decreasing the apoptosis of H₂O₂-induced MH7A cells, which indicates that PCA played its protective effect on H₂O₂-induced MH7A cells through Nrf2/Keap1 signaling pathway. Lentiviral silencing of Nrf2 (siNrf2) mRNA expression has been achieved in RA-FLSs^[14] and can also be used to inhibit the Nrf2/Keap1 signaling pathway in this study.

Conclusion

PCA reduced H₂O₂-induced viability, migration, invasion, and oxidative stress of MH7A cells by activating the Nrf2-Keap1 signaling pathway, which could be reversed by ML-385. There is an existing limitation and the present study is based on cell lines. An animal model for RA is existed, where PCA may be applied to conduct the preclinical study and test its therapeutic efficacy in vivo in our future study.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Financial support and sponsorship

This study was financially supported by the National Natural Science Foundation of China (No. 81973769).

Conflicts of interest

There are no conflicts of interest.

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