Cinnamaldehyde Induces the Expression of MicroRNA-1285-5p and MicroRNA-140-5p in Chondrocytes to Ameliorate the Apoptosis and Inflammatory Response

Abstract

Objective

Cinnamaldehyde (CA) is an active ingredient of Wenyang Tongluo capsule. This study was performed to investigate the function of CA on human chondrocytes.

Design

Different doses of CA were used to treat C28/I2 cells, which were stimulated by interleukin-1β (IL-1β), and then the viability and apoptosis of the cells were examined by cell counting kit-8 and flow cytometry. Interleukin-6 (IL-6), interleukin-20 (IL-20), and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to measure miR-1285-5p, miR-140-5p, IL-20, and high-mobility group box 1 (HMGB1) messenger RNA (mRNA) expression. Western blot assay was performed to detect IL-20, HMGB1, IKBα, phospho-IKBα, IKKα/β, and phospho-IKKα/β expression. Moreover, the relationships between miR-1285-5p and IL-20, as well as miR-140-5p and HMGB1, were validated by dual-luciferase reporter assay.

Results

CA promoted the viability and inhibited the apoptosis of C28/I2 cells stimulated by IL-1β and repressed IL-6, IL-20, and TNF-α levels. CA increased miR-1285-5p and miR-140-5p expression levels. MiR-1285-5p and miR-140-5p promoted the viability and inhibited the apoptosis and inflammation of C28/I2 cells. IL-20 was a target gene of miR-1285-5p, and HMGB1 was a target gene of miR-140-5p. Overexpression of IL-20 or HMGB1 could reverse the effect of CA on C28/I2 cells treated with IL-1β. In addition, HMGB1 increased phospho-IKBα and phospho-IKKα/β expression in IL-1β- and CA-treated C28/I2 cells.

Conclusions

CA protects chondrocytes via regulating miR-1285-5p/IL-20 axis and miR-140-5p/HMGB1/nuclear factor kappa B pathway.

Introduction

Osteoarthritis (OA) is a slowly progressive degenerative joint disease, with an increasing morbidity with aging and a higher morbidity in women than in men. ¹ The main clinical symptoms of OA include chronic pain, joint instability, stiffness, joint deformity, and narrowing of the joint space. ² Articular cartilage is composed of chondrocytes and extracellular matrix, which are rich in collagen and proteoglycan, and chondrocytes are the only cell type in articular cartilage, and chondrocyte apoptosis is positively correlated with the severity of cartilage destruction and matrix degradation. ³ Interleukin-1β (IL-1β) is an important proinflammatory factor that is often used to induce chondrocyte apoptosis and cartilage matrix degradation, thus mimicking OA in vivo and in vitro models. ⁴ Current clinical pharmacological treatments for OA are mainly analgesics such as nonsteroidal anti-inflammatory drugs, but they only relieve pain, and abuse of medication is common.^4,5 Therefore, the development of novel efficient and safe drugs is important for OA treatment.

Cinnamaldehyde (CA), also known as 3-phenylprop-2-enal, is an important bioactive ingredient extracted from Ramulus Cinnamomi and has been found to play a key regulatory role in a variety of diseases. For example, in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, CA can exert neuroprotective effects by modulating neuroinflammation, inhibiting oxidative stress, and improving synaptic connection. ⁶ In stress urinary incontinence model, CA can reduce nitric oxide level and nitric oxide synthase level in lipopolysaccharide-induced RAW 264.7 cells in a dose-dependent manner and increase urethral contractility. ⁷ In an animal model of ulcerative colitis, CA effectively attenuates the symptoms of colitis induced by dextran sulfate sodium salt, including weight loss, shortened colon length, and inflammatory cell infiltration, and CA inhibits the production of proinflammatory factors and NOD-like receptor (NLR) family pyrin domain containing 3 inflammasome in colonic tissues. ⁸ In addition, several studies have recently reported the anti-inflammatory effects of CA in OA cartilage degeneration.^9,10 However, the potential mechanisms of CA in treating OA have not been fully elucidated. In this study, we used IL-1β to induce the injury of human chondrocyte C28/I2 to establish an in vitro model of OA and explored the effects of CA on chondrocyte viability and apoptosis as well as inflammatory responses, and the underlying molecular mechanisms.

Method

Cell Culture and Treatment

Human chondrocyte cell line C28/I2 was purchased from Merck Millipore (Temecula, CA). C28/I2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) and incubated in an incubator (5% CO₂, 37 °C). When C28/I2 cell fusion reached 70%-80%, the cells were stimulated with 10 ng/ml of IL-1β for 24 hours to construct an in vitro model of OA.

The Preparation of CA

CA (C₆H₅CH=CHCHO) (molecular weight: 132.16, purity ≥95%) was purchased from Sigma-Aldrich (St. Louis, MO; Cat. No. W228613), and its molecular structure is shown in Figure 1A . CA was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and prepared into solutions at concentrations of 5, 10, and 20 μg/ml and stored at −20 °C. When C28/I2 cells reached 70%-80% fusion, the cells were treated with 10 ng/ml of IL-1β and different concentrations of CA (0, 5, 10 and 20 μg/ml) for 24 hours, and the control group was treated with 0.1% DMSO.

Figure 1.

Effect of CA on the viability and apoptosis of C28/I2 cells induced by IL-1β. (A) Molecular structure of CA. (B) IL-1β-induced C28/I2 cells were treated with 5, 10, and 20 μg/ml of CA, and cell survival rate was assayed by CCK-8 assay. (C and D) IL-1β-induced C28/I2 cells were treated with 5, 10, and 20 μg/ml of CA, and apoptosis was analyzed by flow cytometry. (E-G) IL-1β-induced C28/I2 cells were treated with 5, 10, and 20 μg/ml of CA, and the levels of IL-6, IL-20 and TNF-α in the cells were measured by ELISA. CA = cinnamaldehyde; IL-1β = interleukin-1β; CCK-8 = cell counting kit-8; IL-6 = interleukin-6; IL-20 = interleukin-20; TNF-α = tumor necrosis factor-α; ELISA = enzyme-linked immunosorbent assay; FITC = fluorescein isothiocyanate. *P < 0.05; **P < 0.01; ***P < 0.001.

Cell Transfection

C28/I2 cells were inoculated at a density of 2 × 10⁵ cells/ml on 6-well plates and cultured at 37 °C. Interleukin 20 (IL-20) and high-mobility group box 1 (HMGB1) overexpression plasmids, miR-1285-5p mimics, miR-140-5p mimics, and the negative control (miR-NC) were purchased from Genomeditech (Shanghai, China). When the cell fusion reached 60%, the above vectors were transfected into C28/I2 cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the transfection efficiency.

qRT-PCR

Total RNA from C28/I2 cells was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions, and RNA was reverse-transcribed using the ABI Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qRT-PCR was then performed using SYBR Green PCR Kit (TaKaRa, Shiga, Japan) on an ABI PRISM7500 Sequence Detection System (Applied Biosystems). U6 small nuclear 1 (U6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized gene expression levels using the 2^−ΔΔCt method. The primer sequences used in this study are as follows: miR-1285-5p: Forward: 5′-CGCAGGATCTCACTTTGTTG-3′; Reverse: 5′-CAGTGCGTGTCGTGGAGT-3′; miR-140-5p: Forward: 5′-TGCGGCAGTGGTTTTACCCTATG-3′; Reverse: 5′-CCAGTGCAGGGTCCGAGGT-3′; IL-20: Forward: 5′-ATGAAAGCCTCTAGTCTTGCCT-3′; Reverse: 5′-GCCCCGTATCTCAGAAAATCC-3′; HMGB1: Forward: 5′-AAAGCGGACAAGGCCCGTTAT-3′; Reverse: 5′-AAGAGGAAGAAGGCCGAAGGAG-3′; U6: Forward: 5′-CTCGCTTCGGCAGCACA-3′;Reverse: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH: Forward: 5′-GAAGGTGAAGGTCGGAGTC-3′; Reverse: 5′-GAAGATGGTGATGGGATTTC-3′.

Cell Counting Kit-8 (CCK-8) Assay

CCK-8 kit (Dojindo, Tokyo, Japan) was used to detect the viability of C28/I2 cells. The cells were inoculated in 96-well plates with 5,000 cells per well, followed by the addition of 10 μl of CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) and incubated at 37 °C with 5% CO₂ for 2 hours. Optical density values at 450 nm per well were measured using a microplate reader (Molecular Devices, Sunnyvale, CA).

Flow Cytometry Assay

The apoptosis of C28/I2 cells was detected using the Annexin V-FITC and propidium iodide (PI) Apoptosis Detection Kit (BD Pharmingen, San Diego, CA). C28/I2 cells were trypsinized with 0.25% trypsin, centrifuged, and then the cells were collected. The cells were washed twice with phosphate-buffered saline (PBS) containing 5% bull serum albumin and then incubated for 20 minutes with 5 µl of Annexin V-FITC solution and 10 µL of PI solution in dark, and after the cells were washed by PBS, the apoptosis was detected by a flow cytometer (BD Biosciences, San Jose, CA) and the data were analyzed using FlowJo 10.0 software (Tree Star, Ashland, OR) to calculate the apoptosis rate of each group. The “Q2+Q3” cell percentage was the apoptosis rate.

Enzyme-Linked Immunosorbent Assay (ELISA)

C28/I2 cells were placed in 6-well plates, cultured for 48 hours, and then chondrocytes were lysed and the supernatant was collected. The levels of interleukin-6 (IL-6), IL-20 and tumor necrosis factor-α (TNF-α) in C28/I2 cells were measured using the corresponding ELISA kits (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer’s instructions.

Luciferase Reporter Assay

Bioinformatics was used to predict the complementary binding sites between miR-1285-5p and IL-20 3′-untranslated region (3′-UTR), as well as miR-140-5p and HMGB1 3′-UTR, respectively. The sequences of IL-20 3′-UTR were subsequently amplified to construct Psi-CHECK2/IL-20 3′-UTR wild-type reporter plasmids (IL-20-WT) and Psi-CHECK2/ IL-20 3′-UTR mutant reporter plasmids (IL-20-MUT). Similarly, the Psi-CHECK2/HMGB1 3′-UTR wild-type reporter plasmids (HMGB1-WT) and Psi-CHECK2/HMGB1 3′-UTR mutation reporter plasmids (HMGB1-MUT) were constructed. Then, the above luciferase reporter plasmids were co-transfected into C28/I2 cells with miR-NC and miR-1285-5p mimics or miR-NC and miR-140-5p mimics. After 24 hours of transfection, the luciferase activity was detected using the Dual-Luciferase^® Reporter Assay System (Promega, Madison, WI). Relative luciferase activity = (Firefly luciferase activity / Renilla luciferase activity) × 100%.

Western Blot Assay

Total proteins were extracted from cells using the radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) and quantified using the bicinchoninic acid kit (Sangon Biotech, Shanghai, China). The protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Subsequently, the membranes were blocked with 5% skimmed milk and then incubated with anti-IL-20 antibody (1:1,000, MA5-26604; Thermo Fisher), anti-HMGB1 antibody (1:1,000, ab190377; Abcam Inc., Cambridge, UK), anti-GAPDH antibody (1: 1,000, ab8245; Abcam Inc.), anti-phospho-IKBα (p-IKBα) antibody (1:1,000, ab133462; Abcam Inc.), anti-IKBα antibody (1:1,000, ab32518; Abcam Inc.), anti-IKKα/β antibody (1:1,000, ab178870; Abcam Inc.), and anti-phospho-IKKα/β antibody (p-IKKα/β; 1:1,000, ab194528; Abcam Inc.) overnight at 4 °C and the membranes were subsequently incubated with horseradish peroxidase–conjugated secondary antibody (1:2,000, ab6702/ab205719; Abcam Inc.) for 2 hours at room temperature. The protein bands were then developed using the ECL luminescence reagent (7Sea PharmTech, Shanghai, China), with GAPDH as the internal reference.

Microarray Analysis

GSE175961 dataset was downloaded from the Gene Expression Omnibus (GEO) database, and the online analysis tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r) provided by the GEO database was adopted to analyze the differentially expressed microRNAs (miRNAs) in cartilage tissue of OA patients and healthy controls. Log₂|fold change| <1 and P < 0.05 were used as the screening criteria.

Venn Diagram

GeneCards database (https://www.genecards.org/) was used to identify the target genes associated with OA. TargetScan database (http://www.targetscan.org/vert_72/) was applied to predict the downstream target genes of miR-1285-5p. StarBase database (https://starbase.sysu.edu.cn/) was used to predict the downstream target genes of miR-140-5p. The related genes of OA were screened from the GeneCards database. And the Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to plot the overlapping genes between the TargetScan database and the GeneCards database, as well as the StarBase database and the GeneCards database.

Bioinformatics Analysis Based on Annotation, Visualization, and Integrated Discovery (DAVID) Database

DAVID database (https://david.ncifcrf.gov/home.jsp), an online website for gene function annotation and analysis, was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, with “P < 0.05” as inclusion criteria.

Statistical Analysis

GraphPad Prism 7.0 was used for statistical analysis, and all experiments were conducted 3 times, and the results were expressed as “mean ± standard deviation”; Student’s t test and 1-way analysis of variance (ANOVA) were used for comparisons. P < 0.05 signified statistical significance.

Results

Effect of CA on Proliferation and Apoptosis of C28/I2 Cells Which Were Treated With IL-1β

C28/I2 cells were treated with IL-1β and different doses of CA (0, 5, 10, and 20 μg/ml) for 24 hours, and the cell viability was detected by CCK-8 assay. The results showed a significant decrease in C28/I2 cell survival rate after stimulation with IL-1β compared with the control group, whereas cell viability was significantly promoted after treatment with 5, 10, and 20 μg/ml of CA compared with the IL-1β group in a time- and concentration-dependent manner ( Fig. 1B ). In addition, flow cytometry showed that the apoptosis rate of C28/I2 cells was significantly higher after stimulation with IL-1β compared with the control group, while the apoptosis rate was significantly lower after treatment with different concentrations of CA ( Fig. 1C and D ). The results of ELISA showed that IL-1β significantly increased the levels of IL-6, IL-20, and TNF-α in C28/I2 cells compared with the control group, while the levels of IL-6, IL-20, and TNF-α were significantly decreased in cells treated with 5, 10, and 20 μg/ml of CA ( Fig. 1E-G ). The above results indicated that the in vitro cell model of OA was successfully constructed and CA significantly promoted the survival rate of C28/I2 cells, as well as inhibited the apoptosis and secretion of proinflammatory factors.

CA Promotes MiR-1285-5p and MiR-140-5p Expression Levels

To investigate the mechanism of CA with the in vitro model of OA, miRNAs differentially expressed in cartilage tissues of OA patients and healthy controls were analyzed with a dataset GSE175961, and miR-1285-5p expression was found to be downregulated (log2fold change = −5.05, P < 0.001) ( Fig. 2A and B ). MiR-1285-5p expression was subsequently examined by qRT-PCR assay, which showed that miR-1285-5p expression was downregulated in C28/I2 cells treated with IL-1β compared with the control group, while miR-1285-5p expression level was significantly increased after treatment with different concentrations of CA ( Fig. 2C ). Moreover, miR-140-5p expression was also examined, and the results showed that miR-140-5p expression was downregulated in C28/I2 cells treated with IL-1β compared with the control group, while miR-140-5p expression was significantly increased after treatment with different concentrations of CA ( Fig. 2D ), indicating that for C28/I2 cells, CA could promote miR-1285-5p and miR-140-5p expression levels.

Figure 2.

CA promotes miR-1285-5p and miR-140-5p expressions. (A) Volcano maps of miRNAs differentially expressed in cartilage tissue of OA patients and healthy controls in GSE175961. (B) MiR-1285-5p expression in cartilage tissue of OA patients and healthy controls in GSE175961. (C) IL-1β-induced C28/I2 cells were treated with 5, 10, and 20 μg/ml of CA. qRT-PCR assays were performed to detect miR-1285-5p expression in the cells. (D) IL-1β-induced C28/I2 cells were treated with 5, 10, and 20 μg/ml of CA. qRT-PCR assays were performed to detect miR-140-5p expression in the cells. CA = cinnamaldehyde; OA = osteoarthritis; IL-1β = interleukin-1β; qRT-PCR = quantitative real-time polymerase chain reaction. **P < 0.01, ***P < 0.001.

MiR-1285-5p and MiR-140-5p Attenuate IL-1β-Induced Injury of C28/I2 Cells

To explore the function of miR-1285-5p and miR-140-5p in the in vitro model of OA, miR-1285-5p mimics and miR-140-5p mimics and negative control miR-NC were transfected into C28/I2 cells, respectively, to construct miR-1285-5p and miR-140-5p overexpression models, and qRT-PCR validated the successful transfection ( Fig. 3A ). CCK-8 assay suggested that the cell survival rate is higher in the miR-1285-5p overexpression group compared with that in the miR-NC group, and similarly, the cell survival rate in the miR-140-5p overexpression group was higher compared with that in the miR-NC group ( Fig. 3B ). Flow cytometry showed a significant decrease in apoptosis in both the miR-1285-5p overexpression and miR-140-5p overexpression groups compared with the miR-NC group ( Fig. 3C ). Furthermore, the results of ELISA showed that the levels of IL-6, IL-20, and TNF-α in cells in the miR-1285-5p mimics group and the miR-140-5p mimics group were significantly decreased compared with the miR-NC group ( Fig. 3D-F ), indicating that overexpression of miR-1285-5p or miR-140-5p significantly promoted the survival rate of C28/I2 cells treated with IL-1β and inhibited apoptosis and inflammatory responses.

Figure 3.

MiR-1285-5p and miR-140-5p attenuate IL-1β-induced injury in C28/I2 cells. (A) MiR-1285-5p mimics and miR-140-5p mimics and negative control miR-NC were transfected into C28/I2 cells, respectively, and the transfection efficiency was verified by qRT-PCR. (B) CCK-8 assay was conducted to detect cell viability after transfection with miR-1285-5p mimics and miR-140-5p mimics. (C) Flow cytometry was executed to analyze the apoptosis after transfection with miR-1285-5p mimics and miR-140-5p mimics. (D-F) ELISA was implemented to detect the levels of IL-6, IL-20, and TNF-α in cells transfected with miR-1285-5p mimics and miR-140-5p mimics. qRT-PCR = quantitative real-time polymerase chain reaction; CCK-8 = cell counting kit-8; IL-6 = interleukin-6; IL-20 = interleukin-20; TNF-α = tumor necrosis factor-α; IL-1β = interleukin-1β; PI = propidium Iodide. *P < 0.05; **P < 0.01; ***P < 0.001.

IL-20 Is a Downstream Target of MiR-1285-5p

To investigate the downstream regulatory mechanism of miR-1285-5p in OA, the GeneCards database was used to analyze genes associated with OA, and the TargetScan database was used to predict the messenger RNAs (mRNAs) containing complementary binding sites to miR-1285-5p, and 452 overlapping genes were screened by plotting a Venn diagram ( Fig. 4A ). In addition, these 452 genes were subjected to GO and KEGG pathway enrichment analysis with DAVID database. GO analysis showed that the biological process (BP) terms of these target genes were significantly enriched in the negative regulation of cell proliferation, apoptotic signaling pathway, negative regulation of tumor necrosis factor production, positive regulation of interleukin-6 secretion, and so on. Cellular components (CC) were significantly enriched in extracellular space, cell surface, integral component of the plasma membrane, and so on. Molecular function (MF) was significantly enriched in protein binding, growth factor binding, and so on ( Fig. 4B ). The results of KEGG analysis showed that these target genes were closely associated with focal adhesion, apoptosis, NF-kappa B signaling pathway, and so on ( Fig. 4C ). IL-20 is one of the important target genes, and IL-20 3′-UTR contains a potential binding site complementary to miR-1285-5p ( Fig. 4D ). In addition, dual-luciferase reporter gene experiments showed that miR-1285-5p mimics significantly reduced the luciferase activity of IL-20-WT, while they had no effect on the luciferase activity of IL-20-MUT ( Fig. 4E ). In addition, qRT-PCR and Western blot assays showed that IL-20 expression was upregulated in IL-1β-induced C28/I2 cells compared with the control group, while miR-1285-5p overexpression significantly decreased IL-20 expression in C28/I2 cells ( Fig. 4F ).

Figure 4.

IL-20 is a downstream target of miR-1285-5p. (A) The target genes screened from GeneCards and TargetScan databases were plotted in a Venn diagram, and the intersecting target genes were analyzed. (B and C) The 452 intersecting target genes in the Venn diagram were subjected to GO and KEGG pathway enrichment analysis. (D) IL-20 3′-UTR contains a potential binding site complementary to miR-1285-5p. (E) Dual-luciferase reporter gene assay confirmed that miR-1285-5p bound directly to IL-20. (F) MiR-1285-5p mimics were transfected into C28/I2 cells and IL-20 expression was detected using qRT-PCR and Western blot assays. IL-20 = interleukin-20; GO = Gene Ontology; KEGG = Kyoto Encyclopedia of Genes and Genomes; qRT-PCR = quantitative real-time polymerase chain reaction; IL-1β = interleukin-1β. **P < 0.01, ***P < 0.001.

HMGB1 Is a Downstream Target of MiR-140-5p

The downstream mechanism of miR-140-5p in OA pathogenesis was also investigated. The OA-related genes from the GeneCards database and the mRNAs containing complementary binding sites with miR-140-5p were plotted by a Venn diagram, and 336 genes were screened out ( Fig. 5A ). GO analysis showed that the BP terms of these target genes were significantly enriched in negative regulation of cell proliferation, growth, protein secretion, and so on. The CC terms were significantly enriched in focal adhesion, extracellular bodies, cell surface, and so on. The MF terms were significantly focused on homologous protein binding, chromatin binding, and so on ( Fig. 5B ). The KEGG analysis unveiled that these target genes were closely associated with apoptosis, phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, transforming growth factor-beta (TGF-beta) signaling pathway, and other pathways ( Fig. 5C ). Notably, HMGB1 3′-UTR contained a potential binding site complementary to miR-140-5p ( Fig. 5D ). Dual-luciferase reporter gene assay showed that miR-140-5p mimics significantly reduced the luciferase activity of HMGB1-WT, while they had no effect on the luciferase activity of HMGB1-MUT ( Fig. 5E ). In addition, qRT-PCR and Western blot assays showed that HMGB1 expression was upregulated in C28/I2 cells treated with IL-1β compared with controls, while miR-140-5p overexpression significantly decreased HMGB1 expression in C28/I2 cells ( Fig. 5F ).

Figure 5.

HMGB1 is a downstream target of miR-140-5p. (A) The target genes screened from GeneCards and StarBase databases were plotted in Venn diagrams, and the intersecting target genes were analyzed. (B and C) The 336 intersecting target genes in the Venn diagram were subjected to GO and KEGG pathway enrichment analysis. (D) HMGB1 3′-UTR contains a potential binding site complementary to miR-140-5p. (E) Dual-luciferase reporter gene assay confirmed that miR-140-5p bound directly to HMGB1. (F) MiR-140-5p mimics were transfected into C28/I2 cells and HMGB1 expression was detected using qRT-PCR and Western blot assays. HMGB1 = high-mobility group box 1; GO = Gene Ontology; KEGG = Kyoto Encyclopedia of Genes and Genomes; 3′-UTR = 3′-untranslated region, qRT-PCR = quantitative real-time polymerase chain reaction; IL-1β = interleukin-1β; WT = wild type; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; PI3K-Akt = phosphoinositide 3-kinase-protein kinase B; TGF-beta = transforming growth factor-beta. **P < 0.01, ***P < 0.001.

CA Attenuates IL-1β-Induced Injury of C28/I2 Cells by Modulating IL-20 and HMGB1

Next, IL-20 and HMGB1 overexpression plasmids were transfected into C28/I2 cells, respectively. Western blot assay indicated that CA could inhibit the expression of IL-20 and HMGB1, and the transfection was successful ( Fig. 6A and B ). CCK-8, flow cytometry, and ELISA revealed that IL-20 or HMGB1 overexpression plasmids could reverse the effect of CA on cell survival rate, apoptosis, and inflammation ( Fig. 6C-G ). Hence, it was concluded that CA attenuated IL-1β-induced injury of C28/I2 cells by IL-20 and HMGB1.

Figure 6.

CA attenuates IL-1β-induced injury of C28/I2 cells by regulating IL-20 and HMGB1. (A) Western blot assay was used to detect IL-20 expression in cells transfected with IL-20 overexpression plasmid, and then treated with IL-1β and CA. (B) Western blot assay was used to detect HMGB1 expression in cells transfected with HMGB1 overexpression plasmid, and then treated with IL-1β and CA. (C-G) CCK-8 assay, flow cytometry, and ELISA were used to measure cell survival rate in cells transfected with HMGB1 overexpression plasmid, and then treated with IL-1β and CA. CA = cinnamaldehyde; IL-1β = interleukin-1β; IL-20 = interleukin-20; HMGB1 = high-mobility group box 1; CCK-8 = cell counting kit-8; ELISA = enzyme-linked immunosorbent assay; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL-6 = interleukin-6; TNF-α = tumor necrosis factor-α; OE = overexpression. *P < 0.05, **P < 0.01, ***P < 0.001.

CA Could Regulate Nuclear Factor-Kappa B (NF-κB) Pathway via HMGB1

In addition, to identify the signaling pathway which was activated by IL-20 and HMGB1 in OA, we detected the expression levels of NF-κB pathway–related proteins. The results showed that HMGB1 overexpression could elevate the expression of p-IKBα and p-IKKα/β which were inhibited by CA; however, IL-20 had no effect on p-IKBα and p-IKKα/β expression ( Fig. 7 ).

Figure 7.

CA could regulate NF-κB pathway by HMGB1. Western blot assay was used to detect IKBα, p-IKBα, p-IKKα/β, and IKKα/β expression. CA = cinnamaldehyde; NF-κB = nuclear factor-kappa B; HMGB1 = high-mobility group box 1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; OE = overexpression. NS P > 0.05, ***P < 0.001.

Discussion

CA is a natural compound with various pharmacological effects such as antibacterial, anti-inflammatory, and antioxidant effects.^11-13 In OA, it was found that trans-cinnamaldehyde (TCA) decreases the protein expression levels of interleukin-8, TNF-α, and prostaglandin E2 in chondrocytes by regulating the PI3K/Akt pathway. ⁹ It is also found that TCA at a concentration of 10 μg/ml significantly reduces matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)-4, and ADAMTS-5 expression levels in the in vitro model of OA induced by IL-1β. Furthermore, TCA significantly reduces cartilage degradation in a rat model of OA and exerts a protective effect on chondrocytes by inhibiting NF-κB and p38-mitogen-activated protein kinase 8 pathways, suggesting that TCA may act as an anti-inflammatory agent for OA. ¹⁰ In this work, we found that 5, 10, and 20 μg/ml of CA significantly promoted the viability and inhibited apoptosis and inflammatory responses of C28/I2 cells, which were treated with IL-1β, suggesting a protective effect of CA on IL-1β-induced C28/I2 cells.

MiRNA is a highly conserved endogenous noncoding RNA of about 25 nt in length, which can modulate the expression of target genes by binding to the 3′-UTR of target mRNA, thus affecting cell proliferation, differentiation, apoptosis, senescence, and other biological processes and participating in the pathogenesis and progression of various diseases. ¹² A growing number of studies have found that miRNAs are involved in the progression of OA, such as miR-29b-3p, ¹⁴ miR-146a, ¹⁵ and miR-27a. ¹⁶ However, the role of miR-1285-5p in OA is unknown. Reportedly, miR-1285-5p is involved in the pathological processes of a variety of diseases. For example, in non–small cell lung cancer, miR-1285-5p promotes the proliferation, migration, and invasion of non–small cell lung cancer cells by targeting cadherin 1 and Smad4. ¹⁷ In breast cancer, miR-1285-5p significantly inhibits the proliferation of breast cancer cells by targeting transmembrane protein 194A. ¹⁸ In prostate cancer, miR-1285-5p is a downstream target of hsa_circ_0001206, and transfection of miR-1285-5p mimics partially reverses the inhibitory effects of hsa_circ_0001206 overexpression on the proliferation, migration, and invasion of prostate cancer cells in vitro and tumor growth in vivo. ¹⁹ In addition, previous studies have shown that miR-140-5p is involved in the pathological progression of OA. For example, miR-140-5p expression is downregulated in OA cartilage tissue, and downregulation of miR-140-5p significantly reverses the inhibitory effect of knockdown of H19 on chondrocyte matrix degradation and calcification markers. ²⁰ It is also found that miR-140-5p expression is downregulated in cartilage tissues of OA patients, and miR-140-5p overexpression inhibits apoptosis and promotes proliferation and autophagy of human primary chondrocytes by downregulating fucosyltransferase 1. ²¹ In this work, we found that miR-1285-5p and miR-140-5p were downregulated in IL-1β-induced C28/I2 cells, and overexpression of miR-1285-5p and miR-140-5p promoted the viability of C28/I2 cells and inhibited apoptosis and inflammatory responses. These data support that miR-1285-5p and miR-140-5p are 2 protective factors for chondrocytes in the pathogenesis of OA.

IL-20, a member of the IL-10 family, is a proinflammatory factor that regulates inflammation, angiogenesis, chemotaxis, and apoptosis, and it is involved in the pathogenesis of OA. ²² For example, IL-20 is found to induce the expression of proinflammatory factors and OA-related genes in OA synovial fibroblasts and chondrocytes by activating mitogen-activated protein kinase 1 and mitogen-activated protein kinase 8 signaling, thus promoting OA progression. ²³ It is also reported that IL-20 level is elevated in the serum of OA patients and may be involved in the systemic inflammatory response. ²⁴ In this work, we found that IL-20 was a downstream target of miR-1285-5p, and IL-20 expression was upregulated in IL-1β-induced C28/I2 cells, and miR-1285-5p overexpression inhibited IL-20 expression. In addition, HMGB1 is a highly conserved nonhistone DNA-binding protein expressed in the nucleus of eukaryotic cells. ²⁵ In OA, HMGB1 expression is found to be upregulated in mouse chondrocytes treated with lipopolysaccharide, and HMGB1 is a downstream target of miR-410-3p, which targets HMGB1 and regulates chondrocyte apoptosis and inflammation through the NF-κB signaling pathway. ²⁶ It is also found that HMGB1 expression is upregulated in IL-1β-induced C28/I2 and that HMGB1 is a downstream target of miR-140-5p, which inhibits IL-1β-induced inflammation, MMP expression, and apoptosis in chondrocytes through regulation of HMGB1. ²⁷ In this work, we found that HMGB1 was a downstream target of miR-140-5p and CA could inhibit p-IKBα and p-IKKα/β expression levels by regulating HMGB1, indicating that in OA, CA could regulate miR-1285-5p/IL-20 axis and miR-140-5p/HMGB1/NF-κB pathway.

In conclusion, this study confirms that CA can promote the viability of C28/I2 chondrocytes and inhibit apoptosis and inflammatory response by regulating miR-1285-5p/IL-20 axis and miR-140-5p/HMGB1/NF-κB pathway, thereby inhibiting OA progression. Our study suggests that CA is a promising natural drug for OA treatment.