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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 4 | Page : 228-238 |
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Ring-finger protein 5 attenuates oxygen-glucose deprivation and reperfusion-induced mitochondrial dysfunction and inflammation in cardiomyocytes by inhibiting the S100A8/MYD88/NF-κB axis
Xuesi Chen, Yingjie Wu, Yingchun Bao
Department of Cardiovascular, The Affiliated People's Hospital of Ningbo University, Ningbo, Zhejiang, China
| Date of Submission | 05-Dec-2022 |
| Date of Decision | 20-Mar-2023 |
| Date of Acceptance | 30-Mar-2023 |
| Date of Web Publication | 27-Jun-2023 |
Correspondence Address:
Dr. Yingjie Wu
Department of Cardiovascular, The Affiliated People's Hospital of Ningbo University, No. 251, Baizhang East Road, Ningbo 315000, Zhejiang
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-22-00140
Mitochondrial dysfunction is closely intertwined with the progression of heart failure (HF). Ring-finger protein 5 (RNF5) is an E3 ubiquitin ligase, whose deletion induces the enhanced S100A8 expression. S100A8 regulates the mitochondrial dysfunction and S100A8/myeloid differentiation factor 88 (MYD88)/nuclear factor-kappa B (NF-κB) pathway promotes an inflammatory response; however, whether RNF5 modulated mitochondrial dysregulation and inflammation through the S100A8/MYD88/NF-κB axis remains unknown. Here, H9c2 cells were stimulated with oxygen-glucose deprivation/reperfusion (OGD/R) to build a HF model in vitro. RNF5 level was assessed in gene expression omnibus database and in OGD/R-induced H9c2 cells with reverse transcriptase quantitative polymerase chain reaction and western blot. The RNF5 level was overexpressed via transfecting RNF5 overexpression plasmids into H9c2 cells. The role and mechanism of RNF5 in OGD/R-elicited H9c2 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, spectrophotometry, flow cytometry, mitochondrial membrane potential (MMP) measurement, enzyme-linked immunosorbent assay and western blot assays. The RNF5 expression was downregulated both in silico and in OGD/R-stimulated H9c2 cells. OGD/R treatment caused a decrease in the cell viability, the MMP level, and the translational expression of mito-cyt-c and NF-κB-cyto, and an elevation in the concentrations of lactate dehydrogenase and creatine kinase myocardial band, the apoptosis rate, the inflammatory factor release, and the relative protein expression of cyto-cyt-c, S100A8, MYD88 and NF-κB-nuc in H9c2 cells. Upregulation of RNF5 reversed these indicators in OGD/R-stimulated H9c2 cells. Altogether, based on these outcomes, we concluded that RNF5 impeded mitochondrial dysfunction and inflammation through attenuating the S100A8/MYD88/NF-κB axis in OGD/R-stimulated H9c2 cells.
Keywords: Heart failure, inflammation, mitochondrial dysfunction, ring-finger protein 5, S100A8/MYD88/NF-κB axis
How to cite this article:
Chen X, Wu Y, Bao Y. Ring-finger protein 5 attenuates oxygen-glucose deprivation and reperfusion-induced mitochondrial dysfunction and inflammation in cardiomyocytes by inhibiting the S100A8/MYD88/NF-κB axis. Chin J Physiol 2023;66:228-38 |
How to cite this URL:
Chen X, Wu Y, Bao Y. Ring-finger protein 5 attenuates oxygen-glucose deprivation and reperfusion-induced mitochondrial dysfunction and inflammation in cardiomyocytes by inhibiting the S100A8/MYD88/NF-κB axis. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:228-38. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/228/379840 |
| Introduction | |  |
Heart failure (HF) resulted from unremitting pressure overload is a common clinical disease, which has been a dominating reason of morbidity, hospitalizations, and mortality among cardiovascular causes, thereby contributing to a prominent public health matter and financial load globally.[1] HF has been estimated to be a 1%–2% prevalence with an increasing tendency.[2] Despite advances in treatment of HF such as guideline-directed therapies, the rehospitalization rates and overall mortality remain high and the prognosis is still unfavorable with an approximately 50% 5-year mortality rate.[3],[4] The potential mechanism of HF has been the star of cardiovascular study, whose resolution benefits to the exploitation of new therapeutic targets. Mitochondrial dysfunction and energy deficiency have been evidenced to be closely intertwined with the progression of HF.[5] Experts in cardiovascular diseases, HF and mitochondria research areas convened by the National Heart, Lung, and Blood Institute have proposed short- and long-term recommendations for the prevention and therapy of HF through utilizing mitochondria-based strategies.[6] Meanwhile, mitochondria have been also reported to be a target for the treatment of HF by using traditional Chinese medicines.[7] Therefore, seeking target for mitochondrial dysfunction can promote the clinical development of HF prevention and treatment.
As an E3 ubiquitin ligase, ring-finger protein 5 (RNF5) primarily situated at the endoplasmic reticulum (ER) and mitochondria is the composition of the UBC6e-p97 complex, which is involved in ER-associated degradation, a pathway implicated with sustaining protein homeostasis.[8] Evidence has pointed to RNF5 may be a therapeutic target in a wide variety of disease models, such as tumors,[9],[10] inflammatory bowel disease,[11] virus-related disease,[12],[13] cystic fibrosis,[14] nonalcoholic fatty liver disease[15] and hepatic ischemia-reperfusion injury.[16] Moreover, RNF5 has been confirmed to be upregulated in the heart of mice with cardiac hypertrophy, and function verification shows that RNF5 suppresses cardiac hypertrophy through facilitating stimulator of interferon genes degradation via K48-linked polyubiquitination.[17] Deletion of RNF5 can induce enhanced expression of S100A8,[11] a type of inflammatory factor that modulates apoptosis and autophagy in the early stage of myocardial infarction via the mitogen-activated protein kinases and phosphatidylinositol 3-kinase-protein kinase B signaling,[18] and contributes to cardiomyocyte death and mitochondrial dysfunction during ischemic/reperfusion injury.[19] In addition, S100A8/MYD88/NF-κB pathway induces cardiac hypertrophy caused by thyroid hormone and promotes an inflammatory response.[20] Collectively, based on these findings, it is reasonable to assume that the RNF5 may regulate inflammatory response and mitochondrial dysregulation in HF involving in the S100A8/MYD88/NF-κB axis.
Thus, to verify this hypothesis, H9c2 cells were stimulated with oxygen-glucose deprivation/reperfusion (OGD/R) to construct a HF model in vitro. RNF5 expression was analyzed in silico and in OGD/R-induced H9c2 cells. Meanwhile, the role of RNF5 in inflammatory factor release and mitochondrial dysregulation was addressed in OGD/R-elicited H9c2 cells. Further, the potential mechanism involving in S100A8/MYD88/NF-κB axis was also discussed in vitro.
| Materials and Methods | | |
Analysis of ring-finger protein 5 expression in silico
The GSE84796 microarray dataset was extracted from the Gene Expression Omnibus (GEO) database from the Agilent GPL14550 platform, which included seven control samples from the free wall of the human left ventricle taken from the healthy heart of an organ donor, and ten patient samples from the human left ventricular free wall heart tissue from patients with end-stage HF at the time of heart transplantation. The differently expressed genes were determined in control and HF groups by employing the LIMMA package in R language.[21]
Cell culture and treatment
Rat cardiomyocyte H9c2 cells (CL-0089) were prepared from Procell (Wuhan, China), which were maintained in Dulbecco's Modified Eagle's Medium (DMEM, PM150210, Procell) provided with 10% fetal bovine serum (FBS, Gibco, Rockville, MD, USA) and 1% penicillin/streptomycin (PB180120, Procell) with 5% carbon dioxide (CO2) at 37°C. OGD/R-challenged H9c2 cells were treated as the previous description.[22] Briefly, after cells reached confluence at 70-80%, H9c2 cells were transferred into fresh DMEM without glucose (PM150270, Procell) and hatched in an anaerobic chamber (95% N2 and 5% CO2) for 4 h at 37°C. Next, the culture medium was substituted by normal DMEM and cells were cultured in a normoxic chamber (95% air and 5% CO2) for 24 h at 37°C as reperfusion. H9c2 cells that were invariably incubated with normal DMEM at 37°C were acted as control cells. To address the role of RNF5 and S100A8 in OGD/R-induced H9c2 cells, pcDNA vector plasmids containing the RNF5 sequences (RNF5) and/or the S100A8 sequence (S100A8), as well as the empty vector plasmids (negative control, NC) were accessed into H9c2 cells through Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Transfection assay was conducted 24 h before OGD/R induction.
Reverse transcriptase quantitative polymerase chain reaction
H9c2 cells from OGD/R-treated group or control group were administrated with Trizol (15596026, Invitrogen) to yielded the total ribonucleic acid (RNA), which was subsequently conducted with reverse transcription by a PrimeScript RT reagent Kit (RR037A, Takara, Dalian, China). RT-quantitative polymerase chain reaction (RT-qPCR) was executed on the A PIKORed 96 (Thermo Fisher Scientific, Waltham, MA, USA) using the TB Green TM Premix Ex TaqTM Ⅱ (Tli RNaseH Plus) (RR820A, Takara). The conditions of RT-qPCR were 95°C for 5 min with 40 cycles for predenaturation, 95°C for 30 s for denaturation, 61°C for 40 s for annealing/extension. The mRNA expression of RNF5 was obtained by a 2−ΔΔCt means. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) worked as an internal reference gene. RNF5 and GAPDH primers were: RNF5-F: 5'-GAATGCCCGGTGTGTAAAGC-3'; RNF5-R: 5'-GGGGTGGAGTTTTCAATCTGG-3'; GAPDH-F: 5'-ACGGCAAGTTCAACGGCACAGTCA-3'; GAPDH-R: 5'-CCACGACATACTCAGCACCAGCATCA-3'.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide analysis
40,000 H9c2 cells were distributed into 96-well plates and cultured at 37°C in 5% CO2. After different treatments, H9c2 cells were administrated with 10 μl MTT solution (M1020, Solarbio, Beijing, China) for 4 h, and then the culture supernatant was thrown away before 100 μl dimethyl sulfoxide (D8371, Solarbio) addition to each well for the dissolution of crystals. The absorbance was obtained with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm.
Examination of activity of creatine kinase myocardial band and lactate dehydrogenase
Following the different treatments, H9c2 cells were collected via centrifugation for 2 min at 250 × g at room temperature. The levels of creatine kinase myocardial band (CK-MB) and lactate dehydrogenase (LDH) in the supernatant were determined by CK-MB isoenzyme Assay Kit (H197-1-1) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and LDH activity assay kit (BC0685, Solarbio) following the operation instruction. The absorbance was gained under a microplate reader (Thermo Fisher Scientific) at 340 nm (for CK-MB) and 450 nm (for LDH), respectively.
Flow cytometry
The apoptosis of H9c2 cells was measured by the flow cytometry assay[23] with an Annexin V-FITC/PI Apoptosis Detection Kit (CA1020, Solarbio). In brief, 24-well plates were spread with 2.5 × 105 H9c2 cells per well and hatched with 5% CO2 at 37°C. After the different treatments, cells were yielded, washed with phosphate buffer saline (P1020, Solarbio), mixed by 0.5 mL of bind buffer, and stained with Annexin V/FITC 5 μL and 5 μL propidium iodide for 15 min without light at room temperature. The H9c2 cell apoptosis was gained on a FACScan flow cytometry with CellQuest software (BD Biosciences, NJ, USA).
Detection of mitochondrial membrane potential
The level of mitochondrial membrane potential (MMP) was measured by the MMP Assay Kit with JC-1 (M8650, Solarbio) following the operating manual. After the different administrations, H9c2 cells were gathered and mixed with 1 ml DMEM with JC-1 fluorescent dye and hatched for 20 min at 37°C. Following the centrifugation at 4°C for 5 min at 600 × g, the supernatant was abandoned and then cells were resuspended in 1× JC-1 buffer. The cell nuclei were dyed with 4',6-diamidino-2-phenylindole (DAPI, C0065, Solarbio). The pictures were observed by a fluorescent microscope, and five fields were randomly chosen for the quantification.
Enzyme-linked immunosorbent assay
Concentrations of interleukin (IL)-6, IL-1β, IL-8, and monocyte chemoattractant protein (MCP)-1 in the supernatant were measured after H9c2 cells were treated as different indications by using commercial enzyme-linked immunosorbent assay (ELISA) kits, including Rat IL-6 ELISA KIT (SEKR-0005, Solarbio), Rat IL-1β ELISA KIT (SEKR-0002, Solarbio), Rat CXCL1/KC (IL-8) ELISA KIT (SEKR-0014, Solarbio) and Rat MCP-1 ELISA KIT (SEKR-0024, Solarbio) in line with the operating manual. The absorbance was read with a microplate reader (Thermo Fisher Scientific) at 450 nm.
Western blot
According to the previous report,[24] total proteins from H9c2 cells were prepared by RIPA buffer (R0010, Solarbio) and their concentrations were confirmed with the BCA Protein Assay Kit (PC0020, Solarbio) in keeping with the manufacturer's specifications. 20 μg protein samples were dissolved and electrically shifted onto a polyvinylidene fluoride membrane (IPVH00010, EMD Millipore, Billerica, MA, USA). Following the block with 5% skim milk (D8340, Solarbio) for 60 min at room temperature, the membranes were introduced with primary antibodies for diverse proteins (RNF5, 1:1000, ab128200, Abcam, Cambridge, UK; Cytochrome C (cyt-c), 1:1000, ab90529, Abcam; S100A8, 1:500, K002925P, Solarbio; myeloid differentiation factor 88 (MYD88), 1:1000, ab131071, Abcam; nuclear factor-kappaB (NF-κB), 1:1000, #8242, Cell Signaling Technology, Inc., Danvers, MA, USA; GAPDH, 1:2500, ab9485, Abcam) overnight at 4°C. Subsequently, the membranes were hatched with goat anti-rabbit Immunoglobulin G (IgG) H&L (horseradish peroxidase) (1:20000, ab6721, Abcam) for 2 h at room temperature and exposed by an electrochemiluminescence assay (PE0010, Solarbio). The band intensity was determined via ImageJ software (National Institutes of Health, Bethesda, MD, USA). To detect the protein level of mitochondrial cytochrome c (mito-cyt-c), mitochondria were segregated from H9c2 cells by Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China) based on the operating manual. The cytochrome c oxidase subunit IV (COX IV) antibody (1:1000, ab16056) acted as mitochondrial loading control. In addition, the translational expression of NF-κB (NF-κB-nuc) in cell nuclei was examined after the nuclei were extracted from H9c2 cells via Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime) in line with the operation instruction. The Lamin B1 antibody (1:1000, ab16048) served as the nuclear envelope marker.
Statistical analysis
The form of mean ± standard deviation was used to present all the results. Statistical differences were determined by the Student's t-test between the two groups and differences were tested by the one-way analysis of variance for over two groups followed by post hoc Bonferroni test by SPSS 26.0 software (IBM, Armonk, New York, NY, USA). P < 0.05 indicated the statistically significant difference.
| Results | | |
Ring-finger protein 5 was low expressed in OGD/R-elicited H9c2 cells
To address the role of RNF5 in OGD/R-constructed HF, the level of RNF5 was firstly detected both in GEO (GSE84796) database and in OGD/R-stimulated H9c2 cells. Data from [Figure 1]a revealed that the RNF5 level in the human left ventricular free wall heart tissue from patients with end-stage HF was significantly lower than that in the free wall of the human left ventricle from the healthy heart. Meanwhile, both the relative mRNA and protein expressions of RNF5 were consistently reduced in OGD/R-induced H9c2 cells relative to these in unstimulated cells [Figure 1]b and [Figure 1]c. Thus, RNF5 was downregulated both in silico and in OGD/R-triggered H9c2 cells. | Figure 1: RNF5 level was decreased both in silico and in OGD/R-induced H9c2 cells. (a) The RNF5 expression was analyzed in the human left ventricular free wall heart tissue from patients with end-stage heart failure (ten cases) and the free wall of the human left ventricle from the healthy heart (seven cases). Data were from the GSE84796 microarray dataset. (b) The relative mRNA level of RNF5 was measured by RT-qPCR. The data were exhibited after normalized with GAPDH. (c) The relative protein expression of RNF5 was detected by western blot. The data were exhibited after normalized with GAPDH. **P ˂ 0.01 and ***P ˂ 0.001 versus Control. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, RT-qPCR: Reverse transcriptase-quantitative polymerase chain reaction, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
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Overexpression of ring-finger protein 5 enhanced OGD/R-challenged H9c2 cell viability
Since RNF5 was downregulated in OGD/R-elicited H9c2 cells, RNF5 expression was elevated through the transfection of RNF5 overexpression plasmids into H9c2 cells. As demonstrated in [Figure 2]a, the RNF5 level was markedly increased following transfection of RNF5 overexpression plasmids compared with that after transfection of empty vector plasmids without OGD/R treatment, indicating a favorable transfection efficiency. OGD/R administration induced a prominent reduction in the RNF5 level both transfection of RNF5 overexpression plasmids and empty vector plasmids into H9c2 cells. In addition, the overexpression of RNF5 partly rescued the OGD/R challenged the reduction in the RNF5 level of H9c2 cells. Similarly, OGD/R stimulation caused a remarkable diminishment in the cell viability of H9c2 cells whether transfected with over-expressed plasmids or empty vector plasmids. Furthermore, upregulation of RNF5 partly recovered the OGD/R triggered the declination in the cell viability of H9c2 cells [Figure 2]b. However, an opposite result was observed in the concentrations of LDH and CK-MB, as well as the apoptosis rate [Figure 2]c and [Figure 2]d. Taken together, overexpression of RNF5 increased cell viability and inhibited cell apoptosis rate in OGD/R-stimulated H9c2 cells. | Figure 2: Upregulation of RNF5 enhanced cell viability and suppressed cell apoptosis rate in OGD/R-induced H9c2 cells. (a) The RNF5 expression was determined by western blot. The data were exhibited after normalized with GAPDH. (b) The cell viability of H9c2 cells was examined by MTT assay. (c) The concentrations of LDH and CK-MB were measured by commercial kits. (d) The apoptosis rate of H9c2 cells was analyzed by flow cytometry assay. ***P ˂ 0.001 versus Control + NC; #P ˂ 0.05, ##P ˂ 0.01 and ###P ˂ 0.001 versus Control + RNF5; $$P ˂ 0.01 and $$$P ˂ 0.001 versus OGD/R + NC. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NC: Negative control, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, LDH: Lactate dehydrogenase, CK-MB: Creatine kinase myocardial band.
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Ring-finger protein 5 improved mitochondrial function of OGD/R-induced H9c2 cells
To explore the function of RNF5 in mitochondrial function, the level of MMP was detected in H9c2 cells via analyzing the ratio of the red and green fluorescence intensity. The MMP level was prominently reduced after H9c2 cells were stimulated with OGD/R, and overexpression of RNF5 also partly restored the OGD/R-triggered the decline in the MMP level [Figure 3]a. On the other hand, OGD/R introduction observably leaded to an arresting reduction in the translational expression of mito-cyt-c and a noteworthy enhancement in the relative protein expression of cyto-cyt-c in H9c2 cells, while overexpression of RNF5 partly reversed these changes in OGD/R-induced H9c2 cells [Figure 3]b. Therefore, RNF5 ameliorated mitochondrial function of OGD/R-elicited H9c2 cells. | Figure 3: RNF5 improved mitochondrial function of OGD/R-induced H9c2 cells. (a) The level of MMP was determined by the commercial MMP Assay Kit with JC-1. The cell nuclei were stained with DAPI. The images were observed by using a fluorescent microscope, and five fields were randomly chosen for analysis. (b) The relative protein expressions of mito-cyt-c and cyto-cyt-c were examined by western blot. COX IV and GAPDH acted as mitochondrial loading control and cytoplasmic loading control, respectively. ***P ˂ 0.001 versus Control + NC; ##P ˂ 0.01 and ###P ˂ 0.001 versus Control + RNF5; $$P ˂ 0.01 and $$$P ˂ 0.001 versus OGD/R + NC. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, MMP: Mitochondrial membrane potential, DAPI: 4',6-diamidino-2-phenylindole, NC: Negative control.
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Overexpression ring-finger protein 5 downregulated OGD/R-triggered inflammatory factor release in H9c2 cells
Additionally, the role of RNF5 in inflammatory factor release was also assessed in H9c2 cells through detecting the concentrations of inflammatory factors, including IL-1β, IL-6, IL-8 and MCP-1. As demonstrated in [Figure 4], OGD/R management triggered a prominent elevation in the concentrations of IL-1β, IL-6, IL-8, and MCP-1 in H9c2 cells, and upregulation of RNF5 significantly counteracted the OGD/R-elicited the diminishment in the concentrations of IL-1β, IL-6, IL-8, and MCP-1 in H9c2 cells. Hence, overexpression RNF5 reduced OGD/R-triggered inflammatory factor release in H9c2 cells. | Figure 4: Overexpression RNF5 attenuated OGD/R-induced inflammatory factor release in H9c2 cells. The concentrations of IL-6, IL-1β, IL-8, and MCP-1 in H9c2 cells were measured by commercial kits. ***P ˂ 0.001 versus Control + NC; #P ˂ 0.05 and ##P ˂ 0.01 versus Control + RNF5; $$P ˂ 0.01 and $$$P ˂ 0.001 versus OGD/R + NC. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, MCP-1: Monocyte chemoattractant protein 1, IL: Interleukin, NC: Negative control.
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Ring-finger protein 5 inhibited the expression of S100A8/MYD88/NF-κB axis
Furthermore, the potential mechanism associated with the role of RNF5 in OGD/R-constructed HF, the relative expressions of proteins involved in the S100A8/MYD88/NF-κB axis were examined by western blot. The translational expressions of S100A8 and MYD88 in H9c2 cells were significantly enhanced with OGD/R induction, while upregulation of RNF5 partly neutralized the OGD/R-induced the elevation in the relative protein expressions of S100A8 and MYD88 in H9c2 cells [Figure 5]a. Additionally, OGD/R challenge elicited a prominent diminishment in the relative protein expression of NF-κB-cyto in H9c2 cells and a significant elevation in the translational expression of NF-κB-nuc, while overexpression of RNF5 partly inverted these alterations in OGD/R-induced H9c2 cells [Figure 5]b. Altogether, RNF5 repressed the expression of S100A8/MYD88/NF-κB axis. | Figure 5: RNF5 restrained the expression of S100A8/MYD88/NF-κB axis. (a) The relative protein expressions of S100A8 and MYD88 in H9c2 cells were determined by western blot. The data were exhibited after normalized with GAPDH. (b) The relative protein expressions of NF-κB-cyto and NF-κB-nuc were examined by western blot. Lamin B1 and GAPDH acted as the nuclear loading control and the cytoplasmic loading control, respectively. *P ˂ 0.05, **P ˂ 0.01 and ***P ˂ 0.001 versus Control + NC; ##P ˂ 0.01 and ###P ˂ 0.001 versus Control + RNF5; $$P ˂ 0.01 and $$$P ˂ 0.001 versus OGD/R + NC. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, MYD88/NF-κB: Myeloid differentiation 88/Nuclear factor-kappa B, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, MCP: Monocyte chemoattractant, NC: Negative control.
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Ring-finger protein 5 decreased OGD/R-induced mitochondrial dysfunction and inflammation in H9c2 cells through inhibiting the S100A8/MYD88/NF-κB axis
To confirm the direct role of S100A8/MYD88/NF-κB axis in OGD/R-induced H9c2 cells, the rescue experiment was conducted through the overexpression of S100A8. The results showed that the relative protein expression of S100A8 was significantly increased in OGD/R-induced H9c2 cells compared to that in unstimulated cells, which was markedly counteracted with the overexpression of RNF5, but further significantly enhanced with the overexpression of S100A8 [Figure 6]a. Overexpression of RNF5 and S100A8 together in OGD/R-induced H9c2 cells markedly elevated the relative protein expression of S100A8 compared to that of overexpression of RNF5 alone, and significantly decreased the relative protein expression of S100A8 compared to that of overexpression of S100A8 alone [Figure 6]a. Overexpression of S100A8 observably enhanced the relative protein expression of MYD88 and NF-κB-nuc but reduced that of NF-κB-cyto in OGD/R-induced H9c2 cells, which were markedly reversed with the additional overexpression of RNF5 [Figure 6]a and [Figure 6]b. Additional overexpression of S100A8 also notably increased the relative protein expression of MYD88 and NF-κB-nuc but decreased that of NF-κB-cyto in OGD/R-induced H9c2 cells transfected with the RNF5 overexpression plasmids [Figure 6]a and [Figure 6]b. Moreover, overexpression of RNF5 alone and overexpression of S100A8 alone prominently enhanced and reduced the MMP level in OGD/R-induced H9c2 cells, respectively, which were both markedly reversed with overexpression of RNF5 and S100A8 together [Figure 6]c. Besides, overexpression of RNF5 alone observably declined the concentrations of IL-1 β, IL-6, IL-8, and MCP-1 in OGD/R-induced H9c2 cells, and overexpression of S100A8 alone significantly elevated the concentrations of IL-1β, IL-6, IL-8, and MCP-1 in OGD/R-induced H9c2 cells compared with these in OGD/R-induced H9c2 cells transfected with NC [Figure 6]d. Both these changes were notably inverted by the overexpression of RNF5 and S100A8 together [Figure 6]d. Taken together, RNF5 attenuated OGD/R-induced mitochondrial dysfunction and inflammation in H9c2 cells by inhibiting the S100A8/MYD88/NF-κB axis. | Figure 6: RNF5 dampened OGD/R-induced mitochondrial dysfunction and inflammation in H9c2 cells by inhibiting the S100A8/MYD88/NF-κB axis. (a) The relative protein expressions of S100A8 and MYD88 in H9c2 cells were examined by western blot. The data were shown after normalized with GAPDH. (b) The relative protein expressions of NF-κB-cyto and NF-κB-nuc were determined by western blot. Lamin B1 and GAPDH acted as the nuclear loading control and the cytoplasmic loading control, respectively. (c) The level of MMP was quantified by the commercial Mitochondrial Membrane Potential Assay Kit with JC-1. The cell nuclei were stained with DAPI. The images were observed by using a fluorescent microscope, and five fields were randomly chosen for analysis. (d) The concentrations of IL-6, IL-1β, IL-8 and MCP-1 in H9c2 cells were measured by commercial kits. **P ˂ 0.01 and ***P ˂ 0.001 versus Control; #P ˂ 0.05, ##P ˂ 0.01 and ###P ˂ 0.001 versus OGD/R + NC; $$P ˂ 0.01 and $$$P ˂ 0.001 versus OGD/R + RNF5; &P ˂ 0.05, &&P ˂ 0.01 and &&&P ˂ 0.001 versus OGD/R + S100A8. The experiments were repeated for three times. RNF5: Ring-finger protein 5, OGD/R: Oxygen-glucose deprivation/reperfusion, MYD88/NF-κB: Myeloid differentiation 88/Nuclear factor-kappa B, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, MMP: Mitochondrial membrane potential, DAPI: 4',6-diamidino-2-phenylindole, MCP-1: Monocyte chemoattractant protein 1, IL: Interleukin, NC: Negative control.
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| Discussion | | |
In the current study, H9c2 cells were stimulated with OGD/R to build a HF model in vitro. RNF5 level was downregulated both in silico and in OGD/R-elicited H9c2 cells. OGD/R treatment exhibited an inhibitory role in cell viability and the MMP level, but a promoted function on the HF markers, apoptosis and inflammatory factor release. Mechanically, OGD/R stimulation enhanced the expression of S100A8 and MYD88, as well as the transfer of NF-κB from cytoplasm to nuclei. However, all of these results were inverted by the gain-of-function studies in OGD/R-stimulated H9c2 cells. Collectively, based on these results, we concluded that RNF5 reduced OGD/R-triggered mitochondrial dysfunction and inflammation in H9c2 cells through inhibiting the S100A8/MYD88/NF-κB axis.
RNF5 is expressed in a wide spectrum of human tissues, and also dysregulated in various disease models, thereby serving as a potential therapeutic target in diverse syndromes. For instance, RNF5 is expressed in neuroblastoma and melanoma cells, whose upregulation indicates a better prognosis. The application of the RNF5 activator inhibits growth of neuroblastoma and melanoma cells both in vitro and in vivo.[9] Declined RNF5 level is demonstrated in intestinal epithelial cells and stroma in the injured area of colitis patients, and loss of RNF5 aggravates severity of colitis and intestinal inflammation in inflammatory bowel disease mice.[11] RNF5 level is enhanced in the hearts of mice with cardiac hypertrophy, and both loss-of-function and gain-of-function assays reveal RNF5 weakens pathological cardiac hypertrophy.[17] Here, the results conformably demonstrated that RNF5 was downregulated in the human left ventricular free wall heart tissue from patients with end-stage HF in silico and in OGD/R-induced H9c2 cells. Thus, the outcomes suggested that RNF5 might act as a biomarker for the diagnosis and treatment of HF.
Cardiomyocyte injury or death is the dominating cause of HF.[25] In the current study, OGD/R administration induced a conspicuous diminishment in cell viability, and a prominent elevation in the concentrations of CK-MB and LDH as well as the apoptosis rate in H9c2 cells. CK-MB and LDH are widely distributed in myocardial cells. Although in the normal physiological condition they cannot traverse cytoplasmic membranes, CK-MB and LDH are secreted in impaired or dead cells.[26] Thus, elevated CK-MB and LDH concentrations in the supernatant of the OGD/R-stimulated H9c2 cells suggested the damage of H9c2 cells. In addition, cardiomyocyte apoptosis also acts as a decisive role in the progression of HF.[27] An enhancement in apoptosis rate implied an apoptosis occurred in the OGD/R-challenged H9c2 cells. However, gain-of-function studies inverted the outcomes in cell viability, LDH and CK-MB concentrations and apoptosis rate in the OGD/R-treated H9c2 cells, indicating that overexpression of RNF5 protected H9c2 cells from OGD/R-triggered injury.
Mitochondrial dysfunction is a booster of the development of HF, thus mitochondrial therapies have been highlighted to be one of momentous approaches for the treatment of HF.[6],[28] MMP is an important parameter for monitoring mitochondrial health.[29] Here, the level of MMP was determined by JC-1 methods in the form of the red/green fluorescence intensity ratio. JC-1 preferentially accumulates in mitochondria in the form of red polymers dependent on the MMP to entry and label mitochondria, but when mitochondria are damaged, JC-1 exists in the cytoplasm as a green monomer owing to breakdown of inner and outer membrane potential.[30] Additionally, in the normal state, cyt-c is present in the space between the inner and outer mitochondrial membranes, which then is released from the mitochondria into the cytoplasm upon the stimulations, such as apoptosis signals.[31] In this study, OGD/R treatment caused a diminishment in the MMP level and the relative protein level of mito-cyt-c accompanied with the enhanced the relative protein expression of cyto-cyt-c in H9c2 cells, suggesting a mitochondrial dysfunction in the OGD/R-treated H9c2 cells. While upregulation of RNF5 reversed these results in the OGD/R-stimulated H9c2 cells. Taken together, RNF5 improved mitochondrial function of OGD/R-triggered H9c2 cells. However, a series of mitochondrial abnormalities have been evidenced to be strongly associated with HF, such as abnormal mitochondrial dynamics, enhanced reactive oxygen species formation, damaged mitochondrial oxidative phosphorylation and changed metabolic substrate use,[28] thus some other related indicators should be detected in the further study. In addition, mitochondrial dysfunction is also one of cause of apoptosis initiation, therefore, the further studies should be focused on the connection between mitochondrial dysfunction and apoptosis in the future.
Secretion and release of pro-inflammatory factors and chemokines have been highlighted to be sophisticated with the pathogenesis of HF.[32] Levels of IL-6, tumor necrosis factor alpha (TNF-α) and MCP-1 have been revealed to be significantly increased in OGD/R-induced H9c2 cells.[33] Also, OGD/R treatment causes a prominent upregulation in the levels of IL-1β, IL-6, IL-8, TNF-α and MCP-1 in H9c2 cells.[34] IL-1β, IL-6 and IL-8 are representative pro-inflammatory mediators,[35],[36],[37] and IL-1β can further trigger the extra release of IL-6 and IL-8.[38] MCP-1 also known as chemokine (C-C motif) ligand 2 is a chemokine secreted by astrocytes that promotes inflammation.[39] Similar to these findings, the concentrations of IL-1β, IL-6, IL-8, and MCP-1 in H9c2 cells were also increased following the OGD/R challenge. Overexpression of RNF5 counteracted the promotions of these pro-inflammatory factors release. Therefore, RNF5 inhibited the release of pro-inflammatory mediators in OGD/R-triggered H9c2 cells.
Mechanically, OGD/R exposure elevated the level of S100A8 and MYD88, and the transfer of NF-κB from cytoplasm to nuclei, and gain-of-function studies showed that overexpression of RNF5 reversed these results. Overexpression of S100A8 in both OGD/R-induced H9c2 cells and OGD/R-induced H9c2 cells with overexpression of RNF5 enhanced the relative protein expression of MYD88 and NF-κB-nuc, but reduced that of NF-κB-cyto, which indicated that S100A8 was upstream of MYD88 and NF-κB in OGD/R-induced H9c2 cells. Thus, RNF5 inhibited the level of S100A8/MYD88/NF-κB axis in OGD/R-triggered H9c2 cells. S100A8/MYD88/NF-κB axis has been demonstrated to be involved in the cardiac hypertrophy and enhanced an inflammatory response.[20] S100A8/A9/MYD88/NF-κB pathway is also complicated with the inflammasome activation.[40] Consistent with our results, loss of RNF5 also lead to an elevated expression of S100A8 in the inflammatory bowel disease.[11] It has been revealed that S100A8 is a pivotal molecule in the modulation of mitochondrial dysfunction and inflammatory response in myocardial infarction[41] and in allergy.[42] Along with these findings, our results showed that gain-of-function experiments of S100A8 reversed the ameliorative role of RNF5 in mitochondrial function and the release of pro-inflammatory mediators in OGD/R-elicited H9c2 cells. Collectively, these data indicated that RNF5 decreased OGD/R-induced mitochondrial dysfunction and inflammation in H9c2 cells through inhibiting the S100A8/MYD88/NF-κB axis.
| Conclusion | | |
In conclusion, the expression level of RNF5 was downregulated both in silico and in OGD/R-elicited H9c2 cells. Overexpression of RNF5 reversed the OGD/R-stimulated the suppressive role in cell viability and the MMP level and the enhanced function on the HF markers, apoptosis and inflammatory factor release. Thus, RNF5 hindered mitochondrial dysfunction and inflammation via inhibiting the S100A8/MYD88/NF-κB axis in OGD/R-challenged H9c2 cells. Although our study clarified the role of RNF5 in the HF cells model, the role of RNF5 should be further elucidated in the HF animal model. Briefly, combined with more preclinical trials, our results boost the development of the treatment for HF.
Availability of data and materials
All the data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
Xuesi Chen designed the study, completed the experiment and supervised the data collection; Yingjie Wu analyzed the data, interpreted the data; Yingchun Bao prepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.
Ethics approval
There are no relevant experiments covered in this paper.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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