2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells
Summary (2 min read)
Introduction
- Arsenic (As) is an important environmental contaminant affecting more than 140 million people worldwide through contaminated drinking water (Rodriguez-Lado et al. 2013).
- One of the main aims of the current study was to further elucidate a potential relationship between HO-1 production and the renal protection by antioxidant TMP in arsenic nephrotoxicity, which is not well understood.
Cell culture and treatment
- The human proximal tubular cell line HK-2 (American Type Culture Collection, Manassas, VA, USA) was grown in culture medium (keratinocyte serum-free 1 3 medium + 5 ng/ml epidermal growth factor and 50 μg/ ml bovine extract + 100 U/ml penicillin and 100 μg/ ml of streptomycin) at 37 °C and 5 % CO2 humidified environment.
- NAC, TMP and other inhibitors were added into media 30 min before As.
Intracellular ROS detection
- Dihydroethidium (DHE, Invitrogen, Eugene, OR) method to detect intracellular superoxide production was used.
- Samples were analyzed in triplicate and repeated 3 times.
- To further confirm apoptotic cell death, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining was performed using a ClickiT Alexa Fluor 488 Imaging Assay (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions except that PI replaced Hoechst 33342 to mount cells and label all nuclei.
- In brief, cells were grown on 6-well plate and incubated with 200 nM MitoTracker Green for another 20 min at 37 °C after 6-h Percentages of normal and abnormal (non-) mitochondrial network morphologies were counted.
Western blotting
- After the various treatments, whole cell lysates were prepared by incubation in RIPA buffer .
- For nuclear transcription factor Nrf2 immunoblotting analysis, nuclear extracts were prepared using methods described previously (Schreiber et al. 1989).
- Protein concentrations were determined with Bio-Rad DC protein assay (Bio-Rad Laboratories, Calif., USA) using bovine serum albumin as the standard.
- The resulting protein samples underwent SDS-PAGE gel electrophoresis and were transferred to PVDF membrane.
Statistical analysis
- All comparisons were made using either one-way ANOVA or a two-tailed t test analysis depending on how many conditions were compared in each experiment.
- One-way ANOVA was followed by Tukey’s post hoc test.
Results
- 2,3,5,6‑Tetramethylpyrazine (TMP) inhibited arsenic‑induced ROS‑dependent HO‑1 expression Arsenic (As) has been identified as inducer of heme oxygenase-1 (HO-1) expression in many cells and tissues (Teng et al. 2013; Li et al. 2013).
- To further investigate the relationships among mitochondrial alterations, TMP prevented As‑induced HO‑1 activation through inhibiting the activation of MAPKs/AP‑1 pathways Transcription factor AP-1 interacts with the corresponding binding site in the HO-1 gene promoter region and mediates HO-1 expression (Zhang et al. 2006).
- Meanwhile, as an important member of AP-1 family, nuclear phospho-c-Jun protein expression increased after As exposure in a dose-dependent manner, while both 50 and 100 μM TMP efficiently inhibited As-induced phospho-c-Jun up-regulation; 100 μM TMP demonstrated higher efficiency (Fig. 5c, d).
- TMP prevented As‑induced up‑regulation of ARS2 expression in HK‑2 cells 5 TMP prevented arsenic-triggered activations of p38 MAPK, JNK and c-Jun pathways in HK-2 cells.
Discussion
- The authors previous study (Gong et al. 2014) identified that sodium arsenite at a clinically relevant dose also might be a risk factor for kidney, while TMP could prevent such an As-induced nephrotoxicity by reducing ROS production, preventing mitochondria dysfunction and suppressing activation of pro-inflammatory signals, including β-catenin, NF-κB, TNF-α and cyclooxygenase-2 (COX2).
- Taken together, their present data demonstrated that the regulation mechanisms of As-induced HO-1 expression were performed through multiple signal pathways, Nrf2, NF-κB, AP-1, p38 MAPK and JNK.
- In current study, another novel finding is the demonstration that TMP could also suppress the activations of Nrf2, AP-1, JNK and ERK after As exposure, accordingly, block HO-1 protein expression in HK-2 cells.
- To their knowledge, this is the first report demonstrating that ARS2 involved in As-induced nephrotoxicity and it was regulated by p38 MAPK, ERK and NF-κB.
- Compliance with ethical standards Conflict of interest.
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Cites background from "2,3,5,6-Tetramethylpyrazine (TMP) d..."
...…100 µM for 6 h)-induced nephrotoxicity by targeting HO-1 and ARS2, which was further evidenced by the findings that TMP (20 mg/kg/day i.p. for 7 days) relieves gentamicin-induced AKI by enhancing Hax-1 mitochondrial localization in HO-1-dependent mechanisms (Sue et al., 2009; Gong et al., 2016)....
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References
84 citations
"2,3,5,6-Tetramethylpyrazine (TMP) d..." refers background or result in this paper
...Two additional transcription factors, Nrf2 and NF-κB, control HO-1 gene and protein expression (Garnier et al. 2013; Wang et al. 2012; Lim et al. 2014)....
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...Indeed, while up-regulation of HO-1 is regarded as cytoprotective under various experiment conditions, it can be non-sufficient to fully recover cells from oxidative damage, as shown in our study and other publication (Wang et al. 2012)....
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...4b and 5, arsenic exposure resulted in MAPK family activations, which is very consistent to the other studies (Wang et al. 2012; Kang et al. 2003), but such activation seemed to be cell and species dependent (Wang et al....
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...2003), but such activation seemed to be cell and species dependent (Wang et al. 2012)....
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...TMP prevented As‑induced HO‑1 through inhibiting the activation of Nrf2 and NF‑κB pathways Two additional transcription factors, Nrf2 and NF-κB, control HO-1 gene and protein expression (Garnier et al. 2013; Wang et al. 2012; Lim et al. 2014)....
[...]
82 citations
"2,3,5,6-Tetramethylpyrazine (TMP) d..." refers background in this paper
...Kidney is one of the targeted organs of arsenic cytotoxicity that could cause renal dysfunction, proteinuria and chronic kidney disease (CKD) (Yu et al. 2013; Michael 2013; Ruiz-Hernandez et al. 2015; Zheng et al. 2014; Chen et al. 2014)....
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...AG Aminoguanidine AKI Acute kidney injury ARS2 Arsenic response protein 2 As Arsenic Bay Bay 11-7082 CKD Chronic kidney disease DHE Dihydroethidium FACS Fluorescence-activated cell sorter HO-1 Heme oxygenase-1 MAPK Mitogen-activated protein kinase Nrf2 Nuclear factor erythroid derived-2 NAC N-acetylcysteine * Xuezhong Gong shnanshan@hotmail.com 1 Department of Nephrology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 274 Zhijiang Middle Road, Shanghai 200071, China 2 Center for Radiological Research, College of Physician and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA 3 Department of Radiation Oncology, College of Physician and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA 1 3 NF-κB Nuclear factor-κB PARP Poly ADP-ribose polymerase PI Propidium iodide ROS Reactive oxygen species SB SB203580 SDH Sucinate dehydrogenase SP SP600125 TMP Tetramethylpyrazine TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling U0 U0126 ZnPP Zinc protoporphyrin...
[...]
76 citations
"2,3,5,6-Tetramethylpyrazine (TMP) d..." refers background in this paper
...Based on the very limited published data, ARS2 has been shown to be essential for the development of plants and mammals and also acts as a transcriptional regulator of Sox2 in neural stem cell (Kiriyama et al. 2009; Wilson et al. 2008; Andreu-Agullo et al. 2012)....
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...However, the precise biological functions of ARS2 in mammalian are largely unknown (Wilson et al. 2008; Andreu-Agullo et al. 2012)....
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75 citations
"2,3,5,6-Tetramethylpyrazine (TMP) d..." refers background in this paper
...TMP prevented As‑induced HO‑1 through inhibiting the activation of Nrf2 and NF‑κB pathways Two additional transcription factors, Nrf2 and NF-κB, control HO-1 gene and protein expression (Garnier et al. 2013; Wang et al. 2012; Lim et al. 2014)....
[...]
...Two additional transcription factors, Nrf2 and NF-κB, control HO-1 gene and protein expression (Garnier et al. 2013; Wang et al. 2012; Lim et al. 2014)....
[...]
73 citations
"2,3,5,6-Tetramethylpyrazine (TMP) d..." refers background in this paper
...Kidney is one of the targeted organs of arsenic cytotoxicity that could cause renal dysfunction, proteinuria and chronic kidney disease (CKD) (Yu et al. 2013; Michael 2013; Ruiz-Hernandez et al. 2015; Zheng et al. 2014; Chen et al. 2014)....
[...]
...AG Aminoguanidine AKI Acute kidney injury ARS2 Arsenic response protein 2 As Arsenic Bay Bay 11-7082 CKD Chronic kidney disease DHE Dihydroethidium FACS Fluorescence-activated cell sorter HO-1 Heme oxygenase-1 MAPK Mitogen-activated protein kinase Nrf2 Nuclear factor erythroid derived-2 NAC N-acetylcysteine * Xuezhong Gong shnanshan@hotmail.com 1 Department of Nephrology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 274 Zhijiang Middle Road, Shanghai 200071, China 2 Center for Radiological Research, College of Physician and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA 3 Department of Radiation Oncology, College of Physician and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA 1 3 NF-κB Nuclear factor-κB PARP Poly ADP-ribose polymerase PI Propidium iodide ROS Reactive oxygen species SB SB203580 SDH Sucinate dehydrogenase SP SP600125 TMP Tetramethylpyrazine TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling U0 U0126 ZnPP Zinc protoporphyrin...
[...]
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Frequently Asked Questions (2)
Q2. What are the future works mentioned in the paper "2,3,5,6‐tetramethylpyrazine (tmp) down‐regulated arsenic‐induced heme oxygenase‐1 and ars2 expression by inhibiting nrf2, nf‐κb, ap‐1 and mapk pathways in human proximal tubular cells" ?
Further studies focusing on the potential function of ARS2 in As-induced nephrotoxicity is worthy of attention. Although further studies are required, the authors can still propose TMP could be effective in the treatment of arsenic-induced nephrotoxicity. In summary, the present study further confirmed that arsenic treatment at clinically relevant dose results in renal damage, additionally, the activations of Nrf2, AP-1, MAPK family and ARS2 involved in such a nephrotoxicity.