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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...…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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...Since the As dose range of 2.0–10 μM is successfully applied for treating acute promyelocytic leukemia (APL) and multiple myelomas (Ivanov and Hei 2004, 2005; Shen et al. 1997), we therefore choose this dose range for our present study....
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...Arsenic (As) is an important environmental contaminant affecting more than 140 million people worldwide through contaminated drinking water (Rodriguez-Lado et al. 2013)....
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...The critical role of HO-1 has also been reported in several organ injuries (Wang and Dore 2007; Billings et al. 2014)....
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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.