International Immunopharmacology 72 (2019) 445–453 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Protective effects of pyrroloquinoline quinone against oxidative T stress-induced cellular senescence and inflammation in human renal tubular epithelial cells via Keap1/Nrf2 signaling pathway ⁎ Ziqiang Wanga,b, Ning Hana, Kunxiao Zhaoa, Ying Lia, , Yanqing Chia, Baoxing Wanga a Department of Nephrology, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei Province 050051, China b Department of Nephrology, Cangzhou People's Hospital, Cangzhou, Hebei Province 061000, China ARTICLE INFO ABSTRACT Keywords: Oxidative stress-induced cellular senescence and inflammation are important biological events in diabetic ne- Pyrroloquinoline quinone phropathy (DN). Our recent studies have found that pyrroloquinoline quinone (PQQ) has protective effects Cellular senescence against HG-induced oxidative stress damage and apoptosis in HK-2 cells. Nevertheless, whether PQQ has the Inflammation effect of anti-inflammation and anti-senescence in HK-2 cells remains unclear. Here, we showed thatlow-dose High glucose PQQ treatment (100 nM) downregulates the expression of P16, P21, IL-1β, TNF-α and NF-κB in HG cultured HK- Keap1/Nrf2 signaling pathway 2 cells. A low dose of PQQ also upregulated the protein expression of SOD2, CAT and inhibited the generation of Renal tubular epithelial cells ROS. We also indicated that PQQ affected the activity of Keap1/Nrf2 pathway, increased the nuclear accumu- lation of Nrf2 and the downstream pathway protein expression of Keap1/Nrf2 signaling pathway (HO-1, NQO-1, GST and GPx-3). When ML385 was added to inhibit the activity of Keap1/Nrf2 signaling pathway, the effects of PQQ on anti-oxidative stress, anti-inflammation and anti-senescence in HK-2 cells under HG condition were weakened. In conclusion, our results suggest that PQQ could modulate HG-induced inflammation and senescence in HK-2 cells via the inhibition of ROS generation and achieves the protective effects through Keap1/Nrf2 pathway and upregulating the expression of its target protein. 1. Introduction demonstrated that PQQ has multiple physiological functions, including neural and cardiovascular protection [13–15], the promotion of growth Diabetic nephropathy (DN) is one of the most common micro- and reproduction [16–18], and as an antioxidant to protect the cells vascular complications of diabetes mellitus [1–3]. Various studies have from oxidative stress-induced damages [19–21]. In recent years, PQQ illustrated that high glucose (HG) increases the production of in- has become increasingly studied with its role in inflammation and tracellular ROS and aggravates oxidative stress, thus leads to apoptosis, cellular senescence [22,23]. Our recent studies have found that PQQ cellular senescence, inflammation, fibrosis and other damages of renal has protective effects against HG-induced oxidative stress damage and tubular epithelial cells [4–7]. Especially, oxidative stress-induced cel- apoptosis in vitro model of diabetic nephropathy. PQQ lular senescence and inflammation are important biological events of (10 nM–10,000 nM) significantly increased the cell viability at48h renal tubular cells in DN [8–11]. Therefore, early intervention and under HG environment, especially in 100 nM concentration [24]. treatment of renal tubular cell senescence and inflammation may ef- However, whether PQQ has the effect of anti-inflammation and anti- fectively delay the development of DN. senescence in HK-2 cells remains unclear. Pyrroloquinoline quinone (PQQ) is an organic molecule as the third Nuclear factor-E2-related factor 2 (Nrf2), a key modulator of redox redox coenzyme following nicotinamides and flavines. PQQ is water- balance and signaling, which plays an important role in defending soluble, and the chemical properties are similar to the combined attri- against oxidative stress damage and regulating antioxidant genes [25]. butes of ascorbic acid and vitamin B6 [12]. Previous researches have Previous studies have suggested that Keap1/Nrf2/ARE signaling Abbreviations: DN, Diabetic nephropathy; PQQ, pyrroloquinoline quinone; NG, normal glucose; HG, high glucose; NAD+, nicotinamide adenine dinucleotide; HO- 1, heme oxygenase-1; NQO-1, NAD(P)H quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; SA-β-gal, senescence associated-beta-galactosidase; GST, glutathione s-transferase; GPx-3, glutathione peroxidase 3; SOD2, manganese superoxide dismutase; CAT, catalase; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (Y. Li). https://doi.org/10.1016/j.intimp.2019.04.040 Received 26 January 2019; Received in revised form 17 April 2019; Accepted 18 April 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved. Z. Wang, et al. International Immunopharmacology 72 (2019) 445–453 pathway plays a critical role in delaying the progression of DN. Evi- 2.5. Immunofluorescence analysis dence suggests that Nrf2 may also play an important role in the reg- ulation of inflammation and senescence [26–29]. In addition, Nrf2 is The HK-2 cells were grown on coverslips, washed with PBS for three reported to be involved in suppression of pro-inflammatory situation times, fixed in 4% paraformaldehyde for 15 min, permeabilized with via regulation of NF-kB signaling pathways [29,30]. Does PQQ affect 0.3% Triton X-100 and then blocked with goat serum for 30 min at the activity of Keap1/Nrf2 pathway in HK-2 cells? Whether PQQ exerts room temperature. The cells were then incubated in primary antibody anti-oxidative stress, anti-inflammation and anti-aging effects through solution (Nrf2, 1:200) at 4 °C overnight. Next day, the cells were in- the Keap1/Nrf2 pathway remains unclear. cubated with secondary antibodies (1:150) for 1 h at 37 °C and DAPI for In the present study, we investigated the anti-inflammation and 10 min at room temperature. Images were obtained with a fluorescence anti-senescence effect of PQQ in HK-2 cells under HG environment for microscope. 48 h. The effect of PQQ on the activity of Keap1/Nrf2 pathway wasalso further studied. The results indicated that PQQ attenuated oxidative 2.6. Total and nuclear protein extraction stress-induced cellular senescence and inflammation in HK-2 cells. The protective effect of PQQ was mediated by Keap1/Nrf2 pathway. Cells were washed twice with PBS. The total cell protein was ex- tracted using RIPA lysis buffer containing protease and phosphatase 2. Methods and materials inhibitor. Nuclear protein was extracted using nuclear and cytoplasmic extraction kit according to the manufacturer's instructions. The protein 2.1. Chemicals and reagents concentration was determined by a BCA method. The protein samples were denatured in the SDS sample buffer at 100°C for 7min and then FBS and DMEM were purchased from Life Technologies BRL stored at −70 °C until further experimentation. (Gaithersburg, MD). D-glucose, penicillin, streptomycin and pyrrolo- quinoline quinone (PQQ) were obtained from Sigma (St. Louis, Mo, 2.7. Western blot analysis USA). Antibodies for P16, P21, Nrf2, HO-1, NQO1, CAT, GPx-3, GST, Keap1 and Histone H3 were obtained from Abcam (Cambridge, UK). A total of 30 μg protein was separated by 10–12% SDS-PAGE and Antibodies for β-actin and SOD2 were purchased from Proteintech then transfered to PVDF membranes, and then blocked with 5% skim (Chicago, USA). Reverse Transcription System, qPCR Master Mix were milk for 1 h at 37 °C. The membranes were incubated with a primary purchased from Vazyme (New Jersey, USA). The β-Galactosidase antibody overnight at 4 °C. Next day, they were washed with TBST Staining kit and reactive oxygen species assay kit were obtained from (3*15 min) and incubated with secondary antibodies (1:1000). Bands Solarbio (Beijing, China). The Nrf2 inhibitor ML385 was obtained from were detected with ECL detection reagent by the Odyssey Fc System MedChem Express (New Jersey, USA). TRIzol reagent, NE-PER nuclear (LICOR, USA). The protein bands intensity were analyzed using Image J and cytoplasmic extraction kit were purchased from Thermo Fisher software. (Carlsbad, USA). 2.8. RNA extraction and real time-PCR analysis 2.2. Cell culture and treatment Total cellular RNA was isolated using TRIzol reagent. RNA samples (2 μg) were then reverse-transcribed into cDNA using cDNA synthesis The Human proximal tubular epithelial cells (HK−2) were obtained kit. Quantitative real-time PCR was carried out using qPCR Master Mix from American Type Culture Collection (Manassas, USA). The cells on an Agilent Mx3000P QPCR Systems (Agilent, CA), following the were grown in low-glucose DMEM (5.5 mM D-glucose) that was sup- manufacturer's instructions. β-actin was used as an internal control. The plemented with 10% fetal bovine serum, 100 U/mL penicillin, and primers used in the study were as follows: Nrf2 (forward: 5′-TCAGCG 100 mg/mL streptomycin at 37 °C. The cells were stimulated with ACGGAAAGAGTATGA-3′ and reverse: 5′-CCACTGGTTTCTGACTGGA normal concentration D-glucose medium (NG, 5.5 mmol/L), high D- TGT-3′), β-actin (forward: 5′-TGACGTGGACATCCGCAAAG-3′ and re- glucose medium (HG, 30 mmol/L). Cells were incubated with PQQ and verse: 5′-CTGGAAGGTGGACAGCGAGG-3′). The cycling conditions ML385 for the indicated concentration, respectively. were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Data were analyzed using the 2−ΔΔCT method. 2.3. Senescence-associated β-galactosidase (SA-β-gal) staining 2.9. Treatment with a Keap1/Nrf2 pathway inhibitor The HK-2 cells were treated as previously indicated in 6-well plates. Senescent cells were detected using an SA-β-gal staining kit as the Previous studies have demonstrated the discovery and validation of manufacturer's instructions. In brief, cells were washed with PBS and ML385 as a novel and specific Nrf2 inhibitor. ML385 interacts with fixed at room temperature for 15 min in 4% formaldehyde and0.2% Nrf2 and affects the DNA binding activity of the Nrf2-Maf protein glutaraldehyde.