Protective effects of pyrroloquinoline quinone against oxidative stress-induced cellular senescence and inflammation in human renal tubular epithelial cells via Keap1/Nrf2 signaling pathway

https://doi.org/10.1016/j.intimp.2019.04.040Get rights and content

Highlights

  • PQQ could protect HK-2 cells from HG-induced inflammation and senescence.

  • The protective effects of PQQ are associated with inhibition of ROS generation.

  • PQQ affects the Keap1/Nrf2 pathway and promotes the nuclear transcription of Nrf2.

  • PQQ upregulates the protein expression of HO-1, NQO-1, GST and GPx-3.

  • PQQ achieves the anti-inflammation and anti-senescence effects via Keap1/Nrf2 pathway.

Abstract

Oxidative stress-induced cellular senescence and inflammation are important biological events in diabetic nephropathy (DN). Our recent studies have found that pyrroloquinoline quinone (PQQ) has protective effects against HG-induced oxidative stress damage and apoptosis in HK-2 cells. Nevertheless, whether PQQ has the effect of anti-inflammation and anti-senescence in HK-2 cells remains unclear. Here, we showed that low-dose PQQ treatment (100 nM) downregulates the expression of P16, P21, IL-1β, TNF-α and NF-κB in HG cultured HK-2 cells. A low dose of PQQ also upregulated the protein expression of SOD2, CAT and inhibited the generation of ROS. We also indicated that PQQ affected the activity of Keap1/Nrf2 pathway, increased the nuclear accumulation 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.

Introduction

Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus [[1], [2], [3]]. Various studies have illustrated that high glucose (HG) increases the production of intracellular ROS and aggravates oxidative stress, thus leads to apoptosis, cellular senescence, inflammation, fibrosis and other damages of renal tubular epithelial cells [[4], [5], [6], [7]]. Especially, oxidative stress-induced cellular senescence and inflammation are important biological events of renal tubular cells in DN [[8], [9], [10], [11]]. Therefore, early intervention and treatment of renal tubular cell senescence and inflammation may effectively delay the development of DN.

Pyrroloquinoline quinone (PQQ) is an organic molecule as the third redox coenzyme following nicotinamides and flavines. PQQ is water-soluble, and the chemical properties are similar to the combined attributes of ascorbic acid and vitamin B6 [12]. Previous researches have demonstrated that PQQ has multiple physiological functions, including neural and cardiovascular protection [[13], [14], [15]], the promotion of growth and reproduction [[16], [17], [18]], and as an antioxidant to protect the cells from oxidative stress-induced damages [[19], [20], [21]]. In recent years, PQQ has become increasingly studied with its role in inflammation and cellular senescence [22,23]. Our recent studies have found that PQQ has protective effects against HG-induced oxidative stress damage and apoptosis in vitro model of diabetic nephropathy. PQQ (10 nM–10,000 nM) significantly increased the cell viability at 48 h under HG environment, especially in 100 nM concentration [24]. However, whether PQQ has the effect of anti-inflammation and anti-senescence in HK-2 cells remains unclear.

Nuclear factor-E2-related factor 2 (Nrf2), a key modulator of redox balance and signaling, which plays an important role in defending against oxidative stress damage and regulating antioxidant genes [25]. Previous studies have suggested that Keap1/Nrf2/ARE signaling pathway plays a critical role in delaying the progression of DN. Evidence suggests that Nrf2 may also play an important role in the regulation of inflammation and senescence [[26], [27], [28], [29]]. In addition, Nrf2 is reported to be involved in suppression of pro-inflammatory situation via regulation of NF-kB signaling pathways [29,30]. Does PQQ affect the activity of Keap1/Nrf2 pathway in HK-2 cells? Whether PQQ exerts anti-oxidative stress, anti-inflammation and anti-aging effects through the Keap1/Nrf2 pathway remains unclear.

In the present study, we investigated the anti-inflammation and anti-senescence effect of PQQ in HK-2 cells under HG environment for 48 h. The effect of PQQ on the activity of Keap1/Nrf2 pathway was also further studied. The results indicated that PQQ attenuated oxidative stress-induced cellular senescence and inflammation in HK-2 cells. The protective effect of PQQ was mediated by Keap1/Nrf2 pathway.

Section snippets

Chemicals and reagents

FBS and DMEM were purchased from Life Technologies BRL (Gaithersburg, MD). d-glucose, penicillin, streptomycin and pyrroloquinoline quinone (PQQ) were obtained from Sigma (St. Louis, Mo, USA). Antibodies for P16, P21, Nrf2, HO-1, NQO1, CAT, GPx-3, GST, Keap1 and Histone H3 were obtained from Abcam (Cambridge, UK). Antibodies for β-actin and SOD2 were purchased from Proteintech (Chicago, USA). Reverse Transcription System, qPCR Master Mix were purchased from Vazyme (New Jersey, USA). The

PQQ inhibited HG-induced inflammation in HK-2 cells

In order to study whether PQQ could inhibit HG-induced inflammation in HK-2 cells, we tested the protein expression of IL-1β, TNF-α and NF-κB by western blot. As illustrated in Fig. 1A-B, the protein expression of IL-1β, TNF-α and NF-κB were significantly increased after HG stimulation for 48 h. However, PQQ treatment inhibited HG-induced secretion of these inflammatory cytokines (P < 0.05). These findings indicated that PQQ inhibited HG-induced inflammation in HK-2 cells.

PQQ inhibited HG-induced cellular senescence in HK-2 cells

Previous studies have

Discussion

The results suggest that PQQ has protective effects against HG-induced inflammation and cellular senescence in HK-2 cells. The protective effects of PQQ are associated with inhibition of ROS generation and upregulating the level of antioxidants. In this study, we also illustrate that PQQ could affect the activity of Keap1/Nrf2 pathway, promote the translocation of Nrf2 to nucleus and upregulate the protein expression downstream pathway of Keap1/Nrf2 signaling pathway. PQQ achieves the

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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