PTEN inhibitor VO-OHpic protects endplate chondrocytes against apoptosis and calcification via activating Nrf-2 signaling pathway.

www.aging-us.com AGING 2023, Vol. 15, No. 6 Research Paper PTEN inhibitor VO-OHpic protects endplate chondrocytes against apoptosis and calcification via activating Nrf-2 signaling pathway Xingang Cui1, Xiaoyang Liu2, Peng Kong3, Ting Du4, Tao Li2, Guihe Yang1, Weimin Zhang2, Xingzhi Jing2, Wenchao Wang2 1 Department of Spine Surgery, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250000, China Department of Spine Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250000, China 3 Department of Orthopaedics, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250000, China 4 Department of Medical, Yidu Cloud (Beijing) Technology Co. Ltd., Beijing 100191, China 2 Correspondence to: Xingzhi Jing, Wenchao Wang; email: ; , https://orcid.org/0000-0003-0206-895X Keywords: PTEN inhibitor, cartilage endplate, Nrf-2, mitophagy, ferroptosis Received: December 2, 2022 Accepted: March 15, 2023 Published: March 24, 2023 Copyright: © 2023 Cui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Cartilage endplate (CEP) degeneration and calcification is an important contributor to the onset and pathogenesis of intervertebral disc degeneration (IDD). However, the underlying mechanisms of CEP degeneration remain elusive, let alone according treatment strategies to prevent CEP degeneration. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that promotes cell apoptosis, and recent studies indicated that PTEN is overexpressed in degenerated intervertebral disc. However, whether direct inhibition of PTEN attenuates CEP degeneration and IDD development remains largely unknown. In the present study, our in vivo experiments demonstrated that VO-OHpic could attenuate IDD progression and CEP calcification. We also found that VO-OHpic inhibited oxidative stress induced chondrocytes apoptosis and degeneration by activating Nrf-2/HO-1 pathway, thus promoted parkin mediated mitophagy process and inhibited chondrocytes ferroptosis, alleviated redox imbalance and eventually improved cell survival. Nrf-2 siRNA transfection significantly reversed the protective effect of VO-OHpic on endplate chondrocytes. In conclusion, our study demonstrated that inhibition of PTEN with VO-OHpic attenuates CEP calcification and IDD progression. Moreover, VO-OHpic protects endplate chondrocytes against apoptosis and degeneration via activating Nrf-2/HO-1 mediated mitophagy process and ferroptosis inhibition. Our results suggest that VOOHpic may be a potential effective medicine for IDD prevention and treatment. INTRODUCTION the penetration of cartilage endplate, CEP calcification and degeneration could significantly reduce the nutrient supply of intervertebral discs and is the leading cause of IDD [3, 4]. However, relatively few studies have focused on the CEP degeneration, let alone according treatment strategies to prevent CEP degeneration. Intervertebral disc degeneration (IDD) is a major cause of low back pain and affects 80% of the population worldwide, which results in job-related disability and high healthcare costs [1]. Intervertebral disc is the largest avascular tissue which is composed of the nucleus pulposus (NP), the annulus fibrosus (AF), upper and lower cartilage endplate (CEP) [2]. The nutrient and oxygen supply of intervertebral disc is mainly through www.aging-us.com Phosphatase and tensin homolog (PTEN) is encoded by a 200 kb gene located on chromosome10q23, and is reported to be involved in many diseases [5]. Through 2275 AGING converting phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol (4,5)-bisphosphate (PIP2), PTEN can counteract the PI3K/AKT signaling activation and is involved in many pathological and physiological processes [6]. The PTEN/PI3K/AKT signaling pathway plays important roles in many diseases including neurodegenerative diseases, cardiovascular diseases and cancers [7]. Recently, PTEN was reported to be overexpressed in degenerated NP cells and is responsible for NP cells apoptosis [8, 9]. Moreover, the essential role of PTEN in osteoarthritis development has recently been clarified, PTEN could decrease the chondrocytes viability and type II collagen production by inhibiting PI3K/AKT activation [10]. However, to our knowledge, no studies have investigated the role of PTEN in CEP degeneration and calcification. VO-OHpic is a reversible non-competitive PTEN inhibitor which is based on vanadium [11]. PTEN inhibition with VO-OHpic has been reported to be able to attenuate cell apoptosis and cardiovascular diseases development [12]. VO-OHpic has been proved safe and highly selective, it could inhibit PTEN with low nanomolar concentrations. Moreover, previous studies have demonstrated that the inhibition effect of PTEN by VO-OHpic is reversible [7]. In the present study, we aim to investigate whether VO-OHpic could inhibit CEP chondrocytes apoptosis and degeneration and whether VO-OHpic could protect against CEP calcification and IDD development. activation could slow down the IDD development by inhibiting the oxidative stress and inflammatory process in NP cells [19]. Moreover, recent study demonstrated that ferroptosis plays a role in NP cells viability and Nrf-2 could regulate ferroptosis marker, GPX4 and iron storage proteins ferritin light and heavy chains (FTL/FTH1) [20]. There are studies which also indicate that PI3K/AKT pathway is involved in the Nrf-2 activation and translocation [21]. Consistently, PTEN could significantly repress the activity of Nrf-2, while loss of PTEN could lead to the activation of Nrf-2 [22]. In the present study, we investigated whether inhibition of PTEN with VO-OHpic could ameliorate oxidative stress induced CEP chondrocytes apoptosis and degeneration. Also in vivo experiments were conducted to investigate the effects of VO-OHpic in attenuating cartilage endplate calcification and inhibiting IDD development. RESULTS VO-OHpic significantly ameliorated IDD progression and cartilage endplate calcification in vivo We established an IDD mice model by transection of the L4/5 bilateral facet joints, supra- and interspinous ligaments to investigate the effects of VO-OHpic in IDD in vivo. The IDD degeneration was scored by immunohistochemistry staining and micro-CT analysis. As shown in Figure 1A, immunohistochemistry staining showed that mice in IDD group exhibited reduced NP and obvious cleft in annulus fibrosus. Bone marrow and empty bone and chondrocyte lacuna were found in the deep zone of CEP. Compared to mice in IDD group, the CEP in VO-OHpic treatment group was thicker with much less bony tissues. The AF and NP were intact. These results demonstrated that VO-OHpic could inhibit C (...truncated)