Basic research Enoxolone suppresses apoptosis in chondrocytes and progression of osteoarthritis via modulating the ERK1/2 signaling pathway Gang Hong Department of Orthopedics, Beijing Chaoyang Hospital, Capital Medical University, Corresponding author: Beijing, China Dr. Gang Hong Department Submitted: 12 December 2019 of Orthopedics Accepted: 4 January 2020 Beijing Chaoyang Hospital Arch Med Sci Capital Medical DOI: https://doi.org/10.5114/aoms.2020.93211 University Copyright © 2020 Termedia & Banach Beijing 100043, China Phone/fax: +86 010-51718262 Abstract E-mail: HermanBrucefva@ yahoo.com Introduction: Osteoarthritis (OA) is an inflammatory disorder of synovial joints which is mainly treated with therapeutic agents showing side effects associated with the gastrointestinal (GI) and metabolic system. Consequent- ly, there is urgent need for a potent, safe and novel agent for treating OA and related disorders. Enoxolone is a pentacyclic triterpenoid obtained from the herb liquorice. Based on earlier findings, we postulated that enoxolone may produce chondroprotective activity by exerting anti-inflammatory, anti-cata- bolic and oxidative stress-decreasing effects. Material and methods: The chondrocytes were extracted from the femo- ral head articular cartilage of healthy rats. Immunofluorescence staining was done for identification of chondrocytes. Cell viability and proliferation studies were done using Cell Counting Kit-8. Apoptotic cells were identified by TUNEL assay and flow cytometry analysis. Autophagy was assessed by monodansylcadaverine assay. Western blot analysis was done for expres- sion of proteins. Results: In the present study we investigated the protective effect of enoxol- one on interleukin 1β (IL-1β) treated Iry chondrocytes in vitro. Treatment with IL-1β resulted in a significant reduction in cell viability of cells in increasing dose and time. Treatment with enoxolone along with IL-1β caused a sig- nificant decrease in growth inhibition. Also, enoxolone inhibited the IL-1β mediated apoptosis and activation of caspase-3 in cells. We also observed that enoxolone elevated the levels of p-ERK1/2, light chain 3 (LC3)-II and Be- clin-1 (autophagy markers) in chondrocytes. The expression of (LC3)-II and Beclin-1 was decreased when the cells were treated with U0126 (ERK1/2 inhibitor). Conclusions: Our findings demonstrate that enoxolone could suppress in- flammatory signaling and apoptosis via the ERK1/2 pathway in chondro- cytes. Key words: osteoarthritis, enoxolone, chondrocytes, ERK1/2, inflammatory signaling, chondroprotective. Introduction Osteoarthritis (OA) is a chronic disease which features loss of cartilage and joint pain associated with inflammation [1, 2]. OA is associated with alterations in subchondral bone, degeneration of cartilage and inflam- mation of the synovial membrane [3]. Literature suggests that none of Gang Hong the present therapeutic approaches can halt the autophagy in hepatocellular carcinoma [22], but progression of the disease successfully. The major- the role of EN in autophagy of chondrocytes has ity of treatments for OA are focused on controlling not been studied. In the present study we evaluat- the symptoms, including pain, and involve use of ed the role of EN in chondrocyte autophagy. drugs belonging to the non-steroidal anti-inflam- A number of studies have suggested involve- matory (NSAIDs) category, or paracetamol is the ment of signaling pathways responsible for mal- choice in some cases [4, 5]. However, long-term functioning in chondrocytes and synovial cells use of these drugs leads to severe GI disturbanc- causing aging, rheumatoid arthritis and osteo- es, and adverse hepatic and cardiac events; hence arthritis. A study confirmed that extracellular there is an urgent need for agents which provide signal-regulated kinase 1/2 (ERK1/2) is associ- a safe and effective route for treating OA. An agent ated with apoptosis in chondrocytes [23]. There from food-derived sources (nutraceuticals) would is a growing need for novel agents for treating be more advantageous [6, 7]. osteoarthritis which can target chondrocytes via Enoxolone (EN), also called glycyrrhetinic acid, multiple cellular pathways by suppressing inflam- is a pentacyclic triterpenoid obtained from the mation without any side effects. Recently it has herb liquorice. EN is used as a flavoring agent been proved that EN can modulate inflammation in food and is also used to mask the bitter taste via the mitogen-activated protein kinase (MAPK) of drugs such as aloe and quinine. EN has been pathway [24]. EN has also been found to target discovered to show numerous pharmacological the extracellular signal-regulated kinase (ERK) activities including anti-inflammatory, anti-oxi- pathway [25]. Both MAPK and ERK are required dant, gastroprotective, antiviral, cardioprotective, for survival, differentiation and maintenance of anti-tumor, neuroprotective and hepatoprotective chondrocytes [23]. The aim of the present study activities in animal models [8–10]. EN is reported was to evaluate any link between enoxolone and to exert a potent anti-inflammatory effect by re- autophagy in IL-1β stimulated Iry chondrocytes in ducing inflammatory edema in an animal mod- vitro, and if there is a correlation, what pathway el of myocardial damage [11]. EN has also been is involved. found to modulate vascular injury and athero- genesis [12]. Since osteoarthritis is associated Material and methods with inflammatory conditions of synovial joints, Animals we postulated that EN can be a potential can- didate for treating osteoarthritis. Interleukin 1β For the study we used male Sprague-Dawley (IL-1β) is a pro-inflammatory cytokine that has rats weighing 210–220 g, each group compris- been reported to play a vital role in the patho- ing 6 animals. The animals were obtained from genesis and factors contributing to osteoarthri- the animal center of Beijing Chaoyang hospital, tis. Upon release IL-1β induces the synthesis of Capital Medical University, Beijing China. All the some more pro-inflammatory cytokines which are experiments were approved by the Animal Ethical responsible for stimulation of apoptosis in chon- Review Board of Beijing Chaoyang Hospital, Chi- drocytes [13]. Cytokines are reported to be potent na. The ethical approval number was AJ001457. stimulators for generation of catabolic enzymes The animals were housed under controlled con- such as matrix metalloproteinases (MMPs). MMPs ditions of temperature (25°C) in a laminar flow are markers indicating excessive degradation of in a 12 h dark and light cycle. The animals were matrix in osteoarthritis [14]. Interleukin 1β is also provided with free access to food and water. The responsible for suppression of cartilage-specific animals were subjected to isoflurane anesthesia proteoglycans, extracellular matrix and collagen (2.5%) as required. All the animals were sacrificed at the end of the study by cervical dislocation by [15]. These changes in catabolism can provide a trained individual. acceleration of degeneration of cartilage. In the present study we analyzed whether EN could in- hibit apoptotic activity of IL-1β. Reagents Autophagy is a conserved catabolic process; it Enoxolone, collagenase-II, rapamycin and Dul- leads to degradation of non-functional organelles becco’s Modified Eagle Medium F-12 (DMEM/ and damaged proteins [16–18]. Also, autophagy F12) were procured from Sigma-Aldrich USA. Bo- exerts a protective effect in the process of degen- vine serum albumin (BSA) and fetal bovine serum eration of fibro-chondrocytes and chondrocytes (FBS) were obtained from Thermo-Fisher, USA. [19, 20]. Autophagy decreases with increasing Methyladenine (PI3K inhibitor) was procured from age, which may be a factor contributing to osteo- InvivoGen USA, and was used in a solution for arthritis in elderly peoples, and hence activation of 100 mM by dissolving in phosphate buffer saline autophagy is very important for reducing the se- (PBS). IL-1β, Terminal deoxynucleotidyl transfer- verity of OA [21]. EN has been reported to induce ase dUTP nick end labeling and apoptosis assay 2 Arch Med Sci Enoxolone suppresses apoptosis in chondrocytes and progression of osteoarthritis via modulating the ERK1/2 signaling pathway kits were bought from Abcam USA. The antibodies rapamycin (7.5 µM) for defined time intervals. The used for the study were anti-cleaved caspase-3 experimental dilutions of EN (i.e. 5 µM, 10 µM, (1 : 1000), anti-beclin-1 (1 : 1000), anti-Bcl-2 15 µM and 20 µM) were prepared in the culture (1 : 1000), anti-LC-3 (1 : 1000), anti-ERK (1 : 1000), media (DMEM/F12 media as described earlier) anti-GAPDH (1 : 1000) and anti-ERK (1 : 1000), added to predefined wells. In the same way, dilu- which were bought from Cell Signaling Technol- tions for IL-1β (10 ng/ml, 15 ng/ml, 20 ng/ml and ogy, USA. The anti-TAMRA and anti-collagen an- 25 ng/ml) were prepared and added to culture me- tibody were supplied by Abcam USA. DAPI and dia. As the dilutions of EN were done using DMSO, Alexa Fluor 488 Phalloidin were purchased from a control group was created by treating the cells KeyGen Biotech China. The Cell Counting Assay with DMSO only. The cytotoxicity assay was done Kit-8 was supplied by InvivoGen USA. after 12, 24 and 48 h, and absorbances for the experiment were recorded on a UV-visible spec- Isolation, culture and identification trophotometer (Shimadzu-1800, Japan) at 450 nm of chondrocytes three times. The percentage of viable cells was cal- culated using the formula % cell viability = (optical