Ab0034 next Generation Sequencing and Shikonin Effects on Human Primary Articular Osteoarthritis Chondrocytes

Abstract

BackgroundOsteoarthritis (OA) is one of the most common joint disorders, leading to functional disability, especially in people aged over 50 years, causing loss to the economy and affecting social development [1]. Drugs addressing the underlying biological causes of OA are not currently available [2].ObjectivesNaturally occurring naphthoquinone derivatives, such as shikonin and derivatives thereof, were shown to inhibit inflammatory processes and chondrocyte apoptosis by regulating the PI3K/AKT pathway in a rat model of OA [3]. The corresponding regulatory mechanisms in human OA chondrocytes and their influence by shikonin have not yet been investigated. To develop new therapeutic approaches, we investigated the effect of shikonin derivatives on inflammation, MMP expression, the regulation of MAPK signalling, and the gene expression patterns of DNA repair metabolism and the cell cycle in human healthy (HC) and OA chondrocytes (pCH-OA).MethodsViability was analyzed using the CellTiter-Glo® Assay. Inflammatory processes were investigated using a proteome profiler™ assay. Furthermore, we analyzed the effects of the shikonin derivatives by protein expression analysis of the phosphorylation pattern and the corresponding downstream gene regulation using RT-qPCR. Using next generation sequencing (NGS), we have analysed more than 70 key genes of the DNA damage metabolism and cell cycle.ResultsBoth HC and pCH-OA showed a dose-dependent decrease in viability after treatment. The strongest effects were found for shikonin with IC50 values of 1.2 ± 0.1 µM. Shikonin counteracts the inflammatory response by massively reducing the expression of the pro-inflammatory mediators. The phosphorylation level of ERK changed slightly. pJNK and pp38 showed a significant increase, and the downstream targets c/EBPs and MEF2c may play a role in the cartilage homeostasis. STAT3 phosphorylation decreased significantly and has a chondroprotective function through the regulation of cyclin D1 and Sox9. NGS data showed a highly significant difference in gene expression under the influence of shikonin.ConclusionOur results demonstrate for the first time that shikonin derivatives have extensive effects on the inflammatory processes, MAPKs, the IL6/STAT3 downstream regulation and the DNA repair gene expression pattern in healthy and OA chondrocytes.References[1]Sen, R.; Hurley, J.A. Osteoarthritis. InStatPearls;StatPearls Publishing: Treasure Island, FL, USA,2021. Bookshelf ID: NBK482326[2]Chow, Y.Y.; Chin, K.Y. The Role of Inflammation in the Pathogenesis of Osteoarthritis.Mediat. Inflamm.2020, 8293921.[3]Fu, D.; Shang, X.; Ni, Z.; Shi, G. Shikonin inhibits inflammation and chondrocyte apoptosis by regulation of the PI3K/Akt signaling pathway in a rat model of osteoarthritis.Exp. Ther. Med.2016,12, 2735–2740.Acknowledgements:NIL.Disclosure of InterestsNone Declared.