KOR-Based Effect of Smo/Gli1 on the Development of Osteoarthritis Inflammation via Hedgehog (Hh) and Mechanism Study

Abstract

Background: Osteoarthritis (OA) is a chronic progressive joint disease with a complex pathogenesis in which inflammation plays a vital role in its development. This study aims to investigate the function of the Kappa Opioid Receptor (KOR) in the Hedgehog (Hh) signaling pathway, as well as its effects and mechanisms on the development of inflammation in osteoarthritis through the Smoothened (Smo)/Glioma-associated oncogene homolog 1 (Gli1) signaling pathway. Methods: The expression of KOR, Smo, Gli1, and Hh was detected by Western Blotting (WB) and Polymerase Chain Reaction (PCR). Interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were monitored by enzyme-linked immunosorbent assay (ELISA). The expression of apoptotic proteins B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid-specific protease-3 (Caspase-3) was detected by WB, and cell growth was assessed using the Cell Counting Kit-8 (CCK-8) assay. The chondrocyte injury was observed by toluidine blue staining, and apoptosis was detected by flow cytometry. A rat OA model was established using the meniscectomy method, and Hematoxylin and Eosin (HE) staining and Modified Safranin-fast green staining were used to observe the histopathological structure of cartilage in OA rats. The histopathological assessment of the severity of cartilage osteoarthritis lesions was made by Mankin scoring. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was used to identify apoptosis in cartilage tissues. Results: In cellular experiments, the osteoarthritis group showed elevated levels of Smo, Gli1, and Hh, reduced expression of KOR, decreased proliferation of cartilage cells, increased apoptosis, and increased inflammatory response (p < 0.05). The addition of JT09 or cyclopamine amplified cell viability and diminished the expression of pro-apoptotic proteins, which was more effective when dual action was applied to osteoarthritis. In animal experiments, concerning the osteoarthritis group, the addition of JT09 or cyclopamine increased the expression of KOR, and reduced the expression of Smo, Gli1, and Hh. It also increased Bcl-2, declined Bax and Caspase-3, and diminished the inflammatory response, and the effect of both simultaneous actions was significant (p < 0.05). These indicated that JT09 synergistic treatment with cyclopamine declined the inflammatory response in rat osteoarthritis. Conclusions: KOR mediates Hedgehog (Hh) to promote osteoarthritis inflammation progression and exacerbates bone damage through the Smo/Gli1 pathway.