The molecular mechanism investigation of HBP-A slows down meniscus hypertrophy and mineralization by the damage mechanical model

Abstract Objective This study aimed to determine if HBP-A slows down meniscus hypertrophy and mineralization due to abnormal mechanical damage and if the therapeutic effects of HBP-A are mediated through p38-MAPK signaling pathways. Methods In vivo guinea pig study: Male Hartley guinea pigs underwent anterior cruciate ligament transection (ACLT) on the right knee; the left knee served as the control. Three days after molding, high, medium, and low doses of HBP-A were injected into the right knee cavity. The injections were given twice a week for 10 weeks. The width of the medial and lateral meniscus is measured separately using a ruler to assess its hypertrophy. The intensity and area of meniscal calcification were evaluated by Alizarin red and Von Kossa staining. Safranin O/Fast Green staining and OA menisci or cartilage damage scores rated to evaluate degeneration of meniscus and cartilage. Meniscal hypertrophy and calcification-related markers, mtrix metalloproteinase 13 (MMP13), runt-related transcription factor 2 (Runx2), Indian hedgehog (Ihh), alkaline phosphatase (ALP), and ankylosis homolog (ANKH), were detected by immunohistochemistry and RT-qPCR. In vitro rat PMFs study: In vitro isolation and identification of the phenotype of rat primary meniscus fibrochondrocytes (PMFs). 10% stretch force was applied to the isolated PMFs for 24 hours, followed by intervention with 0.3 mg/ml of HBP-A. PMFs proliferation, apoptosis, calcification, and hypertrophy were detected by CCK-8, flow cytometry, Alizarin red, and Toluidine blue staining, respectively. Western Blot and RT-qPCR determine meniscal hypertrophy and calcification related markers with p38 MAPK signaling pathway-related target markers. Results In vivo guinea pig study: Guinea pig's meniscus the width, as well as the area and intensity of meniscus calcification and meniscus and articular cartilage injury score were significantly reduced in the HBP-A intervention group compared to the ACLT group. The expression levels of MMP13, Runx2, Ihh, ALP, and ANKH at the protein and gene level significantly decreased in the HBP-A intervention group compared to the ACLT group. In vitro rat PMFs study: Apoptosis, hypertrophy, and calcification of rat PMFs after 10% stretch force for 24h were significantly improved with 0.3mg/ml HBP-A. Western blot and RT-qPCR showed that hypertrophy, calcification, and p38 MAPK signaling pathway-related markers of PMFs were incredibly depressed in the HBP-A intervention group compared to the 10% stretch force group. Conclusion HBP-A can slow down meniscus hypertrophy and mineralization induced by abnormal mechanical loading, and its mechanism of action may be through the p38-MAPK signaling pathway.