Abstract
Lumbar diseases have become a major problem affecting human health worldwide. Conservative treatment of lumbar diseases is difficult to achieve ideal results, and surgical treatment of trauma, complications, it is imperative to develop a new treatment method. This study aims to explore the regulatory mechanism of cartilage endplate ossification caused by abnormal stress, and design intervention targets for this mechanism, so as to provide theoretical reference for the prevention and treatment of lumbar degeneration.
In vivo, we constructed spinal instability model in mice. In vitro, we used a mechanical tensile machine to simulate the abnormal stress conditions of the endplate cartilage cells. Through the high-throughput sequencing, we found the enrichment of Hippo signaling pathway. As YAP is a key protein in the Hippo signaling pathway, we then created cartilaginous YAP elimination mice (Col2::YAPfl/fl). The lumbar spine model was constructed again in these mice for H&E, SOFG and immunofluorescence staining. In vitro lentivirus was used to knock out YAP, immunofluorescence staining, WB and qPCR were performed. Finally, we conducted therapeutic experiments by using YAP agonist and AAV5 carrying YAP plasmids.
We collected 8w samples from C57/BL6 mice after modeling. We found ossification of the endplate in mice similar to human disc degeneration. High-throughput sequencing of stretched cells demonstrated high enrichment of the Hippo signaling pathway. By immunofluorescence staining, it was confirmed that Col-II decreased and Col-X gradually increased in the endplate cartilage of mice. This was also confirmed at 7 days after an in vitro stretch of 5% and 12%. Meanwhile, we found that cartilaginous YAP elimination mice developed very severe endplate degeneration. However, the endplate was well protected by intraperitoneal injection of YAP agonist or AAV5-YAP endplate injection, and the results in vitro were consistent with that.
In the process of cartilaginous ossification, abnormal stress regulates Col10a1 to promote cartilage endplate ossification through Hippo signaling pathway mediated YAP, and we expect to find potential drug targets for treatment through this mechanism.
