In recent years, more and more scholars have focused on the gene and molecular mechanism of endplate cartilage in the study of intervertebral disc degeneration. Some scholars suggested that curcumin-mediated autophagy could enhance the adaptability of endplate cartilage cells to high-intensity loads, thereby enhancing the to achieve the purpose of reducing intervertebral disc degeneration25. Another scholar7 found that overexpression of histone-lysine N-methyltransferase EZH2 inhibited the expression of type II collagen, aggrecan and Sox-9 in rat endplate chondrocytes and found that MMP-13 expression was increased, and EZH2 silencing reversed this expression. It can be seen that the expression of Collagen II, MMP and Sox-9 in the endplate chondrocytes is very specific. The function of Collagen II is mainly to absorb the stress of the intervertebral disc and maintain the normal shape of chondrocytes, which constitute the cytoskeleton and the main components of the extracellular matrix, when the secretion of Collagen II is reduced, the components of the extracellular matrix are reduced. Changes in MMP-13 matrix metalloproteinases can change the structure of endplate cartilage, and the function of endplate cartilage will decrease, resulting in endplate cartilage degeneration. Sox-9 is an important member of the Sox gene family and plays a key regulatory role in mammalian sex determination and chondrogenesis, which can not only directly bind to the specific DNA sequence that regulates Collagen II, but also upregulate the expression of Collagen II. It can also enhance the activity of the proteoglycan promoter, an important component of cartilage16. Sox-9 can also directly inhibit genes normally expressed by hypertrophic chondrocytes such as Col-10α and MMP-1310. Sox-9 is considered to be an important target in chondrocyte engineering to transform seed cells. In this part, we used Collagen II, Sox-9 and MMP-13 marker genes and proteins in endplate cartilage tissue to verify endplate chondrocytes in normal group and IL-1β group, the results were also consistent with other scholars.
When the membrane protein receptor upstream of Hippo signaling pathway receives extracellular signals, Mst1/2 is activated, Mst1/2 phosphorylates and interacts with salvador to phosphorylate Lats1/2 kinase, the phosphorylated Lats1/2 kinase is activated, and after activation The Lats1/2 kinase inactivates a portion of YAP by phosphorylating downstream ser127, and the phosphorylated YAP is subsequently translocated, sequestered from the nucleus into the cytoplasm and then degraded. Dephosphorylated YAP is activated upon entry into the nucleus, thereby driving growth-promoting genes through the TEA domain transcription factor family, such as connective tissue growth factor (CTGF) and cysteine-rich angiogenesis-inducing factor 61 (Cyr61) involved in cell proliferation. However, one study in nucleus pulposus tissue examination30, YAP expression decreased with age, whereas upstream LATS1 levels increased in young rats, and LATS1 was transcriptionally regulated as a direct target gene of YAP, and the level of LATS1 mRNA increased with the activation of YAP3, it can be seen that the regulation of YAP and LATS1 constitutes a negative feedback loop to ensure that excessive activation of YAP does not occur and maintain the steady state of the Hippo signaling pathway. Another article studied the pathological conditions of inflammatory factors to simulate osteoarthritis, and found that the expressions of Mst1/2 and Lats1/2 did not change significantly under the inflammatory stimulation of TNF-α or IL-1β. However, the expression of Yap1/Taz and its target genes CTGF and Cyr61 was decreased in primary chondrocytes under the action of TNFα4. Paper21 showed that Chsy3 (chondroitin synthase 3) activates YAP1 through chondroitin sulfate -mediated actin tension, although LATS1 knockdown reduced the expression of YAP1 downstream target genes to a certain extent. There were no significant effects on the expression of anabolic and catabolic genes, unlike the typical Hippo pathway. In this paper, qRT-PCR detection showed that MST1/2, LATS1/2, YAP1, and CTGF in the Hippo-Yap signaling pathway were statistically significant. Among them, MST1/2, LATS1/2, and YAP1/ CTGF in the normal group were up-regulated compared with the IL-1β group. As an important transcription factor in the Hippo pathway, YAP1 has been shown to be involved in the regulation of various cellular processes, stem cell proliferation and differentiation. Regardless of changes in the expression of upstream elements, only through phosphorylation or dephosphorylation of YAP1 can it play a role in regulating cells. Therefore, we selected the most important YAP1 in the pathway as our research object.
IL-1β is a potent inflammatory cytokine that plays a key role in the pathogenesis of intervertebral disc degeneration23. Previous studies have shown that IL-1β can induce a decrease in extracellular matrix gene expression in in vitro diagnostics and that IL-1β inhibition may provide an effective therapeutic target for preventing and reversing intervertebral disc degeneration. MiR-210-5p plays an important regulatory role in osteochondral metabolism. Scholars have shown that miR-210-5p can inhibit the oxygen consumption rate of chondrocytes and may be a potential intervention in osteoarthritis24. It can be seen that miR-210-5p and YAP1 play a regulatory role in the pathogenesis and growth of tumors, and also play an important regulatory role in degenerative diseases. In this part, we verified miR-210-5p by luciferase experiments. It had a gene binding site with YAP1, and it was verified that the expression of miR-210-5p in endplate chondrocytes was significantly increased under the action of IL-1β, which was opposite to the trend of YAP expression, which also confirms that miR-210-5p and YAP1 functions in endplate chondrocytes. We further verified the opposite changes in YAP1 by plasmid overexpression and silencing of miR-210-5p, and we found that Collagen II and Sox-9, MMP-13 protein and mRNA phenotypes in IL-1β-induced endplate cartilage degeneration cells corresponding changes also occurred in miR-210-5p, which fully demonstrated the combination of miR-210-5p and YAP1, and we caused changes in the expression of YAP1 through overexpression and silencing. In the degeneration group, we mediated YAP1 by silencing miR-210-5p. Overexpression delayed and reversed the cellular phenotype of degenerated cartilage endplate cells, which was further validated by immunofluorescence.
Some scholars2used the rat tail to study the micro- and nano-scale structural analysis of degenerative intervertebral discs, and assessed the histopathology, glycosaminoglycan (GAG) content, histological score, endplate structure and elastic modulus of the intervertebral disc. The study found that the Pfirrmann grading system combined with the micro- and nano-structure changes of the intervertebral disc more comprehensively reflected the degree of intervertebral disc degeneration, so this grading system could accurately, objectively and comprehensively evaluate the degenerative changes of the caudal intervertebral disc in rats. We chose to use an 18G needle for puncture and 26G needle for drug injection. From the results of X-ray and MRI 8 weeks after acupuncture, it can be seen that the intervertebral disc in the model group was significantly degenerated, the X-ray showed the loss of intervertebral disc height and the formation of marginal osteophytes. Safranin fast green and HE staining clearly showed that the nucleus pulposus tissue of the degeneration group became sparse and the arrangement of the annulus fibrosus was disordered, so the acupuncture rat tail intervertebral disc degeneration model in our group was successful, and the injection dose in this group was 12–14 Week-old rats, the injection dose was 10ul, 4 weeks after the injection, the MRI showed that the model + lentivirus group PGMLV-SC5, T2-weighted images showed that the degeneration of the intervertebral disc was still obvious, the Pfirrmann grade did not change significantly, the intervertebral disc signal was black and low signal, the intervertebral disc and nucleus pulposus were ill-defined with the collapse of the intervertebral space. X-ray showed the imaging manifestations of intervertebral space collapse. However, the edema around the intervertebral space was small. Safranin fast green and HE staining showed that the water content of the nucleus pulposus increased slightly compared with the model group, meanwhile the arrangement of the annulus fibrosus was slightly repaired. Immunohistochemistry showed that the expression of YAP1 was increased, which all indicated that we injected lentivirus into the intervertebral disc of the degenerated segment of the rat, and silenced miR-210-5p. Increased expression of YAP1 delays the progression of intervertebral disc degeneration in rats.
We have verified that YAP1 and its downstream target gene CTGF play a positive regulatory role in endplate chondrocytes and in vivo in rats. Compared with the degeneration model group, the expression of YAP1 and its functional phenotype were increased. In rats, we silenced miR-210-5p by lentivirus to mediate YAP1, which also had the effect of reversing the functional protein phenotype. In this study, we verified by luciferase experiment that miR-210-5p had a targeted binding site with YAP1, and the expression of miR-210-5p in endplate chondrocytes was significantly increased under the action of IL-1β, which was closely related to the expression of YAP. In contrast to the trend, by silencing miR-210-5p, we also changed the functional phenotype of degenerated rat endplate chondrocytes, which was surprising after chronic injection of miR-210-5p in rats. The same changes also occurred, which fully shows that miR-210-5p plays a regulatory role in rat endplate chondrocytes and in vivo. The miR-210-5p/YAP1 regulatory axis may be provide a new way of thinking in the treatment of spinal degenerative diseases.