MMP-10 Deficiency Effects Differentiation and Death of Chondrocytes Associated with Endochondral Osteogenesis in an Endemic Osteoarthritis.

OBJECTIVE: The objective of this study was to determine the matrix metalloproteinase-10 (MMP-10) expression pattern and to assess how it contributes to endochondral osteogenesis in Kashin-Beck disease (KBD). DESIGN: The cartilages of KBD patients, Sprague-Dawley rats fed with selenium (Se)-deficient diet and/or T-2 toxin, and ATDC5 cells were used in this study. ATDC5 cells were induced into hypertrophic chondrocytes using a 1% insulin-transferrin-selenium (ITS) culture medium for 21 days. The expressions of MMP-10 in the cartilages were visualized by immunohistochemistry. The messenger RNA (mRNA) and protein expression levels were determined by real-time polymerase chain reaction (RT-PCR) and Western blotting. MMP-10 short hairpin RNA (shRNA) was transfected into hypertrophic chondrocytes to knock down the gene expression of MMP-10. Meanwhile, the cell death of MMP-10-knockdown chondrocyte was detected using flow cytometry. RESULTS: The expression of MMP-10 was decreased in the growth plates of children with KBD. A decreased expression of MMP-10 also was observed in the growth plates of rats fed with an Se-deficient diet and/or T-2 toxin exposure. The mRNA and protein expression levels of MMP-10 increased during the chondrogenic differentiation of ATDC5 cells. MMP-10 knockdown in hypertrophic chondrocytes significantly decreased the gene and protein expression of collagen type II (Col II), Col X, Runx2, and MMP-13. Besides, the percentage of cell apoptosis was significantly increased after MMP-10 knockdown in hypertrophic chondrocytes. CONCLUSION: MMP-10 deficiency disrupts chondrocyte terminal differentiation and induces the chondrocyte's death, which impairs endochondral osteogenesis in the pathogenesis of KBD.

Effects of feeding status on nucb1 and nucb2A mRNA expression in the hypothalamus of Schizothorax davidi.

NUCB1 and NUCB2, two novel nucleobindins, have attracted extensive attention for their role in the appetite regulation in mammals. However, little is known about the appetite regulation of NUCB1 and NUCB2 in fish species. Therefore, we investigated the role of these peptides in the regulation of feeding in Schizothorax davidi (S. davidi). In this study, full-length cDNA sequences of nucb1 and nucb2A of S. davidi were obtained for the first time. Additionally, the tissue distribution and the effects of different energy status on nucb1 and nucb2A mRNAs abundance were assessed, showing that nucb1 and nucb2A are widely distributed in 18 detected tissues, with the highest expression in the cerebellum. The abundances of nucb1 and nucb2A increased in the hypothalamus at 1 h and 3 h post-feeding. Furthermore, fasting and re-feeding experiments showed that the expressions of nucb1 and nucb2A in hypothalamus significantly decreased after fasting for 7 days, and returned to the control level after re-feeding for 3 or 5 days. In conclusion, the present study suggests that both NUCB1 and NUCB2A are involved in the short-term and long-term appetite regulation, as an anorexigenic factor, in S. davidi. These results can provide a basis for further investigation into the appetite regulatory role of NUCB family in teleost.

Cloning, expression profiling, and effects of fasting status on neuropeptide Y in Schizothorax davidi.

To better comprehend the mechanism that neuropeptide Y (npy) regulates feeding in Schizothorax davidi, we cloned and identified the full-length cDNA sequence of the npy gene in this species using RACE technology. Subsequently, we explored the npy mRNA distribution in 18 tissues and investigated the expression of npy mRNA at postprandial and fasting stages. We found that the npy full-length cDNA sequence is 803 bp. Moreover, npy mRNAs extensively expressed in all detected tissues, with the highest expression in hypothalamus. In postprandial study, the expression of npy mRNA in the hypothalamus was significantly decreased after eating (p < 0.01). In addition, the expression of the npy gene was significantly increased on the fifth day after fasting (p < 0.05). However, after refeeding, the expression of the npy gene was decreased significantly on days 9, 11, and 14 (p < 0.01). Our research suggest that npy may have an orexigenic role in S. davidi. PRACTICAL APPLICATIONS: S. davidi, a coldwater fish native to China, has high economic value, and it has gained great popularity. To date, there is still no large-scale breeding of S. davidi in China. How to strengthen the production performance of S. davidi is a hot research area. Neuropeptide Y (NPY), a 36-amino-acid single-chain polypeptide, is one of the main appetite regulation factors. However, to date, no studies have reported on the biological function of npy in the feeding of S. davidi. In our study, we revealed that the trend of hypothalamic npy expression during the postprandial and fasting stages. The results suggested that npy might be an appetite-promoting factor in this species. Overall, we provide the theoretical basis for how to strengthen the production performance of S. davidi through appetite regulation.