Research on the role and mechanism of indian hedgehog protein in abnormal calcification degeneration of chondrocytes

Abstract

Objective To explore the role and mechanism of indian hedgehog (Ihh) in degenerative and abnormal calcification of chondrocyte. Methods Costal chondrocytes were extracted from C57 mice 6 days after birth, By HE staining, Saffron o solid green staining, collagen type Ⅱ immunohistochemical to identify the chondrocyte phenotype The experiment was divided into the ihh high expression group, and Ihh recombinant protein 5 mg/L was added. Ihh knockout group was added to cyclopamine 20 μmol/L. In the blank control group, phosphate buffer solution (PBS) was added. Using real-time quantitative polymerase chain reaction (Real-time PCR) to detect typeⅡcollagen (COLⅡ), type X collagen (COLX), Runt related transcription factor (Runx2), osteocalcin (OCN), alkaline phosphatase (ALP), aggrecan (AGG), progressive ankylosis (ANKH), extracellular nucleotide phosphatase 1 (ENPP1) factors mRNA expression level. Flow cytometry was used to detect the effect of inhibition of Ihh signaling pathway on chondrocyte apoptosis. Results Compared with the control group, the expression of ALP (3.450±1.357 vs. 1.223±0.740), OCN (2.410±0.395 vs. 1.093±0.453), Runx2 (3.057±1.477 vs. 1.144±0.574) and COLX (3.804±1.400 vs. 1.116±0.511) was increased in the high expression group, and the expression of ANKH (0.255±0.042 vs. 1.101±0.471), AGG (0.574±0.355 vs. 1.007±0.126) and COLII (0.670±0.065 vs. 1.027±0.236), ENPP1 (0.354±0.058 vs. 1.091±0.446) was decreased (P<0.05). When the cyclopamine is added, the opposite result appears(P<0.05). Flow cytometry results showed that Inhibition of Ihh signaling pathway can induce apoptosis (t=9.412, P<0.05). Conclusion Ihh can raise COLX, Runx2, OCN and ALP, inhibit the ANKH, ENPP1, AGG and COLⅡ gene expression, and further promote the cartilage cells anomaly calcified. Key words: Indian hedgehog; Chondrocytes; Calcification