| Deer antler is a traditional and precious Chinese medicinal material in my country.It has a significant effect on improving the functions of the human immune system and nervous system,so it is highly favored by people.Under the existing breeding conditions,it is of great practical significance to explore how to improve the yield and quality of deer antler.Angiogenesis and endochondral ossification are two key physiological processes involved in velvet antler formation.Angiogenesis can transport nutrients to cells in antler tissue and accelerate cell division and growth;at the same time,under the control of various molecules,chondrocytes accelerate the process of antler endochondral ossification and participate in vascular invasion.Therefore,revealing the regulatory mechanism of antler angiogenesis and cartilage development at the molecular level is of great significance to improve the yield and quality of velvet antler.Galectin 1(Gal-1)is an important protein in the regulation of tumor growth.On the one hand,it promotes the migration and proliferation of endothelial cells,and on the other hand,it induces angiogenesis and invasion.It is highly expressed in periosteal stem cells in the raw area.Therefore,it can be speculated that Gal-1 is involved in the angiogenesis and cartilage development of deer antler,and the specific mechanism is still unclear.Vascular endothelial growth factor(VEGF),as an angiogenic growth factor,can effectively regulate angiogenesis.Related studies have found that the expression of VEGF is detected in velvet antler cartilage tissue.There is no report on whether VEGF is involved in the regulation of velvet antler cartilage development.In addition,the rapid growth of deer antler requires a large amount of oxygen to provide energy,but the accumulation of reactive oxygen species does not prevent the growth rate of deer antler,which also indicates that the peroxiredoxins(PRDXs)in deer antler tissue play an important role.By detecting the expression levels of PRDX1-6 in the pre-cartilage layer and cartilage layer,it was found that the expression of PRDX2 increased significantly with the maturation of chondrocytes.In addition to the role of scavenging reactive oxygen species,it has not been reported whether it has other biological functions.In this study,the cells(mesenchymal cells,pre-chondroblasts and chondrocytes)in velvet antler tissue were isolated and cultured,and Gal was studied by RNA interference technology,Western blotting,co-immunoprecipitation,real-time quantitative PCR and immunofluorescence.The roles of Gal-1 and VEGF in the development of deer antler cartilage,and the mechanism of PRDXs involved in regulating deer antler cartilage formation,the following results were obtained:1.The effect and mechanism of Galectin1 on the growth and differentiation of antler chondrocytes(1)The results of western blotting showed that Gal-1 increased significantly during the differentiation of mesenchymal cells into chondrocytes,indicating that Gal-1 may be involved in the maturation of antler chondrocytes.For proliferative chondrocytes,knockdown of Gal-1,down-regulation of Cyclin B1 expression,and up-regulation of P21 expression inhibited cell proliferation.This indicated that Gal-1regulated the proliferation of chondrocytes.(2)Knockdown of Gal-1 in hypertrophic chondrocytes can inhibit the expression of Col10 a and Runx2,and prevent the differentiation of antler chondrocytes into hypertrophic chondrocytes.Further studies showed that Gal-1 regulated the expression of Col10 a through the Wnt10b/β-catenin/Runx2 signaling pathway.(3)After knocking down Gal-1,the expression of VEGF was inhibited,thereby inhibiting the angiogenesis of antler.Co-immunoprecipitation results showed that Gal-1 interacted with Nrp1 to activate the transcriptional activity of YAP1,thereby regulating the expression of VEGF.2.The effect of exogenous VEGF on the growth and differentiation of antler chondrocytes and its mechanism(1)VEGF is expressed in the differentiation process of antler mesenchymal cells like chondrocytes,and the expression in mesenchymal cells is very weak,but with the growth and development of cells,the expression level of VEGF has a significant upward trend.(2)Exogenous addition of VEGF inhibited the expression of Col2a、Sox9 and IGFR1 in chondrocytes,and at the same time caused the cell shape to change from normal spindle shape to irregular round shape.It indicated that VEGF treatment effectively inhibited the growth of antler chondrocytes.(3)VEGF induces intracellular oxidative stress by inhibiting the expression of PRDX2 and PRDX4.In addition,VEGF also promoted the expression of the inflammatory factor IL-6,which eventually led to the degeneration of the antler chondrocyte phenotype.In addition,the addition of melatonin can effectively alleviate VEGF-induced oxidative stress and inflammatory responses(4)The results of western blot showed that melatonin alleviated the VEGF-induced chondrocyte phenotype degradation by activating the AKT/CREB/Sox9 signaling pathway and inhibiting the STAT5/IL-6 signaling pathway.3.The role and regulatory mechanism of PRDX2 in antler chondrocytes(1)Antler chondrocytes can express PRDX1-6 proteases,and the expression level of PRDX2 is significantly increased during the maturation of pre-chondrocytes to chondrocytes.Immunohistochemical results showed that the positive staining of PRDX2 in the antler cartilage layer was more obvious than that in the antler pre-cartilage layer.(2)Down-regulation of PRDX2 can inhibit the expression of cartilage matrix proteins,including Col2 a,Aggrecan and COMP.The results of toluidine blue and alcian blue staining showed that down-regulation of PRDX2 inhibited the synthesis of proteoglycan in chondrocytes.It is suggested that the expression of PRDX2 is involved in regulating the formation of extracellular matrix.(3)Down-regulation of PRDX2 significantly inhibited the expression of CTGF in antler chondrocytes,and CTGF is a growth factor that regulates the formation of extracellular matrix.Therefore,it is speculated that PRDX2 is involved in regulating the formation of extracellular matrix depending on the expression of CTGF.Further results showed that PRDX2 regulates the expression of CTGF by activating the Wnt5a/YAP1 signaling pathway.(4)The down-regulation of PRDX2 promotes the expression of IL-6 in antler chondrocytes.In addition,the JAK2/STAT3 signaling pathway induced by IL-6 is also activated.It is known that the inflammatory response is to inhibit the expression of antler cartilage matrix proteins.Another major factor.Summary: This topic studied the role and regulatory mechanism of Gal-1 and VEGF in the development of antler cartilage.The results show that the two are involved in regulating the angiogenesis of deer antler,but also accelerate the calcification of cartilage,which also explains from the side that deer antler cannot grow infinitely like a tumor.In addition,this study also found that PRDX2 can regulate the formation of extracellular matrix in addition to the ability to scavenge reactive oxygen species,which also provides a theoretical basis for exploring cartilage damage repair. |
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