Mechanisms Of Ihh-PTHrP Signal Axis On The Development Of Femoral Head Necrosis In Broilers

Chicken is one of the highest degree selective breedings. The intensive selection improves production performance and the intensive management and husbrandry system decrease the production cost. This leads to the high incidence of nutrition metabolic disease such as leg disorders although the economic value of broilers significantly is increased. Leg problems in broilers are a kind of disease including femoral head necrosis (FHN), tibial dysplasia (TD) and so on. FHN is characteristiced by lameness. Birds with severe FHN are unable to access feed and water, leading to failure and death, which constitutes a severe welfare problem as well as larg economic losses. The etiology of chicken femoral head necrosis is very complex, and it is still not fully elucidated. Based on lots of epidemiological statistical analysis, experiment research and clinical observation, it is believed that chicken femoral head necrosis is related with many factors inside and outside the body and internal factors are the most important. Moreover, abnormal endochondral ossification is one of the most important etiology leading to chicken femoral head necrosis.Endochondral ossification is regulated by the variety of hormones and cytokines. These factors work together in the growth plate chondrocytes and affect the femoral and tibial longitudinal growth by regulating the chondrocytes proliferation, differentiation and maturation. Among the various regulatory factors, Ihh/PTHrP singal axis plays a critical role. The disorder of signal pathway can lead to the occurrence of femoral head necrosis. However, the regulated function of the signal axis in the pathological mechanism of femoral head necrosis had not been reported. The purpose of this study aimed to build animal model of chicken femoral head necrosis, to clarify the role of the signal axis in the molecular mechanism of chicken femoral head necrosis, to provide a scientific basis for the pathology and diagnosis of the disease by comparing the changes of production performance, bone metabolic index, bone biology index, and genes expression of Ihh and PTHrP in proximal femoral growth plate between induce-affected and healthy birds.1. The regulated roles of ihh/PTHrP signal axis in the pathogenesis of femoral head necrosis in broliers60 broiler chicks (gallus gallus, Ross 308) of both sexes were used in the present study. Chickens were randomly allocated to control and methylprednisolone-treated groups and each group had 30 birds with 3 pens of 10 birds. After the 7 days pre-feeding, the treated-group birds were intramuscularly injected daily with methylprednisolone (20mg/kg of body mass/d) and the control-group birds were injected daily with sterile saline. The changes of body weight, feed intake, liver index, index of bone metabolism, bone biomechanical indicators and histopathological between natural affected, induced and healthy chickens were determinated. The result showed as follows. I) This experiment successfully established the disease model of chicken femoral head necrosis by hormone treatment, and the natural affected and induce-affected chickens almost demonstrated the same histopathological changes.2) The bone index, bone length, bone strength and bone density were significantly declined in affected chickens compared with healthy birds.3) The length of the growth plate and articular cartilage of affected chickens was markedly thinner than the healthy.4) The gene expression levels of Ihh, GLI2, BMP6, MMP9, Bcl-2, VEGF and collagen Ⅱ in affected chickens were significantly decreased compared with the healthy, however the expression of collagen X and PTHrP obviously increased. In conclusion:The FHN modele with pectoral intramuscular methylprednisolone (20mg/kg body weight) treatment was infallible, and the disorder of Ihh-PTHrP signaling axis had important roles in the pathogenesis of FHN.2. The regulation of Ihh/PTHrP signal axis on the proliferation, hypertrophy and maturation of cultured chicken chondrocytesChondrocytes were isolated from proximal tibial growth plate with 42 days of age Ross broilers for primary culture. After adherent culturing for 24h, chondrocytes were incubated with different doses Ihh signal blocker cyclopamine (10-4-10-7 M) for 12h,24h, 48h. Then the activity of alkaline phosphatase was determined. Meanwhile, the expression of PTHrP, Ihh, collagen X, collagen Ⅱ, GLI2, BMP-6 and Bcl-2 was detected by real-time PCR and western blot. The results showed that the gene expression of PTHrP and collagen × increased when Ihh signaling blocked, and the expression of GLI2, BMP-6, Bcl-2 and collagen Ⅱ significantly decreased, but it was worth noting that the expression of Ihh was increased. Based on these results, the Ihh signal was essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling.