Effect Of EGCG On Thiram Induced Tibial Dyschondroplasia In Broiler Chick

Tibial Dyschondroplasia(TD) is a kind of bone diseases most likely happening on broilers because of their overgrowth. A main characteristic of the TD is that there existing cuneiform cartilage which fails to be calcification and vascularizationexisting in the proximal tibial stem epiphyseal growth plate of sick chicken. Two main reasons were found to result in the TD. One is due to the failure of angiogenesis in the tibial growth plate(GP) which thereby resulting in the transportation hurdles of nutrition to chondrocyte. Ultimately, cartilage in the zone of mineralization and calcium deposition fails to complete the calcification. The other reason result in TD is that the chondrocyte in the lesion area undergo proliferation and difference abnormalities, and proteins secretion function in cells was destroyed. These reasons accelerate the degradation efficiency of cartilage extracellular matrix(ECM) which is responsible for the establishment of spatial structure for calcification. Additionally, by combining with the latest research results from others, we find that heat shock protein family(HSPs) play an important role in the broiler TD happen.In mammals, Hsp-90 is a pro-angiogenic factor that can facilitate the angiogenesis and osteogenesis. Whereas in birds, Hsp-90 exhibits in an opposite way, up regulation of Hsp-90 will inhibit the angiogenesis in cartilage. Studies showed that Hsp-90 significantly expresses in the GP of TD broilers, followed by angiogenesis reduction, hypoxia and protein secretion abnormality in the Hypertrophic cartilage zone. Application of 17-DMAG, a specific Hsp-90 inhibitor, were demonstrated to relieve TD symptom obviously. Inhibition of 17-DMAG on broiler TD is likely to related to its regulation on cytokine secretion in chondrocyte, however the exact mechanism is still far from been well known. Epigallocatechin gallate(EGCG) is a kind of polyphenols with the highest contents in green tea. It can inhibit the proliferation of kinds of tumor cells, promoting tumor cell apoptosis. In addition, EGCG can also regulate the expression of HSPs and their client proteins. As an important Hsp-90 inhibitor, it is fully feasible to investigate the effect of EGCG treatment on TD; however, there is not any study on the mechanism of EGCG treatment on TD till now.Based on a large amount of literature, the present study establishes the broiler TD model via thiram induction. Through supplement chicken with EGCG before and during thiram treatment, we investigate the effect of EGCG on TD happen and recovery. Especially, we aim to elucidate the possible mechanism involving the regulation of EGCG on Hsp-90 expression which ultimately mediates broiler TD. For this, we check the gene expression changes of Hsp-90, VEGF and Flk-1 before and after EGCG treatment on TD broilers. We also check the Hsp-90 protein expression by western blot way. The exact results and conclusions in each research part are given as followes: 1. Effect of EGCG on TD broiler growth and growth plate developmentTD suffered broilers induced by thiram present a variety of characteristics including depression, poor appetite, weight loss, abnormality to stand, failure to walk and skeleton deformity and do on. Typical pathological symptoms were observed on thiram induced broiler TD mostly as the same with the nature occurred TD. There exists cuneiform cartilage which fails to be calcification and vascularizationexisting in the proximal tibial stem epiphyseal growth plate of sick chicken. Birds both in EGCG prevention group and EGCG treatment group recover significantly compared to the TD group. The size of the lesion cartilage was also decreased obviously, indicating that EGCG exhibits a positive effect on the TD prevention as well as recovery. 2. Effect of EGCG on the morphological changes of growth plate in TD broilers.Compared to the control group, chondrocytes from the proliferating zone of growth plate in TD broilers differentiated abnormally, angiogenesis and invasion of blood vessels in cartilage decreased. After EGCG application, the differentiation of chondrocytes from the proliferating zone of growth plate in both EGCG prevention group and EGCG treatment group broilers restore to be normalized. The numbers of blood vessels in TD suffered cartilage increased along with the EGCG application, extending to the deep place of proliferation zone cartilage. 3. Effect of EGCG on SOD、AKP、AST activity in TD broilerTo evaluate the effect of EGCG on liver function, we determine the AKP and AST activity in serum and the SOD activity in liver before and after EGCG treatment during TD. As the result showing, both concentrations of SOD and AKP significantly decreased(P<0.05), while AST content increased obviously compared to the control group(P<0.05). After EGCG application on TD broilers, levels of SOD and AKP activities increased greatly in the EGCG prevention group and EGCG treatment group broilers. At the meantime, AST content in the two EGCG treatment groups declined compared to the TD group. All these results indicate the positive role of EGCG in the liver function protection in TD broilers. 4. Effect of EGCG on Hsp-90、VEGF、Flk-1 gene expression in TD broilersWe examined the transcriptional levels of Hsp-90, VEGF and Flk-1 gene to reveal the possible mechanism related to the contribution of EGCG on angiogenesis in TD broilers. Results showed that gene expression of Hsp-90 and VEGF increase 5.5(P<0.05) and 8(P<0.05) times respectively in TD broiler than that in control group. Along with the EGCG treatment before and after the thiram induction, Hsp-90 and VEGF gene expression obviously dropped(P<0.05), both returned to the control level. On the contrary, transcriptional level of Flk-1 m RNA emerged obvious decrease in the TD group chicken, while rose to the normal level again after treating TD broilers with EGCG. These results indicate that EGCG may promote the angiogenesis through regulating the gene expression of Hsp-90, VEGF and Flk-1 in chondrocytes. 5. Effect of EGCG on the protein expression of Hsp-90 in TD broilersAfter determining the Hsp-90 protein production in chondrocytes from each group by western blot way, we found the synthesis of Hsp-90 protein in GP of TD broiler significantly increased compared to the control one. After EGCG treatment, Hsp-90 protein levels both in the EGCG prevention group and EGCG treatment group broilers greatly dropped, even lower than the control group.