| Tibial dyschondroplasia(TD),a common bone disease in broiler chickens,can cause many diseases and sub health status.GSTA3,a member of the GSTs family,is mainly involved in detoxification of the body,and the regulation of signaling pathways related to oxidative stress.Our previous studies have showed that the apoptosis exist in chondrocytes of broiler tibial growth plate in chicken with TD.On the basis of the previous study,our research that the exogenous GSTA3 protein from Recombinant Prokaryotic Expression was injected into broilers is to clarify the effects of growth plate of recombinant GSTA3 on the genes expression that relate to apoptosis,which can provide information about the pathogenesis and control methods of TD.The study used the TD model induced by thiram and prepared GSTA3 recombinant proteins by our previous technology.180 one-day-old AA broilers were divided into 6(A,B,C,D,E and F)groups at random after feeding for one week.D,E and F of above 6 groups were fed with diets containing 100 mg·kg-1 of thiram at 8 or 9 days old.Subsequently,each group mentioned above were divided into control group(A and D),low dose(20 μg · kg-1)injection group(B and E)and high dose(50 μg·kg-1)injection group(C and F),and the low dose injection group and high dose injection group were injected respectively with the different dose of recombinant GSTA3,and control group with PBS by intramuscular injection at 8,10,12 and 14 days old.We collected tibial growth plate at 1,2,4,6,10 and 15 days of feeding thiram and assessed the model by HE method and measuring the TD score.We detected the antibody by ELISA and detect the transcription level of Bcl-2,Bax,Caspase-3,Caspase-6,MDM2,p53,BAG-1,BAG-3,GOS2,HDAC1,H4,PCNA和STAT3 in cartilage cells using Real-time PCR,and analyze the protein levels of Bax and PCNA by immunohistochemistry.The results of ELISA showed that the concentration of Bcl-2 in serum of broiler was increased at 1 and 6 days,but its concentration was significantly decreased in control groups at 15 days(P<0.05).The level of Bc1-2 in serum of broiler was significantly increased in control groups(C)at 2 and 6 days and the level of Bcl-2 in serum of broiler was significantly increased in experiment groups(F)at 4 and 6 days because of the injection of high doses of GSTA3 recombinant protein(P<0.05).Real-time PCR analysis showed due to the injection of high dose of GSTA3 recombinant protein to broiler,the mRNA expression level of Bcl-2 gene of broiler at 2,4,6 and 15 days and the expression level of MDM2 gene of broiler at 1,2,4 and 10 days and H4 gene of broiler at 2,6 and 10 days were significantly up-regulated in broiler growth plate chondrocytes in experiment groups(F)(P<0.05).In contrast to the above gene,we observed significant down-regulated in transcripts of the immune related genes,namely Caspase-6 gene of broiler at 2,10 and 15 days and GS2 gene of broiler at 4 and 10 days(P<0.05).The mRNA transcription levels of BAG-3 gene of broiler at 4 and 6 days and STAT3 gene of broiler at 1,2 and 4 days were significantly up-regulated after the infection by low dose of GSTA3 recombinant protein in broiler growth plate chondrocytes in experiment groups(E),while the expression of Bax gene of broiler at 1,2 and 6 days,Caspase-3 gene of broiler at 1,2,4,6,10 and 15 days,PCNA gene of broiler at 1,4 and 10 days and STAT3 gene of broiler at 6,10 and 15 days were significantly down-regulated(P<0.05).The expression level of p53 gene of broiler at 6,10 and 15 days and BAG-1 gene of broiler at 10 and 15 days were significantly up-regulated,but the expression of HDAC1 gene of broiler at 4 and 10 days was significantly down-regulated after the injection of either high dose or low dose of GSTA3 recombinant protein(P<0.05).The results of immunohistochemistry showed that the Bax expresses in the cytoplasm of hypertrophy zone,and no expression was observed in the proliferative and hypertrophic regions.The protein expression of Bax at 2,4 and 6 days in the hypertrophy area and 6 days in prehypertrophic area was down-regulated by recombinant protein GSTA3.High expression and low expression of PCNA existed respectively in hypertrophy cell nucleus,and proliferation,prehypertrophic area.The protein expression of PCNA in the growth plate proliferation zone and prehypertrophic area at 6,10 and 15 days and the hypertrophy zone of the TD treatment group at 2 days were downregulated by the recombinant protein GSTA3.The apoptosis level of TD growth plate increased significantly,and the recombinant protein GSTA3 involved in the regulation pathway of cartilage cells apoptosis and can reduce the cartilage cell apoptosis... |
