| The growth plate is the main occurrence area of endochondral ossfication of long bones,in which chondrocytes get through a transition phase to bone.During the development of the growth plate,the chondrocytes undergo several periods: the resting period,proliferation period,prehypertrophic period and hypertrophic period.Tibial dyschondroplasia(TD)is a common skeletal disease frequently occurring in the rapid growth stage of broilers.A white,transparent,avascular,non-mineralization cartilage plug appears in the proximal growth plate,which is the most typical characteristic of TD.Recent studies have shown that the lack of vascularization and inadequate calcification are the main causes of TD.The lack of vascularization is due to the reduction of vascular invasion in the hypertrophic area of the growth plate during the occurrence of TD,after that,the oxygen acquisition is reduced and the cell differentiation process is disturbed,then the cartilage plug is formed.The lack of calcification is due to the reduction of matrix metalloproteinases(MMPs)secretion and the degradation rate of extracellular matrix during the occurrence of TD.Because of that,growth plate failed to provide enough space for bone deposition,resulting in a delayed calcification.Our research focused on the degradation of the extracellular matrix and apoptosis.Matrix metalloproteinases can be secreted by a variety of cells,including epithelial cells,vascular endothelial cells,and chondrocytes.Therefore,the abnormal development of chondrocytes may directly affect the secretion of matrix metalloproteinases.During the development of chondrocytes,mature and differentiated chondrocytes need to be eliminated through programmed death to ensure the normal thickness and development of growth plate.We found that a large number of apoptotic cells accumulate in the growth plate of TD-affected chickens.The abnormal apoptosis may play an important role in the development of TD.Chlorogenic acid(CGA)is a kind of carboxyphenolic acid formed by the condensation of quinic acid with caffeic acid.It is widely distributed in the plant kingdom and can be extracted from eucommia,honeysuckle and other plants.CGA is one of the main active ingredients of eucommia extract.Studies have shown that chlorogenic acidhas a variety of pharmacological activities,including antibacterial,antiviral,antitumor,regulating lipid metabolism,eliminating free radicals and exhilarating the central nervous system.In addition,recent studies have shown that chlorogenic acid has positive effects on chondrocytes and bone development.However,there are no reports about the effects of chlorogenic acid on the apoptosis mechanism and TD.In this study,thiram was used to induce TD.150 broilers were randomly divided into the control group,the TD group,and the TD treatment group(CGA group).Thiram was added for TD induction from the fourth day in TD and CGA group and chlorogenic acid was applied by oral administration in CGA group from the eighth day.The relevant data were observed and recorded.After the test,the therapeutic effect of chlorogenic acid was evaluated by the broiler body weight,organ index changes,and tibial parameters.The expression levels of apoptosis-related factors,BECN1(one of the major regulatory proteins of autophagy),and matrix metalloproteinases were measured to explore the role of programmed death in the occurrence of TD,its relationship with the degradation of extracellular matrix and the mechanism of CGA treatment.Our study can provide new perspectives and theoretical basis for the treatment of TD and the application of CGA.The main results are as follows:1.The improvement of chlorogenic acid on clinical symptoms of TD and broilers' growth performanceAfter induction by using thiram,broiler chickens showed obvious clinical symptoms including lameness,listlessness,loss of appetite,bilateral knee joint swelling.After treatment with chlorogenic acid,the condition of the TD-affected chickens was significantly improved and gradually got the ability to stand shortly,their appetite was increased and knee swelling was reduced.Till the 18 th day,clinical symptoms of TD broilers had completely disappeared.After measuring the weight,organ index and tibial parameters of broiler chickens,it was found that the TD group showed a significant downward trend compared with the control group,and the width of the TD tibial growth plate was significantly increased.After the application of chlorogenic acid,all of these parameters were recovered to the control group level.2.Effect of chlorogenic acid on degradation of extracellular matrix of TD growth plateAccording to the HE staining and masson staining of the growth plate,it was foundthat the extracellular matrix of the TD group was less degraded when we compared the same area in TD group with control group,the proportion of extracellular matrix in TD growth plate was significantly higher than that in the control group.In addition,the expression of MMP-9,MMP-10,MMP-13 was detected,in the TD group,the expression levels of MMP-9,MMP-10,MMP-13 were significantly decreased when compared with control group,whereas the expression levels were increased in the CGA group.3.Effect of chlorogenic acid on the apoptosis in TD growth plateAccording to the results of TUNEL staining,it was found that the number of apoptotic cells in the TD group was significantly increased compared with control group,but the number can be significantly decreased after the treatment with chlorogenic acid.After measuring the expression levels of BCL-2,BAX,Cyt c,caspase3,and caspase9,it was found that when compared with the control group,the expression of the anti-apoptotic protein BCL-2 in the TD group was significantly reduced,and the expression levels of BAX,Cyt c,caspase3 and caspase9 in the TD group were significantly increased.After the administration of CGA,the expression of BCL-2 was significantly increased and the rest of the proteins were decreased to the level in control group.4.Effect of chlorogenic acid on BECN1 expression in TD growth plateAccording to the immunohistochemical staining of the growth plate area and the detection of the autophagy marker BECN1,we found that the occurrence of TD was accompanied by a significant decrease of the BECN1 expression,and the CGA can provide positive effect for the increase of BECN.In summary,this test successfully confirmed the therapeutic effect of chlorogenic acid on tibial dyschondroplasia in broilers,and the increase of apoptotic levels of growth plate during the occurrence of TD,which may be related to the accumulation of apoptotic chondrocytes and the decreased degradation rate of extracellular matrix.In addition,the adverse tendency between the autophagy level and apoptosis level during the occurrence of TD may implies that autophagy was involved in the regulation of apoptosis.Reducing apoptosis by increasing the level of autophagy can be a new perspective for treating TD. |
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