Mechanism Of High-fat Diet Inducing Femoral Head Necrosis In Broilers

The rapid development of the economy and the continuous improvement of farming technology have enabled the rapid development of the broiler industry,and the scale of broiler farming has continued to expand.At the same time,the incidence of metabolic diseases and leg diseases in broilers gradually increase.Femoral head necrosis(FHN)is one of the most common leg diseases.Broilers with femoral head necrosis walk hard,unable to eat and drink normally,with a result of slow growth and reduced production performance,causing huge economic losses.Abnormal lipid metabolism is one of the pathogenic factors leading to femoral head necrosis in broilers,but its pathogenesis remains to be studied.Chondrocyte is the major cell type in articular cartilage,and its cell activity and survival status directly affect the morphological and physiological functions of articular cartilage.Endoplasmic reticulum stress(ERS)is one of the pathways for inducing apoptosis.It has been reported that ERS mainly induces apoptosis of chondrocytes cultured in vitro via PERK-eIF2α-ATF4-CHOP pathway.In this experiment,a high-fat diet was used to establish a model of hyperlipidemia and femoral head necrosis in broilers.The serum lipid levels,liver function index,organ index,lipid metabolism-related gene expression levels were detected in the experimental chickens.And the expression of endoplasmic reticulum stress and apoptosis-related genes in articular cartilage was explored in the FHN broilers to explore the mechanism of endoplasmic reticulum stress on femoral head necrosis induced by hyperlipidemia under the treatment of high-fat diet.At the same time,the articular chondrocytes cultured in vitro were treated with free fatty acids to detect the changes of gene expression and apoptosis of articular chondrocytes,and to elucidate the mechanism and pathway of endoplasmic reticulum stress-induced apoptosis of articular chondrocytes.1.Mechanism of high-fat diet-induced hyperlipidemia in broilers and induction of femoral head necrosisIn this study,the high-fat diet was fed to establish a model of hyperlipidemia and femoral head necrosis in broilers.To explore the mechanism of endoplasmic reticulum stress on femoral head necrosis induced by hyperlipidemia under the treatment of high-fat diet,the serum lipid levels,liver function index,organ index,and the mRNA expression of lipid metabolism-related genes in the experimental chickens were detected,and the morphological changes of articular cartilage tissue were observed in the FHN broiler and the expression of endoplasmic reticulum stress and apoptosis-related genes were determined.100 broilers were randomly divided into 2 groups of 50 each.The broilers in the experimental group began to feed the high-fat diet from the 1st day until the end of the experiment;the broilers in the control group only received the basal diet.Observe the behavioral status of broilers at 21st,28th,35th and 42th day,and collect blood,liver,fat,bone and other samples to evaluate the broiler’s mobility,organ index,liver function,lipid metabolism,femoral head cartilage morphology.The expressions of endoplasmic reticulum stress-related genes in articular chondrocytes were evaluated.The results showed that during the whole feeding cycle,the incidence of femoral head necrosis in the broiler chickens in the experimental group was significantly higher than that in the control group.The broilers with femoral head necrosis showed symptoms such as lameness and reluctance to walk,decreased organ index,abnormal lipid metabolism,and liver tissue.Pathological changes were evaluated,such as internal fat deposition.Morphological observation showed that the structure of articular cartilage was disordered and the content of extracellular matrix was decreased in broilers with femoral head necrosis.Real-time quantitative PCR showed that the expression of collagen-2,aggrecan and anti-apoptosis gene Bcl-2 decreased in femoral head chondrocytes.The expression levels of endoplasmic reticulum stress-related genes PERK,ATF4,CHOP,IRE1,TRAF2,ASK-1,JNK,caspase-9,and caspase-3 increased.It indicated that high-fat diet could cause hyperlipidemia,abnormal liver function and femoral head necrosis in broilers,and the occurrence of femoral head necrosis was related to endoplasmic reticulum stress and chondrocyte apoptosis.2.Mechanism of Free Fatty Acid Inducing Apoptosis of Articular Chondrocytes in VitroIn this study,the articular chondrocytes of broilers were successfully isolated and cultured,and the primary harvested cells were identified by toluidine blue staining,alcian blue staining and polymerase chain reaction.Subsequently,CCK-8 kit was used to detect the cellular viability of chondrocytes which were under the treatment of PA and SA with different concentrations and time.The mechanism of endoplasmic reticulum stress and mRNA expression of apoptosis-related genes were detected by real-time fluorescent quantitative PCR to understand the mechanism of chondrocyte apoptosis induced by free fatty acids.The results showed that the activity of articular chondrocytes decreased and the morphology changed after PA and SA treatment.The anti-apoptotic genes Bcl-2,Bcl-10 and endoplasmic reticulum stress-related genes PERK,ATF4,CHOP were up-regulated in the chondrocytes of experimental group with the expression levels of TRAF2,ASK-1,JNK,caspase-9 and caspase-3 increasing,and the mRNA expression of proapoptotic gene Bid and endoplasmic reticulum stress related-gene IRE-1,decreased,indicating that endoplasmic reticulum stress and apoptosis occurred in the chondrocytes under the action of free fatty acid,and the main pathway to induce apoptosis may be the PERK-eIF2α-ATF4-CHOP pathway.