| Tibial dyschondroplasia(TD)is a metabolic cartilage disease commonly seen in birds during their rapid growth period.TD usually causes bone malformations,movement disorders and growth retardation in animals,reducing the production performance and affecting the animal welfare and the economic benefits in the poultry industry.There is various etiology of TD,but most studies focused on the failure of vascular invasion and incomplete calcification in the growth plate cartilage,which are mainly manifested in irregular and pale cartilage embolism,intracartilage vascular necrosis,a large number of abnormal chondrocytes and serious disorder of extracellular matrix in the proximal growth plate.Therefore,finding a natural drug that can improve these pathological characteristics is very important for preventing the occurrence and treating the development of TD in production.Ginsenoside Rg1(Rg1)is one of the main pharmacological active components of ginseng,which has strong antioxidant,anti-inflammatory,and anti-apoptotic activities.It is widely used to treat diseases related to articular cartilage,inflammation,cardiovascular,liver,and immune system.However,as a commonly used antioxidant and anti-apoptotic traditional Chinese medicine,whether Rg1 has an improvement effect on the resistance of broilers to TD has attracted little attention.Therefore,this study established the model of TD induced by thiram in both broiler chickens(in vivo)and chondrocytes(in vitro).From the perspectives of chondrocyte apoptosis,extracellular matrix disorder,growth plate angiogenesis,and LncRNA differential expression analysis,we evaluate the therapeutic effects and potential mechanism of Rg1 in regulating thiram induced TD in broiler chickens.1.Evaluation of the therapeutic effect of Rg1 on TD induced by thiram in broilersIn this study,based on previous research in our laboratory,we systematically evaluated the therapeutic effect of Rg1 on thiram induced TD in fast growing AA broilers from multiple aspects,mainly including the improvement of clinical symptoms,the changes of growth performance,tibial growth parameters,tibial growth plates index,and blood biochemistry,and the pathological evaluation of various tissues and organs.The results showed that Rg1 could significantly improve the symptoms of poor mental state,rough hair,swelling joint,decreased appetite,and delayed movement of chicken TD induced by thiram in clinic.From day 7 to day 18,the body weight and average daily gain of broilers treated with thiram significantly decreased,leading to a decline in production performance.In addition,the morphometric parameters(bone weight,bone length and bone diameter)and mechanical parameters(bone density and bone strength)of tibia in the TD group significantly decreased at day 18.After Rg1 administration,both the growth performance and the morphometric and mechanical parameters were significantly restored in broilers.At the same time,we systematically analyzed the pathological damage caused by thiram of tissues and organs such as the brain,liver,kidney,spleen,and heart of broilers.From the staining of pathological tissue sections,we can observe that the main pathological changes caused by thiram occur in liver tissue.In TD broilers,liver cells are arranged in disorder,with focal liver cell degeneration/necrosis,accompanied by inflammatory cell infiltration and bleeding.After treatment with Rg1,the pathological damage of the liver was also improved.Therefore,these studies provide preliminary evidence for whether Rg1 has a protective effect on TD in broilers.2.The mechanism of Rg1 improved chondrocyte apoptosis and matrix disturbance induced TD by thiram in broilersChondrocyte apoptosis and extracellular matrix disorder are the main pathological features in TD.As a chemical and biologically active substance with antioxidant and antiapoptotic properties,whether Rg1 can improve chondrocyte apoptosis and matrix disorder of the growth plate in TD still needs further confirmation.However,the changes of the main components(collagen and aggrecan)and their microstructure in the growth plate matrix have not yet been studied.Therefore,this study was to explore the effects and its potential mechanisms of Rg1 on the chondrocytes and the main components of extracellular matrix in the tibial growth plate of broiler chickens induced by thiram in vivo and in vitro.The results showed that the chondrocytes in the tibial growth plate of broilers treated with thiram showed significant necrosis,mainly manifested by the concentration of the nuclei and cytoplasm,and even the disappearance of the nuclei,with the cytoplasm being eosinophilic.The TUNEL staining results further showed that the number of apoptotic chondrocytes in the growth plate was significantly increased after treatment with thiram,and the mitochondrial pathway was involved in this process.After treatment with Rg1,the apoptosis of chondrocytes was significantly reduced.We isolated and cultured the primary chondrocytes from the growth plate in broiler chickens,and observed the effects of Rg1 on the apoptosis of chondrocyte and experssion of the mitochondrial pathway related proteins induced by thiram.Therefore,Rg1 can improve the apoptosis of chondrocytes induced by thiram through mitochondrial pathways.In addition,this study explored the changes of the main components of extracellular matrix by scanning electron microscopy and Raman spectroscopy.The results showed that the collagen distribution in the growth plate,which showing a greater degree of disorder,winding and fracture by scanning electron microscopy.The results from Raman spectroscopy showed that the correlation peak between collagen and aggrecan increased to a certain extent.At the same time,there were significant changes in the related genes and proteins expression of matrix degradation related enzymes and major component.These results indicate that the structure and composition of extracellular matrix in growth plates of broilers induced by thiram n have significantly changed,which may be one of the important reasons for the develpment of TD in broilers.The treatment of Rg1 significantly improved the extracellular matrix disorder in TD.3.The mechanism of Rg1 improved the blood vessel damage induced TD by thiram in broilersAlthough many studies have suggested that blood vessels play a key role in the occurrence of TD,the methods of its treatment are still unknown.Therefore,this study investigated the protective effect of Rg1 on the blood vessel damage induced TD by thiram in broilers.Firstly,the histological results showed that the density of blood vessels in the tibial growth plate of the TD animals was reduced when compared with that of the control group,especially in the proliferative and hypertrophic areas.Blood routine tests were conducted on blood samples from broilers in different groups.It was found that the indicators of blood routinethe(such as red blood count,hemoglobin,and hematocrit)significantly decreased at day 7 in the TD group.Based on the above results,we speculated that the reduction of tibial cartilage vessels was closely related to the lack of tibial blood cells in broilers.Rg1 significantly improved the number of vascular invasions and the state of blood cells.To further explore the potential mechanism of vascular invasion,we studied the role of HIF-1α/VEGFA pathway in angiogenesis in vitro and in vivo.From the results,thiram significantly inhibited the genes and proteins expression of HIF-1α,VEGFA and VEGFR2 in the growth plate cartilage or chondrocytes,while these factors were significantly upregulated after Rg1 treatment.Therefore,Rg1 can promote the generation of blood vessels via the VEGF signaling pathway in the growth plate,thereby improving the cartilage damage induced TD by thiram in broiler chickens.4.Screening and verification of differential long non-codingRNA of TD in broilers by high-throughput sequencing technologyTD is one of the common cartilage diseases in poultry,but the molecular mechanism of long non-codingRNA(LncRNA)involved is still unclear.Therefore,this study was based on high-throughput sequencing screening and functional annotation methods to study the differential LncRNAs of the tibial growth plate in the TD broiler,and to find some key LncRNAs that may be related to the occurrence of TD.The results showed that a total of 137 differentially expressed LncRNAs(Fold Change ≥ 2 and P < 0.05)were identified in this study,of which 100 LncRNAs were significantly upregulated and 37 LncRNAs were significantly downregulated.The results of KEGG enrichment analysis of these differentially expressed LncRNAs showed that apoptosis,necroptosis,animal autophagy,p53 signaling pathway,Wnt signaling pathway,Fox O signaling pathway,and TGF-β signal pathways play the key role in the occurrence of TD.The differentially expressed LncRNA involved in these pathways provides a molecular basis for in-depth understanding of the pathogenesis of TD in the future,and also provides a new idea for screening specific molecular markers for effective diagnosis of TD in broiler chicken in clinic. |
