Mechanism Of Total Flavonoids Of Rhizoma Drynariae In Regulating Tibial Dyschondroplasia In Broiler Via Gut-Pancreas Axis-mediated Glucose Homeostasis

Broilers are a common food animal and are popular for short growth cycle.The current prevalence of intensive farming has prompted the poultry industry to focus excessively on the growth rate of broilers,which has resulted in bones that cannot cope with the increased weight,ultimately increasing the incidence of leg disease in broilers.Tibial dyschondroplasia(TD)is one of the common leg diseases in broiler,with clinical symptoms of slow movement and walking supported by both or one wing.According to statistics,TD accounts for 30% of the incident bone disease in broilers worldwide.In addition,skeletal growth and development problems caused by TD in broilers have resulted in economic losses of up to $150 million in the United States.Therefore,the prevention and treatment of TD broilers should be taken seriously in the poultry industry.There are many causes of TD,with high blood glucose levels,variations in the gut microbiota and vascular distribution largely influencing the development of TD in broilers.However,the specific pathogenesis of TD in chickens are still unknown.Total flavonoids from Rhizoma Drynariae(TFRD)extract is derived from the roots of Drynaria roosii Nakaike.It has been indicated to be useful in the treatment and prevention of bone disorders.Previous studies have also found that TFRD has an ameliorative effect on TD in broiler chickens,but the specific mechanism of protection is still unknown.240 1-day-old AA broilers were selected as experimental animals.The chickens were divided into CON,TD and TFRD groups.Broilers were acclimatised for 3 days,then fed 50 mg/kg of thiram for 4 days to establish the TD model and treated with 500 mg/kg of TFRD until the end of 21 days.The specific pathogenesis of TD broilers and the specific protective effect of TFRD on TD broilers were investigated.The main study components and results are as follows:Study 1: Changes in tibial bone development of TD broilers and the therapeutic effect of TFRDBroilers fed thiram for 4 days showed obvious clinical symptoms of TD,with legs splitting or walking on one wing support,indicating successful TD model establishment.Results of general growth performance suggested that TFRD treatment significantly increased body weight and tibia weight(p<0.001)and significantly reduced growth plate width(p<0.001)in TD broilers.Pathological histological results showed that TFRD treatment significantly improved the delayed calcification of tibiae in TD broilers.The above results suggest that TFRD has an improving effect on tibial bone development in TD broilers.Study 2: Changes in glucose metabolism in TD broilers and the therapeutic effect of TFRDIn order to investigate whether the changes in blood glucose were related to TD.The blood glucose levels of broilers were first measured by the kit,and the results showed that the levels of blood glucose in TD broilers were significantly higher compared to the CON group(p<0.001),After TFRD treatment,the blood glucose of TD broilers decreased to normal levels.The result of q RT-PCR showed that TFRD treatment significantly reduced elevated GLUT8 and GLUT12 m RNA levels in TD broilers(p<0.001).Secondly,pathological histological assessment of the pancreas showed a large amount of inflammatory infiltration in the pancreas of TD broilers,while the broilers in the TFRD group had normal pancreatic development and no inflammatory infiltration.Finally,the results of pancreatic function showed that the levels of insulin(INS),lysosomal acid lipase A(LIPA)and α-amylase(AMYL)were significantly lower in the TD broilers compared to the CON group(p<0.01).The levels of LIPA and AMYL were significantly higher in broilers in the TFRD group compared to the TD group(p<0.001).These results suggest that TFRD improved pancreatic function and restored elevated blood glucose in TD broilers.Study 3: Effect of gut microbiota on blood glucose levels in broilersThe relationship between gut microbiota and blood glucose was investigated by faecal microbiota transplantation(FMT).60 1-day-old AA broilers were selected and divided into CON and TD groups.Broilers were acclimatised for 3 days and fed 50mg/kg of thiram for 4 days to establish the TD model.FMT was started from day 7.Gavage was performed for one week.The faecal microbiota were extracted from CON broilers(donor)and transplanted by gavage into TD broilers(recipient,15birds/FMT-Con group).Faecal microbiota were extracted from TD broilers(donors)and transplanted into CON broilers by gavage(recipients,15 birds/FMT-TD group).The results revealed that the FMT restored disturbances in the gut microbiota of TD broilers.Plasma GLU levels and m RNA expression of GLUT8 and GLUT2 were significantly lower in broilers of the FMT-Con group compared to the TD group(p<0.001).This suggests that the recovery of gut microbiota disorders in TD broilers was accompanied by a reduction in blood glucose levels.Study 4: Probing the effect of TFRD on glucose metabolism in TD broilers based on the gut-pancreas axisTo investigate the specific mechanism by which gut microbiota affects blood glucose levels.The intestinal barrier was first tested and the results showed that TFRD treatment significantly increased intestinal villus height and intestinal wall thickness(p<0.001)and significantly increased the expression of the tight junction proteins Occludin and Claudin-1(p<0.001)in broilers of the TD group.This suggests that TFRD improves intestinal barrier function in TD broilers.The results of the gut microbiota analysis then showed that the abundance of Blautia and Coprococcus was significantly higher in the TD group compared to the CON group.TFRD treatment reduced the abundance of Blautia in the TD group.Blautia and Coprococcus are genera rich in metabolic butyric acid(BA).Therefore,analysis of short-chain fatty acids showed that TFRD treatment reduced the abundance of BA in the TD group.Finally,the butyric acid receptor GPR109 A was tested and the results showed that TFRD treatment reduced the levels of GPR109 A in the pancreas of TD broiler chickens(p<0.001).These results suggest that butyric acid metabolised by the gut microbiota Blautia acts on the GPR109 A receptor on the pancreas to regulate blood glucose levels.Study 5: Probing the effect of TFRD on bone metabolism in TD high-glucose broilers based on PI3K/AKT/VEGFA signalling pathwayTo investigate the potential regulatory mechanisms of tibial bone damage in TD broilers,transcriptome sequencing was performed on tibiae.Differentially expressed tibial genes were mapped to the KEGG database for functional clustering analysis.The results showed that the focal adhesion pathway was most clearly differentially expressed.Focal adhesion kinases play a crucial role in vascular morphogenesis and repair,and were involved in vascular endothelial cell motility,proliferation,adhesion and apoptosis through activation of PI3K/AKT.The distribution of blood vessels in the tibial growth plate of broiler chickens was then examined by histopathology.The results showed that the density,number and distribution of blood vessels in the tibial growth plate were more in the CON group.No vascular infiltration was seen in the corresponding area of the tibia in the TD group.The distribution,number and density of blood vessels in the TFRD group were significantly richer than those in the TD group.Finally,the PI3K/AKT/VEGFA signalling pathway was examined by Western blot(WB)and q RT-PCR techniques.The results showed that the expression of PI3K and AKT was extremely significantly lower(p<0.001)and the expression of VEGFA was higher in the TD group compared with the CON group.Compared with the TD group,the expression of PI3 K,AKT and VEGFA in the TFRD group were significantly higher in the TFRD group compared to the TD group(p<0.001).These results suggest that TFRD promotes the vascular distribution of tibial growth plate and improves the expression of PI3K/AKT/VEGFA signalling pathway in TD broilers.In summary,the broiler gut microbiota affects the occurrence of TD by regulating glucose metabolism mediated by the gut-pancreas axis.Specifically,the Blautia metabolite BA modulates glucose homoeostasis by acting on the GPR109 A receptor in the pancreas.Disturbed glucose metabolism inhibited the expression of the PI3K/AKT/VEGFA signalling pathway in the tibial growth plate,thereby exacerbating TD lesions.TFRD not only directly promotes strong bones,but also inhibits TD lesions in broilers by regulating gut microbiota,lowering blood glucose and promoting vascular renewal through the above-mentioned pathways.Our results suggest that gut-pancreas axis-mediated glucose metabolism may be a new target to solve bone metabolism-related diseases and point to a possible role for TFRD as a potentially safe and harmless herbal extract in poultry production.