Study On The Mechanism Of QilingJiaogulan Powder In The Prevention And Treatment Of Broiler Ascites Syndrome Based On Network Pharmacology

Broiler ascites syndrome(AS),also known as broiler pulmonary hypertension syndrome(PHS),is a nutritional metabolic disease that occurs in fast-growing broilers,resulting in poor development and substandard slaughter weight of broilers,have brought serious economic losses to the chicken industry.Due to the inducing factors of broiler AS are complex and diverse,and its pathogenesis is still not completely clear.Therefore,it is still an important research direction to further explore the pathogenesis of broiler AS and screen effective drugs for prevention and treatment of broiler AS.A large number of studies have proved that traditional Chinese medicine compounds can effectively prevent and treat broiler AS from different angles.Network pharmacology is a modern research method to study the potential active ingredients,mechanism of action and compatibility rules of traditional Chinese medicine compounds,which can be used to elucidate the effective components and pharmacological mechanisms of traditional Chinese medicine compounds efficiently.Qiling Jiaogulan Powder is a classic traditional Chinese medicine compound for the treatment of broiler AS in the volume of Chinese medicine published in the“quality standard of veterinary drugs”(2017 edition).The mechanism of Qiling Jiaogulan Powder in the treatment of broiler AS has not been reported in the literature.In this study,an experimental model of broiler AS was constructed by multi-factor method of low temperature(9~11 ℃),high-energy feed(adding 3% lard,4% fish meal),and high salt drinking water(0.12% Na+),to study the prevention and treatment effects of Qiling Jiaogulan Powder.And based on the network pharmacology method,to explore the pathogenesis of AS in broilers and the pharmacological mechanism of Qiling Jiaogulan Powder in the prevention and treatment of broiler AS.This study provides a rich research basis for the pathogenesis of AS in broilers,and provides a more in-depth theoretical basis for the study of the pharmacological mechanism of Qiling Jiaogulan Powder.1.The effect of Qiling Jiaogulan Powder in the prevention and treatment of broiler ASIn this study,240 7-day-old Ross broilers were adaptively raised to 14-day-old,and randomly divided into 6 groups: control group,AS model group,high,medium,low-dose groups of Qiling Jiaogulan Powder(4 g/kg,2 g/kg,1 g/kg Qiling Jiaogulan Powder),and L-Arg group(10 g/kg L-arginine).The resultes showed that: the clinical symptoms and pathological necropsy changes of broilers in the model group were consistent with the symptoms of broiler AS.AHI is an important index for evaluating the occurrence of AS in broilers,and the AHI and the incidence of broiler AS were significantly higher than the control group.The body weight of broilers was decreased,while the feed-to-weight ratio was significantly increased.The levels of SOD,GPX,and CAT in the serum of broilers were decreased,while the MAD level was significantly increased.HE staining showed that the lung lesions observed by HE staining in the model group were consistent with the pathological changes in the lungs of broilers when AS occurred.These results indicated that in this experiment,the broiler AS experimental model constructed by the multi-factor method was successful.Compared to the model group,Qiling Jiaogulan Powder improved the clinical and necropsy symptoms of broilers,decreased AHI index and the incidence of AS,increased body weight,decreased the ratio of feed to gain,increased the levels of SOD,CAT and GPX in serum of broilers,and significantly decreased the level of MDA.HE staining observed that Qiling Jiaogulan Powder alleviated the lung lesions of broilers to varying degrees.The above test results showed that Qiling Jiaogulan Powder could effectively prevent and treat AS in broilers.2.To study the pharmacological mechanism of Qiling Jiaogulan Powder in the treatment of broiler AS based on network pharmacology.The results of network pharmacology showed that: 49 eligible active ingredients of Qiling Jiaogulan Powder were obtained,and 167 core targets for the interaction between Qiling Jiaogulan Powder and broiler AS were obtained.GO enrichment analysis of core targets obtained 117 biological processes,37 molecular functions,and 15 cell components.KEGG pathway enrichment screened out 35 pathways,which were mainly related to FOXO signaling pathway,TGF-βsignaling pathway,MAPK signaling pathway,Cell cycle,Adherens junction,Apoptosis,etc.The network pharmacology study showed that 167 core targets were involved in hypoxic stress,cell proliferation,apoptosis,cell cycle progression,vascular endothelial function and other reaction processes,which led to the occurrence of AS in broilers.The FOXO signaling pathway,TGF-βsignaling pathway and MAPK signaling pathway,Cell cycle,Adherens junction,Apoptosis,etc are involved in the occurrence and development of broiler AS by regulating these reaction processes.The components of Qiling Jiaogulan Powder have good binding activity with the core targets,to treat AS in broilers mainly through the FOXO signaling pathway,TGF-β signaling pathway,MAPK signaling pathway,Cell cycle,Adherens junction,Apoptosis,etc.3.Based on the FOXO3 a signaling pathway to study the pharmacological mechanism of Qiling Jiaogulan Powder in the prevention and treatment of broiler ASNetwork pharmacology studies screened out that the FOXO signaling pathway plays an important role in the mechanism of Qiling Jiaogulan Powder in the treatment of broiler AS,and FOXO3 a is one of the important transcriptional regulators of the FOXO family,which plays an important role in regulating cell differentiation,cell proliferation,apoptosis,DNA damage repair,oxidative stress,metabolism,and follicle maturation.Therefore,in this study,the lungs of broilers at 21 d,28 d,35 d and 42 d were collected as the research objects,and based on the FOXO3 a signaling pathway,the pharmacological mechanism of Qiling Jiaogulan Powder in preventing and treating broiler AS was studied.⑴ The gene and protein expression levels of FOXO3 a signaling pathway related factors were detected by q PCR,IHC and ELISA.The results showed that compared to the control group,the m RNA levels of FOXO3 a and TP53 were significantly decreased in the model group,the protein expression levels of FOXO3 a and TP53 were decreased,the m RNA levels of BCL2,CDK2 and CCND1 were significantly increased,the protein levels of BCL2,CDK2 and CCND1 were increased.Compared to the model group,the m RNA levels of FOXO3 a and TP53 were significantly increased in the Qiling Jiaogulan Powder groups,the protein expression levels of FOXO3 a and TP53 were increased,while the m RNA and protein expression levels of BCL2,CDK2 were significantly decreased,the m RNA and protein expression levels of CCND1 were decreased.The results showed that Qiling Jiaogulan Powder can enhance the activity of FOXO3 a signaling pathway,induce cell apoptosis;block the cell cycle process,and then inhibite cell proliferation.⑵ The effects of Qiling Jiaogulan Powder on the activities of Casepase-3 and Casepase-9and oxidative stress indexes in broiler lungs was detected by enzyme-labeled instrument.Compared to the control group,the activities of Casepase-3 and Casepase-9 were decreased in the model group,the levels of SOD,GPX and CAT were decreased,however the level of MDA was significantly increased.Compared to the model group,Qiling Jiaogulan Powder increased the activities of Casepase-3 and Casepase-9,increased the levels of SOD,GPX and CAT in the lung,and significantly decreased the levels of MDA.These results showed that Qiling Jiaogulan Powder could increase the activities of Casepase-3 and Casepase-9,up-regulate the levels of SOD,GPX and CAT,and decrease the content of MDA in the lung,and protecte the lung from oxidative stress injury.4.Based on the TGF-β1/SMAD2 signaling pathway to study the pharmacological mechanism of Qiling Jiaogulan Powder in the prevention and treatment of broiler ASThe results of network pharmacology showed that the TGF-β signaling pathway was one of the important mechanisms of Qiling Jiaogulan Powder in the treatment of AS in broilers,and the TGF-β1/SMAD2 signaling pathway is closely related to pulmonary hypertension.Therefore,in this study the lungs and pulmonary arterioles of broilers were collected as the research objects to investigate the involvement of the TGF-β1/SMAD2 signaling pathway in the pathogenesis of AS in broilers,and the pharmacological mechanism of Qiling Jiaogulan Powder in the prevention and treatment of broiler AS.⑴ The results of Masson trichrome staining and HYP content detection in the lung showed that: compared to the control group,the lung tissue structure of the broiler chickens was disordered in the model group,and a large number of blue collagen fibers were formed,and the HYP content was significantly increased.Compared to the model group,Qiling Jiaogulan Powder groups improved the above-mentioned lung lesions,the content of HYP in Qiling Jiaogulan Powder groups were significantly lower.These results indicated that Qiling Jiaogulan Powder improved pulmonary fibrosis.⑵ The results of Weigert and Van-Gieson staining and pathological image analysis of pulmonary arterioles showed that compared to the control group,the model group had more collagen fibers deposited in pulmonary arterioles,medium film thickening,intimal hyperplasia,and lumen stenosis and other lesions,and the relative media thickness(%)and relative media area(%)of pulmonary arterioles in the model group were significantly increased.Compared to the model group,Qiling Jiaogulan Powder groups alleviated the above-mentioned lung lesions to varying degrees,decreased the relative medial thickness(%)and the relative media area(%)of pulmonary arterioles.The above results indicated that Qiling Jiaogulan Powder alleviated AS-induced pulmonary arteriole remodeling in broilers.⑶ The gene and protein expression levels of TGF-β1/SMAD2 signaling pathway related factors were detected by IHC,q PCR and ELISA.Compared to the control group,the expression level of a-SMA protein was significantly increased in the model group,the m RNA and protein levels of TGF-β1,SMAD2,COL1A1 were significantly increased,the m RNA and protein expression levels of MMP2 were increased.Compared to the model group,Qiling Jiaogulan Powder decreased the protein expression level of a-SMA,significantly decreased the m RNA and protein levels of TGF-β1,SMAD2,COL1A1 and the m RNA expression level of MMP2,decreased the protein level of MMP2.The results of this experiment showed that Qiling Jiaogulan Powder inhibited the TGF-β1/SMAD2 signaling pathway,and down-regulated the expression levels of a-SMA,COL1A1 and MMP2,alleviated pulmonary fibrosis,improved pulmonary arteriole remodeling.Conclusion: This study shows that 167 core targets are involved in hypoxic stress,cell proliferation,apoptosis,cell cycle progression,vascular endothelial function,inflammation and other reaction processes,which lead to the occurrence of AS in broilers.FOXO signaling pathway and TGF-β signaling pathway are closely related to the pathogenesis of AS in broilers.The compatibility of single herbs of Qiling Jiaogulan Powder and its active ingredients have good binding activity with the core target,which can effectively prevent and cure broiler AS.Its pharmacological mechanism is mainly related to regulating the FOXO3 a signaling pathway and TGF-β1/SMAD2 signaling pathway,protecting broiler chicken lung damage,inhibiting oxidative stress,inducing apoptosis,blocking cell cycle,and reducing pulmonary arteriole remodeling.