| Aim: In livestock farming,breeders add excessive antibiotics to feed in order to prevent livestock from dying from disease and causing losses,so that livestock can improve their resistance and thus become less sick.However,when livestock containing antibiotic residues are consumed by people,the accumulation of antibiotics in the human body can cause a variety of human harm,such as children’s bone development being affected,severe allergic reactions,kidney damage,and even the development of drug-resistant bacteria.In order to avoid the overuse of antibiotics in livestock farming,the application of Chinese medicine as a feed additive is also an important preventive measure.The search for safe and effective "natural combination compounds" in Chinese medicine with few side effects as feed additives can effectively improve the immune function of livestock and poultry,prevent inflammatory diseases and bacterial infections,and thus reduce the use of antibiotics.Eucommiae Folium(EF)and Acanthopanax Senticosus(AS)are both liver and kidney tonic and strengthen the muscles and bones,as well as lowering blood pressure,sedating,regulating immunity and strengthening resistance.The extraction of Chinese medicinal ingredients is inextricably linked to the study of their processes.Based on the project management institute on wet ultra-micro pulverization extraction(WUPE)and membrane coupled purification(clarification by microfiltration membrane(CMM)is used in this paper,CMM is a type of membrane-coupled purification.),on the basis of the research on EF and AS livestock breeding applications,this thesis screens the extraction process using cell viability and expression of inflammatory substances in cell culture broth,and the extracts from the optimal process are investigated for their immunological,anti-inflammatory and antibacterial effects.In addition,Network Pharmacology(NP)will be used to predict the targets and pathways related to the effects of EF,AS and intestinal diseases,with a view to providing new research directions and ideas for the application of Chinese medicine in livestock farming.Methods:(1)The screening process of EF-AS extract preparation was guided by the immuno-anti-inflammatory effect.The screening conditions included the drug ratio of EF to AS,extraction method,extraction time and before and after by 0.2 μm CMM.Firstly,the extracts under each condition were applied to RAW264.7 cells to observe whether there was a proliferative effect;secondly,the inflammation model was established using Lipopolysaccharide(LPS),with L-NAMC hydrochloride(L-NAMC)as the positive drug group,EF-AS was administered for 1 h for pre-protection and then co-incubated with LPS for 24 h.The viability of RAW264.7 cells and the detection of inflammatory substances in the cell culture medium were measured.(2)The investigates the effect of anti-inflammatory immune activity of EF-AS extracts clarified by WUPE and CMM on RAW264.7 cells.The experiments started with screening the high,medium and low concentrations of EF-AS extracts under this preparation method.Before inflammation modelling,it was divided into normal group,EF-AS 10 μg/ml,1 μg/ml,0.1 μg/ml dose group;in the inflammation model,it was divided into normal group,LPS group,EF-AS 10μg/ml+LPS,1 μg/ml+LPS,0.1 μg/ml+LPS dose group,L-NAMC+LPS group.Firstly,the proliferation and phagocytosis of RAW264.7 cells by EF-AS extract was investigated,and the nitric oxide(NO)content of the culture medium was analysed.Secondly,in the cell inflammatory model,reflect immune Anti-inflammatory effectsthe of the content of NO,Tumor Necrosis factor-α(TNF-α),nterleukin-1β(IL-1β),interleukin-6(IL-6),Lactate dehydrogenase(LDH),Reactive oxygen species(ROS),Myeloperoxidase(MPO)and Glutathione(GSH)in the cell supernatant were detected by the kit.(3)The investigate the modulatory effects of EF-AS extracts on pro-inflammatory M1-like and anti-inflammatory M2-like macrophages after clarification and preparation by WUPE and CMM.10 ng/ml LPS was used to polarise M1-like macrophages for 48 h and 10 ng/ml interleukin-10(IL-10)for 48 h to polarise M2-like macrophages.The effects of EF-AS extracts on the proliferation,phagocytosis and phenotypic markers of polarized M1-like and M2-like cells were observed.(4)The deals with the antibacterial effect of EF-AS extracts on Staphylococcus aureus(SA).The bacteriostasis effect of EF-AS extracts under different treatment methods was determined by the paper slide method.Minimum Inhibitory Concentration(MIC)of EF-AS extracts was determined by two-fold dilution test tube method.(5)The NP approach was used to predict the inflammation-related targets and mechanisms of EF-AS for the treatment of intestinal diseases.The EF active ingredients and their targets of action were obtained by searching the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database,while the AS active ingredient was analysed in combination with BATMAN,chemistry specialised databases and Swiss ADME database,and the resulting AS active ingredients were then searched for their targets in the Swiss Target Prediction database.The intestinal disease genes were analysed in combination with both Gene Cards and OMIM databases and screened for their common targets of action with EF and AS active ingredients.The common targets obtained were uploaded to the String database for Protein-protein interaction(PPI)analysis,and PPI networks were constructed to predict key target proteins.Cytoscape 3.7.1 software was selected to complete the "EF-AS-active ingredient-target-intestinal disease" network diagram,followed by analysis of GO function and KEGG pathway enrichment to predict the mechanism of action of EF-AS in the treatment of intestinal diseases.Results:(1)The screening process of EF-AS extract preparation was guided by the immuno-anti-inflammatory effect.The experimental results show that:(1)compared with the normal group,LPS at 0.5 μg/ml,1 μg/ml and 2 μg/ml can cause damage to RAW264.7 cells,and the survival rate of RAW264.7 cells was about 50% at LPS of 1 μg/ml.(2)When the EF and AS drug ratio of 3:2 had a proliferative effect on RAW264.7 cells;compared with the model group,it had obvious protective effect on the inflammatory model of RAW264.7 cells stimulated by LPS,and was significantly better than the groups with the ratio of 3:4 and 3:8;at the same time,NO,TNF-α,IL-1β and IL-6 inflammatory substances expression was significantly reduced.(3)The EF-AS extracts extracted by WUPE had more obvious pharmacological effects than the heated extraction(HE).(4)With the same extraction method,extraction for 10 min had neither toxic nor significant proliferative effects on cells compared to extraction for 5 min,whereas in the inflammation model,extraction for 10 min had a highly significant difference compared to extraction for 5 min and reduced expression in every inflammatory substance assay.EF-AS extracts filtered through.(5)RAW264.7 cells were better regulated by EF-AS extracts after clarification by microfiltration than before clarification.Therefore,the EF-AS extracts in the subsequent study were prepared by WUPE extraction for 10 min at a drug ratio of 3:2 and filtered through a 0.2 μm CMM.(2)The investigates the effect of anti-inflammatory immune activity of EF-AS extracts clarified by WUPE and CMM on RAW264.7 cells.The experimental results showed that:(1)EF-AS extract concentrations of 100 μg/ml,10 μg/ml,1 μg/ml and 0.1 μg/ml all had proliferative effects on RAW264.7 cells,and we set 10 μg/ml,1 μg/ml and 0.1 μg/ml as high,medium and low dose groups.(2)Immunomodulation of RAW264.7 cells by EF-AS extract,which has a significant proliferative and increase in the phagocytic effect on this cell without increasing NO expression.(3)EF-AS increased cell viability,phagocytosis and GSH content in the inflammatory model of RAW264.7 cells,significantly decreased inflammatory substances NO,TNF-α,IL-1β,IL-6,and significantly decreased LDH,MPO and ROS content.(3)The investigate the modulatory effects of EF-AS extracts on pro-inflammatory M1-like and anti-inflammatory M2-like macrophages after clarification and preparation by WUPE and CMM.The results show that: EF-AS extract reduced M1-like macrophage survival while M2-like cells were unaffected;EF-AS extract affected marker i NOS decreased in M1-like cells and marker Arginase increased in M2-like cells;EF-AS extract not only increases the phagocytosis of M1-like cells,but also has the same tendency to have an effect on M2-like macrophages.(4)The deals with the antibacterial effect of EF-AS extracts on SA.The results show that:EF-AS extracts prepared by WUPE and CMM restrict the growth of SA.The MIC result was0.0912 mg/ml.(5)The NP approach was used to predict the inflammation-related targets and mechanisms of EF-AS for the treatment of intestinal diseases.A total of 3 potential active compounds of EF and 20 potential active compounds of AS,495 target genes corresponding to the active components of EF and AS,1951 targets related to intestinal diseases,225 targets shared by drugs and diseases.The GO enrichment analysis identified 2597 gene functions,the enrichment analysis of KEGG pathway identified 165 related signal pathways,concluding that EF-AS may be used for the treatment of intestinal diseases through human cytomegalovirus infection,TNF signaling pathway,IL-17 signaling pathway,HIF-1 signaling pathway,etc.Conclusions: Comparison of WUPE and CMM with conventional heating and filtration methods,the EF-AS extract prepared by the former was more effective in enhancing the immuno-anti-inflammatory effect of RAW264.7 cells and moderately inhibiting the growth of SA than the traditional heated filtration method.The principle of action may be related to the enhancement of the phagocytic ability of EF-AS extract on RAW264.7 cells and the reduction of the expression of inflammatory substances and oxidants correlated,suggesting that WUPE and CMM may be more favourable for the extraction and the isolation of immuno-anti-inflammatory and antibacterial components of EF and AS.NP predicted the targets and mechanisms related to EF-AS for the treatment of intestinal diseases,and found that EF and AS may regulate multiple pathways such as TNF signaling pathway,IL-17 signaling pathway and HIF-1 signaling pathway through multiple targets such as AKT1 and TP53 to improve the inflammatory conditions of intestinal diseases,which share the same pathway of action with the results of cellular experiments.Therefore,EF and AS could be developed for use as animal feed additives to reduce antibiotic use. |
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