Inhibitory Effect Of Quercetin Mediated AMPK/SIRT1 Signaling Pathway On Inflammation Induced By Mycoplasma Gallisepticum

Mycoplasma Gallisepticum(MG)is an avian pathogen present worldwide that can cause respiratory and reproductive diseases in the host,leading to problems such as slow growth and reduced egg production,and once secondary or mixed infections occur,they can lead to a significant increase in mortality and bring serious economic losses to the poultry farming industry.Antibiotic treatment is most often used in the clinic to alleviate symptoms to a certain extent,but the emergence of major problems such as the generation of drug-resistant strains,drug residues and decreased immunity in sick chickens has aggravated the clinical control of the disease.AMPK,as an important regulator of cellular energy metabolism,can rely on the SIRT1 pathway to exert anti-inflammatory effects,and has achieved some results in the study of inflammatory response against pathogen infection,and the anti-inflammatory effects of this pathway can help provide new targets for the study of infectious inflammation.Quercetin(Que)has been found to be a natural anti-inflammatory agent in recent studies,and our group found that quercetin can exert anti-inflammatory effects through the AMPK/SIRT1 pathway,but it is unknown whether quercetin can exert anti-MG-induced inflammatory damage through mediating AMPK/SIRT1 pathway activity.Therefore,in this study,bioinformatics methods such as Alpha Fold2,molecular docking,and molecular dynamics simulation were used to explore the binding mode between chicken AMPKγ1 subunit and quercetin in order to predict the mode of action of quercetin and chicken AMPKγ1 subunit,as well as the binding energy and stability between them.To further investigate the mechanism of quercetin’s action in preventing MG-induced inflammatory damage,the molecular mechanism of quercetin’s anti-MG-induced inflammatory response was further verified by immunohistochemistry,HE staining,Western blot and ELISA using laying hens and primary chicken embryo fibroblasts,respectively.(1)In this study,we firstly performed global molecular docking of quercetin,positive control small molecule ZMP(i.e.AMP analogue,the conversion product of the 5-hydroxyl group of AICAR phosphorylated by adenosine kinase in vivo)with the active structure of mammalian AMPK(PDB ID: 4CFH)by Auto Dock Vina,and the docking results showed that both quercetin and ZMP bound to the mammalian The docking results showed that both quercetin and ZMP bind to the γ subunit of the mammalian AMPK structure.Based on this,this experiment used Alpha Fold2 to predict and obtain the model of chicken AMPKγ1 subunit online,and the molecular docking results showed that quercetin,ZMP and chicken AMPKγ1 subunit binding sites were similar,both bound near the active center of the structural domain,and both formed hydrogen bonds with His119 and Arg120,the key residues near the active center of chicken AMPKγ1 subunit.Further 100 ns molecular dynamics simulations of the ligand-receptor docking complex were performed by Gromacs(version 2021.2),and the stability of quercetin-chicken AMPKγ1 subunit complex binding was confirmed by RMSD and RMSF results;by MMPBSA free binding energy analysis,the binding energy of quercetin to chicken AMPKγ1 subunit was lower,indicating its complex The visualization results further confirmed that quercetin bound to the chicken AMPKγ1 subunit,which was similar to the binding site of ZMP.The above results suggest that the stable binding of quercetin to AMPK may be a way to activate AMPK.(2)It is well known that Mycoplasma Gallisepticum infection mainly triggers pathological phenomena such as inflammatory damage to trachea,bronchi and lung tissues,among which lung tissues are one of the main targets of MG infestation.In order to clarify whether quercetin has the pharmacological effect of anti-MG-induced inflammatory damage,this experiment was conducted in 9-day-old laying hens,and the MG infection model was successfully established by unilateral air sac injection of MG R strain culture and nasal and eye drops.The results of HE staining,immunohistochemistry and Western blot experiments confirmed that quercetin had the effect of reducing inflammatory cell infiltration in lung tissues and promoting the structural integrity of alveoli to improve lung inflammation damage,and quercetin could cause an increase in p-AMPK level in lung tissues.The results confirmed that quercetin had an inhibitory effect on MG-induced inflammatory injury,and it was hypothesized that its anti-inflammatory effect was associated with the promotion of AMPK phosphorylation.(3)In order to elucidate the molecular mechanism of quercetin’s anti-MG-induced inflammatory damage,this experiment used primary chicken embryonic fibroblasts as the research object,and screened the optimal MOI(viral infection complex),time point of MG infection and the safe use concentration of quercetin and instrumental drug by CCK8 method,and successfully established a model of MG infection in chicken embryonic fibroblasts,and the experimental group was treated with 600 MOI MG for 6 h after the action on cells The experimental group was treated with quercetin for 12 h,while the AMPK activator AICAR and the inhibitor Compound C were used to interfere with the AMPK activity in chicken embryo fibroblasts,and the changes of the pathway protein activity were detected by Western blot,and it was found that quercetin could significantly activate the MG-induced AMPK/SIRT1 pathway in chicken embryo fibroblasts;further The results of Western blot assay confirmed that p-AMPK/AMPK and SIRT1 expression were significantly increased and p-P65/P65 and IL-1β expression were significantly decreased in MG-induced cells treated with quercetin.This result confirmed that quercetin’s anti-inflammatory effect against MG-induced inflammatory response was mediated by activating AMPK/SIRT1 signaling pathway,thereby inhibiting the protein expression of NF-κB,which in turn reduced the expression of inflammatory factors,i.e.quercetin mediated the activity of AMPK/SIRT1/NF-κB signaling pathway and thus inhibited the inflammatory response induced by Mycoplasma fowleri.In summary,this study used bioinformatics to verify that quercetin has similar binding sites to AMPK activators,and to elucidate the mechanism by which quercetin mediates AMPK/SIRT1/NF-κB pathway activity and thus anti-MG-induced inflammatory damage at animal,cellular and molecular levels.This study provides new ideas for the search and development of non-antibiotic drugs against MG infection,and lays the foundation for the development of anti-inflammatory drugs targeting AMPK.