To Explore The Efficacy Of P-Hydroxybenzaldehyde In The Treatment Of Crohn’s Disease And The Regulation Of NF-κB/AMPK/PPARγ Pathway Based On Network Pharmacology

Objectives: Crohn ’s disease(CD)is a chronic non-specific inflammatory disease of the digestive tract.Its etiology is still unknown.It has a long course of disease and the possibility of lifelong recurrence.The extraintestinal manifestations of CD are similar to those of ulcerative colitis(UC),but the incidence of CD is higher than that of UC.At present,5-ASA,glucocorticoids,and anti-tumor necrosis factor-α(TNF-α)are the first-line treatments for CD.If other related therapies such as immunosuppression are used for a long time,it may not only increase the risk of infection,but also include some other serious side effects that damage the liver and bone.Based on the above reasons,it is urgent to develop a natural resource or new therapy that can prevent and treat CD.Nostoc commune is a cyanobacterial organism.As an important biological resource,it is often used to prevent and treat diseases.Recent studies have also found that Leigongjun is effective for gastrointestinal diseases.Based on our previous study,we found a component,p-hydroxybenzaldehyde(HD).This component can improve the intestinal inflammatory response,which has a certain effect on UC.We assume that there are many similarities between UC and CD,and there may be related improvements for CD.Through the above ideas,from network pharmacology,in vivo experiments and in vitro experiments,the mechanism and protective effect of HD on CD were explored,which provided experimental basis for the application of HD in the prevention and treatment of CD,and also provided reference for the further development of potential drugs or related foods for the treatment of CD.Methods: At present,the research on the mechanism of action in the treatment of inflammatory bowel disease,Crohn ’s disease and other diseases is more extensive.By introducing the research of network pharmacology,the research direction before the experiment is guided,and the experimental results can also be mutually confirmed.Multiple databases were used to search for compound components,drug targets,drug similarity and oral bioavailability,etc.,to screen out the active components and potential targets of HD,and then screen and integrate the targets of CD,and draw the relevant expression map.Cytoscape software was used for analysis,and enrichment analysis maps were drawn to explore the relationship between p-hydroxybenzaldehyde and Crohn ’s disease.In our previous study,we found that HD(40 mg/kg)had a significant effect on improving UC in mice.Therefore,in view of the similarity between CD and UC,TNBS was used to induce a mouse CD model and the same dose of HD was used for experiments.In this pre-experiment,it can be found that HD has a good improvement effect.In view of this,40 mg/kg was used as the high-dose group.Using the method of multiple reduction,20 mg/kg and 10 mg/kg were selected as the middle-dose group and the low-dose group.The rats were divided into HD high dose group(HD-H,40 mg/kg),HD medium dose group(HD-M,20 mg/kg),HD low dose group(HD-L,10 mg/kg),SASP group(SASP,300 mg/kg),normal group(N)and model group(M).Anesthesia was performed after emptying the intestine by enema.The previously configured 2.5% TNBS 50% ethanol solution was gently injected into 100μL.After injection,the enema tube was pulled out and the mice were suspended.After giving TNBS once,the mice in HD group and SASP group were given the corresponding therapeutic drugs(0.1 m L/10 g)by gavage for 7 days.Four hours after the completion of the administration on the7 th day,all mice were sacrificed to collect colon tissue.Appropriate amount of frozen colon tissue was ground to prepare tissue homogenate.The prepared tissue homogenate was centrifuged for 10 min,4°C,12000 r/min,and the supernatant was separated.Next,the expression levels of TNF-α,IL-6 and IL-1β related proteins and MPO activity in the separated supernatant were detected.The expression of related proteins in NF-κB/AMPK/PPARγ signaling pathway was detected.In vitro,in order to further study the effect of HD on CD,we selected Caco-2 cells for experiments.The expression of related proteins in NF-κB/AMPK/PPARγ signaling pathway was detected by TNF-α-induced Caco-2cell model.Results: Through network pharmacology,the intersection target of HD and CD can be established.Through the query of relevant databases,the screening of genes and the use of software such as Cytoscape,it is proved that HD has a target for CD.By analyzing the results to determine the direction of further experimental research.After using TNBS,mice showed some typical symptoms of Crohn ’s disease,which proved successful modeling.After intragastric administration of HD,it was found that it could significantly improve colon shortening,weight loss and DAI score.The colon tissue of the mice was observed under a microscope.It was found that the colon tissue structure of the HD group was relatively intact,most of the goblet cells were intact,and edema and inflammatory cell infiltration were rare,which proved that HD could improve the histopathological morphology of CD mice.HD inhibited the expression of TNF-α,IL-6 protein and mRNA in a dose-dependent manner.In each group,HD(40 mg/kg)could basically restore the expression of TNF-α and IL-6 to normal levels.The MPO activity of the HD group was significantly inhibited in a dose-dependent manner,indicating that the infiltration of neutrophils was significantly improved.HD can inhibit the activation of NF-κB and improve the related diseases by activating PPARγ to promote AMPK phosphorylation.Conclusions: The mechanism may be related to the activation of PPARγ/AMPK signaling pathway and the inhibition of NF-κB signaling pathway to regulate the release of pro-inflammatory factors.