The Effects And Mechanism Of Bilobalide On DSS-induced Mouse Model In Ulcerative Colitis

Research Background:Inflammatory bowel disease(IBD)refers to a range of intestinal inflammation-related diseases,including ulcerative colitis(UC)and Crohn’s disease(CD).Among them,the main manifestation of UC is chronic non-specific inflammation of the colon and rectum,which can involve any location of the colon and rectum.It generally starts from the inflammation of the rectal mucosa and continues to extend to the proximal colon.At present,the pathogenesis of UC is still unclear,but more and more studies have shown that the occurrence of UC is caused by multiple factors,including genetic susceptibility,epithelial barrier defects,immune response dysregulation,and environmental factors.With the development of UC,most patients may even develop life-threatening complications such as intestinal perforation,toxic megacolon,and cancer.Therefore,the development of corresponding specific drugs for UC is an important direction of current research.So far,the treatment of UC is mainly drug therapy,however,the drugs currently used in clinical practice sometimes have limited efficacy and may cause serious side effects.Current clinical data show that the incidence of UC occurs in all age groups and genders,and its incidence is increasing year by year.In thepast 30 years,the incidence of UC in China has also increased with the improvement of people’s living standards.UC has the characteristics of repeated attacks and prolonged non-healing as a chronic non-specific intestinal inflammation.Therefore,in view of the current status of UC in China,early research on related drugs and treatment strategies is the focus of future research.As an important natural medicine resource library,natural products contain biologically active substances that can have therapeutic effects on various diseases.Moreover,natural products themselves have the advantages of novel structure and less side effects,and their acquisition methods are relatively simple and economical.Therefore,screening out drugs for relieving UC from natural products has good application prospects.Based on the above viewpoints,it is of great significance to find a new natural medicine that can effectively control UC with less toxic and side effects.In the previous work,the RAW264.7 cell model was used to screen several natural products(Bilobalide,Loganin and Panaxatriol),and the changes of pro-inflammatory cytokines were detected by q RT-PCR assay.All of the drugs in the study are effective on LPS-induced inflammation,but considering factors such as drug efficacy,drug side effects,and economy,the natural product Bilobalide was finally selected as the research object for relieving UC.Research Objectives:To explore the mechanism of natural product Bilobalide in inhibiting the inflammatory response of UC,and to clarify whether Bilobalide can play a therapeutic role in UC by protecting the intestinal epithelial barrier and regulating the homeostasis of intestinal flora.Experimental Methods:(1)Screening of natural products: q RT-PCR technology was used to detect the effects of natural products(Bilobalide,Loganin and Panaxatriol)on LPS-induced related pro-inflammatory cytokines.(2)In vivo experiment: Firstly,we used DSS to induce the mouse model of UC,and at the same time,the mice were given the protective treatment of Bilobalide.After the modeling,the colon tissues and feces of the mice were collected to evaluate the length and histological characteristics of the colon.Secondly,the collected colon tissue was ground to collect the supernatant and extract proteins.The expressions of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α in the supernatant were detected by ELISA,and MAPK and AKT/NF-κB pathway proteins were detected by Western Blot.In addition,the tight junction proteins ZO-1,Occludin and Claudin-3 related to intestinal barrier integrity in the intestinal tissues of mice were also examined by immunofluorescence and Western Blot.Finally,16 S r DNA high-throughput sequencing analysis was performed on the collected feces to test the effect of Bilobalide on intestinal flora.(3)In vitro experiment: First,RAW264.7 cells and MC38 cells were induced by LPS and with Bilobalide protection at the same time.The expression of i NOS,COX-2,IL-1β,IL-6 and TNF-α were detected by q RT-PCR assay.Then cell proteins were extracted,and MAPK and AKT/NF-κB pathway proteins were detected by Western Blot.In addition,we also verified the expression of tight junction protein in MC38 cells by immunofluorescence and Western Blot.Experimental results:In the natural product screening experiment,it was found that Bilobalide had the best inhibitory effect on pro-inflammatory cytokines compared with Loganin and Panaxatriol,and had no obvious effect compared with 5-aminosalicylic acid,a commonly used drug for the treatment of UC.Therefore,Bilobalide was selected for follow-up experiments.In animal experiments,we found that Bilobalide treated mice had increased colon length,normalized colon histological features,and down-regulated MPO and the expression of pro-inflammatory cytokines IL-1β,IL-6,and TNF-α compared with DSS treated inflammatory model mice.We also found that the activation of MAPK and AKT/NF-κB signaling pathways was inhibited and inflammation was reduced in the Bilobalide group compared with the DSS group.In addition,we found that Bilobalide up-regulated the expression of tight junction proteins ZO-1,Occludin and Claudin-3 to protect the integrity of the intestinal epithelial barrier.Finally,through stool sequencing analysis of mice,we found that Bilobalide can affect the overall richness of intestinal flora,promote the proliferation of probiotics(including Lactobacillus),enhance beneficial metabolism,maintain the homeostasis of intestinal flora,and thus achieve the effect of relieving UC.At the cellular level,consistent with studies in vivo,Bilobalide can alleviate and protect RAW264.7 cells against LPS-induced inflammatory damage and effectively reduce the expression of relevant inflammatory cytokines,thus inhibiting inflammation.Consistent with the results of in vivo experiments,Bilobalide inhibited the activation of MAPK and AKT/NF-κB signaling pathways in LPS-induced RAW264.7 cells.In addition,we validated the effect of Bilobalide on the intestinal epithelial barrier in MC38 cells and found that Bilobalide increased the expression of tight junction protein between cells following an inflammatory response.Conclusion:In summary,this experiment preliminarily screened Bilobalide from some anti-inflammatory natural products as the follow-up research object,and found that Bilobalide can alleviate DSS-induced colitis in mice and LPS-induced cellular inflammation by inhibiting MAPK and AKT/NF-κB signaling pathways.At the same time,Bilobalide can also improve the intestinal epithelial barrier damaged by DSS and LPS,and maintain the integrity of the intestinal epithelial barrier;and bilobalide can improve the intestinal flora,reshape the composition of the flora,and promote the colonization of probiotics to improve the role of UC.