Study On The Lactoferrin-modified Patchouli Alcohol Liposomes For Colitis Treatment

Inflammatory bowel disease（IBD）is a chronic,recurrent and idiopathic inflammation of the gastrointestinal tract that causes long-term,even irreversible,damage to the structure and function of the gastrointestinal tract.IBD is common in North America and Europe but has become a global disease in the past 20 years.The etiology and pathogenesis of IBD are still unknown,which may be related to gene susceptibility,imbalance of intestinal microbial homeostasis,and impaired intestinal mucosal barrier function.Medication for IBD includes salicylate,corticosteroids,immunosuppressants,and immunomodulators.However,the use of these drugs can cause serious side effects and complications,such as an increased incidence of malignancies or infections.Therefore,it is necessary to find a new method to treat inflammatory bowel disease.When inflammation occurs,macrophages will be polarized into M1pro-inflammatory macrophages,which will up-regulate inflammatory cytokines such as TNF-α,IL-6,IL-12,IL-1βand increase reactive oxygen species（ROS）to aggravate the occurrence of inflammation.Therefore,macrophages are an important therapeutic target for regulating inflammation.Objective:In this study,lactoferrin-modified liposomes were constructed,which contained patchouli alcohol with anti-inflammatory activity,delivered drugs to M1-type macrophages,regulated macrophages,reduced the production of pro-inflammatory cytokines,improved the condition of inflammatory bowel disease,and studied the relevant treatment mechanism.Methods:DSPE-PEG₂₀₀₀-NHS modified drug-carrying liposomes were prepared by film dispersion.Lactoferrin can be conjugated with DSPE-PEG₂₀₀₀-NHS to prepare lactoferrin-modified liposomes.The particle size and potential of liposomes were measured by laser particle size analyzer,and the appearance of liposomes was observed by transmission electron microscopy（TEM）.The drug loading and encapsulation rate of liposomes were determined by GC-MS.The stability of liposomes in vitro,drug release and modification of lactoferrin were investigated.In the cell experiment,BMDM derived from mouse bone marrow was differentiated into M1-type macrophages to investigate the uptake of liposomes by m1-type macrophages.After the co-incubation of M1-type macrophages with the liposome drug delivery system,the mRNA levels of pro-inflammatory factors（IL-1β,IL-6,IL-12,etc.）were detected by q-PCR,and the levels of cellular inflammatory factors were determined by ELISA.The levels of reactive oxygen species（ROS）in M1 macrophages were detected by flow cytometry,and the protein phosphorylation levels of MAPK（p38,Erk,JNK）signaling pathway and NF-κB signaling pathway were detected by Western blot.Animal experiments were conducted to investigate the therapeutic effect of liposomes on colitis in mice induced by Dextran sodium sulfate（DSS）.Results:1.Liposome（LF-lipo）modified by lactoferrin was prepared.The liposomes were uniform in size,about 140 nm,and stable.2.The results of cell experiments showed that liposomes could effectively reduce the mRNA level of proinflammatory cytokines in M1-type macrophages,reduce the release of proinflammatory cytokines,and effectively reduce the level of ROS.Western blot results showed that liposomes inhibited activation of the MAPK pathway and down-regulated phosphorylation of proteins associated with the NF-κB signaling pathway.3.The results of animal experiments showed that liposomes have the ability to target the colon,can effectively treat colitis in mice,improve the quality of life of mice,and have good safety.Conclusion:The LF-lipo prepared in this study can effectively target the colon and has a good therapeutic effect on colitis.The mechanism is related to the inhibition of MAPK（p38,Erk,JNK）signaling pathway and NF-κB signaling pathway,thereby inhibiting the production and release of proinflammatory cytokines in M1 macrophages and reducing the inflammation in the colon.