| Ulcerative colitis(UC)is a common inflammatory disease of the colon,with a high incidence and a trend of increasing incidence among young people in recent years.It is characterized by diarrhea,mucosal ulcers and colonic bleeding,but the specific pathogenesis is not clear,including genetic factors,dietary factors,physiological factors,psychological factors and other aspects,clinically extremely difficult to cure,and easy to recur,most patients need lifelong medication,greatly reducing the patient’s quality of life,and the long-term risk of developing colon cancer.Currently,the drugs commonly used to treat ulcerative colitis include aminosalicylates,corticosteroids,immunomodulators and anti-inflammatory small molecule drugs.However,a small portion of aminosalicylates are absorbed in the small intestine,which is a strong irritant to the gastrointestinal tract,and some people have allergies to these drugs,so the efficacy of these drugs is very limited.Corticosteroids are mainly used for the treatment of acute or severe colitis,and are not suitable for long-term maintenance treatment,as long-term use can cause a decline in the patient’s systemic immunity,which may eventually lead to serious infections.Immunomodulator drugs are mainly used for short-term maintenance treatment after hormone therapy is ineffective,but they are toxic to the liver and kidneys,can cause certain damage,and are not suitable for long-term use.Therefore,there is an urgent need to develop drugs with high efficacy and low side effects to replace it.Patients with UC are often accompanied by disruption of the three major barriers of the colon:loss of the mucus layer,damage to the protective mucosal layer,and massive accumulation of inflammatory cells.Disruption of the first two barriers results in exposure of the extracellular environment to the hostile colonic luminal environment.Harmful substances and pathogenic microorganisms in the colonic lumen enter the colonic tissue through the mucus layer and damaged mucosa,causing a large number of inflammatory macrophages to accumulate in this tissue area,releasing a large amount of inflammatory cytokines such as tumor necrosis factor-α(TNF-α)and reactive oxygen species,which in turn leads to further development of colitis symptoms.In addition,the intestinal microflora of patients with colitis is often in a state of imbalance,and harmful bacteria,mainly Gram-negative bacteria,proliferate.Lipopolysaccharide(LPS),a major component of the cell wall of Gram-negative bacteria,is shed from the cell wall when the bacteria die under the attack of the body’s immune cells,and the shed lipopolysaccharide then acts on colonic epithelial cells and macrophages to increase the permeability of the mucosal barrier by reducing the expression of tight junction proteins between colonic epithelial cells on the one hand.On the other hand,it stimulates macrophages to produce inflammatory cytokines to further damage the colonic tissue,and this vicious circle makes the patient’s colitis symptoms develop further.As a result,inflammatory tissue sites in the colon are often characterized by damage to the colonic mucosa,loss of the colonic mucus layer,massive release of inflammatory cytokines,increased reactive oxygen and reactive nitrogen species,increased free LPS,and a disturbed metabolic environment due to disturbances in the intestinal microflora.Traditional studies on the therapeutic aspects of colitis have generally been limited to the level of anti-inflammation and mucosal repair.Although the relationship between colitis and intestinal microflora has been revealed in the literature in recent years,there have also been studies to promote the penetration of nanoparticles in mucus to facilitate the entry of nanoparticles into the colonic tissue and improve drug utilization.However,few studies have been conducted to treat multiple aspects of colitis in a comprehensive and systematic manner,and the triggering factors of colitis are multifaceted,and it is difficult to achieve the expected efficacy if only a few of these aspects are treated to break this positive feedback regulation.It has been shown that Epigallocatechin gallate(EGCG),a natural compound rich in tea leaves,has antibacterial,anti-inflammatory,antioxidant and mucosal repair promoting functions.It works mainly through the redox reaction of phenolic hydroxyl groups in its structure with free radicals,inhibiting peroxidase activity and down-regulating the expression of pro-inflammatory cytokines such as TNF-αbased on the nuclear factor kappa-B(NF-κB)pathway.However,its application is greatly limited by its poor in vitro stability,high susceptibility to oxidation,and low oral utilization.In recent years,nano-drug delivery technology has developed rapidly.Compared with traditional delivery systems,nano-drug delivery systems can significantly improve the utilization of drugs in living organisms,slow down the oxidation of drugs and prolong the duration of action of drugs in vivo,and can be modified on their surfaces by controlling the size and material properties,thus providing them with a variety of functions.As a natural macromolecular bioprotein from the silkworm,it is easy to obtain,simple to manufacture,biocompatible and biodegradable,and its special amphiphilic molecular structure allows it to self-assemble into nanoparticles by desolventization,which can be used as a natural nano-drug carrier material.It has been reported that Cathelicidin-BF(CBF),an antibacterial peptide from silver-ringed snake,has good anti-inflammatory,broad-spectrum antibacterial and LPS adsorption effects,and has no toxic side effects on human body,and its related preparations have been used clinically,which has great potential for clinical application.Therefore,if EGCG is encapsulated by filipin protein to make nanoparticles and modified with antimicrobial peptide CBF on its surface,it can not only solve the shortcomings of EGCG which is unstable and easy to oxidize in vitro,but also improve the bioavailability of EGCG and achieve the functions of anti-inflammation,antioxidation,mucosal repair,LPS removal and regulation of intestinal microbial environment.This is a very comprehensive and systematic therapeutic strategy for the treatment of colitis,and has a high prospect of clinical translational application because the nanoparticles are made of natural materials with high biosafety.In this study,we successfully prepared CBF antimicrobial peptide surface-modified silk protein-loaded EGCG nanoparticles and performed a series of physicochemical characterization to demonstrate that EGCG was successfully wrapped and CBF antimicrobial peptide was successfully modified on the nanoparticles.In vitro drug release experiments demonstrated that the nanoparticles with multiple secondary structures of filipin have excellent responsive release under different stimulation conditions(p H,ROS,GSH),which provides a therapeutic idea for specific drug release in acidic and high ROS colonic inflammatory microenvironment.It has also been demonstrated by in vitro experiments that it has good antibacterial,antioxidant and LPS scavenging functions.In antibacterial experiments on Staphylococcus aureus as a representative of Gram-positive bacteria and Escherichia coli as a representative of Gram-negative bacteria,we found that Staphylococcus aureus was more sensitive to EGCG compared to Escherichia coli.The concentration of 0.156 mg/m L EGCG completely killed S.aureus,while the inhibition rate of E.coli at this concentration did not exceed 30%.Anti-oxidation experiments showed that at a concentration of 2.5 mg/m L nanoparticles,the scavenging rate of ABTS~+and PTIO~+could reach more than 90%and40%,respectively.The LPS removal assay showed that 2 mg of nanoparticles modified with CBF antimicrobial peptide could adsorb and remove close to 200 ng of LPS,while the control blank nanoparticles had essentially no removal ability.In an in vitro cellular assay,the nanoparticles were effectively internalized by RAW264.7 and CT-26 cells with good cytocompatibility,and could better promote the healing of cell-scarred mock wounds.In addition,the nanoparticles can reduce TNF-αand interleukin-12(IL-12)and increase interleukin-10(IL-10)expression,which can have a good anti-inflammatory effect.In addition,the nanoparticles can induce M1 type macrophages to polarize to M2 type to a certain extent,and remove intracellular reactive oxygen species(ROS)/reactive nitrogen species(RNS)to protect cells from oxidative damage.Finally,in vivo animal experiments demonstrated that the nanoparticles had good biosafety at the animal level,and there were no significant differences in the liver and kidney-related indexes such as blood routine and alanine aminotransferase(ALT),aspartate transaminase(AST),creatinine(CRE)and blood urea nitrogen(BUN)compared with healthy mice.Creatinine(CRE)and blood urea nitrogen(BUN)were not significantly different from those of healthy mice.In an animal model of ulcerative colitis,the nanoparticles showed excellent therapeutic effects,not only in reducing the symptoms of Dextran sulfate sodium salt(DSS)induced colitis in mice,but also in mice with severe ulcerative colitis symptoms.The nanoparticles significantly improved the loss of colonic mucus barrier induced by DSS and also repaired the colonic mucosal barrier by upregulating the expression of ZO-1,MUC2,Occludin and other tight junction proteins,as shown by Periodic Acid-Schiff stain(PAS)and immunofluorescence section staining.The results showed that the abundance and diversity of intestinal microflora in mice with colitis decreased significantly compared with healthy mice,and the ratio of beneficial to harmful bacteria was imbalanced.The better the treatment effect,the closer the intestinal flora structure was to that of healthy mice.In summary,can EGCG nanoparticles loaded with silk proteins modified by CBF antimicrobial peptide surface achieve effective treatment of ulcerative colitis through anti-inflammation,anti-oxidation,repair of mucosal damage,promotion of macrophage phenotypic polarization,scavenging of LPS and restoration of intestinal microflora balance,and the nanoparticles have good biosafety and great potential for clinical translation. |
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