Therapeutic Effect And Mechanism Investigation Of Sericin From Different Sources Against Ulcerative Colitis

Ulcerative colitis(UC)is a chronic,nonspecific and recurrent intestinal disease that is mainly found in the large intestine.Recent years have seen the increasing of the incidence of UC,which becoming one of the world's refractory diseases with the improvement of our people's living standards.UC patients usually exhibit many clinical symptoms,such as abdominal pain,diarrhea,weight loss and hematochezia,which seriously threaten public health.It is believed to be induced by multiple factors(e.g.,genetic susceptibility,dietary,and microbial factors),and is characterized by damage of epithelial barrier,imbalance of inflammatory homeostasis,and accumulation of a large amount of reactive oxygen species(ROS).Hence,the treatment for UC not only means relieving inflammation but also repairing damaged colonic mucosa.So far,UC treatments are mainly restricted to reducing the degree of inflammation by using anti-inflammatory drugs or immunosuppressors.Although these drugs can generate the theraputic effect on UC,their further application is limited because of their nonspecific effects on the immune system,resulting in allergic reactions,nausea,elevated liver tests,pancreatitis and other life-threatening short-term and long-term debilitating side effects.In addition,anti-inflammatory drugs(5-aminosalicylic acid)with locally activity and minimal systemic absorption are require d to take frequent high dose administration to exert desirable clinical efficacy.Therefore,it is urgent to identify new therapeutics with the properties of mucosal healing,anti-inflammation,and anti-oxidation for UC treatment.Silk,as known as natural silk,is the continuous long fiber formed by solidification of silk liquid when the matured silkworm spin cocooned.Like wool,it is one of the earliest animal fibers used by humans.Silk consists of two parts,namely the outer sericin and the inner silk fibroin.Sericin,an important component of silk,boas ts a long history,but it is usually discarded as waste in the silk processing because the molecular space structure of sericin represents random curl,loose and disordered organization and low mechanical strength.Recent years have witnessed growing attention by researchers in the field of biomedicine in order to better exploit and utilize various natural resources.Its variable amino acid composition and various functional groups enable it to gain attractive bioactivity,including good biocompatibility,anti-inflammation,oxidation resistance,facilitation of cell proliferation and adhesion.However,its potential for treating UC has not yet been explored,and there are no reports on comparing the therapeutic effects of three kinds of sericin from different source s against UC.The purpose of this study was to compare biological compatibility,uptake rate,anti-inflammatory ability,anti-oxidation properties and the ability of the mucosal repair of three different sources sericin(including Silkworm sericin(SS),Tussah sericin(TS)and Castor silk sericin(CSS)),and the theraputic effect of three kinds of sericin on UC induced by dextran sulfate sodium(DSS)by oral administration of hydrogel(chitosan/alginate)-embedding SS,TS or CSS molecules and further discover the underlying mechanism.The research content is divided into the following three parts:In the first section,the extraction and characterization of sericin of different sources.(1)The SS,TS and CSS were extracted from silkworm cocoon,tussah cocoon and castor silkworm cocoon by the way of sodium carbonate degumming,respectively;(2)UV absorption curves of three kinds of sericin were determined by UV-Vis spectrophotometer.In the second section,cell experiments in vitro of different sources of sericin.(1)The toxicity of SS,TS and CSS to colitis cell lines(macrophage Raw264.7 and colonic epithelial cells Caco2)was determined by the way of MTT;(2)Laser scanning confocal microscopy(CLSM)and flow cytometry(FCM)were used to qualitatively and quantitatively detect the internalization of SS,TS and CSS by Raw264.7 cells and Caco2 cells;(3)The expression level of main proinflammatory factor TNF-? secreted by Raw264.7 cells before and after SS,TS and CSS treatment was determined by ELISA kit;(4)Superoxide anion probe(dihydropyridine,DHE)was used to determine the ROS levels of in Raw264.7 cells before and after SS,TS and CSS treatment;(5)The mucosal healing effects of SS,TS and CSS on caco2 cells were examined by the cells scratch assay.In the third section,therapeutic effect evaluation of sericin of different sources for UC.(1)Vivo or ex vivo imaging technology was used to verify the distribution of SS,TS or CSS in the gastrointestinal tract and five viscera of mice after oral administration;(2)The oral administration effects of sodium alginate/chitosan-embedded SS,TS or CSS hydrogel for UC were detected by using a UC mouse model induced by Dextran sodium sulfate(DSS),and its underlying mechanisms were further discovered.The results show that the purified SS presented low cytotoxicity for Raw264.7 cells and Caco2 cells compared with TS and CSS,and the cell viability was still more than 80% even Raw264.7 cells and Caco2 cells were treated with a concentration of 200 ?g/mL of SS molecules for 48 h.And SS could be most efficiently internalized by Raw264.7 cells and Caco2 cells.After SS,TS and CSS were internalized by Raw264.7 cells,SS most significantly down-regulate the expression of main pro-inflammatory factors TNF-?,and there were no significant difference in the expression levels of pro-inflammatory factors TNF-? between SS treatment group with a concentration of 100 ?g/mL and negative control group.In addition,three kinds of sericin displayed a dose-dependent anti-oxidation and mucosal healing effect.Finally,animal experiments confirmed that oral administration of hydrogel(chitosan/alginate)-embedding SS molecules could effectively aggregate to colonic lesions and attenuated UC-induced symptoms(e.g.,body weight loss,colon shortening,increase of myeloperoxidase activity,and histopathological appearance),which was supported by signs of mucosal healing,elevation of the amounts of tight junction proteins(zonula occludens-1)and colonic mucous.Taken together,our results demonstrated that SS molecules showed the most lowest cytotoxicity,accelerating the mucosal healing,down-regulating the production of pro-inflammatory factor,and eliminating the intracellular reactive oxygen,and it could most effectively relieve UC symptoms.So,SS is worthy of be exploited as a promising oral natural therapeutic for UC treatment with the aid of hydrogel.