Development Of Amyloid-EGCG Hydrogels And Its Effects On Mitigating Colitis And Modulating Gut Microbiota

Inflammatory bowel disease(IBD),a lifelong chronic inflammatory disease of the gastrointestinal tract,has become one of the major challenges for public health worldwide.Dietary or food functional component interventions may be an ideal way to prevent or treat IBD.It can and relieve colitis symptoms and reduce the adverse reactions of conventional medicines.The most abundant and most active catechin in green tea is(-)epigallocatechin3-gallate(EGCG).EGCG has the potential to inhibit inflammation and improve intestinal health,which is expected to ameliorate IBD symptoms.However,tea polyphenols are unstable and their bioavailability is poor,which limits their applications in food processing,functional food and medicine.At present,some oral gastrointestinal delivery systems based on nanomaterials have been developed to improve the stability and bioavailability of EGCG,but most of the carrier loading capacities are low,requiring repeated administration in vivo animal studies to achieve appropriate efficacy.Assembly of proteins into flexible biocolloid,amyloid fibrils with a high aspect ratio,has become a new templating strategy for high-end technical materials and bio-nanotechnology.At the same time,protein amyloid fibrils are components of the diet.Therefore,the 3D supramolecular hydrogels were prepared by simply mixing of EGCG with templating HEWL amyloid fibrils,greatly increasing the loading capacities of EGCG.Firstly,the hydrogels were characterized,and the interaction between polyphenols and amyloid fibrils and the evolution law of the soft substance formed across multiple scales were studied.Then the stability of EGCG,gastrointestinal release and distribution of hydrogels were analyzed.Finally,the effects of the hydrogels on the amelioration of colitis and its modulation of gut microbiota in colitis mice were investigated.The main results are as follows:1.Development and structural characterization of HEWL amyloid-EGCG hydrogelsIn this section,HEWL amyloid fibrils were prepared under acidic conditions by heating.After simply mixed with EGCG,3D supramolecular hydrogels were developed,which significantly increasing loading capacities(up to 4.0 wt%)compared to that of other delivery systems and improving the stability of EGCG.The hydrogels showed translucency and birefringence under polarized light,indicating that the formation of the hydrogels did not affect the nematic orientation of the protein amyloid fibrils and phase separation did not occur.In addition,the formation mechanism of hydrogels was further explored from the aspects of molecular interaction and multi-scale structural evolutions.The polyphenols directly combined with HEWL amyloid fibrils,and the equilibrium dissociation constant(Kd)was 3.41×10-7,indicating a strong affinity between EGCG and HEWL amyloid fibrils.Microstructure analysis revealed that EGCG was adsorbed on the surface of amyloid fibrils and continuously deposited to form hybrid nanofilaments,which were further self-assembled into 3D supramolecular hydrogels.Rheological experiments showed that the hydrogels stiffness increased with increasing EGCG content from 0.5 wt%to 4.0 wt%.2.Stability of EGCG after encapsulation,in vitro simulation of gastrointestinal digestion and in vivo gastrointestinal tract distribution,and long-term oral toxicity of hydrogelsIn this section,the properties of amyloid-EGCG hydrogels were further studied.Firstly,the amyloid-EGCG hydrogels(pH 6.0)was prepared to evaluate the thermal and storage stability of EGCG.The results showed that after encapsulated by hydrogels,the stability of EGCG in storage conditions in a close-to-neutral environment(pH 6.0)and during thermal treatment at different temperatures was appreciably or substantially enhanced.The gastrointestinal digestion and intestinal distribution of hydrogels were studied by simulating gastrointestinal digestion in vitro and MicroPET/CT imaging in vivo.The results showed that the amyloid-EGCG hydrogels could resist the digestion in the environment of stomach and small intestine,effectively avoiding the loss of EGCG in the stomach and small intestine.The hydrogels could be retained in the gut,especially in the colon for a long time,making it an ideal payload to target the inflammation in the gut.Finally,long-term oral intervention was used to evaluate hepatorenal toxicity in normal mice.The results showed that long-term oral administration of the hydrogels was not toxic to mice.Moreover,the hydrogels could significantly increase the levels of superoxide dismutase(SOD)and catalase(CAT)in the liver and thus enhance the antioxidant activity of the body to some extent.3.The effects of HEWL amyloid-EGCG hydrogels on the amelioration of dextran sulfate sodium induced colitis miceIn this section,the effects of oral amyloid-EGCG hydrogels on the amelioration of acute colitis mice induced with dextran sulfate sodium(DSS,2%)for 7 days were studied.The results showed that the hydrogels significantly alleviated the inflammation of the colon,restored the weight of the mice,and decreased the disease activity index(DAI)scores.In addition,it could improve colon morphology and mitigated DSS-induced colon shortening.Treatment with hydrogels significantly reduced the secretion of inflammatory factors in serum.Meanwhile,treatment with amyloid-EGCG hydrogels could repair colon epithelial cell damages and significantly increase claudin-1 and Occludin levels in colon tissues,improving the integrity of the intestinal barrier.Oral administration of the hydrogels also effectively suppressed the overexpression of inflammation-related genes levels in colon tissues.According to the previous section,the amyloid-EGCG hydrogels could be retained in the gut for a long time,which may cover the epidermal layer and act as a protective barrier to prevent the invasion of pathogens and bacteria in the intestinal cavity.At the same time,the polyphenol EGCG in hydrogels may help to recover epithelial cell injury and repair damaged barrier integrity by interference of the nuclear factor κB(NF-κB)signaling in intestinal epithelial cells,thereby reducing intestinal inflammation.4.Modulation effects of amyloid-EGCG hydrogels on gut microbiota in DSS-induced colitis miceIn this section,DSS-induced colitis mice were used as models to study the effects of hydrogels on modulating gut microbial composition and short chain fatty acids(SCFAs)levels by high-throughput sequencing of gut microorganisms and the determination of SCFAs in cecum contents.The results showed that the hydrogels significantly regulated gut microbial dysbiosis and reduced the accumulation of microorganisms in colitis,especially the facultative anaerobes(Aestuariispira and Escherichia)in the Proteobacteria.In addition,hydrogels significantly increased the total SCFAs levels related to the regulation of gut microbiota.In general,the hydrogels could regulate gut microbiota dysbiosis and selectively block the abnormal bloom of the commensal Enterobacteriaceae in the inflammatory environment.The mechanism of its selective modulation of gut microbiota would be further studied in our future studies.