| 1 Background and ObjectiveCrohn's disease(CD)is a kind of chronic idiopathic autoimmune disease,characterized by gastrointestinal inflammation.Genetic susceptibility,gut microbiota and abnormal activation of immune response are all associated with the pathogenesis of CD.Abnormal mesenteric lymphatics is a common pathetic alteration of CD,and is also an important underlying mechanism of submucosal edema and intestinal inflammation.Lymphagiogenesis and dilated mesenteric lymphatic vessels are observed in CD patients according to previous studies,and decreased lymphatic density is associated with postoperative recurrence.The results of in vivo experiments revealed that downregulating VEGFR-3 signaling pathway spoiled mesenteric lymphatics with aggravation of colitis.By contrast,upregulating intestinal lymphatics via VEGF-C administration markedly ameliorated experimental colitis.Taken that,we speculate that the early intervention on mesenteric inflammation in CD patients will better manage disease progress.To target mesenteric lymphatics,biomaterials can be used to modulate drug release.In recent years,a series of mesentery-targeting strategies have been developed for the need of daily management of diabetes mellitus,HIV infection and so on.These strategies mainly focus on targeting specific cells or receptors,inducing endocytosis by enterocytes,ultimately fulfilling specific absorption and local drug release by mesenteric lymphatics.Regrettably,the translation of basic science to clinical practice is still confronted with many problems,the most important one among which is the drug transport efficiency is relatively lower than expected.Given these problems,we investigated dietary lipid(long chain fatty acid)to better target the mesenteric lymphatics.After digestion,triglycerides(TGs)are hydrolysed to fatty acids and 2-monoglyceride,absorbed into enterocytes,re-synthesized to TGs,combined with lipoproteins and trafficked to mesenteric lymphatics.According to previous reports,intestinal lymphatic transport of model drugs occurs after oral administration of a unitdose lipid-based formulation to fasted dogs.Moreover,bile acid increases intestinal lymphatic drug transport in the fasted rat.It has been proved glycocholic acid exhibit specific high-efficiency intestinal uptake and lymphatic transport via intestinal bile acid-binding protein.As for that,we aimed at harnessing long chain fatty acid,specifically absorbed by MLVs,to construct a nano-carrier to target mesenteric lymphatics.This topic first investigated the feasibility of targeting MLTs to treat CD.Using Il-10-/-experimental colitis,based on histopathology and molecular biology,investigating pathetic alteration,we aim at developing a MLVs-targeting material to validate the MLTs as a potential therapeutic target for CD treatment.2 Materials and MethodsPart I Construction of chylomicrons-simulating nano-material,characterization,and targeting efficiency1.Materials.Using MSN as mother nucleus,decorated with ?-?'-dilaurin on the surface,the obtained NPs were named as LMSN,LMSN was coated with enteric coating(Eudragit co-polymer L100),the final product was named as ELMSN.2.Characterization.TEM was used to study morphology of NPs at different stage.Nitrogen adsorption experiment was carried out to investigate the pore distribution and nitrogen adsorption/desorption isotherms.Drug release profiles were studied in gastrointestinal-simulating fluids.The cellular uptake,endocytosis and exocytosis were studied using transwell membrane and sorts of inhibitors(i.e.Genistein).3.Targeting efficiency.IVIS and fluorescence sections were made to study the biodistribution of MSN and ELMSN in the GI tract after oral administration.Pharmacokinetics in rats were investigated with HPLC after oral administration with NPs carrying LAQ.Part II Influence on mesenteric inflammation,lymphatic drainage and the underlying mechanism with MLV-targeting drug release1.Morphology of MLVs and related tissues.Visualization of MLVs via BODITY administration fulfilled MLVs' counting under fluorescent stereoscope.Immunofluorescence of tight junction protein of LECs revealed the integrity of lymphatic endothelium.Morphology of surrounding MATs was studied by H&E sections.2.Function of MLVs.LYVE-1 stained sections were used to assess LVs density in the mucosa and submucosa of mice.Western blot was used to study LVs-related growth proteins(VEGF-C/VEGFR3).Lymphatic drainage was determined by intrarectal administration of Evans blue dye.3.Mesenteric inflammation.q RT-PCR was used to determine TNF-?,IL-1?,IFN-?,IL-6 m RNA level in the mesentery.Adipokines like adiponectin,leptin and resistin m RNA level were also studied.Part III Therapeutic effect on colitis in Il-10-/-experimental colitis by MLVstargeting drug release1.Body weight,colon length and disease activity index [DAI].To assess severity of colitis,body weight was measured on day 0,7,14,28 during treatment.DAI was recorded by scoring of stool consistency and fecal blood.Fecal blood was confirmed using the Hemoccult Sensa.The colon length was measured at sacrifice time.2.Levels of pro-inflammatory cytokines and related proteins.ELISA kit was used to quantify TNF-? and IL-6 concentration in plasma on week 0,2,4.q RT-PCR was used to determine TNF-?,IL-1?,IFN-?,IL-6 m RNA level in the colon.Western blot was used to determine expression of non-phosphorylated and phosphorylated forms of p65 and I?B in proximal colons.3.Alteration of gut microbiota.16 Sr RNA gene sequencing technology was used to determine the richness and distribution of gut flora.Combined gas liquid chromatography was used to quantify fecal SCFAs.3 ResultPart I Construction of chylomicrons-simulating nano-material,characterization,and targeting efficiency1.MSN and LMSN demonstrated uniform spherical appearance under TEM.After coated with enteric coating,the structure of mesopore disappeared.In the gastric and enteric-simulating fluid,the drug release profiles revealed that enteric coating could prevent premature drug release from ELMSN.2.Either LMSN or MSN could penetrate Caco-2 cells transwell membrane,and this confirmed the NPs could be efficiently absorbed by enterocytes.NPs entered the Caco-2 cells mainly through clathrin mediated and caveolae-mediated endocytosis.Exocytosis was related to lysosomes and Golgi apparatus.3.IVIS revealed that retention and uptake of ELMSN in the GI tract were notably enhanced compared with MSN.Fluorescent sections demonstrated that ELMSN mainly distributed in duodenum and ileum.FITC-labeled ELMSN successfully entered the mesenteric lymphatic vessels under stereoscope.4.LAQ@ELMSN notably enhanced drug concentration in plasma and in mesenteric lymphatics compared to free drug administration.Cycloheximide [CXI],a lymph flow inhibitor,which blocks lymphatic uptake by interference with the secretion of chylomicrons in enterocytes,was employed to explore the mechanism of lymphatic transport.The results revealed that free LAQ administration is mainly absorbed into blood,while selective preference into lymphatic vessels is concluded for LAQ@ELMSNPart II Influence on mesenteric inflammation,lymphatic drainage and the underlying mechanism with MLV-targeting drug release1.MLVs-targeting drug delivery inhibited lymphagiogenesis.Lymphagiogenesis was observed in experimental colitis by immunohistochemistry.The upregulated proinflammatory cytokines and NF-?B related proteins were reversed by LAQ@ELMSN administration.Downregulation of TNF-VEGF-C/VEFGR3 pathway suppressed lymphangiogenesis.2.LAQ@ELMSN promoted lymphatic function.MLVs-targeting drug delivery markedly increased functional MLVs.The lymphatic drainage was also improved according to Evans blue administration,which might be contributing to inhibition of NF-?B-i NOS signaling pathway.3.LAQ@ELMSN restored microstructure of leaky MLVs.Fluorescence intensity and distribution of tight junction proteins significantly restored by LAQ@ELMSN administration.Moreover,inflammation in surrounding MAT was suppressed as hypertrophy was reversed and inflammation-related adipokines were downregulated.Part III Therapeutic effect on colitis in Il-10-/-experimental colitis by MLVstargeting drug release1.LAQ@ELMSN deeply ameliorated colonic inflammation.Body weight,colon length and DAI were improved after LAQ@ELMSN administration.Levels of proinflammatory cytokines and related proteins were markedly downregulated.2.LAQ@ELMSN demonstrated extended amelioration on intestinal inflammation after drug withdrawal.LAQ@ELMSN-treated group significantly postponed the upregulation of the pro-inflammatory cytokines.3.LAQ@ELMSN altered gut microbiota and metabolites by LAQ@ELMSN.Chao 1 alpha diversity analysis revealed a notable increase in the diversity and taxa richness of gut microbiota.The content of SCFAs showed a corresponding increase after LAQ@ELMSN treatment.4 Conclusion1.The construction of ELMSN fills the gap in targeting MLTs for treating Crohn'slike colitis.Existing evidence has proved that MLTs play an important role in the pathogenesis of CD,which can be a potential therapeutic target for CD treatment. This study aims to develop a MLVs-targeting nano-carrier to increase drug concentration in MLTs which is meaningful for controlling mesenteric inflammation.2.MLTs can be a neo-therapeutic target for CD treatment.Lymphangiogenesis and vessel dilation as well as rupture of microstructure have been reported associated with early-onset submucosa edema.The current theories on pathogenetic pathways of CD are mainly based on the “outside-to-inside” model,that is,the sequence of dysbiosis,dysregulated mucosal innate and adaptive immunity,transmural disease,then abnormal mesentery,and extraintestinal manifestations.Some investigators suggest that lymphangitis in fact can be a primary culprit for CD,which is in consistent with the “inside-out” paradigm of the process of CD.3.MLTs are correlated with gut microbiota modulations.MLVs transport bacterial fragment and other antigens.MLVs act as communicating bridge between immune system and gut flora and help maintain homeostasis of gut microbiota.The function of MLVs is damaged in CD patients which will result in disturbance of gut flora.Improved lymphatic drainage exerts positive effect on taxa richness and relative metabolites,and is meaningful for studying correlation between MLTs and gut microbiota. |
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