| Background and aimsInflammatory bowel disease(IBD)is a chronic autoimmune disease with peptic ulcer as its main pathological manifestation.The key target for patients to improve their symptoms and hinder the inflammatory-cancerous transformation is to induce complete healing of the intestinal mucosa at the site of the lesion,but at the same time,the weakness of clinical IBD treatment lies in the impaired repair of the intestinal mucosa.Therefore,achieving complete intestinal mucosal healing is the only way to meet the clinical needs of IBD and overcome the bottleneck in IBD treatment.The repair of the intestinal mucosa is dependent on the rapid proliferation and renewal of intestinal stem cells(ISC)and their effective differentiation.During this process,ISC are closely regulated by the immune microenvironment.Macrophages are the gatekeepers of the intestinal immune homeostasis,and their maintenance of the inflammatory "promotion-dissipation" homeostasis plays a decisive role in the repair process mediated by ISC,as reflected by the M1/M2 polarisation homeostasis of macrophages coupled to the epithelial tissue damage-repair process.Previous studies have shown that Dihydroartemisinin(DHA)has been shown to have a clear role in a variety of animal models of IBD induced by trinitrobenzene sulfonic acid(TNBS),Oxazolone(OXA)and other autoimmune diseases such as SLE.In addition,DHA has shown clear efficacy in various animal models of IBD induced by TNBS,Oxazolone(OXA)and other autoimmune disease models such as SLE,and can exert anti-colon inflammatory-cancer effects in models of inflammatory-cancer transformation based on the modulation of macrophage polarization.In addition,in vivo pharmacokinetic studies of DHA have shown that it is mainly distributed in the gastrointestinal tract,whether administered orally or by injection.Therefore,DHA is an excellent candidate for the study of the "immune homeostasis-damage balance" theory to promote intestinal mucosal healing.Secondly,in the context of IBD disease,there are still gaps in research to describe the profound systemic effects of DHA-induced M2 immune remodelling in macrophages,potential drug targets and drug mechanisms of action,and the dependence of its mediated immunity on the promotion of mucosal repair needs to be revealed.The dependence of its mediated immunity on the promotion of mucosal repair needs to be revealed.Based on cutting-edge research,it has been shown that the remodelling of the metabolic pattern of immune cells determines the polarised state of the immune phenotype on the one hand and has a key impact on the output of their functions on the other.Holistic changes in macrophage metabolism are the underlying logic for the transformation of their immune phenotype,activating macrophage function and supporting their production of key regulatory factors.Specifically:M1 macrophages use aerobic glycolysis as their main energy supply pathway.Their tricarboxylic acid cycle is blocked and metabolites such as citrate and succinate accumulate,a metabolic pattern that facilitates the rapid production of adenosine-triphosphate(ATP)and the accumulation of large amounts of immunocidal mediators that sustain the proinflammatory immune phenotype.In contrast,M2 macrophages have an oxidative phosphorylation mode as their main energy supply,maintaining an intact tricarboxylic acid cycle with high levels of glutamine and fatty acid oxidation.This metabolic mode is effective in neutralizing the pro-inflammatory response and reshaping inflammatory homeostasis,and will also provide sufficient and efficient energy supply and product support for tissue repair.In summary,we propose the following hypothesis:in IBD models,DHA can promote the formation of repair-related immune microenvironment in lesions based on the mechanism of inducing macrophage M2 immunometabolism,promote proliferation and differentiation of ISC and intestinal epithelial repair,and improve intestinal function.The effective completion of this topic will contribute to the improvement and complementation of future clinical and drug development for IBD in the following aspects:(1)Based on the balanced characteristics of immune microenvironment regulation,the previous understanding of the purely anti-inflammatory activity of DHA is expanded.(2)The potential value and feasibility of DHA in the treatment of intestinal mucosal repair is demonstrated.(3)Based on the decisive role of metabolism in immune regulation in the chain of "metabolism-immunity-repair",the research method and technology system were developed to provide technical support for the development of Chinese medicine for the treatment of IBD mucosa healing.Research strategies1 DHA-specific efficacy study for intestinal mucosal repair and functional reconstruction in mice with IBDThis part of the study evaluates the efficacy of DHA in promoting intestinal repair from the perspective of disease scoring,intestinal tissue healing and intestinal function reconstruction in a comprehensive and three-dimensional manner,supporting the hypothesis that DHA promotes deep intestinal mucosal healing.(1)Construction of a DSS-specific injury model with a focus on tissue repair and disease scoringIn this section,a graded dose of DHA(2.5mg/kg/day,5mg/kg/day,10mg/kg/day)and the first-line clinical drug Prednisolone(PLN)(5mg/kg/day)were used to induce intestinal injury in mice as a model of IBD.(5mg/kg/day)and the first-line clinical drug Prednisolone(PLN)(5mg/kg/day),and two dosing regimens were designed:(i)a full dosing regimen:except for the NC group,all mice were grouped by body weight and dosed during the 3%DSS injury phase,the autonomic repair phase and the 1.5%DSS recurrence phase to investigate the best efficacy of DHA at the disease stage;(ii)a repair phase only dosing regimen:except for the NC group,all mice were dosed according to the end of the injury phase.The mice were grouped according to the DAI disease score at the end of the injury period and then administered only during the autonomous repair period.In both models,the daily DAI disease score was used as an indicator of disease severity.(2)Histological level evaluation of DHA to accelerate intestinal mucosal healing during the repair period in miceColons and faeces from mice administered only during the repair period were examined at the mid and end of the repair period;the level of calprotectin(CALP)in the faeces of each group of mice was measured to indicate the level of deep inflammation in the intestinal mucosa and the risk of recurrence.The level of histological healing of the injured colon was evaluated according to the length of the colon,the inflammatory infiltrate observed in the colonic hematoxylin-eosin staining(HE staining)and the integrity of the crypt.(3)Evaluation of DHA for recovery of intestinal function in miceThe recovery of the intestinal barrier function was evaluated by the permeability test of Fluorescein isothiocyanate isomer(FITC)in mice at the end of the recurrence period;the recovery of the colonic secretion function was evaluated by the area of acidic secretory fluid stained with Alizarin in mice at the middle of the repair period.The restoration of colonic motility and resorption function was evaluated by the number of fecal pellets excreted and the fecal water content of each group of mice at different stages of the repair period.2.Exploring the pharmacology of DHA for intestinal mucosal repair based on macrophage metabolic remodelingIn this part of the study,we first clarify the essential role of macrophages in the DHA-mediated intestinal mucosal repair process through the platform of the peritoneal macrophage transplantation model and the "macrophage-ISC" co-culture unit,and answer the question "Can DHA promote intestinal mucosal healing based on macrophages?".Afterwards,we will use mouse intestinal macrophage transcriptome sequencing to examine gene-wide and unbiased DHA-induced phenotypic and metabolic alterations in macrophages,and analyse specific pathways to answer the question "What are the functions of macrophages that DHA focuses on?"(1)Clarify that macrophages are effective target cells for DHA in miceUsing a repair-phase only administration model,the DHA-treated induced peritoneal macrophages were transplanted by infusion as the repair-phase administration route,and it was clear that the DHA-induced macrophages had the function of promoting the repair of the injured colon.Direct intervention of IEC-6 epithelial cells or intestinal organoids using DHA was compared with intervention of IEC-6 cells or intestinal organoids by DHA-induced macrophage medium to compare the proliferation capacity of epithelial cells,growth and outgrowth of organoids in both cases to clarify the importance of macrophages in DHA-mediated regeneration of intestinal mucosa.Normal and DHA-induced RAW264.7 cells were used to co-culture with fluorescent organoids,and fluorescent organoids co-cultured with different macrophages were transplanted back into colon-injured mice using organoid transplantation to observe the colonisation and growth area of organoids at the injury site to reflect the functional effects of DHA-induced macrophages on ISC regeneration of the intestinal epithelium.(2)DHA-induced phenotypic shift in macrophage polarization assayMacrophages from the mid-repair phase of the intestinal lamina propria of mice in the M and DHA-H groups in a repair-only administration model were extracted and transcriptome sequencing was performed to analyze the shift in the polarization phenotype of mouse intestinal macrophages after DHA intervention from the perspective of overall network changes.RAW264.7 macrophages were used,given lipopolysaccharide(LPS)(100ng/mL)and interferon γ(IFN-γ)(20ng/mL)for modelling,various doses of DHA(0.5μM,1μM,2μM)and PLN(25μg/mL)for administration,using the expression of Arginasel(Arg-1)and Resistin-like α(RELMα),markers of M2 polarization repair,was measured by Western blot at to determine the shift in M2 polarization in each cell group.(3)DHA-induced metabolic pattern shifts in macrophagesIntestinal lamina propria macrophages in the mid-repair phase of DSS-only administration model in M and DHA-H group mice were extracted and transcriptome sequencing was performed to systematically analyze the transcriptomic network of DHA remodeling immunometabolism.Using RAW264.7 macrophages,given LPS(100ng/mL)and IFN-γ(20ng/mL)for modelling,and DHA(2μM)for administration,the effect of DHA on cellular energy metabolism was analysed using the Seahorse XF Energy Metabolism Analyzer to measure the metabolic pattern shift in cellular ATP production by RAW264.7 following DHA intervention.Using RAW264.7 macrophages,LPS(100ng/mL)and IFN-γ(20ng/mL)were given for modelling,each dose of DHA(0.5μM,1μM,2μM)and PLN(25μg/mL)was administered,and the level of lactate produced in each group of cells was measured using a lactate content kit;the cells in each administration group were observed to respond to the glucose analogue under starvation The relative mRNA content of Glucose transporter-1(Glut-1)was measured by RT-qPCR to evaluate the glucose requirement and utilization in each group of cells.3.Molecular targeting of DHA based on "metabolism-immunity-repair".This part of the study firstly screens and docks the targets of DHA in macrophages and then completes the evaluation of the efficacy of DHA in a "macrophage-ISC" coculture unit under the condition that the key target molecule of DHA regulates immune metabolic remodeling is missing,and analyzes the differences to answer the question"What is the dependence of DHA-regulated immunometabolic remodelling on intestinal mucosal healing?" This question was answered.(1)Predictive screening and virtual docking of DHA drug targetsThe Dassault Systemes BIO VIATM Discovery Studio 2020(DS)software was used to perform virtual screening of DHA targets under CentOS 7 and to analyse the interactions between the resulting target molecules to focus on the core molecules;and to predict the interaction pattern,spatial structure characteristics,key binding sites and binding energy of the screened molecules with DHA.The results will be used to predict the interaction pattern,spatial structure,key binding sites and binding energies of the selected molecules.(2)DHA target binding validationMolecular simulation techniques were used to simulate the complex formed by DHA and the screened molecule 11-beta-hydroxysteroid dehydrogenase isozyme 1(11βHSD-1)for 100ns molecular dynamics simulations,and the stability of the complex was analyzed and confirmed by plotting root mean square error(RMSD)and The stability of the complexes was analyzed and confirmed by plotting root mean square error(RMSD)and root mean square deviation(RMSF);drug affinity responsive target stability(DARTS)technique was used to validate the binding of DHA to the molecule in RAW264.7 cells.11βHSD-1 binding in RAW264.7 cells.(3)Functional validation of DHA targets(ⅰ)DHA-based 11βHSD-1 induced immunometabolic remodeling in macrophages:inhibition of 11βHSD-1 activity in RAW264.7 cells using 11βHSD-1 inhibitor(BVT14225)was followed by administration of LPS(100ng/mL)and IFN-γ(20ng/mL)modeling,each dose of DHA(0.5μM,1μM,2μM)and PLN(25 μg/mL)were administered,and the expression levels of M2 polarization marker proteins such as Arg1,ATP production from glycolysis and oxidative phosphorylation,respectively,intracellular lactate content,relative mRNA content of Glut-1 and phagocytosis 2NBDG levels during starvation were measured in each group of macrophages to determine the ability of DHA to regulate the shift of macrophages to M2 polarization and metabolic shift.(ⅱ)DHA-based 11βHSD-1 induction of macrophages for ISC proliferation and differentiation:normal and DHA-induced RAW264.7 cells were used to construct a"macrophage-organoid" co-culture system,and the proliferation marker G proteincoupled receptor 5(Lgr5),Ki67 protein(Ki67)and differentiation marker mucin 2(Lgr5)were detected by immunofluorescence in the co-cultured organoids.The expression of Leucine rich repeat containing G protein-coupled receptor 5(Lgr5),Ki67 protein(Antigen identified by monoclonal antibody Ki 67,Ki67)and Mucin 2,an indicator of differentiation,was measured by immunofluorescence,The expression of macrophages was used to determine whether macrophages promote ISC proliferation and differentiation.After inhibiting 11βHSD-1 activity in RAW264.7 cells using BVT14225,the proliferation and differentiation status of intestinal organoids was investigated after subculture with them to determine whether DHA promotes ISC proliferation and differentiation based on activation of macrophage 11βHSD-1.4.Exploring the mechanism of DHA-regulated macrophage "metabolism-immunityrepair" couplingIn this part of the study,based on preliminary screening and literature search,we have conducted a preliminary exploration of the DHA-regulated "metabolismimmunity-repair" coupling mechanism in macrophages and validated it in the absence of key molecular targets to answer the question "What is the DHA-regulated"metabolism-immunity-repair" coupling mechanism?repair" coupling mechanism?This question was answered.RAW264.7 macrophages were administered with LPS(100ng/mL)and IFN-γ(20ng/mL),DHA(0.5μM,1μM,2μM)and PLN(25μg/mL),and the relative mRNA content of Sedum heptanone glucokinase(CARKL)in each cell group was measured by RT-qPCR.The expression of signal transducer and activator of transcription 6(STAT6),phosphorylated-STAT6(p-STAT6),signal transducer and activator of transcription 3(STAT3),p-STAT3 and other related proteins was measured by Western blot.Signal transducer and activator of transcription 3,STAT3),p-STAT3 and other related proteins.After inhibition of 11βHSD-1 activity in RAW264.7 cells using BVT14225,the cells were then molded and administered as described above,and RTqPCR and Western blot were used to examine the expression of each group of cells.Western blot to detect the relative mRNA content of CARKL and the expression of STAT6,p-STAT6,STAT3,p-STAT3 and other related proteins in each group of cells.The transcriptome was sequenced to analyze the transcript levels of Wnt pathway ligands and other cellular pathways related to repair in the mid-repair stage of intestinal lamina propria macrophages from M and DHA-H mice in the DSS-only repair administration model.Results1 DHA has been shown to accelerate intestinal mucosal healing and improve the function of diseased colon in a specific model of intestinal mucosal repair in mice(1)DHA accelerates intestinal mucosal healing during repair in mice at the overall disease score and histopathological levelThe DAI scores for the full course of administration indicated that the DHA administration groups exerted their efficacy in promoting intestinal mucosal healing and accelerating intestinal barrier reconstruction mainly during the repair phase of the model.Similarly,in the repair phase administration model,each DHA administration group was still able to accelerate the reduction of disease scores in the repair phase and inhibit the increase of DAI scores in the relapse phase.In terms of the level of repair of the colonic tissue structure,a significant reduction in inflammatory infiltration within the colonic mucosa,a high number of crypt foci,structural integrity and accelerated recovery of colonic length were evident in each DHA administration group at the midrepair stage(Day 10).At the middle of the repair period(Day 10),the level of calprotectin in the faeces of the mice in each DHA administration group decreased,indicating that the deep mucosal damage was alleviated.At the end of repair(Day14),the colon length of all groups was restored to the same level,but the model group still had inflammatory infiltration in the colon mucosa,and the number of crypt foci was low and the structure was abnormal.The mucosal tissues of the mice in the DHA administration groups were basically normalized.The above results indicated that the doses of DHA could promote the recovery of intestinal mucosa in mice after injury from the structural tissue level.(2)DHA promotes functional recovery of the damaged intestine in miceWe examined the function of the colonic barrier(FITC leakage),the level of mucosal secretory proteins in the colon during the repair period(Alizarin staining),the level of water absorption in the colon(fecal water content in mice)and the motility of the colon(number of feces expelled in 1h).In addition,the above results showed a significant advantage of each DHA administration group compared to the glucocorticoid group,suggesting that DHA is different from the single antiinflammatory activity of glucocorticoids,but instead has a significant advantage in promoting mucosal healing and accelerating disease remission.2 DHA targets macrophages,enhances their oxidative phosphorylation and induces a metabolic remodelling towards the M2 type(1)Macrophages are the necessary anchor point for DHA to promote mucosal repairThe peritoneal macrophage back-transplantation assay demonstrated the efficacy of DHA-induced macrophages in promoting intestinal mucosal healing in vivo.Based on THP-1/IEC-6 co-culture and "macrophage-like organ" culture units,it was shown that DHA promotes ISC proliferation,differentiation and functional maturation dependent on the induction of macrophages.(2)DHA promotes a shift in macrophage polarization towards the M2 phenotypeFrom the perspective of macrophage polarization phenotype,GSEA analysis of mouse intestinal macrophage sequencing results showed that intestinal macrophages were generally polarized towards the M2 phenotype in the DHA-administered group compared to the DSS model group.The expression of M2 polarization molecules,such as the promotion of cytosolic burial and exosomal release,was also detected in vitro using RAW264.7 cells in the DHA-dosed group compared to the model group.(3)DHA enhances oxidative phosphorylation in macrophages,enabling them to reprogram their immune metabolismThe sequencing results of mouse intestinal macrophages by GO and KEGG enrichment analysis showed that DHA had a significant effect on macrophage metabolism compared to the DSS group,especially on sugar and energy metabolism:DHA had a tendency to inhibit macrophage glycolysis,upregulate intracellular oxidative phosphorylation and promote ATP synthesis and transport.Our results using the Seahorse cell energy metabolism meter showed that DHA significantly induced a decrease in glycolytic capacity and an increase in oxidative phosphorylation capacity in macrophages compared to the model group.In addition,RAW264.7 cells showed reduced glucose uptake and lower intracellular lactate content after DHA induction.3 11βHSD-1 in macrophages is a molecular target for the efficacy of DHA(1)Screening of DHA targets and validation of molecular dockingDassault Systemes BIO VIATM Discovery Studio 2020(DS)software was used for target screening of DHA and the results were used to predict the interactions between the resulting molecules using the String database,focusing on the core molecule 11βHSD-1.The results showed that the relative binding energy of DHA and 11βHSD1 was-8.6kcal/mol,indicating that the two molecules could easily form a complex.And according to the docking scoring and analysis,Pi-Sigma bond,Pi-Alkyl bond,Alkyl bond and van der Waals force existed between DHA and 11βHSD-1.Molecular dynamics simulations showed a smooth vibration of the RMSD curve,indicating that the complex formed by the 11βHSD-1 molecule and DHA is able to exist in a stable state;a high degree of flexibility in specific regions was observed in the RMSF curve,indicating that the atoms of the complex formed have a certain degree of freedom and are able to perform certain functions.HSD-1 molecule and protect the 11βHSD-1 molecule from degradation by streptase.(2)DHA induces macrophage metabolic remodeling for ISC regeneration dependent on activation of 11βHSD-1In terms of immunometabolic remodeling in macrophages,after inhibiting 11βHSD-1 activation in macrophages,DHA no longer had induced macrophages to upregulate the expression of Arg-1,RELMα and other M2 repair markers,reduced macrophage glycolysis levels,decreased macrophage glucose uptake and intracellular lactate content,and promoted oxidative phosphorylation levels.In promoting ISC regeneration,inhibition of 11βHSD-1 activation in macrophages resulted in DHA no longer having pharmacological efficacy in promoting organoid proliferation and differentiation,suggesting that DHA-dependent 11βHSD-1 regulates immunometabolic remodeling and intestinal mucosal healing.41 1βHDS-1/CARKL/STATs/Wnts is a key DHA-mediated "etabolic-immunerepair" signaling pathwayDHA significantly induced upregulation of CARKL transcription in macrophages,promoted downstream STAT3/STAT6 phosphorylation and transcribed various prorepair ligands of the Wnt family,and the above effects were lost after 11βHSD-1 blockade in macrophages.Together with the results of the full study,it was demonstrated that DHA could achieve metabolic remodeling,immunophenotypic polarization,and ultimately initiate repair in macrophages through the 11βHSD1/CARKL/STATs/Wnts signaling pathway.ConclusionIn summary,we conclude the following.(1)DHA is able to promote deep healing of the intestinal mucosa and has medicinal effects to improve intestinal function.(2)Macrophages are the target cells for DHA to achieve the proliferation and differentiation of ISC that maintain the integrity of the intestinal mucosa.(3)DHA induces lower levels of glycolytic metabolism in macrophages,enhances macrophage oxidative phosphorylation and induces polarization of macrophages towards the M2 repair phenotype.(4)11βHSD-1 is a target molecule of DHA that regulates immunometabolic remodelling for intestinal mucosal healing.11βHSD-1/CARKL/STATs/Wnts is a key DHA-mediated "metabolic-immune-repair" signalling pathway. |
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