The Mechanism Of Artesunate Modulating Ulcerative Colitis Via ERS-UPR Signaling Pathway

Ulcerative colitis(UC)is a non-specific and recurrent inflammatory disease of the colonic mucosa that affects the rectum and colon.The pathogenesis of UC has not been fully elucidated,and it is currently believed to be the result of the interaction of multiple factors,such as gene intestinal flora,host immune system and environment.In UC,the intestinal barrier is destroyed,leading to the translocation of intestinal flora and overactivation of the immune system,which in turn causes inflammation.The endoplasmic reticulum is connected with other organelles by means of a huge inner membrane system.Therefore,the endoplasmic reticulum can sense a variety of constantly changing physiological signals,thereby adjusting its various functions to maintain cell homeostasis.Disruption of the balance of correctly folded proteins in the endoplasmic reticulum can trigger endoplasmic reticulum stress(ERS),and consequent unfolded protein response(UPR).Increasing studies have shown that the ERS-UPR signaling pathway is involved in the occurrence and development of UC.Continued and unresolved ERS can induce epithelial cell apoptosis,damage the intestinal barrier,activate intestinal inflammation and induce immune deficiency,leading to UC.Studies have shown that a variety of artemisinins(ARTs)have protective effect on experimental colitis,but whether ARTs have inhibitory effects on ERS-UPR signaling pathways,and whether the protective effect of ARS on UC depends on these pathways is not clear.As a water-soluble artemisinin derivative,Artesunate(ARS)was widely used clinically.Thus,this study selected ARS as the research object to study the regulatory effect of ARS on ERS-UPR signaling pathways in mouse UC model and in vitro.In this study,the expression of key genes and proteins in ERS-UPR signaling pathway,inflammatory signaling pathway and intestinal immune response,as well as changes of immune cells were detected,to clarify the potential protective mechanism of ARS in intestinal inflammatory diseases(IBD),which providing a reference for the application of ARTs in IBD.1 The protective effects of ARS on DSS-induced UC in miceIn this study,mice colitis was induced by drinking water with dextran sulphate sodium salt(DSS)for 7 days to imitate the active phase of UC in humans.During the induction of model,the mice were treated with 30 mg/kg ARS once a day via intraperitoneal injection.The protective effect of ARS on colitis was evaluated by the changes of body weight,colon length and disease activity index(DAI),as well as the pathological changes of colon tissue,which were observed via HE staining and electron microscopy.The results showed that DSS stimulation caused body weight loss,DAI increase and colon shortening in mice,as well as intestinal mucosal epithelial cell erosion,goblet cell reduction and crypt destruction accompanied by inflammatory cell infiltration.while ARS treatment can significantly alleviate the above Symptoms and pathological changes.Immunofluorescence and Western blot analysis showed that ARS significantly inhibited DSS-induced loss of mucin 2(Muc2)and tight junction protein Claudin-1.Moreover,ARS increased Bcl-2/Bax ratio and decreased the expression of cleaved-caspase-3.These results suggested that ARS had obvious protective effect on intestinal barrier in UC mice.Subsequently,we detected the phosphorylation of nuclear factor-?B(NF-?B)and the mRNA expression of inflammatory cytokines IL-1?,IL-6 and TNF-? respectively by Western blot and RT-qPCR.The results showed that ARS notably inhibited the activation of nuclear factor-?Ba(I?B?)NF-?B p65 and the expression of IL-1?,IL-6 and TNF-? in colon tissue of UC mice,which confirmed the anti-inflammatory effect of ARS in colon tissues.The above data suggest that ARS can play a protective role in UC mice through maintaining the important proteins expression of intestinal mucosal barrier,inhibiting cell apoptosis and inflammatory response,and alleviating colonic lesions and clinical symptoms.2 The Inhibitory effects of ARS on ERS signaling pathways in colon tissueWe detected the expression of ERS marker proteins in mice colon tissue.And the Western blot and immunofluorescence results showed that DSS exposure significantly increased the expression of GRP78 and CHOP,and activated the UPR sensing proteins Perk,IREla and ATF6 and their cascade signaling pathways.However,ARS significantly inhibited the severity of ERS by blocking the activation of the PERK-eIF2?-ATF4-CHOP and IRE1?-XBP1 signaling pathways in UPR.To investigated whether ERS was involved in the effect of ARS on maintaining colon barrier and inhibiting NF-?B activation,we co-treated 4-PBA and 2-DG respectively with ARS in DSS-induced UC mice.The results showed that on the basis of ARS,4-PBA co-treatment had no significant effect on the expression of caspase12 and the Bcl-2/Bax ratio,but it could further alleviate the activation of PERK-eIF2?-ATF4-CHOP and IRE1?-XBP1 signaling pathways;2-DG significantly reversed the inhibitory effect of ARS on the above-mentioned signal pathways.This indicated that 4-PBA and 2-DG can be used to control the severity of ERS.Compared with the ARS alone treatment group,4-PBA could strengthen the beneficial effect of ARS on the barrier proteins Cluadin-1 and Muc2,and the inhibitory effect on the expression of NF-?B,IL-1?,IL-6 and TNF-?.At the same time,4-PBA strengthened the relieving effect of ARS on the clinical symptoms and pathological changes of UC mice.However,2-DG significantly reversed the regulation effect of ARS on the above indicators.These results indicated that excessive ERS is an important cause of the occurrence and development of UC challenged by DSS.ARS could inhibit the development of colon inflammation and maintain intestinal barrier function via inhibiting the overactivation of PERK-eIF2?-ATF4-CHOP and IRE 1 ?-XBP 1 signaling pathways.This revealed the mechanism of ARS inhibiting colon inflammation from the perspective of ERS-UPR signaling pathway.3 Inhibitory effect of ARS on ERS in intestinal epithelial cellsIn this study,IEC-6 cells were pretreated with ARS and stimulated by chlamycin to induce ERS in order to investigate the inhibitory effect of ARS on ERS in IEC-6 cells and the regulatory effect of ERS on Claudin-1 and NF-?B.First,the working concentrations of ARS and tunicamycin(Tm)were determined by CCK8 to be 1 ?m and 2 ?m respectively.Then,the cells were stimulated with Tm at a concentration of 2 ?M for different times to observe the expression of ERS marker proteins GRP78 and CHOP,as well as the expression of Claudin-1 and NF-?B activation.Western blot results showed that as time went by,the expression of GRP78 and CHOP and the ratio of p-p65/p65 and p-I?B/I?B gradually increased,and the effect was most significant at 12 h.In addition,Tm stimulation for 12 h could significant reduce the expression of Claudin-1.This indicated that ERS could activate NF-?B and inhibit the expression of Claudin-1 in IEC-6 cells.ARS treatment could significantly inhibit the activation of IRE1?-XBP1s and PERK-eIF2?-ATF4-CHOP signaling pathways,and alleviate the regulation of NF-?B and Claudin-1 by ERS.Furthermore,siRNAPERK and siRNA IRE1?transfection significantly inhibited the activation of the PERK-eIF2?-ATF4-CHOP and IRE1?-XBP1s signaling pathways,respectively.The PERK-eIF2?-ATF4-CHOP pathway mainly regulated the expression of Claudin-1,while the IRE1?-XBP1s signaling pathway was mainly responsible for regulating the activation of NF-?B.4 The immunoregulatory effect of ARS in UC miceWe stimulated mice with DSS for 7 days to induce UC,and then treated them with 30 mg/kg ARS for 5 days via intraperitoneal injection to observe the regulatory effect of ARS on mouse immune cells and the therapeutic effect of UC.In order to explore the regulatory effects of ARS on Th17,Treg and macrophages in UC,we used flow cytometry to detect the ratio of Th17 and Treg cells in CD4+cells in mouse spleen,and macrophages M2/M1 in colon tissue.The expression of the four types of cell marker genes and secreted cytokines in colon tissue was detected by RT-qPCR technology.Compared with the DSS group of mice,ARS treatment could significantly inhibit the ratio of Th17 cells in CD4+cells,as well as the expression of IL-17A,IL-17F and RORyt mRNA.In addition,ARS treatment significantly increases the mRNA expression of TGF-?.Moreover,ARS Increased the ratio of M2/M1 macrophages and the expression of Arg-1 and IL-10 mRNA in UC mouse,and inhibited the mRNA expression of pro-inflammatory factors iNOS,IL-1?,IL-6 and TNF-?.After ARS treatment,the clinical symptoms and colonic histopathological changes of UC mice had been significantly alleviated.The above results indicated that ARS could protect DSS-induced UC by regulating the balance of Th17/Treg cells and the polarization of macrophages.5 The inhibitory effect of ARS on ERS modulates intestinal immunityIn order to investigate whether the inhibitory effect of ARS on ERS is involved in the regulation of colonic immunity and the therapeutic effect on UC,we used 4-PBA and 2-DG respectively co-treatment with ARS in DSS-induced UC mice.The results showed that on the basis of ARS treatment,4-PBA co-treatment could further inhibit the ratio of Th17 cells in spleen CD4+cells,as well as the mRNA expression of IL-17A,IL-17F and RORyt in the colon,but it has a negative effect on Treg cells and TGF-?.Compared with the ARS treatment group,the co-treatment of 4-PBA and ARS can significantly increase the ratio of M2/M1 type of macrophages in colon tissue,and 4-PBA can strengthen the effect of ARS on Arg-1 and IL-10.In addition,4-PBA can enhance the promoting effect of ARS on the expression of ARG-1 and IL-10 mRNA,and inhibit the mRNA expression of iNOS IL-1? IL-6 and TNF-?.In addition,4-PBA promotes the alleviation of ARS on the clinical symptoms and pathological changes of UC mice.On the contrary,2-DG Significantly offset the regulatory effect of ARS on the above indicators.The above results indicated that ERS is involved in the regulation of Th17/Treg cell balance and macrophage polarization.