Effect And Mechanism Of Secreted GRP78 On The Allevation Of DSS-induced Ulcerative Colitis In Mice

?Background?The 78 k Da glucose-regulated protein 78(GRP78),also known as Binding Immunoglobulin Protein(BIP)and Heat Shock Protein A5(HSPA5),is the member of HSP70 family.GRP78 is widely distributed in cells.In the endoplasmic reticulum,GRP78 acts as an essential chaperone protein to aid in the correct folding of proteins.Under endoplasmic reticulum stress,such as hypoxia,infection,etc.,the expression of GRP78 is up-regulated to assist the correct folding of proteins to avoid the accumulation of misfolded and denatured proteins in the endoplasmic reticulum and to prevent cell apoptosis.Up-regulated GRP78 can be transferred to other parts of the cell and even be secreted outside the cell to become secreted GRP78(s GRP78).s GRP78 has an immunoregulatory property and is included in resolution-associated molecular patterns(RAMPs).Our previous studies also showed that s GRP78 cloud induce BMDCs to become tolerant DCs,induce spleen CD19+ B cells to become CD19hiPD-L1hi/Fas Lhi B cells.s GRP78 impairs production of LPS-induced cytokines by interaction with CD14.Ulcerative colitis(UC)is closely related to the disorder function of immune cells in the colon tissue.Macrophages and intestinal epithelial cells are two key cells in the development of UC.Whether s GRP78 could play parts in the resolution of ulcerative colitis is worthy of study.Based on the previous study,we investigated the inflammatory-resolution role of s GRP78 in DSS-induced ulcerative colitis and the effects of s GRP78 on LPS induced activation of macrophages and intestinal epithelial cells.?Methods?1.Expression and purification of mouse s GRP78Mouse recombinant GRP78 was prepared from the extract of Escherichia coli BL21 which was transformed by a plasmid encoding the full length of mouse GRP78.The endotoxin in the sample was removed,and the protein concentration was determined by BCA method.The protein samples were filtered to remove bacteria,and stored at-80?;the protein samples were identified by SDS-PAGE and western blot.2.Establishment of DSS-induced ulcerative colitis mice model.C57BL/6J mice were randomly divided into untreated group,control group(DSS + NS)and experimental group(DSS + s GRP78(10 ?g/g)).2.5% DSS water was given at the beginning of the establishment and changed every two days.After five days,all groups were replaced with sterile water.The mice were sacrificed on the eighth day.Pathological changes in colon tissue were assessed by HE staining.Cytokine levels were detected by CBA or ELISA.Flow cytometry was used to detect infiltration,proportion and phenotype of lymphocytes in mesenteric lymph nodes,spleen and peritonea.Western blot was used to detect the activation of signaling molecules and the expression of tight junction proteins in intestinal epithelial cells.3.Evaluating the effect of sGRP78 on polarization of macrophagesBMDMs were divided into 3 groups: untreated group,LPS group and s GRP78(10 ?g/ml).Peritoneal macrophages were divided into 5 groups: untreated group,LPS group(100 ng/ml),LPS group(100 ng/ml)+ s GRP78(10 ?g/ml),s GRP78(10 ?g/ml)and IL-4(20 ng/ml).IL-6,TNF-?,IFN-? and i NOS were detected by q RT-PCR or CBA method.BMDMs were stimulated with LPS for 18 hours.Then cells treated by LPS,GRP78 and IL-4,respectively.Inflammatory cytokines were measured by CBA method and by q RT-PCR.4.Evaluating the effect of sGRP78 on mouse intestinal epithelial cellsIsolated intestinal epithelial cells were treated with LPS(10 ?g/ml)and sGRP78 at increasing concentration.The expression of TLR4,i NOS and COX2 and phosphorylation levels of signaling molecules were detected by western blot.Cytokine levels were detected by q RT-PCR and CBA method.?Results?1.s GRP78 ameliorates the disease activity of DSS-induced ulcerative colitis in mice(1)Compared with the untreated group,2.5% DSS can cause weight loss,loose stools,bloody stools,shortened colon and even death in mice.HE staining showed that the pathological score at the end of the colon was also significantly increased,as well as the production of cytokines,such as IL-6,TNF-? and IFN-?.The clinical symptoms,histological scores,and expression of inflammatory cytokines were significantly decreased after intraperitoneal injection of s GRP78.s GRP78 treatment increases the frequency of Tregs in the mesenteric lymph nodes and spleen,decreases the proportion of CD45+ lymphocytes in colon tissue and the proportion of macrophages in the peritoneal,mesenteric lymph nodes and spleen significantly.The expression of CD80 and CD86 on macrophages are also down-regulated.(2)TLR4/NF-?B,TRIF/IRF3,and TLR4/MAPKs signaling pathways are activated in colon tissues of DSS mouse.After intraperitoneal injection of s GRP78,the expression of p-p65,p-IRF3,p-P38,p-ERK and p-JNK in colon tissues were significantly down-regulated,as well as the expression of TLR4,COX2 and i NOS.2.sGRP78 inhibits M1 polarization of macrophages induced by LPSCompared with the negative control,LPS can induce BMDMs and peritoneal macrophages to secrete a large mount of inflammatory cytokines such as IL-6,TNF-? and IFN-?.However,s GRP78 can not stimulate the secretion of the above inflammatory cytokines.Furthermore,s GRP78 can decrease the inflammatory cytokine release induced by LPS.3.s GRP78 on LPS-induced primary intestinal epithelial cells in mice(1)Compared with the negative control,LPS can induce IECs to secrete a large number of cytokines such as IL-6,TNF-? and IFN-?,while s GRP78 can significantly inhibit the secretion of these cytokines.(2)Western blotting results showed that LPS could induce the activation of TLR4/NF-?B,TRIF/IRF3 and TLR4/MAPKs signaling pathways in mice primary intestinal epithelial cells,while s GRP78 could down-regulate the activation of these signaling pathways,as well as the expression of COX2 and i NOS.?Conclusions?This study showed that s GRP78 could ameliorate the ulcerative colitis activity in DSSinduced mice through suppressing the inflammatory cytokine release by macrophages and intestinal epithelial cells and suppressing the activation of TLR4 related signaling pathways:(1)s GRP78 can alleviate enteritis(eg.body weight,bloody stool and survival time,infiltration of lymphocytes);inhibit the activation of TLR4/NF-?B,TRIF/IRF3 and TLR4/MAPKs signaling pathways in colon tissues,thereby downregulating the expression of inflammatory proteases or inflammatory cytokines.(2)Macrophages may play a very important role in the process of s GRP78 regression of inflammation.(3)s GRP78 can partially reverse the M1 polarization of macrophages induced by LPS and inhibits the activation of TLR4/NF-?B,TLR4/MAPKs and TRIF/IRF3 pathways in intestinal epithelial cells,thereby ameliorating the inflammatory state.