The Effect And Mechanism Of Glycyrrhizin On Murine Hepatic Ischemia-reperfusion Injury The Effect And Mechanism Of RAGE On Murine Experimental Colitis

Background:Gasdermin D(GSDMD),a genetic substrate for inflammatory caspases,plays a central role in pyroptosis of macrophages and release of interleukin-1?(IL-1?),but was mainly referred to microbial infection.High mobility group box-1(HMGB1),served as an alarm molecule during various pathological process,has been widely recognized to be involved in liver ischemia-reperfusion(I/R).Glycyrrhizin,a natural anti-inflammatory and antiviral triterpene in clinical use,was recently referred to have ability to prevent I/R induced liver injury by inhibiting HMGB1 expression and activity.However,the mechanisms responsible for damage amelioration subsequently to HMGB1 inhibition during liver I/R remain enigmatic.Objective:This study was designed to explore the functional role and molecular mechanism of glycyrrhizin in the regulation of I/R induced liver injury.Methods and Results:1.Glycyrrhizin ameliorates I/R induced liver tissue damageWe analyzed the effects of Glycyrrhizin on mouse livers subjected to 90 min of warm ischemia followed by 6 h of reperfusion.Serum ALT and AST levels,a measure of hepatocellular injury,were detected.Histological grading of liver I/R is determined by Suzuki's score and H&E staining.Neutrophil infiltration is shown by MPO activity and Ly6G stained immunohistochemistry of liver tissues2.The mechanism of glycyrrhizin in the regulation of I/R induced liver injury2.1 Glycyrrhizin inhibits liver I/R induced HMGB1,NLRP3 activation and Kupffer cells deathThe location and expression of HMGB1 and NLRP3 were detected by immunohistochemistry staining.F4/80,a surface glycoprotein expressed by murine macrophages,was observed by immunofluorescent staining of liver sections after 9 h reperfusion,indicating the death of Kupffer cells which developed typical pyroptosis morphology with cell swelling and membrane rupture2.2 Glycyrrhizin decreases liver I/R induced pyroptotic Kupffer cells deathTo further clarify the way of Kupffer cells death,we analyzed the production of IL-1?,the mechanism by which the cytokine was released from cells could be pyroptosis,in serum-free culture supernatants after 12h incubation of Kupffer cells obtained from mice after operation by ELISA.The protein levels of HMGB1,caspase-1,IL-1? and GSDMD,in precursor and/or activated format,in kupffer cell lysates were determined by western blot2.3 Glycyrrhizin prevents H/R induced kupffer cells pyroptosis via endogenous HMGB1To elucidate the molecular mechanisms of glycyrrhizin mediated inflammatory response in Kupffer cells pyroptosis after liver I/R,we used a hypoxia-reoxygenation(H/R)-stimulated cell culture system.Glycyrrhizin,HMGB1 neutralizing antibody,or recombinant HMGB1 was further added to investigate the mechanism by which HMGB 1 may exert in H/R induced kupffer cells pyroptosis.IL-1? release was examined by ELISA in culture supernatants HMGB1,caspase-1,IL-1? and GSDMD expression were examined by Western blot in cell lysates.Propidium iodide(PI)was observed by flow cytometry3.The role of GSDMD in mediating H/R induced kupffer cells pyroptosis3.1 GSDMD knockdown protects kupffer cells against H/R induced pyroptosisSmall interfering RNA(siRNA)technology was used to elucidate GSDMD involved in H/R induced kupffer cells pyroptosis.Kupffer cells were transfected with small interfering RNA(siRNA)before H/R stimulation.IL-1? release is examined by ELISA in culture supernatants of cells with indicated treatments.Protein expression of NLPR3,activated caspase-1,IL-1?and GSDMD are examined by western blot in cell lysates with indicated treatments3.2 Activation of GSDMD is controlled by caspase-1 in H/R induced kupffer cells pyroptosis The above results demonstrated that GSDMD contributes to the H/R induced inflammation response,we next investigated the role of caspase-1 on GSDMD by using a caspase-1 inhibitor Z-YVAD-FMK in our model.Caspase-1 activation in our in vitro H/R model was further confirmed by both western boltting and FLICA fluorescent staining assay.IL-1?release is examined by ELISA in culture supernatants and protein expression of caspase-1,IL-1? and GSDMD are examined by Western blot in cell lysatesConclusions:1.Glycyrrhizin attenuates tissue damage during liver ischemia-reperfusion injury in mice2.Glycyrrhizin inhibits GSDMD-mediated kupffer cells pyroptosis induced by liver I/R3.Glycyrrhizin suppresses H/R induced kupffer cells pyroptosis via endogenous HMGB14.GSDMD knockdown protects kupffer cells against H/R induced pyroptosisBackground:Mucosal barrier function serves the essential function of coexisting with the intestinal microbiota tolerating commensal bacteria while fighting pathogens and preserving the ability to absorb nutrients to maintain homeostasis.Pattern recognition receptors(PRRs),such as TLRs and NLRs,are important in general for sustaining barrier homeostasis by regulating bacterial clearance,epithelial proliferation and mucosal healing,and even the composition of the microbiota.The receptor for advanced glycation end products(RAGE)is also considered a PRR.Clinical investigations indicate an upregulation of RAGE in patients suffered from inflammatory bowel disease,and especially the ulcerative colitis,however the underlying mechanisms are still unclear and need to be elucidatedObjective:This study was designed to explore the functional role and molecular mechanism of RAGE in the regulation of DSS induced murine epithelial barrier injury and colitisMethods and Results:1.Establishment of inducible RAGE gene knockout mouse modelWe crossed CreERT2+/-RAGEfl/fl with CreERT2+/+RAGEfl/-to generate adequate CreERT2+/-RAGEfl/fl mice.Then intraperitoneal(I.P.)injection of 2 mg Tamoxifen was used for five consecutive days to achieve ubiquitous CreERT2 activity in 7-week-old maleCreERT2-/+RAGEfl/fl mice.2.RAGE deficiency leads to more severe DSS-induced colitis2.1 Establishment of DSS-induced murine colitis modelAcute colitis located in distal colon was induced by oral gavage of DSS to study the contribution of RAGE in the innate immune mechanisms of colitis.A DSS concentration of 2.5%(w/v)in the drinking water for 7 days induces strong colitis,but low mortality rates.2.2 RAGE expression in colon tissue is upregulated after DSS administrationAcute colitis was induced by oral gavage of 2.5%DSS for seven consecutive days in wide type male mice.Mice were sacrificed and colon tissues were collected on day 0,4 and 7.After fixed in 4%formalin and embedded in paraffin,tissue paraffin sections were immunohistochemistry stained for RAGE.2.2 RAGE deficiency leads to more severe DSS-induced colitisAcute colitis was induced by oral gavage of 2.5%DSS for seven consecutive days in wide type and RAGE-/-male mice.The percentage of body weight decrease was calculated and compared.Mice were sacrificed and colon length were measured,followed by colon tissues were harvested and routinely stained for hematoxylin and eosin on day 0,4 and 7.3.RAGE deficiency aggravates tissue injury due to epithelium regeneration inhibition3.1 RAGE deficiency aggravates epithelium barrier injuryTo further investigate the mechanism underlying the more severe colitis resulted from RAGE knockout,we detected variation of colon permeability ex vivo by applying FITC-dextran.Expression of tight junction proteins were detected by immunohistochemistry staining assay.3.2 RAGE deficiency may inhibit IL-22 mediated epithelium regenerationUsing ELISA to measure the levels of cytokines in tissue homogenates.Using flow cytomety to detect IL-22+ cells in colonic lamina propria in order to explore the main source of IL-22.Using IHC staining to test p-STAT3,a key event which could be activated by IL-22 to promote epithelial cells proliferation.Using IHC staining to test Ki-67.3.3 RAGE deficiency may inhibit production of IL-1? derived from macrophagesUsing immunofluorescent staining to label IL-1?+ CD68+ macrophages.Using flow cytomety to detect the recruitment of F4/80 positive macrophages in both lamina propria and mesenteric lymph nodes.3.4 IL-22 ameliorates DSS-induced colon tissue injury in RAGE-/-miceAcute colitis was induced by oral gavage of 2.5%DSS for seven consecutive days in RAGE-/-male mice with or without IL-22 treatment.The percentage of body weight decrease was calculated and compared.Mice were sacrificed and colon length were measured,followed by colon tissues were harvested and routinely stained for hematoxylin and eosin on day 7.Conclusions:1.The expression levels of RAGE in colon tissues are upregulated after DSS administration.2.RAGE plays protective roles in maintaining mucosal barrier function.3.RAGE-/-may inhibits epithelial regeneration in dependent of IL-22 production.4.RAGE-/-inhibits production of IL-1? derived from macrophages.