Study On Mechanisms Underlying IL-33 Ameliorates Experimental Colitis Via Regulating Autophagy In Mice Investigation On The Role And Mechanisms By Which Glycyrrhizin Alleviates Con A-induced Acute Kidney Injury By Promoting The Expression Of IL-25

Background:Interleukin-33?IL-33?is a newly discovered member of IL-1 cytokine family.It is widely expressed in endothelial cells,epithelial cells,macrophages and other cell types,and participates in the pathogenesis and pathological process of infectious diseases,inflammatory diseases,autoimmune diseases and other diseases.IL-33 is a bifunctional molecule,which not only participates in the regulation of gene expression as a nuclear transcription factor,but also releases into extracellular space as a cytokine to promote Th2immune response through IL-33/ST2 signaling pathway.Unlike conventional cytokines,IL-33 can actively and rapidly release as an endogenous danger signal?DAMP?or"alarmin"after cell injury,activating the immune system and amplifying inflammation in the early stage of infection and tissue injury.In recent years,it has been found that the expression of IL-33/ST2 in inflammatory bowel disease,especially in the intestinal mucosa of patients with ulcerative colitis,has increased significantly,suggesting that IL-33 plays an important role in inflammatory bowel disease.However,the role of IL-33in inflammatory bowel disease is still controversial,and the exact role and mechanism of IL-33 in the intestinal inflammatory environment are still poorly understood.Autophagy is an evolutionarily conserved mechanism of cell survival,which maintains cell homeostasis by degrading damaged or toxic proteins through lysosome pathways.Autophagy is also considered to be an important effector and regulator of inflammatory response,which plays a crucial role in regulating moderate inflammatory response and maintaining cell and tissue balance.It has been proved that many cytokines can regulate autophagy,and autophagy also affects the production and secretion of many cytokines,but the relationship between IL-33 and autophagy remains to be clarified.TLR4 is a classical inflammatory signaling pathway and an autophagy initiation sensor and there is a relationship between IL-33 and TLR4 pathway.Our previous studies have shown that IL-33 improves experimental colitis in mice by promoting Th2/Foxp3+regulatory T cell response.However,the effect of IL-33 on cell autophagy in intestinal tissue of mice and the role and mechanism of autophagy in experimental enteritis of mice remain unclear.Objective:To explore the effect of alarmin IL-33 on autophagy in experimental enteritis in mice and the role and mechanism of both in acute intestinal inflammatory response,so as to provide experimental basis for exploring the role and mechanism of cytokines and autophagy in the pathogenesis and pathological process of inflammatory diseases,and to find new potential biological targets for the treatment of inflammatory bowel diseases.Methods:I.Experiments in vivo1.The mice were randomly divided into normal control group?PBS?,TNBS enteritis group,ethanol control group,PBS control group?TNBS+PBS?,IL-33 treatment group?TNBS+IL-33?,3-MA intervention group?TNBS+IL-33+3-MA?.2.Establishment of acute enteritis model and drug treatment:acute enteritis model of mice was induced by 2.5%TNBS?100?L/mouse?.The control group was given 50%absolute ethanol of the same volume as the model control group,and the control group was given sterile PBS of the same volume as the normal control group.Other groups were given corresponding reagents or drugs after TNBS modeling:IL-33 treatment group mice were intraperitoneally injected with IL-33?2?g/mice,once a day?;PBS control group mice were intraperitoneally injected with sterile PBS?the same volume as IL-33,once a day?;3-MA group mice were intraperitoneally injected with 3-MA?24 mg/kg?immediately after intraperitoneal injection of IL-33.3.After TNBS modeling and drug treatment,the body weight,fecal occult blood and disease activity index of mice in each group were observed at the same time on day 0,1,2,3 and 4,respectively.The gross pathological changes of colon and histopathological changes of colon were observed on day 4 to evaluate the morbidity and therapeutic effect of enteritis in mice.4.On the 4th day of TNBS modeling and drug treatment,total proteins were extracted from colon tissue of mice.The expression of autophagy marker protein LC3II/I and autophagy-related protein P62 was detected by Western Blot method,and the effect of IL-33 on autophagy status of colon tissue cells was evaluated.5.Colon tissues of mice were Co-stained with Beclin-1 and F4/80 immunofluorescent antibodies.The expression and localization of Beclin-1 in colon tissues were detected by laser confocal microscopy,and the effect of IL-33 on macrophage autophagy in colon tissues was evaluated.6.The levels of pro-inflammatory cytokines TNF-alpha,IFN-gamma,anti-inflammatory cytokines IL-5 and IL-13 in colon tissues and serum were detected by ELISA,and the effects of IL-33-induced autophagy on the levels of cytokines were observed.7.TNBS enteritis model was established by TLR4 gene knockout mice and treated with IL-33.The contents of LC3II/I and P62 in colon tissue of mice were detected by Western Blot.The role of TLR4 in IL-33-induced autophagy of macrophages in vivo was observed.II.Experiments in vitroBone marrow-derived macrophages from wild-type,TLR4 knockout and IL-33 knockout mice were cultured in vitro.They were divided into blank control group,PBS control group,IL-33 stimulation group and EBSS starvation group.The following experiments were carried out respectively:1.In vitro experiment 1:On the 6th day of primary culture of wild macrophages,after transfection of macrophages with GFP-LC3 plasmid for 24 hours,IL-33 stimulation group was stimulated with IL-33?100ng/mL?for 24 hours,and other groups were added corresponding reagents or drugs as control.Laser confocal microscopy was used to detect the formation of autophagies in macrophages.2.In vitro experiment 2:On the 7th day of primary culture of wild macrophages,IL-33stimulation group was stimulated with IL-33?100ng/mL?,and other groups were added with corresponding reagents or drugs as control.Western Blot was used to detect the content of autophagy marker protein LC3II/I and P62.3.In vitro experiment 3:primary cultured wild type and TLR4 gene knockout mice macrophages were stimulated by IL-33?100ng/mL?in the IL-33 treatment group,and the other groups were added corresponding reagents or drugs as control.The content of autophagy marker protein LC3II/I and P62 was detected by Western Blot,and the role of TLR4 in IL-33-induced autophagy of macrophages in vitro was observed.4.In vitro experiment 4:primary cultured wild type and IL-33 gene knockout mice macrophages were stimulated by rapamycin?500ng/mL?for 24 hours after maturation.The expression of autophagy marker protein Beclin-1 was detected by Western Blot,and the effect of IL-33 gene knockout on autophagy of macrophages was observed.Results:1.Compared with the normal control group and the ethanol control group,the weight of mice in TNBS enteritis group decreased significantly,and the disease activity index,colon gross pathological score and colon histopathological score increased significantly?P<0.001?,showing obvious acute intestinal inflammation symptoms,which confirmed the successful establishment of TNBS-induced acute enteritis model in mice.2.Compared with TNBS enteritis group and PBS control group,the weight of mice in IL-33 treatment group increased rapidly,and the disease activity index,colon gross pathology and colon histopathology improved significantly,and the related scores decreased significantly?P<0.01,P<0.05?,indicating that IL-33 treatment could significantly improve the symptoms of acute intestinal inflammation in mice.3.Western Blot test showed that compared with the normal control group,the expression of LC3 II increased and p62 decreased in the IL-33 treatment group,while the LC3II/beta-actin ratio increased in the IL-33 treatment group?P<0.01?,indicating that IL-33could induce autophagy of colon tissue cells in mice.4.The gross and histopathological observation of colon after autophagy blockade showed that compared with IL-33 treated mice,the symptoms of acute intestinal inflammation in the 3-MA intervention group were significantly worse,and the gross pathological and histopathological scores of colon were significantly higher?P<0.01,P<0.05?,indicating that blocking IL-33-autophagy pathway could reduce the number of IL-33 pairs.The alleviating effect of acute enteritis suggests that IL-33 can alleviate acute enteritis at least partly by inducing autophagy.5.Immunofluorescence staining showed that the expression of Beclin-1 in F4/80 positive cells in colon tissue of mice treated with IL-33 was significantly increased compared with that of TNBS enteritis group,suggesting that IL-33 treatment could significantly enhance the autophagic activity of macrophages in colon tissue.6.The expression of GFP-LC3 in primary cultured macrophages was observed by confocal laser microscopy.The results showed that there were many bright green fluorescent spots in macrophages treated with IL-33 compared with blank control group and PBS control group,indicating that the autophagic activity of macrophages treated with IL-33 increased significantly.7.Western Blot was used to detect the expression of LC3II/I in primary cultured macrophages.The results showed that compared with blank control group and PBS control group,the LC3II/beta-actin ratio of macrophages treated with IL-33 increased significantly?P<0.001?.These results together with results 6 suggest that IL-33 can induce autophagy in primary cultured macrophages.8.Western Blot was used to detect the expression of Beclin-1 in primary cultured IL-33^-/-macrophages.The results showed that compared with wild macrophages,the expression of Beclin-1 in IL-33^-/-macrophages decreased significantly,and the ratio of Beclin-1/beta decreased significantly?P<0.05?,indicating the sensitivity of IL-33^-/-macrophages to rapamycin-induced autophagy decreased significantly,which suggests that IL-33 is a key regulator of macrophage autophagy.9.Western Blot was used to detect the expression of LC3II/I in colon tissues of wild-type and TLR4-knockout mice.The results showed that compared with wild-type mice,the expression of LC3II in colon tissues of TLR4^-/-mice was dramatically decreased,and the ratio of LC3II/beta-actin was significantly decreased?P<0.01?.It suggested that the sensitivity of colon tissues of TLR4-knockout mice to IL-33-induced autophagy was significantly decreased,indicating that TLR4 signalling was essential for the induction of macrophage autophagy by IL-33.10.Western Blot was used to detect the expression of LC3II/I in primary cultured macrophages generated from wild type and TLR4 knockout mice,respectively.The results showed that compared with wild type mice,the expression of LC3II in TLR4^-/-macrophages decreased,and the ratio of LC3II/beta-actin decreased significantly?P<0.001?.These results together with result 9 showed that IL-33 could induce macrophages'autophagy through TLR4 signalling pathway.11.The levels of related cytokines by IL-33 treatment in colon tissue and serum was detected by ELISA.The results showed that the levels of pro-inflammatory cytokines TNF-alpha and IFN-gamma in serum of mice in IL-33 treatment group were significantly lower than those in PBS control group?P<0.05?,while the levels of anti-inflammatory cytokines IL-5 and IL-13 were significantly higher than those in PBS control group?P<0.05?.These results suggest that IL-33 can promote the balance of pro-inflammatory cytokines and anti-inflammatory cytokines.12.The effect of 3-MA blocking autophagy on the levels of cytokines in colon tissue and serum was detected by ELISA.The results showed that the levels of pro-inflammatory cytokines TNF-alpha and IFN-gamma in colon tissue and serum of mice in 3-MA intervention group were significantly higher than those in IL-33 treatment group?P<0.05?,while the levels of anti-inflammatory cytokines IL-5 and IL-13 were significantly lower than those in IL-33 treatment group?P<0.05?.The difference was statistically significant?P<0.05?,indicating that blockade of autophagy could reduce the balance effect of IL-33on pro-inflammatory cytokines and anti-inflammatory cytokines.These results suggests that IL-33 promotes cytokine balance by inducing autophagy.Conclusion:1.IL-33 alleviates TNBS-induced experimental colitis in mice by enhancing autophagy.2.IL-33 is a key regulator of macrophage autophagy.3.IL-33 induces macrophage autophagy through TLR4 signaling pathway.4.IL-33 regulates the balance of pro-and anti-inflammatory cytokines by inducing autophagy.5.IL-33-TLR4-Autophagy is an important mechanism of IL-33 regulating acute intestinal inflammatory response in mice.Background: Acute Kidney Injury?AKI?is increasing in both developing and developed countries,and there is no effective treatment for it up till now.It is particularly noteworthy that AKI has a high incidence in the context of acute liver failure?ALF?and is associated with poor prognosis and high mortality.Therefore,AKI research in the context of ALF has become an urgent and meaningful research field and it includes establishing experimental animal models of liver-kidney co-injury,exploring the pathogenesis of AKI in the experimental animal model of "ALF+AKI",and screening drugs that have therapeutic effects on both ALF and AKI.The therapeutic effects of Glycyrrhizin?GL?in the treatment of acute liver injury and chronic hepatitis has been confirmed by a large number of studies.Because of the significant protective effect of GL on liver function and the preliminary therapeutic effect on kidney diseases,it is suggested that GL may be a candidate drug for co-injury of liver and kidney.However,the role and mechanism of GL in acute kidney injury need to be further studied.Monocyte/macrophage system plays an important role in the homeostasis and immune inflammation of kidney tissue.The different polarization directions of pro-inflammatory M1 and anti-inflammatory M2 macrophages determine the process of renal tissue damage,repair and renal interstitial fibrosis.Therefore,the treatment based on promoting the polarization of M2 macrophages is a promising strategyfor the treatment of kidney diseases.Interleukin-25?IL-25?can target macrophages and transmit negative regulatory signals to macrophages,and inhibit the production of pro-inflammatory cytokines.However,the role and mechanism of IL-25 in renal diseases remain to be elucidated.Objective: To investigate the therapeutic effect of glycyrrhizin on acute kidney injury induced by Con A and to investigate its cellular and molecular mechanisms,so as to provide experimental basis for clinical application of glycyrrhizin in treating acute kidney injury.Methods: Part I: The role of Glycyrrhizin in Con A-induced Acute Kidney Injury in Mice 1.Model establishment and drug treatment: C57BL/6 mice of the same batch were randomly divided into four groups: PBS control group,Con A model group,GL control group and GL treatment group.Con A was injected into the tail vein of Con A model group mice?15mg/kg?;the same volume of PBS into the tail vein of PBS control group mice;GL control group mice intraperitoneal injection of GL?200mg/kg?;GL treatment group mice intraperitoneal injection of GL?200mg/kg?as pretreatment and then intraperitoneal injection of Con A.2.After 10 hours of drug treatment,the gross pathological changes of liver and kidney were observed,the liver and kidney indices were weighed and calculated,and samples of serum,urine,liver tissue and kidney tissue were collected for examination.3.HE staining and PAS staining were used to observe the histopathological changes of liver and kidney.4.Alanine aminotransferase?ALT?and aspartate aminotransferase?AST?levels of serum were measured by automatic biochemical analyzer to evaluate the degree of liver injury and the effect of drugs.5.The levels of serum creatinine?SCr?,urea nitrogen?BUN?and urinary creatinine?UCr?were measured by ELISA to evaluate the degree of renal injury and the effect of drugs.6.The expression of IL-25 in kidney tissue was detected by ELISA and immunohistochemistry to observe the effect of GL treatment on the expression of IL-25 in kidney tissue.Part II: Glycyrrhizin promotes IL-25 expression and induces polarization of M2 macrophages to alleviate acute kidney injury in mice 1.Animal grouping and drug treatment: C57BL/6 mice of the same batch were randomly divided into four groups: PBS control group,Con A model group,IL-25 control group and IL-25 treatment group.The IL-25 treatment group was pretreated by intraperitoneal injection of IL-25?100 ?g/mouse?and then injected Con A?15 mg/kg?,while the IL-25 control group was intraperitoneally injected with the same volume of IL-25.2.10 hours after the last administration,the gross pathological changes of the kidneys,liver and kidney indices were observed and measured,and the serum,urine and kidney tissue samples were collected.3.PAS staining was used to observe the pathological changes of kidney tissue and evaluate the effect of IL-25 in acute kidney injury.4.The levels of serum creatinine?SCr?,urea nitrogen?BUN?and urinary creatinine?UCr?were measured by ELISA,and the effects of IL-25 on renal function in AKI mice were evaluated.5.Immunofluorescence staining was used to detect the expression of CD206 in kidney tissues and to observe the effect of IL-25 on macrophage polarization in kidney tissues.6.Primary cultured mouse peritoneal macrophages were stimulated with Con A or IL-25 for 10 hours.The levels of IL-1beta and IL-10 in the supernatant were detected by ELISA to observe the effects of IL-25 on the secretion of pro-inflammatory?IL-1beta?and anti-inflammatory?IL-10?cytokines by macrophages.7.Primary cultured mouse peritoneal macrophages were stimulated with Con A or IL-25for 10 hours.The expression of CD206 was detected by immunofluorescence assay and TLR4 by flow cytometry to observe the effect of IL-25 on the expression of CD206 and TLR4 in macrophages.8.Peritoneal macrophages of primary cultured TLR4-/-mice and TLR2-/-mice were stimulated with IL-25 and Con A for 10 hours respectively.The expression of CD206 in macrophages was detected by immunofluorescence assay,and the signal pathway of IL-25-induced macrophage polarization was analyzed.Results: 1.Successful establishment of liver-kidney co-injury model in mice: Gross pathological observation showed that compared with PBS control group,the Con A model group had obvious inflammation in liver and kidney;liver and kidney index were significantly increased?P<0.05?;liver and kidney histopathological damage score increased significantly?P<0.001?;serum ALT and AST levels increased significantly?P<0.01?,serum muscles increased significantly?P<0.01?.The levels of SCr and BUN increased significantly?P<0.01?and UCr decreased significantly?P<0.01?.These results indicated that Con A caudal vein injection could cause acute liver and kidney injury in mice at the same time.2.Compared with the Con A model group,the kidney index of the GL treatment group significantly reduced?P<0.05?,the renal histopathological damage score?P<0.05?,the serum creatinine?SCr?,urea nitrogen?BUN?levels significantly decreased?P<0.05?,and the urinary creatinine?UCr?significantly increased?P<0.05?,indicating that GL could significantly improve the pathological damage of acute kidney injury in mice.3.Compared with Con A model mice,the expression of IL-25 in renal homogenate and tissue sections of GL treated mice increased significantly?P<0.01?.Compared with Con A model mice,GL treatment also recovered the decrease of IL-25 induced by Con A?P<0.01?,suggesting that the therapeutic effect of glycyrrhizin on acute kidney injury inmice could be related to the induction of IL-25 production.4.Compared with the Con A model group,the renal histopathological damage score of mice in IL-25 treatment group was significantly lower?P<0.01?;the levels of serum creatinine?SCr?,urea nitrogen?BUN?were significantly lower?P<0.05?;and the levels of urinary creatinine?UCr?were significantly higher?P<0.01?.These results suggest that IL-25 has protective effect on acute kidney injury in mice.5.After stimulation with Con A and IL-25 respectively,the IL-10 level in the cell culture medium of IL-25 treatment group was significantly higher than that of PBS control group?P<0.01?,while the IL-1beta level had no significant change?P>0.05?.Compared with Con A alone,the level of IL-10 and IL-1beta in Con A+IL-25 group increased significantly?P<0.01?and decreased significantly?P<0.05?,suggesting that IL-25 treatment could promote the secretion of inhibitory cytokine IL-10 by macrophages and inhibit the secretion of inflammatory cytokine IL-1beta by macrophages,suggesting that IL-25 at least partially regulates the balance of cytokines in acute kidney injury and thus plays an anti-inflammatory role.6.Immunofluorescence staining showed that compared with PBS control group,IL-25 alone significantly increased the expression of CD206 in kidney tissue?P<0.01?;Con A + IL-25 significantly enhanced the expression of CD206 in kidney tissue?P<0.01?compared with Con A model group.IL-25 treatment also significantly increased the expression of CD206 in primary cultured mouse peritoneal macrophages?P<0.01?,suggesting that IL-25 promoted the polarization of M2 macrophages in both mouse kidney tissues and primary cultured macrophages.7.After stimulation with Con A and IL-25 respectively,compared with PBS control group and IL-25 control group,the expression of TLR4 on the surface of macrophages in Con A group increased significantly?P<0.01?.Compared with Con A group,the expression of TLR4 in macrophages induced by Con A was significantly reduced by IL-25?P<0.01?,suggesting that IL-25 promoted the polarization of M2 macrophages by down-regulating TLR4 on the surface of macrophages.8.Compared with wild type and TLR2-/-mice,M2 macrophages in TLR4-/-mice increased significantly,suggesting that TLR4?not TLR2?signal regulates the polarization of M2 macrophages.In addition,IL-25 can induce polarization of TLR2-/-mouse macrophages to M2,but not to M2,suggesting that up-regulation of M2 macrophages induced by IL-25 is a TLR4 dependent manner.IL-25 promotes the polarization of M2 macrophages by down-regulation of TLR4.Conclusion: 1.Glycyrrhizin has a significant therapeutic effect on Con A-induced acute kidney injury,and is a potential candidate drug to effectively prevent acute kidney injury and improve renal function.2.The expression of IL-25 in kidney tissue of mice was promoted by glycyrrhizin,and the downward trend of IL-25 induced by Con A was reversed by glycyrrhizin.3.The expression of TLR4 in macrophages was up-regulated by Con A,while IL-25 could down-regulate the expression of TLR4 in macrophages induced by Con A,which showed an antagonistic effect on Con A-induced inflammation in AKI.4.TLR4 is the key signal molecule of M2 macrophage polarization.IL-25 induces macrophage polarization to M2 in a TLR4?not TLR2?dependent manner by down-regulating TLR4 expression.5.IL-25 promotes the balance of macrophage-derived pro-inflammatory?IL-1beta?and anti-inflammatory?IL-10?cytokines.6.The GL/IL-25/M2 axis is an important regulatory mechanism of inflammation during acute kidney injury,and may become a candidate target for acute kidney injury.