| Liver fibrosis is a pathological process that triggers a sustained injury repair reaction in chronic inflammatory injury of the liver,resulting in abnormal deposition of extracellular matrix in liver tissue,which further causes abnormal changes in liver structure and liver function.The imbalance of various immune factors during liver fibrosis promotes the occurrence and development of liver fibrosis.Studies have shown that macrophages have a high degree of plasticity and polarized into different phenotypes,then performed different functions in different immune internal environments.For example,lipopolysaccharides?LPS?and IFN-?can promote the differentiation of macrophages into classically activated M1;M1 macrophages can produce inflammatory factors to promote the body's inflammatory response;IL-4 and IL-13 can induce macrophages differentiate into selectively activated M2-polarized,and M2-polarized macrophages can produce anti-inflammatory factors,reduce inflammation,reduce oxidative stress,inflammatory damage and promote tissue repair.Kupffer cells?KCs?are the largest group of tissue macrophages in the body and are important functional cells of the liver's innate immune system.Clinical studies and animal experiments have confirmed that in alcoholic and nonalcoholic fatty liver disease,M1-polarized KCs cells can promote steatosis and apoptosis of hepatocytes,inflammatory cells are recruited,leading to fibrosis;but once M1type KCs are restricted,it can delay the progression of alcoholic liver disease.The study found that the balance of M1/M2 type KCs plays an important regulatory role in the development of alcohol or non-alcoholic liver injury.There are dynamic equilibrium changes in the number of M1 and M2 types of KCs at different stages of liver fibrosis.Interleukin-22?IL-22?belongs to the IL-10 family and has dual anti-inflammatory and pro-inflammatory effects.Studies in vitro and in the liver have confirmed that IL-22 has hepatocyte protection.Our previous study found that IL-22 can exert anti-liver fibrosis by inhibiting the activity of M1 type KCs,and the expression of JAK2/STAT3signaling pathway is significantly increased during this process,suggesting that JAK2/STAT3 pathway is likely to be involved in IL-22 inhibition.M1 type KCs activity process,as an important immune factor regulating the occurrence,development and outcome of liver fibrosis,does IL-22 exert anti-liver fibrosis by regulating the activity of M1 type KCs,and what is the mechanism?This is rarely reported at home and abroad,and it is also the interest of this research.Therefore,the aim of this study was to investigate the role of IL-22 in inhibiting the progression of liver fibrosis during the regulation of M1/M2 KCs polarization,and its relationship with JAK2/STAT3 signaling pathway and its mechanism.PART ?:CORRELATION BETWEEN IL-22 AND KCS AND LIVER FIBROSISOBJECTIVE:To detect the degree of hepatic fibrosis and the expression of IL-22 and M1/M2 KCs in liver tissue by collecting liver tissue samples from patients with different degrees of hepatic fibrosis,and to analyze the relationship between IL-22,M1/M2 type KCs and liver fibrosis.METHODS:Liver tissue samples from patients with different degrees of chronic hepatitis B complicated with hepatic fibrosis were collected.Hepatic fibrosis was observed by HE and Sirius Red staining.The degree of liver fibrosis was assessed according to the Ishak scoring system.The distribution of IL-22 in different liver tissues was detected by immunofluorescence staining;the M1 type KCs?F4/80+/iNOS+?and M2 type KCs?F4/80+/CD206+?were detected by immunofluorescence double staining technique.Characteristic phenotype,observe the number and distribution of different phenotypes of KCs in the liver,were used confocal microscopy to take pictures and analyzed images using Image J?National Institutes of Health,Bethesda,MD?systemsoftware.RESULTS:?1?According to the results of HE and Sirius Red staining,combined with the Ishak scoring system,different degrees of liver tissue specimens were divided into:normal group?control,n=5?,G1S1?n=4?,G2S2?n=5?.G3S3?n=6?and G4S4?n=6?groups,the degree of liver fibrosis was assessed by Ishak scoring system between groups,and the results of Sirius Red staining were statistically different between groups?P<0.01?;IL-22 fluorescent staining is mainly distributed in the hepatic sinus and hepatic lobular central vein or portal area.The positive rate is the lowest in the control group,and gradually increases in the early/intermediate liver fibrosis,the highest expression in G2S2;and then gradual reductionafter G3S3,and G4S4 was significantly reduced;the fibrosis group was statistically different from the control group?p=0.000?;?3?M1 type KCs?F4/80+/iNOS+?and M2 type KCs?F4/80+/CD206+?is also mainly distributed in the hepatic sinus and hepatic lobule central vein or portal area.The positive rate of double staining is extremely low in control liver tissue,while it is gradually increased in G1S1 and G2S2,while in G3S3 and G4S4 gradually reduced,there was a statistically significant difference between the G2S2 and G3S3 groups compared with the control group?P=0.000?;?4?M2/M1 ratio exhibits change peak during the progress of liver fibrosis,wherein the ratio of the highest G2S2,with the control group having a significant difference?P=0.000?.It can be seen that IL-22,M1/M2-KCs are mainly distributed in the hepatic sinus and hepatic lobular central vein or portal area,and the number is gradually increased in the early stage of fibrosis,but gradually decreased in the late stage of fibrosis.During the progression of liver fibrosis,the expression of IL-22 was significantly positively correlated with M1-KCS and M2-KCs?r=0.901,P=0.000;r=0.935,P=0.000,respectively?.CONCLUSIONS:IL-22,M1/M2-KCs are mainly distributed in the hepatic sinus and hepatic lobular central vein or portal area,and the number is gradually increased in the early and middle stages of fibrosis,and gradually decreased in the late stage of fibrosis;during the progression of liver fibrosis,there was a significant positive correlation between the expression of IL-22 and M1/M2-KCS.PART ?:IN VIVO STUDY ON THE MECHANISM OF IL-22 INREGULATING KCS POLARIZATION ON LIVER FIBROSISOBJECTIVE:To elucidate the mechanism of IL-22-induced KCs polarization affecting the occurrence and development of hepatic fibrosis by using mouse liver fibrosis model.METHODS:?1?BALB/C mice were injected intraperitoneally with CCl4to construct a liver fibrosis model?CCl4 was dissolved in edible olive oil and formulated into 20%CCl4 solution for use:0.4 ml/100g dose,2 times/week,for6 weeks?,CCl4 was administered intraperitoneally with rmIL-22 and anti-IL22after 4 weeks of administration,grouped into:normal group?normal,n=60?;negative control group?control,n=40?;liver fibrosis group?CCl4,n=40?;4 liver fibrosis+IL-22 group?CCl4+rmIL22,rmIL-22,100ng/mouse,5 times/week,n=40?;5 liver fibrosis+anti-IL22?CCl4+anti-IL22,anti-IL22,150 mg/mouse,3times/week,n=40?;mice were sacrificed at the 4th and 6th week of the experiment,and peripheral blood and liver tissues were taken.Separation of KCs were also taken by liver tissue;?2?Hepatic tissue fibrosis was detected by HE and Sirius Red staining,and the degree of liver fibrosis and differences of each group were evaluated according to Ishak scoring system;?3?M1 type KCs?F4/80+/iNOS+?and M2 type KCs?F4/80+/CD206+?characteristic phenotype,observe the number and distribution of different phenotypes of KCs in the liver were detected by immunofluorescence double staining technique;using Image J system software analysis these images;?4?ELISA and real-time PCR were used to detect the expression levels of related factors of JAK/STAT3 pathway and relevant collagen protein/inflammatory factors in peripheral blood and KCs.RESULTS:?1?Compared with the normal group,the liver fibrosis model was established at 4w when CCl4 was intervened,and cirrhosis was achieved at8w.Under HE staining,hepatocytes in CCl4 group showed edema,degeneration and necrosis.A large number of inflammatory cells infiltrated,and a large amount of collagen was deposited.The lesions were obvious in the portal area.Corresponding lesions showed different degrees of red in the intrahepatic portal area under Sirius Red staining.Green collagen?Col-?1,Col-3A1?was deposited,and the degree of liver fibrosis was significantly aggravated in the 6w CCl4group compared with the 4w CCl4 group?P=0.001?.The degree of hepatic fibrosis was lower in the CCl4+rmIL-22 group than in the 6w CCl4 group?P=0.014?,while the degree of liver fibrosis was significantly aggravated in the CCl4+anti-IL22 group compared with the 4w CCl4 group?P=0.000?.?2?The phenotypic results of M1 and M2-KCs in immunofluorescence double staining showed that the number of M1-KCs cells in each model group was significantly higher than that in control group?P=0.000?,and the increase in CCl4+anti-IL22group was the most significant.The number of M2-KCs cells in each model group?except CCl4+anti-IL22 group?was significantly higher than that in the control group?P=0.000?.In the CCl4+rmIL-22 group,the increase was most significant;in addition,the M2/M1 ratio was highest in the CCl4+rmIL-22group compared with the control group?P=0.000?,while the CCl4+anti-IL22group was the lowest?P=0.031?.?3?Peripheral blood ELISA results showed that the expressions of IL-1?,IL-6,TGF-?1,Col-?1 and Col-3A1 were increased in different models compared with control group?P<0.01?.Among them,the increase of CCl4+anti-IL22 group was the most obvious,while that of CCl4+rmIL-22 group was lower.?4?RT-PCR detection of KCs showed that KCs derived from CCl4+rmIL-22 group expressed higher levels of STAT3,CD206,IL-22R1,IL-10R2?P<0.01?;all model groups?except CCl4+rmIL-22 group?,the expression of Erk,Akt,IL-1?,IL-6 and TGF-?1 was significantly higher than that in the control group?P<0.01?.In addition,although the JAK2 gene expression in each model group was not significant difference compared with the control group,but iNOS expression was significantly higher than the control group?P<0.01?.CONCLUSIONS:IL-22 plays the inhibiting role inCCl4-induced liver fibrosis mouse model;its possible mechanism is to activate the STAT3/IL-10R2signaling pathway,promote the polarization of M2-KCs,and then inhibit the proinflammatory factors secretion from KCs,and changes the microenvironment in the body,further inhibit the progression of liver fibrosis.PART ?:IN VITRO STUDY ON THE MECHANISM OF IL-22 INREGULATING KCS POLARIZATION FOR INHIBITINGHSC ACTIVITYOBJECTIVE:To observe the role of IL-22 in the regulation of KCs polarization and signaling pathway in vitro,and to construct a co-culture system of KCs and HSC cells to verify the role of IL-22 in regulating KCs polarization inhibition of HSC activity.METHODS:First,the human mononuclear cell line U937 was used for in vitro culture,LPS was induced into M1-KCs macrophages,and rmIL-22 and wp1066?JAK2/STAT3 inhibitor?were intervened in the latter,grouped as:M0,M0+DMSO,M1,M1+wp1066,M1+IL-22,M1+wp1066+IL-22,M1+anti-IL10R2,M1+anti-IL10R2+IL-22;ELISA,flow cytometry?FACS?,qRT-PCR and Western Blotting technique was used to detect the change of M1/M2polarization in KCs.Secondly,the primary HSC and KCs of liver were isolated by collagenase in situ perfusion and density gradient centrifugation.The positive selection and purification of KCs were taken by F4/80 strain.KCs purity and phagocytic activity were detected;KCs isolated from each group?normal,control,CCl4,CCl4+rmIL-22,CCl4+anti-IL22?were co-cultured with activated HSC;FACS was used to detect M1/M2 polarization ratiobefore and after co-culture.The expression of?-SMA of HSC was detected by immunofluorescence;the expressions of Col-?1,Col-3A1from HSC were detected by ELISA.RESULTS:?1?U937 was induced into M0-KCs at 200 ng/mL PMA.The latter was polarized to M1-KCs under the induction of 100 ng/mL LPS.The polarizability of M1-KCs was 78.5%by FACS.The activity of M0-KCs phagocytosis the self-fluorescent latex beads was 94.5%.?2?Different concentrations of rmIL-22 and wp1066 were used to interven M0-KCs,cck-8and FACS were used to detect cell proliferation rate and apoptosis,respectively.20ng/mL IL-22 and 2.5?M wp1066 had no significant changes in KCs cell proliferation or apoptosis;?3?FACS detectedthe degree of p-STAT3 activation of different concentrations of rmIL-22?10,20,40 ng/mL?against M1-KCs.The results showed that the concentration of rmIL-22 was not significantly correlated with the activation of p-STAT3,and the effect of 20 ng/mL was the best.The peak activation time was within 15-30 min,and it was significantly weakened after 3 h;?4?ELISA showed that IL-1?,IL-6 and iNOS were the highest in M1group.After adding rmIL-22 and/or wp1066,the expressions were reduced by different degrees.The level of expression was lowest under co-intervention?P<0.01 compared with M1?,following by rmIL-22 alone?P<0.01 compared with M1?,the intervention effect by wp1066 alone was weak?P<0.01 compared with M1?.In addition,CD206 expression was lower in M1.With the intervention of rmIL-22 and/or wp1066,the expression of CD206 increased to different degrees.Among them,intervention of rmIL-22 alone had the highest expression?P<0.01 compared with M1?.The intervention effect by rmIL-22 and wp1066 was the second?P<0.01 compared with M1?,and the effect of wp1066alone was weaker?P<0.05 compared with M1?.?5?RT-PCR detection of gene expression results showed that JAK2,STAT3,CD206,IL-22R1 and IL-10R2genes were significantly increased in rmIL-22 environment?P<0.01?,while IL-22 was blocked at wp1066,the levels were decreased?except CD206??P<0.01?.Interestingly,the expression of Erk,Akt,iNOS,IL-1?and IL-6 genes was significantly decreased in IL-22 environment?P<0.01?.After wp1066blocked IL-22,the expression of thesegenes was significantly restored?P<0.01?.?6?FACS detected the expression of p-STAT3 in M1-KCs after rmIL-22intervention.The results showed that the expression of p-STAT3 was peaked within 30 min after rmIL-22 intervention,and then decreased after 3 h,the decrease was significant?P<0.01?.Secondly,the effect of rmIL-22 on p-STAT3activation was observed significantly inhibition under the environment of wp1066?P<0.01?;in addition,KCs phenotypic polarization results showed that after 30 min of rmIL-22 intervention,the polarization of M1-KCs was significantly decreased,while M2-KCs was significantly increased?P<0.01?;However,wp1066 can inhibit the polarization of M1-KCs to some extent?P<0.01?;under intervention of wp1066 and rmIL-22,it can promote M1-KCs polarization trend was further attenuated,and M2-KCs were further enhanced?P<0.01?.The results also showed that under the intervention of anti-IL10R2,the effect of rmIL-22 on KCs polarization was completely blocked,suggesting that IL-10R2 is a key role receptor for the action of IL-22 on KCs.?7?The results of WB test further showed that the expression of p-STAT3 was significantly increased in M1-KCs after rmIL-22 intervention 30 min?P<0.01?,while the expression of p-JAK2 was not obvious.In addition,during the intervention of rmIL-22,the expression of p-Erk and p-Akt was inhibited?P<0.01?.Secondly,wp1066 significantly inhibited the expression of JAK2 and p-STAT3?P<0.01?.Under the intervention of wp1066,rmIL-22 was significantly inhibited to activated p-STAT3?P<0.01?;again,after the action of anti-IL10R2 on KCs,JAK2 and p-STAT3 activation wasblocked by rmIL-22?P<0.01?;finally,wp1066 and anti-IL10R2 can block the effect of rmIL-22 on the Erk and Akt pathways.?8?Isolation of primary HSC and KCs from liver.Using F4/80+positive selection,KCs with a purity of 86.3%were obtained,and the 2h phagocytic activity was 98.5%.The isolated HSC was resuspended in20%serum DMEM.In the medium,after appressing,culturing,and changing the solution,cell proliferation occurred about 5 days,and was collected and frozen for use.The positive rate of?-SMA was 96.5%by immunofluorescence staining.?9?The KCs isolated from each group?control,6w CCl4,CCl4+rmIL-22,CCl4+anti-IL22?were co-cultured with activated HSC for 24 h?divided into 5 groups:normal,control,6w CCl4,CCl4+rmIL-22,CCl4+anti-IL22?;FACS detectedthe M2/M1 polarization ratio of KCs before and after co-culture.It showed that the M2/M1 ratio??of CCl4+rmIL-22group were significantly higher than those of other groups?P=0.000?;After co-culture,the proportion of M1-KCs in each group was significantly higher than that before co-culture,while the proportion of M2-KCs was decreased.?10?Immunofluorescence detection of HSC expression of?-SMA showed that the expression of?-SMA was significantly decreased in the CCl4+rmIL-22 group compared with the other groups?P<0.01?;there was no difference between the other groups.?11?After co-culture,the Col-?1 and Col-3A1 secreted by HSC in CCl4+rmIL-22 group were significantly lower than those in other groups?P<0.01?,while the other groups were not statistically different.?12?FACS detection of HSC apoptosis after co-culture showed that no significant HSC apoptosis was observed between the groups.CONCLUSIONS:In vitro studies have shown that IL-22 can inhibit the polarization of M1-KCs by up-regulating the STAT3-IL10R2 pathway,and down-regulate the Erk1/2 and Akt pathways to promote the polarization of M2-KCs,and change the polarization balance of M1/M2-KCs and the production of pro-inflammatory factors.In addition,experiments based on the in vivo model to establish in vitro co-culture confirmed that the M2/M1-KCs ratio increased,which significantly inhibited the secretion activity of HSC. |
PDF Full Text Request