| Part 1Role of CHI3L1 in acute liver injury induced by thioacetamide Objective To investigate the role of Chitinase-3-like-1(CHI3L1)in acute liver injury caused by thioacetamide(TAA).Methods Serum ALT,AST and CHI3L1 levels were detected by chemical analyzer and ELISA in 30 patients with clinical liver dysfunction and 10 healthy subjects.Acute liver injury model was established in C57BL/6 mice with different doses of TAA(80mg/kg,150mg/kg).After 3 days,the expression of CHI3L1 m RNA in liver was detected by q RT-PCR,and the levels of ALT,AST and CHI3L1 in serum were also detected.Three groups of acute TAA liver injury models were established in mice with C57BL/6 background,respectively,such as CHI3L1 gene knockout(CHI3L1-KO)group,wild type(WT)group and tail vein injection of recombinant CHI3L1 protein(r CHI3L1)group.Serum ALT and AST levels of the three groups were detected,and the degree of liver injury was evaluated by histopathology(quantified by Suzuki’s score).The m RNA expression of IFN-γ、IL-4、IL-17 A and Foxp3 in liver was detected by q RT-PCR,and the proportion of CD4+IFN-γ+,CD4+IL-4+,CD4+IL-17+Th cells and CD4+Foxp3+Treg cell in liver was detected by flow cytometry.Rusults There was a negative correlation between serum ALT and CHI3L1 level in patients with liver injury,the same as AST and CHI3L1.Serum CHI3L1 level in liver injury patients was lower than that in healthy subjects(p<0.01).After they were treated with liver protection drugs,serum CHI3L1 level significantly increased(p<0.01).Administration of TAA by intraperitoneal injection significantly increased serum ALT and AST levels in mice(p<0.01),and ALT and AST levels in the 150mg/kg group were significantly higher than those in the80mg/kg group(p<0.01),suggesting that TAA liver injury was obviouslyconcentration-dependent.Levels of intrahepatic CHI3L1 m RNA and serum CHI3L1 were significantly decreased(p<0.05 and p<0.01),also showing a concentration dependence.Serum ALT and AST levels in CHI3L1-KO group were significantly higher than those in WT group(p<0.01),which were higher than those in r CHI3L1group(p<0.01).Histopathological analysis also showed that liver tissue damage was the most severe in CHI3L1-KO group,and the least in r CHI3L1 group.The intrahepatic IFN-γ m RNA of CHI3L1-KO group was significantly higher than that of WT group(p<0.01),and Foxp3 m RNA was significantly lower compared with WT group(p<0.01),but there was no difference in the expression levels of IL-4 and IL-17 A m RNA.The m RNA expression levels of IFN-γ、IL-4 and IL-17 A in r CHI3L1 group were all significantly lower than those in WT group(p<0.01),but the level of Foxp3 m RNA in the liver of r CHI3L1 group was higher than that of WT group(p<0.01).The proportion of CD4+IFN-γ+ Th cell in the liver of CHI3L1-KO group was significantly higher than that of WT group(p<0.05).As for CD4+Foxp3+ Treg cell,the proportion was significantly lowered(p<0.05).No significant differences were detected in the proportion of CD4+IL-4+and CD4+IL-17+Th cells between the two groups.Treatment with r CHI3L1 could decrease the proportion of CD4+IFN-γ+,CD4+IL-4+,and CD4+IL-17+Th cells in liver(p<0.05)and increase the proportion of CD4+Foxp3+Treg cells(p<0.05).Conclusions CHI3L1 can reduce the number of Th1,Th2 and Th17 cells and increase the number of Treg cells in the liver,thereby reducing the expression of th1/2/17 cytokines in the liver and playing a protective role in the model of TAA-induced acute liver injury.Part 2The mechanism of CHI3L1 in regulating the differentiation of Th1 cells in TAA-induced liver injury Objective To investigate the protective mechanism of CHI3L1 in TAA-induced acute liver injury.Methods The spleen na?ve CD4+T cells(Th0)of CHI3L1-KO and WT mice were extracted,and Th1/2/17 and Treg cells differentiation systems were constructed under specific skewing conditions.Flow cytometry was used to detect the proportion of CD4+IFN-γ+,CD4+IL-4+,CD4+IL-17+Th cells and CD4+CD25+Foxp3+Treg cells and the expression of T-bet and GATA3.Recombinant CHI3L1 was added into the Th1/Treg differentiation system in the na?ve CD4+T cells of WT mice,and the proportion of CD4+IFN-γ+Th cells and CD4+CD25+Foxp3+Treg cells were detected by flow cytometry.The m RNA expression of p-STAT3 was detected by q RT-PCR in Th1 cells differentiated from Th0 of CHI3L1-KO and WT mice,and the protein expression of p-STAT3 was detected by western blot.In addition of the three groups built before,r CHI3L1+IFN-γ group,CHI3L-KO+ anti-IFN-γ group and CHI3L1-KO +WP1066(p-STAT3 inhibitor)group were set up.Serum levels of IFN-γ,ALT and AST in each group were detected.The degree of liver injury was evaluated by histopathology(quantified by Suzuki’s score).Results The proportion of Th1 cells differentiated from CHI3L1-KO naive CD4+T cells was significantly higher than that from WT naive CD4+T cells(p<0.01).No difference was observed in the proportion of differentiation of Th2/17 and Treg cells.In Th1 cell differentiation system,r CHI3L1 significantly reduced the percentage of Th1 cells differentiated from na?ve CD4+T cells(p<0.01).However,there was no significant change in the proportion of Treg cells after adding r CHI3L1 with different concentrations into the system where Th0 differentiated into Treg cells.In Th1 cells differentiated from CHI3L1-KO naive CD4+T cells,the expression of T-bet was significantly higher than that of WT group(p<0.01),and the m RNA and protein expressions of p-STAT3 were also significantly higher compared with WT group(p<0.05),while the expression of GATA3 did not change.Serum IFN-γ level in CHI3L1-KO group was significantly higher than that in WT group(p<0.01),which was higher than that in r CHI3L1 group(p<0.05),but the level in r CHI3L1+IFN-γ group was significantly higher than that in r CHI3L1 group(p<0.05).The ALT,AST and Suzuki’s scores in the r CHI3L1+ IFN-γ group were significantly higher than those in the r CHI3L1 group(p<0.05),while the serum level of ALT and AST were significantly lower in the CHI3L1-KO+anti-IFN-γ group than those in the CHI3L1-KO group(p<0.05).When compared with CHI3L1-KO group,levels of serum ALT and AST in CHI3L1-KO+WP1066 group were significantly decreased(p<0.01),accompany with the Suzuki’s scores(p<0.05),the proportion of CD4+IFN-γ+Th cells in liver(p<0.01),and the level of serum IFN-γ(p<0.05).Conclusions Through inhibiting STAT3 phosphorylation and T-bet transcription activity,CHI3L1 could inhibit the differentiation from Th0 to Th1 cells,therefore reduces the serum level of IFN-γ and attenuates TAA-induced liver damage. |
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