The Role Of GITR In The Mechanism Of Acute Autoimmune Hepatitis Induced By ConA In Mice

Part 1 The feature of liver injury and activation of HSC in acute autoimmune liver lesions induced by ConA in miceObjective:To study the pathogenic feature of liver injury, activation of HSC and its possible role in the liver injury induced by ConA.Methods:Mice were randomly divided into control group and ConA treatment group. Animals of ConA treatment group were exposed to ConA 20 mg/kg through vena caudalis injection; meanwhile the control group received the same volume of NS injection. After injection for 2h,8h,24h, and 48h, animals were sacrificed. Samples of blood, liver and spleen were harvested. The level of transaminases, albumin, and globulin were determinate with application of automatic biochemical analyzer, and the pathologic changes after mouse vena caudalis injection of ConA at different time were studied by HE stained paraffin sections. Serum concentrations of TGF-β1 and TGF-β3 were detected with application of ELISA; Immunohistochemistry was taken to detect intra-hepatic expression of HSC activation markersα-SMA and Vimentin; Meanwhile, the protein expressions of TGF-β1, TGF-β3,α-SMA and Vimentin in liver were detected by Western-blot.Results:(1) 2h after ConA injection, the transaminases started to increase, when 8h after ConA injection, the serum transaminases significantly heightened (P<0.05) and reached the peak level. The transaminases remained significantly higher levels than control group, both P<0.05, but were lower than 8h, both P<0.05.There were no significant differences in the level of albumin and globulin between the treatment groups and control group, there was an increasing tendency in albumin concentrations after ConA injection, while there was a decreasing tendency in globulin levels in ConA treatment groups. (2) After ConA injection, both liver index and spleen index of the mice increased significantly, and both reached the peak level at 8h. (3) Pathology:Mice of control group existed normal and clear hepatic lobules, hepatic cord arranged neatly, no congestion of the central venous; group of at 2h after ConA treatment showed mild congestion, mild infiltration with lymphocyte in portal area and the hepatic cord appeared slightly disordered arrangement, but the hepatic cells morphology was still normal; 8h group showed significant liver congestion, disappearance of the liver cord and sinus, steatosis of the hepatic cells, and infiltration of numerous inflammatory cells within the portal area; 24h group showed liver congestion, degeneration of the liver cells, and moderate infiltration of inflammatory cells; 48h group showed mild degeneration of liver cells, and mild infiltration of inflammatory cells in portal area. (4) After injection of ConA, immunohistochemistry and Western-blot results showed that HSC activation markersα-SMA and Vimentin expression was significantly higher than the mice of control group, of which the peak expression reached at 8h after ConA injection. (5) Serum TGF-(31 expression tended to increase at each time point after injection of ConA which peaked at 8h, significantly different with the normal group, P<0.01, hepatic protein expression of TGF-β1 was consistent with the serum level; (6) From 2h to 8h after injection of ConA, serum and hepatic expressions of TGF-β3 tended to increase, of which the peak expression reached at 8h,compared with control group, P<0.01, but at 24h and 48h after ConA injection, TGF-β3 expression began to decline, and significantly lower than normal group, P<0.05.Conclusion:(1) ConA injection could successfully induce autoimmune liver injury in mice, and existed apparent timeliness, in which the most serious liver lesions took place at 8h after ConA injection, and after 24 hours the liver appeared to proceed with self-repairing. (2) There existed early activation of HSC and expression imbalance of TGF-β1 and TGF-β3 in ConA-induced acute autoimmune liver injury, which may be associated with the degree of liver dysfunction and the mechanisms of progression to cirrhosis.Part 2 The influence of GITR/GITRL in the pathogenesis of autoimmune injury induced by ConA in miceObjective:To investigate the influence of GITR-GITRL in the mechanism of autoimmune hepatitis induced by ConA in mouse.Methods:Animals were randomly divided into control group, model group and Fc-GITR intervention group. Model group suffered from ConA (20mg/kg) injection to inducing acute autoimmune liver injury; Fc-GITR intervention group received intraperitoneal injection of Fc-GITR (5μg/mouse) 1 minute prior to intravenous injection of ConA (20mg/kg). At 8h,24h after ConA injection, the mice of three groups were anaesthetized. Samples of blood, liver and spleen were harvested. The level of transaminases, albumin, and globulin were determinate with application of automatic biochemical analyzer, the pathologic changes of three groups at different time were studied by HE stained paraffin sections, and expression of GITR and GITRL in the liver of control and model group were detected with the method of real-time PCR and western-blot.Results:(1) Transaminase levels in mice of model group at 8h and 24h after ConA injection were significantly higher than control group, while the transaminase levels in mice of Fc-GITR intervention group were significantly lower than model group, and there were no significant differences between Fc-GITR intervention group and control group. There were no significant differences with albumin or globulin levels between three groups. (2) Hepatic index, spleen index and the mortality rate of mice in Fc-GITR intervention group were significantly lower than the model group. (3) Pathology:Histological injuries of the liver in Fc-GITR pretreatment group were significantly reduced than the model group at each time after ConA injection. (4) Gene and protein expressions of GITR and GITRL were significantly higher than those in the control group at 8h and 24h after ConA injection.Conclusion:Intervention of the GITR signaling could reduce the acute autoimmune liver injury induced by ConA, which indicated that GITR-GITRL may be involved in the pathogenesis of acute autoimmune hepatitis induced by ConA.Part 3 The influences of GITR/GITRL on HSC activity in ConA induced acute autoimmune hepatitis of mice and in vitroObjective:To study the influences of GITR/GITRL on HSC activity in ConA-induced acute autoimmune hepatitis in mice and in vitro.Methods:(1) In vivo:Animals were randomly divided into control group, model group and Fc-GITR intervention group. Model group suffered from ConA (20mg/kg) injection to inducing acute autoimmune liver injury; Fc-GITR intervention group received intraperitoneal injection of Fc-GITR (5μg/mouse) 1 minute prior to intravenous injection of ConA (20mg/kg). At 8h,48h after ConA injection, the mice of three groups were anaesthetized. Samples of blood, liver and spleen were harvested. Serum and liver expression of TGF-β1 and TGF-β3 were detected by ELISA and Western-blot respectively; Immunofluorescence and Western-blot methods were taken to detect the HSCs activation marker proteins (Vimentin) in the mice of three groups. (2) In vitro:The spleen mononuclear cells (SPMC) in mice of different groups were separated with Percoll lymphocyte separator liquid, and then co-cultured with the primary mouse HSCs strain, HSC proliferation and activation were observed; Pre-treated the SPMC that separated from model group mice with Fc-GITR, and then co-cultureed with HSCs, HSC proliferation and activation were observed.Results:(1) Vimentin expressions of Fc-GITR intervention group were significantly lower than the model group. (2) Serum and hepatic expression of TGF-β1 was significantly reduced in Fc-GITR intervention group compared with the model group, while Fc-GITR intervention had no significant effect on TGF-β3 protein expression. (3) In vitro:Activation of HSC co-cultured with SPMC of model group was significantly higher than co-cultured with those of the control group; while activation of HSC co-cultured with SPMC of Fc-GITR intervention group was significantly lower than co-cultured with those of the model group; Activation of HSC co-cultured with Fc-GITR pretreated SPMC from the model group was significantly decreased than those co-cultured with no Fc-GITR pretreated SPMC from the model group.Conclusion:GITR-GITRL Signal intervention significantly reduced HSC activation in ConA-induced autoimmune liver injury and in vitro, which indicated that GITR signaling may be involved in initiation of HSC activation in ConA-induced hepatitis.