| BackgoundLiver transplantation is the only effective treatment for end-stage liver disease.However,the lack of donor liver is still an important factor limiting the development of clinical liver transplantation.At present,with the aging of the population becoming more and more serious,the age of liver transplantation donors is increasing,and the aged donor liver has become an important source of liver transplantation.Although the application of aged donor liver has been initially recognized,it is still an urgent problem to explore the post-operative management of aged donor liver,especially the maintenance of immune tolerance.Kupffer cells are the largest macrophage population in the body and the main phagocyte in the liver.The migration from M1 type to M2 type after phagocytizing apoptotic cells is an important mechanism of inducing liver transplantation immune tolerance,which is related to the change of intracellular calcium concentration.This suggests that the change of calcium flow may be the key bridge between the efferocytosis and the shift of cell polarity.Recent studies have found that GPR37 can positively regulate the efferocytosis and affect the intracellular calcium concentration of macrophages.However,the specific mechanism needs to be further studied.Therefore,it is of great clinical significance to study the role of GPR37mediated efferocytosis in the M2 polarization of KCs,and to clarify the mechanism of the shift of the polarity of KCs to M2 type after phagocytosis of apoptotic cells.PartⅠEffect of GPR37 on the formation of immune tolerance microenvironment after liver transplantation from aged ratsObjective:To observe the effects of overexpression of GPR37 on the degree of acute rejection,the ability of apoptotic cell clearance and the level of M2 polarization of Kupffer cells in liver of aged rats after liver transplantation.Methods:（1）According to the improved"double cuff method",male Lewis rats were used as donors and male BN rats as recipients to establish the acute rejection model of rat liver transplantation,（2）According to the situation of liver donors,they were randomly divided into 4 groups（n=30/group）:control group（Ctrl group）,aged rat group（Aged group）,GPR37 empty vector group（AAV-Ctrl group）,GPR37overexpression group（AAV-OE group）,（3）The recipient rats in each group were randomly divided into three groups,10 rats in each group.10 rats were used for liver and blood sample collection,10 rats were used for tissue protein extraction,10 rats were used for liver KCs isolation and culture,（4）Western blot was used to detect the expression of GPR37 in liver tissue and KCs,（5）The expression level of GPR37 in liver tissues of each group was detected by immunohistochemistry,（6）The expression of GPR37 in KCs was detected by immunofluorescence staining,（7）He staining was used to detect the pathological changes of liver tissue in each group,and the acute rejection after liver transplantation was scored by RAI,（8）The levels of ALT and AST in serum were detected by automatic biochemical analyzer to evaluate the changes of liver function,（9）TUNEL staining was used to detect the number of apoptotic cells in liver tissue of rats in each group to evaluate the ability of removing apoptotic cells in liver tissue,（10）Immunohistochemistry was used to detect the number of CD163 and i NOS positive KCs in liver tissue of rats in each group to evaluate the polarization state of KCs in liver tissue of rats in each group,（11）ELISA and RT-PCR were used to detect the expression levels of IL-6,TNF-αand IL-10 in serum of rats in each group,（12）After treatment with cytochalasin D,TUNEL staining and immunohistochemistry were used to detect the number of apoptotic cells,CD163 and i NOS positive KCs in liver tissue of each group,so as to evaluate the effect of GPR37 mediated apoptotic clearance on M2 polarization of KCs.Results:（1）Western blot results showed that compared with the ctrl group,the expression of GPR37 in aged group was significantly decreased,and the expression of GPR37 in KCs of the aged group was significantly decreased,（2）The results of immunohistochemistry showed that the expression of GPR37 in liver tissue of the aged group was significantly lower than that of the ctrl group,（3）Immunofluorescence results showed that compared with the ctrl group,the expression of GPR37 in KCs was significantly decreased in the aged group,（4）The results of HE staining showed that the acute rejection like changes of donor liver in AAV-OE group were significantly improved,and the RAI score was significantly decreased than those in the aged group,（5）The results of automatic biochemical analyzer showed that the levels of ALT and AST in the AAV-OE group were significantly lower than those in the aged group,（6）TUNEL staining showed that the number of TUNEL positive cells in the liver of AAV-OE group was significantly lower than that of aged group,suggesting that the number of apoptotic cells was decreased,（7）The results of immunohistochemistry showed that the number of i NOS positive KCs decreased significantly and the number of CD163 positive KCs increased significantly in the AAV-OE group compared with the aged group,suggesting that the degree of M2 polarization of KCs increased,（8）The results of ELISA and RT-PCR showed that the expression levels of IL-6 and TNF-αin serum of AAV-OE group were significantly lower than those of aged liver group,and the expression levels of IL-10 were significantly higher than those of aged liver group,（9）After treatment with cytochalasin D,the number of TUNEL positive cells in AAV-OE group was significantly increased,（10）After treatment with cytochalasin D,the number of i NOS positive KCs increased significantly in AAV-OE group,while the number of CD163 positive KCs decreased significantly in AAV-OE group.Conclusion:（1）The results showed that the expression of GPR37 in KCs from aged rats decreased significantly,（2）overexpression of GPR37 in KCs from aged rats improved the degree of acute rejection after liver transplantation,reduced the level of apoptosis after liver transplantation,promoted KCs M2 polarization in liver tissue,（3）the decrease of apoptotic cells and the increase of KCs M2 polarization induced by overexpression of GPR37 were related to efferocytosis.PartⅡMechanism of GPR37 on M2 polarization of Kupffer cells from aged rat liver donorsObjective:To establish the co-culture system of KCs and apoptotic cells,and further explore the specific mechanism of GPR37 promoting M2 polarization of KCs through the pathway of efferocytosis in vitro.Methods:（1）KCs were isolated and cultured according to the improved"three-step"method,namely,type IV collagenase digestion,gradient centrifugation and selective adherence,（2）According to the source of KCs,they were divided into four groups（n=30/group）:control group（Ctrl group）,aged liver group（Aged group）,GPR37 empty vector group（AAV-Ctrl group）,GPR37 overexpression group（AAV-OE group）,（3）Spleen T lymphocytes were isolated and treated with H2O2 to induce T cell apoptosis.Co-culture system of KCs-apoptotic cell was established according to the ratio of 1:10,（4）Western blot was used to detect the expression levels of caspase3,c-casepase3,Bax,Bid,CD163,CD206,i NOS and CD86 in KCs,（5）The co-localization of PKH26 and F4/80,CD163 and F4/80 in KCs were detected by immunofluorescence staining,（6）Fluo-4AM labeled calcium probe was used to detect the concentration of calcium in KCs,（7）Western blot was used to detect the expression levels of PI3K,AKT,p-AKT,STAT3 and p-STAT3 in KCs,（8）After IPI-549 or DMSO treatment,the expression levels of PI3K,AKT,p-AKT,STAT3 and p-STAT3 in KCs were detected by Western blot,the degree of STAT3 incorporation,co-localization of CD163 and F4/80in KCs were detected by immunofluorescence staining,（9）After GSK2141795 or DMSO treatment,the expression levels of PI3K,AKT,p-AKT,STAT3 and p-STAT3 in KCs were detected by Western blot;the nuclear penetration of STAT3,the co-localization of CD163 and F4/80 in KCs were detected by immunofluorescence staining,（10）After Stattic or DMSO treatment,Western blot was used to detect the expression levels of PI3K,AKT,p-AKT,STAT3 and p-STAT3 in KCs and immunofluorescence staining was used to detect the degree of STAT3 incorporation,CD163 and F4/80 co-localization in KCsResults:（1）Western blot results showed that the expressions of c-casepase3,Bax and Bid in AAV-OE group were significantly lower than those in aged group,and the expressions of c-casepase3,Bax and Bid in AAV-OE group were significantly higher after blocking the efferocytosis,（2）The results of immunofluorescence showed that the co-localization expression of PKH26 and F4/80 in KCs in AAV-OE group was significantly higher than that in the aged group,and the co-localization expression of PKH26 and F4/80 in KCs in AAV-OE group was significantly decreased after blocking the efferocytosis,（3）Western blot results showed that compared with the aged group,the expression of i NOS and CD86 in KCs in AAV-OE group was significantly decreased,and the expression of CD206 and CD163 was significantly increased,the expression of i NOS and CD86 in KCs in AAV-OE group was significantly increased,and the expression of CD206 and CD163 was significantly decreased after blocking the efferocytosis,（4）The results of immunofluorescence showed that the co-localization expression of CD163 and F4/80 in KCs in AAV-OE was significantly higher than that in aged group,and the co-localization expression of CD163 and F4/80 in KCs in AAV-OE group was significantly decreased after blocking the efferocytosis,（5）The results of Fluo-4AM staining showed that the intracellular calcium concentration of KCs in AAV-OE group was significantly higher than that in aged liver group.After blocking the efferocytosis,the intracellular calcium concentration of KCs in AAV-OE group was significantly decreased,（6）Western blot analysis showed that the expression of PI3K,p-AKT and p-STAT3 in KCs in AAV-OE group was significantly higher than that in the aged group.After BAPTA-AM blocked calcium flow,the expression of PI3K,p-AKT and p-STAT3 in KCs in GPR37 overexpression group was significantly decreased,（7）Western blot analysis showed that the expression of p-AKT and p-STAT3 in KCs in AAV-OE group decreased significantly after inhibition of IPI-549,immunofluorescence analysis showed that after inhibiting AKT phosphorylation,the nuclear entry of STAT3and the co-localization of CD163 and F4/80 in KCs in AAV-OE group decreased,（8）Western blot analysis showed that the expression of p-AKT and p-STAT3 in KCs in AAV-OE group decreased significantly after inhibiting AKT phosphorylation;immunofluorescence analysis showed that after inhibiting AKT phosphorylation,the nuclear entry of STAT3 and the co-localization of CD163 and F4/80 in KCs in AAV-OE group decreased,（9）Western blot analysis showed that the expression of p-STAT3 in KCs in AAV-OE group was significantly decreased after p-STAT3 phosphorylation was inhibited,immunofluorescence analysis showed that the degree of STAT3 entry and co localization of CD163 and F4/80 in KCs in AAV-OE group were decreased after p-STAT3 phosphorylation was inhibited.Conclusion:（1）GPR37 mediated enhancement of KCs efferocytosis function is necessary for them to play the role of apoptosis clearance,and is conducive to the increase of its M2 polarization,（2）Calcium-dependent PI3K-AKT-STAT3 signaling pathway shows enhanced activity during phagocytosis,which promotes M2 polarization of KCs. |
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