The Role And Mechanism Of Galectin-9 In Myocardial Infarction

Part Ⅰ Galectin-9’s role in ventricular remodeling after myocardial infarctionPurpose: Although Galectin-9’s role in many inflammatory diseases has been widely confirmed,it is unknown whether it plays a role in ventricular remodeling after myocardial infarction.The purpose of this part of the experiment is to use mice to establish a MI model to investigate the effects of Galectin-9 intervention on injury,inflammation,myocardial apoptosis and myocardial fibrosis after myocardial infarction.Methods: Six to eight weeks of C57 BL / 6 male mice were selected to build MI models,which were observed and counted at time points such as 1 day,3 days,7 days,and 28 days after MI according to different research purposes.In order to study the role of Galectin-9 in ventricular remodeling after myocardial infarction,C57 BL / 6 mice were randomly divided into the following three groups:(1)the sham operation group,according to the MI model construction method,only puncture without ligation.(2)the Galectin-9 treatment group,0.5ug of Galectin-9 was injected intraperitoneally for three consecutive days before MI.(3)the PBS-treated group,200 ul of PBS was injected intraperitoneally for three consecutive days before MI.In addition to the above operations,(2)(3)two groups of mice were given intraperitoneal injections of 0.5ug Galectin-9 and 200 ul PBS three times a week before the designated observation time point.From the first day after surgery,observe the survival of the mice in each group and take the following detection methods at different time points: take the mouse heart for TTC staining to calculate the size of the MI area,use HE and Masson staining to detect the degree of myocardial injury and fibrosis,Use immunohistochemistry to detect the aggregation of inflammatory cells such as MPO + neutrophils,Mac3 + macrophages and CD3 + T cells,and use terminal deoxynucleotidyl transferase-mediated d UTP Nick-End Labeling TUNEL The degree of death was measured by cardiac echocardiography in mice using left ventricular end diastolic diameter(LVEDD),left ventricular end systolic diameter(LVESD),ejection fraction(EF%),and short-axis shortening rate(FS%)to evaluate cardiac function.RT-The expression of IL-1β,IL-6,TNF-α and IL-10,TGF-beta and other inflammatory factors were detected by PCR.Results: First of all,we found that Galectin-9 can effectively reduce the degree of myocardial damage.Compared with the PBS control group,Galectin-9 can significantly reduce the area of myocardial infarction and improve cardiac function.Secondly,the intervention of Galectin-9 can reduce the aggregation of inflammatory cells such as neutrophils,macrophages and T lymphocytes in the early stage of myocardial infarction.Myocardial cell apoptosis can be inhibited in the early and late stages of MI,and myocardial fibrosis can be reduced in the late stage of MI.Finally,Galectin-9 can significantly inhibit the expression of inflammatory factors such as IL-1β,IL-6,TNF-α,and up-regulate the expression of IL-10,while the expression of TGFsignificant change.Conclusion: This study shows that Galectin-9 can reduce the inflammatory response,reduce myocardial cell apoptosis,improve myocardial fibrosis,and maintain cardiac function after myocardial infarction.Therefore,Galectin-9 plays a protective role in myocardial infarction.Part Ⅱ Galectin-9 can induce DC tolerance in vitroPurpose: The role of DC is different from that of tolerated DC.The conversion from DC to tolerated DC is one of the important mechanisms to regulate the immune response.However,it is unknown whether Galectin-9 can induce tolerance in DC.In this experiment,DCs were cultured in vitro to investigate whether Galectin-9 can induce DCs to transform into tolerant DCs.Methods: C57 BL / 6 male mice were selected for 4-6 weeks,and the bone marrow cells of the mice were taken under strict conditions of aseptic operation.IL-4 and GM-CSF were added to the culture medium to stimulate mononuclear cells derived from bone marrow to induce generation of immature DCs.The immature DCs were divided into blank group and Gal-DC group based on the addition or non-addition of Galectin-9.The two groups were stimulated with LPS for 6h or 24 h to induce maturation.The DC phenotypes(CD11c,MHC-II,CD40,CD86)of the two groups were detected by flow cytometry.The levels of cytokines IL-12、 IL-23、 IL-10 and TGF-beta were detected by ELISA in two groups of DC supernatants,and the expression of IDO was detected by RT-PCR.Results: Under the stimulation of LPS,the expression of co-stimulatory molecules such as CD40,CD86 and MHC-II in both groups of cells increased,indicating that LPS can effectively induce DC maturation.However,the expression of costimulatory molecules in Galectin-9 treatment group was much lower than that in single LPS treatment group.The ELISA test results showed that the contents of IL-12 and IL-23 in the supernatant of Galectin-9 treatment group were significantly lower than those in the blank group,while the contents of IL-10 and TGF-beta were significantly increased.The results of RT-PCR showed that the expression of IDO in Galectin-9 treatment group increased significantly.Conclusion: Galectin-9 can induce DC to transform into tolerant DCPart Ⅲ Galectin-9-induced tolerant DC improves ventricular remodeling after myocardial infarction by altering the immune balance of CD4 + T cellsPurpose: In the previous section we have demonstrated that Galectin-9 can protect against myocardial infarction by reducing inflammation and induce the production of tolerated DC.Therefore,exploring whether this tolerated DC can play a role in myocardial infarction can help elucidate the mechanism of Galectin-9’s protective role in myocardial infarction.Methods: 4-6 weeks old mice were selected,and mouse bone marrow cells were collected.GM-CSF and IL-4 were added to the medium to induce im DC production.Then it was stimulated with LPS + Gal-9 or LPS + Gal-9 + TNI to induce the unloaded antigen t DC(Un-t DC)and TNI antigen-loaded t DC(TNI-t DC),and magnetic beads were used to sort the t DC for future use.8-10 weeks old mice were selected,and the left anterior descending coronary artery was ligated to construct a MI model.All MI mice were divided into the following 3 groups:(1)PBS control group: 1 hour before MI surgery,200 ul PBS was injected into mice via tail vein.(2)Un-t DC group: 1 hour before MI operation,1′106 Un-t DC were dissolved in 200 ul PBS and injected into mice.(3)TNI-t DC group: 1 hour before MI operation,1′106 TNI-t DC were dissolved in 200 ul PBS and injected into mice.(2)(3)The two groups were given 1′106 t DC tail vein injections twice a week before the detection time point was reached.Cardiac ultrasound was used to evaluate the left ventricular end diastolic diameter(LVEDD),left ventricular end systolic diameter(LVESD),ejection fraction(EF%),and short-axis shortening rate(FS%)of mice after MI 7 and 28 days to evaluate cardiac function;Flow cytometry was used to detect the number of CD4 + T cells(Th1,Th17,and Treg)in the spleen of mice 7 days after MI;RT-PCR was used to detect the expression of inflammatory factors IFN-γ,IL-17 A,IL-12,and IL-10 in cardiac tissues 7 days after MI,and the expression of IL-1β,IL-6,TNF-αTGF-beta 3 days or 7 days after MI.The neutrophils,macrophages,and CD3 + T cell aggregates were detected by immunohistochemistry after 3 days of MI.Masson staining was used to detect the degree of myocardial fibrosis after 28 days of MI.Results: We found that both Un-t DC and TNI-tDC can improve ventricular remodeling after myocardial infarction and TNI-t DC has a better effect.The specific performance is: compared with the PBS control group,the two groups of t DC-treated mice have better preservation of cardiac function;the number of Th1 and Th17 cells in the spleen is significantly reduced;The expressions of pro-inflammatory factors IFN-γ,IL-17 A,IL-12,IL-1β,IL-6,and TNF-α were significantly reduced,while the expression of IL-10 increased.All of the above changes in the TNI-t DC treatment group were more significant than those in the Un-t DC group,and the number of Treg cells in the TNI-t DC treatment group increased significantly,while Un-t DC failed to increase the number of Treg cells.Conclusion: The adoptive transfer of tolerant DC induced by Galectin-9 into MI mice can improve the immune balance of mouse CD4 + T cells,thereby reducing cardiac inflammation and improving ventricular remodeling after myocardial infarction.