| Objective:To explore the role of MT2 in regulation of macrophage polarization and possible mechanism of MT2 in regulating the inflammatory reaction,and provide a theoretical basis for the mechanism of protective effect of MT2 on sepsis of organ injury.Method:1.The sepsis animal model preparationA random number table was used to divide 24 healthy SPF 6-8 weeks old female C57 BL wild type mice(WT)and 24 corresponding to MT2 knockout mice(KO)into four groups: the control group,the LPS6 hour group(LPS6h),the LPS12 hour group(LPS12h),and the LPS24 hour group(LPS24h).Each group contained 6 wild type mice and 6 knockout mice.The sepsis mice model was prepared by intraperitoneal injection of lipopolysaccharide(10mg / Kg).The control group was injected with an equal amount of saline.2.Effects of MT2 on inflammatory factors and redox levels in mice with sepsisAfter receiving LPS 6h,12 h and 24 h,4% chloral hydrate(0.15 mL / 20g)were injected into the abdominal cavity for blood serum preparation from the heart.The serum levels of MT2,MDA,SOD,pro-inflammatory cytokine TNF-α 、 IL-6 and anti-inflammatory cytokines IL-10、TGF-β were detected by ELISA.3.Effect of MT2 on organ damage and macrophage polarization in mice with sepsisEach group of mice was killed by cervical dislocation,and the liver,lungs and small intestine tissues were extracted.4% paraformaldehyde was fixed,dehydrated,dewaxing and embedding sectioning.HE staining observed the pathological changes of viscera organs.The F4 / 80+ macrophage distribution in various organs were detected by immunohistochemistry.The total RNA of liver was extracted,and the expression level of MT2,M1 macrophage markers(iNOS,IL-6,TNF-α,IL-1β)and M2 macrophage markers(Arg1 、IL-10 、TGF-β 、chi3l3)were detected by qPCR.The peritoneal macrophages in the control group and LPS 24 h group were extracted,and thepercentages of F4/80 + CD11 c + cells(M1 macrophages)and F4/80 + CD11c-CD206+ cells(M2 macrophages)were analyzed by flow cytometry.4.Effect of MT2 on the polarization of mouse bone marrow derived macrophagesMice bone marrow macrophages(from knockout mice and wild mice)were treated with 5μg / L LPS for 24 h,and the culture medium was collected.ELISA was used to detect pro-inflammatory cytokines TNF-α,IL-6 and anti-inflammatory cytokines IL-10 and TGF-β in the culture medium.Cells were collected: total RNA was extracted,the expression of M1 type macrophage markers(iNOS、IL-6、TNF-α、IL-1β)and M2 type macrophage markers(Arg1、IL-10、TGF-β、chi3l3)were detected by qPCR;the percentage of F4/80+ CD11c+ cells(M1 type macrophages)and F4/80 +CD11c-CD206 +cells(M2 type macrophages)were analyzed by using flow cytometry.Result:1.Protective effect of MT2 on LPS induced sepsis viscera injury(1)In control group,the content of MT2 in the serum and the expression of MT2 mRNA in the liver of the wild type were significantly higher than those in the knockout mice,the difference had statistical significance(P < 0.01).After injection of LPS,the contents of MT2 in wild type mice increased significantly,and showed obvious time dependence,there was no obvious effect to knockout mice.(2)Compared with the control group,after injection of LPS,two genotypes of mice showed burnout,decreased appetite,and decreased ability to operate,and showed an aggravating trend with the length of administration was prolonged.There was no difference between the two genotypes of mice.(3)There were no significant differences in the levels of pro-inflammatory cytokines TNF-α 、 IL-6 and anti-inflammatory cytokines IL-10 、TGF-β in the serum of the control group.After LPS exposure,pro-inflammatory cytokines and anti-inflammatory cytokines increased significantly in the two genotypes of mice.The pro-inflammatory cytokines reached a peak at 6h,and decreased with the prolongation of the time of 6h,while the anti-inflammatory cytokines increased with the prolongation of the time.At the same time point,the levels of pro-inflammatory cytokines TNF-α、IL-6 in the knockout mice were significantly higher than those in the wild type mice(P < 0.05),and the contents of anti-inflammatory cytokines IL-10、TGF-β were significantly lower than those in the wild type mice(P < 0.05).(4)In the control group,the contents of MDA and SOD in the serum of both genotypes mice were similar.After the LPS exposure,the MDA content in the both genotypes mice increased with the prolongation of the time,and the SOD activity decreased with the prolongation of the time.At the same time,knockout mice compared with wild-type mice,the MDA content in serum of knockout mice increased significantly,statistically significant differences in 12 h and 24h(P < 0.01 or P < 0.001),while SOD activity decreased significantly,with statistically significant differences in 24h(P < 0.05).(5)HE staining showed that after treatment with LPS,liver,lung,and small intestines from mice of both genotypes showed inflammatory cell infiltration,cell degeneration and necrosis,tissue hyperemia water and other pathological changes,and with an increasing trend as the time prolonged.In comparison with the wild type mice,the pathological changes in the knockout type mice were more obvious than those in the wild type mice.2.The effect of MT2 on the distribution of macrophages in every organ of sepsis mice.The immunohistochemical results showed that the distribution of F4/80+macrophages in various organs of two genotypes was significantly dependent on LPS.At the same time point,F4/80+ macrophages in knockout mice were significantly more invasive than those in wild type mice.3.Effect of MT2 on the polarization of macrophage in mice with sepsis。(1)QPCR results showed that the expression level of M1 and M2 type macrophage markers in mice of both genotypes in the control group was similar,and there was no significant difference.After LPS exposure 6h,the expression of M1 macrophage markers reached the peak,and then decreased gradually with time.The expression of M2 macrophage markers increased gradually with time.At the same time point,the expression level of M1 macrophage markers iNOS,IL-6,TNF-α,IL-1β in the knockout mice was significantly higher than that in wild type mice,and the expression level of M2 type macrophage markers Arg1、IL-10、TGF-β、chi3l3 in wild type mice was significantly higher than that in knockout mice.(2)The results of flow cytometer showed that there was no difference in the content of M1 and M2 macrophages in the mice in the control group.24 hours after LPS exposure,both genotypes of mice M1 andM2 macrophages increased significantly.knockout mice compared with wild-type mice,the increase of M1 macrophages in the knockout mice was significantly higher than that in the wild-type mice,the increase of M2 macrophages was not as obvious as that of the wild-type mice,the difference had statistical significance(P < 0.01).4.The effect of MT2 on the polarization of mouse bone marrow derived macrophages.(1)In the control group,there was no difference in the morphology of the two genotypes of macrophages,most of them were round,with only a few that were spindle and irregular shaped,and the cells had few pseudo foot.After treatment with LPS,both kinds of genotype cells showed a large number of pseudo feet,the cell volume increased,and the number of fusiform and irregular cells increased,but there was no significant difference in morphology between the two genotypes.(2)The results of ELISA detection showed that the contents of anti-inflammatory and pro-inflammatory cytokines in the two types of macrophage culture medium in the control group were similar.After LPS treatment,anti-inflammatory and pro-inflammatory cytokines were increased,the pro-inflammatory cytokines TNF-α 、 IL-6 in the macrophage culture medium of the bone marrow source of the knockout mice were noticeably more than those of bone marrow derived macrophages from the wild type mice,the difference had statistical significance(P < 0.05 or P < 0.01),Anti-inflammatory cytokines IL-10 、TGF-βwere significantly lower than those of marrow-derived macrophages from the bones of the wild-type mice,and the difference was statistically significant(P < 0.05 or P < 0.01).(3)QPCR results showed that after treatment of LPS,M1 type macrophage markers was higher in bone marrow-derived macrophages from the knockout mice than those from the wild type mice(P < 0.05 or P < 0.01),M2 type macrophage markers was significantly less than those in bone marrow-derived macrophages from the wild type mice,the difference was statistically significant(P < 0.01 or P < 0.05).(4)Flow cytometer analysis showed that after LPS treatment,the ratio of M1 macrophages from bone marrow macrophages of the wild type mice was significantly lower than those of the knockout mice(P < 0.05),and the ratio of M2 macrophages was significantly higher than those of the knockout mice bone marrow macrophages(P < 0.05).Conclusion:MT2 has a significant protective effect on septic organ damage caused by LPS,and its mechanism may be related to the regulation of mononuclear macrophage polarization,which in turn influences the balance of inflammation. |
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