| Objective:To study the regulation mechanism of CD11 b on polarization of M1 macrophage and the recruitment of myeloid-derived suppressor cells(MDSCs)and its influence on the pathogenesis of endotoxic shock,which provide a theoretical basis for the intervention and treatment of endotoxin shock.Method:1.C57BL/6 mice were pretreated with the same concentration of CD11 b agonist Leukadherin-1(40 ?g/g,LA1)for 2 hours,and the lethal doses of LPS(25,37.5 and 50 ?g/g)were utilized to observed mortality in mice;mice were pretreated with different concentrations of LA1(10,20 and 40 ?g/g)for 2 hours,and the same concentration of lethal dose LPS(37.5 ?g/g)was used to to observe mortality in mice.2.Mice were pretreated with different concentrations of LA1,and an endotoxic shock model was established with LPS after 2 h.(1)H&E staining was used to observe the injury of liver and lung in mice(2)TUNEL method for detecting apoptosis of liver and lung tissues.(3)Flow cytometry was used to detect the expression of CD86 and CD40 for active markers of M1 macrophages.(4)ELISA to detect the level of IL-6,TNF-?,IL-12 and IL-1? in serum.(5)The mRNA expression of IL-6,TNF-?,IL-12 and IL-1? in mouse peritoneal macrophages wsa detected by qRT-PCR.3.Mouse bone marrow-derived macrophages(BMDMs)were induced with GM-CSF,and BMDMs were treated with different concentrations of LA1.The morphology of the cells was observed by a microscope and the cell viability was measured by CCK8,which determined the appropriate concentration.4.BMDMs were pretreated with different concentrations of LA1,followed by stimulation with LPS/IFN-?.(1)Flow cytometry was used to detect the expression of CD86 and CD40 in BMDMs.(2)ELISA detected the levels of IL-6,TNF-?,IL-12 and IL-1? in culture supernatant of BMDMs.(3)qRT-PCR was used to detect the expression of IL-6,TNF-?,IL-12 and IL-1? mRNA levels in BMDMs.(4)The phosphorylated levels of p38,ERK1/2,JNK and p65 in BMDMs was detected by Western blot.5.Untreated or LA1 treated BMDMs were transferred to the mice respectively,and then a model of endotoxic shock was established by using LPS.(1)H&E staining was used to observe the injury of liver and lung in mice(2)TUNEL method for detecting apoptosis of liver and lung tissues.(3)ELISA to detect the level of IL-6,TNF-?,and IL-12 in serum.6.(1)BMDMs were treated with LPS,and the expression of TLR4 and CD11 b on the cell surface was detected by flow cytometry.(2)Mice were administration of LPS,flow cytometry to detect the expression of TLR4 and CD11 b on the surface of macrophages in peritoneal and peripheral blood.(3)BMDMs were treated with LPS,the internalization of TLR4 and CD11 b was observed by immunofluorescence confocal.7.(1)BMDMs were treated with LA1,and the expression of TLR4 and CD11 b on the cell surface was detected by flow cytometry.(2)Mice were administration of LA1,flow cytometry to detect the expression of TLR4 and CD11 b on the surface of macrophages in peripheral blood.(3)BMDMs were treated with LA1,the internalization of TLR4 and CD11 b was observed by immunofluorescence confocal.(4)CO-IP verifies the interaction between TLR4 and CD11 b.8.Mice were injected LA1 at different time points,and the proportion of MDSCs and their sub-type cells in spleen 9.was detected by flow cytometry.9.MDSCs were induced with GM-CSF and IL-6 in vitro,followed treatment with LA1.qRT-PCR was used to detect the expression of IL-10,NOX-1,iNOS and Arg-1 in mRNA levels.10.After LA1 pretreatment,flow cytometry was used to detect the proportion of MDSCs and their sub-type cells in the spleen of endotoxic shock mice.Result:1.LA1 reduces LPS-mediated mortality in endotoxic shock mice 2.(1)H&E showed that LA1 alleviated the damage of liver and lung tissue in endotoxic shock mice.(2)LA1 attenuated the apoptosis of liver and lung tissue in endotoxic shock.(3)LA1 reduced the expression of CD86 and CD40 in macrophages.(4)LA1 reduced the expression of IL-6,TNF-?,IL-12 and IL-1?in serum.(5)LA1 reduced the mRNA levels of IL-6,TNF-?,IL-12 and IL-1? in macrophages of peritoneal.3.High concentrations of LA1(?40 ?M)attenuate the cell viability of BMDMs,which suitable concentration wsa 20 ?M or less.4.(1)LA1 reduced the expression of CD86 and CD40 in BMDMs.(2)LA1 decreased the expression of IL-6,TNF-?,IL-12 and IL-1? in culture supernatant of BMDMs.(3)LA1 reduced the expression of TNF-? and IL-12 in mRNA levels.(4)LA1 reduced phosphorylated levels of p38,ERK1/2,JNK and p65.5.(1)Transfered LA1-treated BMDMs attenuated the injury of liver and lung in endotoxic shock.(2)Transfered LA1-treated BMDMs attenuated the apoptosis of liver and lung tissue in endotoxic shock.(3)Transfered LA1-treated BMDMs decreased the expression of IL-6,TNF-?,IL-12.6.(1)LPS/IFN-? reduced the expression of TLR4 and CD11 b in BMDMs.(2)LPS decreased the expression of TLR4 and CD11 b in macrophages of peritoneal and peripheral blood.(3)LPS/IFN-? promote internalization of TLR4 and CD11 b,which observed by immunofluorescence microscopy.7.LA1 reduced the expression of TLR4 and CD11 b in BMDMs.(2)LA1 decreased the expression of TLR4 and CD11 b in macrophages of peripheral blood.(3)LA1 promote internalization of TLR4 and CD11 b,which observed by immunofluorescence microscopy.(4)CO-IP confirms the interaction between CD11 b and TLR4.8.LA1 increased the proportion of MDSCs and G-MDSCs in spleen.9.LA1 increased the proportion of MDSCs and G-MDSCs in spleen of endotoxic shock.10.LA1 increased the mRNA levels of IL-10,NOX1 and iNOS in MDSCs.Conclusion: 1.LA1 agonism of CD11 b could inhibit phosphorylation of the signaling pathway proteins p38,JNK,ERK1/2,and p65,thereby inhibiting the activation of macrophage and attenuating LPS-mediated endotoxic shock.2.LA1 agonism of CD11 b could promote the aggregation of MDSCs and G-MDSCs,and attenuate LPS-mediated endotoxin shock. |
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