| Objectives: 13-methylamino-18-thiomatrine(MASM)is a matrine derivative with a variety of biological activities,such as immunosuppression and anti-inflammation.The purpose of this study is to investigate the therapeutic effect of MASM on septic model mice and explore its mechanism.Methods: The experimental septic mouse model was established by cecal ligation and puncture(CLP),while the therapeutic effect of MASM on this model mice was observed.The expression levels of IFN-γ,TNF-α,MCP-1,IL-12p70,IL-1β,IL-10,IL-6 and GM-CSF in mice’s serum were detected by flow cytometry.Western blot was used to detect the expression of LC3 Ⅰ,LC3 Ⅱ and p62 protein in the liver of these mice.The pathological changes of lung,liver and kidney were observed by HE staining.The number and morphology of autophagosome and autophagy lysosome in liver tissue were observed via transmission electron microscope.The effect of MASM on autophagy was investigated by LPS-induced autophagy in macrophage.ELISA and RT-PCR were used to measure the concentration of inflammatory cytokines in supernat of the culture medium,and Western blot was used to detect the phosphorylation of PI3 K,Akt and mTOR in LPS.The effect of MASM on autophagy and its mechanism was evaluated via detecting dynamic protein change of phosphorylation of PI3 K,Akt and mTOR signal pathway treated with LPS.Result:1.The effect of MASM on the survival rate of septic mice.If the septic mice were treated with normal saline,they all died in 120 hours after the CLP of surgery,whereas when the mice were treated with MASM 1mg/kg,3mg/kg or 10mg/kg the survival rate was 8.3%,when MASM was given,the50% and 66.7%,respectively.The results showed that MASM had a therapeutic effect on experimental sepsis in mice and could significantly improve its survival rate(P < 0.01).2.The therapeutic effect of MASM on septic mice.Two hours before the surgery of CLP,6-8 weeks old C57BL/6 mice were randomly divided into CLP model group and MASM 3mg/kg treatment group,tested at 6h,24 h and96h at different time points after modeling.The results of Western blot showed that at 6h and 24 h after modeling,compared with CLP model group,the ratio of LC3 Ⅱ/LC3 Ⅰ in MASM treatment group increased significantly,while the expression level of p62 protein decreased significantly.3.The effect of MASM on the secretion of inflammatory factors in septic mice serum.Compared with Sham group,the inflammatory factors such as IFN-γ,TNF-α,MCP-1,IL-12p70,IL-1β and IL-6 in model mice were significantly increased(P<0.01).When MASM was 3mg/kg and 10mg/kg,serum inflammatory factors IFN-γ,IL-6,TNF-α and IL-1β were significantly inhibited,indicating that MASM can antagonize the release of inflammatory factors caused by sepsis.4.The effect of MASM on the expression of autophagy-related proteins in the liver of septic mice.24 h after modeling,the results showed that MASM(3mg/kg)treatment could promote the transformation of autophagy-related protein LC3 Ⅰ into LC3 Ⅱ in the liver tissue of septic mice,and further inhibit the expression of p62 protein.After modeling,the ratio of LC3Ⅱ/LC3 Ⅰ was significantly higher than that of the model group,and the expression level of p62 protein was significantly lower than that of the model group.It is confirmed that MASM can significantly promote the occurrence of autophagy in sepsis.5.The effect of MASM on histopathological impairment induced by sepsis in mice.HE staining was used to observe the pathological changes of lung,liver and kidney under light microscope.The results showed that in the model group,there were many serious impairments in those organs,thrombosis in lung and kidney,destruction of alveolar structure in lung,thickening of pulmonary septum and infiltration of inflammatory cells in lung and liver.After MASM treatment of lung,liver and kidney there were less impairment in those organs,MASM can reduce inflammatory cell infiltration,tissue hyperemia and necrosis.that indicated MASM treatment can significantly the pathological damage of various tissues caused by sepsis.6.The effect of MASM on the number of autophagosomes and autophagy lysosomes in liver tissue of septic mice.Under transmission electron microscope,the numbers of autophagosomes and autophagy lysosomes in liver tissue of mice in CLP model group were 3 and 6,while those in Sham group were 1 and 2,respectively.There was significant difference between model group and Sham group.In MASM group,the number of autophagosomes in 1mg/kg,3mg/kg and 10mg/kg was 5,7 and 13,and the number of autophagy lysosomes was 11,18 and 39,respectively.It was proved that MASM could increase the number of autophagosomes and autophagy lysosomes in liver tissue of septic mice in a dose-dependent manner.7.The effect of MASM on the proliferation of macrophages induced by LPS.The cells were divided into control group,LPS(500ng/mL)group and LPS(500ng/mL)+MASM group.LPS+MASM group was incubated with MASM 10μM for 4h.After administration at different time points,cell proliferation was measured by CCK8 method.It was found that LPS stimulation and MASM tre atment had no significant effect on macrophage proliferation.8.The effect of MASM on the expression of TNF-a,IL-1β,IL-6 and IL-10 in macrophages induced by LPS.The expression of inflammatory cytokines in macrophages was detected by ELISA.The results shown that LPS could induce the expression of pro-inflammatory cytokines TNF-a,IL-1β and IL-6,and their concentrations increased from 66±10.00 pg/ml and55±15.17pg/ml,57±7.11pg/ml to 265±32.65pg/ml and 118±12.43pg/ml,315±42.51pg/ml,respectively.LPS de creased the expression of anti-inflammatory cytokines from 49±9.27 pg/ml to 30±7.01pg/ml.After administration of MASM,the concentration of TNF-a,IL-1β and IL-6decreased to 165±21.54 pg/ml and 78±15.62pg/ml,205±22.51pg/ml,respectively.At the same time,MASM could increase the expression level of anti-inflammatory cytokine IL-10 and increase its concentration from30±7.01pg/ml to 101±8.36pg/ml.At the same time,MASM could increase the expression level of anti-inflammatory cytokine IL-10.The expression of inflammatory cytokines mRNA in each group was detected by RT-PCR,and the results were consistent with the above.9.The effect of MASM on the signal proteins associated with autophagy treated by LPS.Western blot demonstrated that LPS could increase the phosphorylation levels of PI3 K,Akt and mTOR in PI3K/Akt/mTOR signal pathway of macrophages,while MASM could antagonize the effect of LPS on the phosphorylation of PI3 K,Akt and mTOR in macrophages.It was preliminarily confirmed that MASM could promote autophagy.Conclusion: MASM can reduce the mortality of experimental septic mice,inhibit the secretion of serum inflammatory cytokines and promote the expression of autophagy-related proteins in liver tissue of model mice.These effects are related to the induction of autophagy.MASM may mediate autophagy through PI3K/Akt/mTOR pathway. |
PDF Full Text Request