Protective Effect Of Emodin On Brucella Infection In Mice And Its Possible Mechanism

Objective: By constructing the mice model of Brucella infection,observing the effect of monosomic emodin on infection model treatment.Mouse macrophages were cultured in vitro,to study the regulation of emodin on macrophage function of Brucella infection,and to find new drugs for the treatment of brucellosis and provide theoretical and experimental basis.Methods:1.Experiment in vivo: VITEK 2 Compact automatic microbial analysis system and 16 SrDNA molecular biology method were used to identify the bacteria in laboratory.Mice were injected with different concentrations,prescreening concentrations of Brucella infection in mice infected with SAT and spleen,and then constructing Brucella infection mouse model.Sixty female SPF mice of BALB/c were randomly divided into 6 groups.Those mice were set up as 8mg/m L emodin group,4mg/m L emodin group,2mg/m L emodin group,doxycycline group,PBS group and blank group.After intragastric administration(except for the blank group),The effect of emodin on the mice model of Brucella infection was evaluated by the changes of the colony count,body mass index,liver and spleen index and histopathological changes.2.Experiment In vitro: Bone marrow cells were isolated from BALB/c mice.Than it was induced and differentiated by adding MG-CSF.Cells labeled with mouse F4/80 antibody and CD11 b antibody.Flow cytometry was used to detect the differentiation of bone marrow cells into macrophages(MΦ).MTT method was used to detect the effect of emodin on the survival rate of MΦ,and doxycycline was used as control to compare the IC50 of two drugs.Infection of macrophages with Brucella were 4h(The proportion of cells and bacteria was 100:1).Cell were treated with emodin for 1,6,12,24,48 h,respectively.The effect of emodin on the survival of MΦ was studied by colony counting method.Determination of TNF-α,IL-6,IFN-γ in culture supernatant of MΦ by ELISA.Detection of gene expression level of TNF-α,IL-6,IFN-γ of MΦ in the infected Brucella by q RT-PCR.Results: 1.Results of experiment in vivo: The results were consistent with the results of biochemical and 16 SrDNA identification.It was confirmed that the strain was Brucella.The initial concentration of Brucella infection in mice was 1×105CFU,and A mouse model of Brucella infection was successfully constructed.Effect of emodin on mice infected with Brucella: The log CFU of 8mg/m L and 4mg/m L emodin group were respectively(4.023±0.199)(4.771±0.498).They Significantly lower than PBS group(5.161±0.501).The body mass index of 2mg/m L,4mg/m L and 8mg/m L emodin group was significantly higher than that of PBS group,and spleen coefficient was significantly lower than PBS group.The liver coefficient of 8mg/m L emodin group was significantly lower than that of PBS group,and there was no significant difference with doxycycline group.Compared with PBS group,2mg/m L,4mg/m L,8mg/m L emodin group of mice spleen have difference degrees of improvement.The arrangement of pathological structures was normal.The white pulp and red pulp were clear.Compared with PBS group,the liver cells of mice were treated with 8 mg/m L emodin group.2.Results of experiment in vivo: The percentage of bone marrow cells induced to differentiate into macrophages was 91.28% in the eighth day.Emodin IC50(608.4μg/m L)was significantly higher than doxycycline IC50(225.5μg/m L).Results of colony count showed the lg CFU values of 6,12,24,48 h emodin group were significantly lower than those in blank group.Compared with emodin group,24 h group had the lowest lg CFU value,which was lower than that of doxycycline group.Results of ELISA showed compared with brucella group,emodin increased the content of TNF-α,IL-6,IFN-γ in a dose-dependent manner.The contents of TNF-α 、 IL-6 and IFN-γ were highest in 24 h emodin group.There was no significant difference in the content of IFN-γ in 12 h emodin group(74.233±4.416)compared with 24 h emodin group(78.328±8.932).Results of q RT-PCR showed The relative expression of TNF-α,IL-6 and IFN-γ in emodin group of24 h was higher than that in other groups(1 h,6 h,12 h,48 h).At the same time point,the expression level of TNF-α gene in emodin group was higher than that in brucella group.Except for 1 h and 12 h emodin group,the expression of IL-6 in the other groups was higher than that in the brucella group.6 h,12 h,24 h emodin group was significantly higher than the brucella group IFN-γ gene expression.12 h emodin group had the highest expression level of IFN-γ.There was no significant difference between12 h emodin group and 24 h emodin group.Conclusion: Emodin has protective effects on mice infected with Brucella and it’s in a concentration dependent manner.Its mechanism is related to the increase of TNF-α,IL-6,IFN-γ and the ability of murine macrophages to kill Brucella.