Therapeutic Effect And Mechanism Of Etoposide On Diffuse Alveolar Hemorrhage In Mice With Lupus

BackgroundDiffuse alveolar hemorrhage(DAH)is a clinical syndrome characterized by hemoptysis,iron deficiency anemia,and diffuse alveolar infiltration.A variety of autoimmune diseases can cause DAH and systemic lupus erythematosus(SLE)causes the majority.Although SLE complicated by DAH is rare,the fatality rate is high,ranging from 50%to 70%.DAH is commonly used in clinical remission with corticosteroids,combined with plasma exchange immunoadsorption therapy,about 50%of patients relapse,need to use corticosteroids combined with immunosuppressive agents to maintain,but usually the treatment effect is not good.Therefore,it is very necessary to find a safe and effective treatment for DAH.Etoposide(ET)is an anti-tumor drug that acts on DNA topoisomerase II.It is mostly used clinically for the treatment of lymphoma and haemophilic syndrome.The drug has low toxicity and high safety.In addition,etoposide can be used to treat macrophage activation diseases,such as macrophage activation syndrome.Etoposide can reduce cell damage by reducing TNF-α in the lungs and eliminating activated macrophages and NKT cells.And significantly improve the survival rate of related models.According to existing studies,macrophages play an important role in the pathogenesis of DAH.However,few studies have focused on the therapeutic effect of ET in DAH.In this study,Pristine was used to induce a lupus DAH model in C57BL/6 mice.In this model,the pulmonary capillary inflammation is morphologically similar to the DAH associated with human SLE.In this study,animal experiments and molecular experiments were conducted to explore the possible mechanism of DAH and the anti-apoptotic effect of etoposide on DAH mice.ObjectiveBy constructing a DAH mouse model(single intraperitoneal injection of Pristine),explore the therapeutic effect of ET in DAH mouse model and its possible mechanism,and explore the best treatment plan for DAH mouse model.Methods1.Intervention effect of ET on DAH mice.(1)Study on the improvement of pulmonary hemorrhage in DAH mice by ET intervention in the whole course.Twenty-four female C57BL/6 mice were randomly divided into control group,DAH model group and ET treatment group with 8 mice each.On the day of the experiment,the mice in the DAH model group and the ET treatment group were injected intraperitoneally with 0.5 ml Pristane,the control group was injected with the same amount of PBS,and the mice in the ET treatment group were injected intraperitoneally with ET(10 mg/kg/d)for 14 consecutive days.The mice were dissected on the 15th day,the lung tissue hemorrhage was observed visually,and the pathological changes of the lung tissue were analyzed pathologically by HE staining.(2)The effect of different concentrations of ET treatment on the incidence of DAH in mice.Forty female C57BL/6 mice were randomly divided into control group,3.3 mg/kg group,2.5 mg/kg group,1.7 mg/kg group and 1.0 mg/kg group,each with 8 mice.On the day of the experiment,except for the control group The other 4 groups of mice received a single intraperitoneal injection of 0.5 ml Pristane.The 3.3 mg/kg group was intraperitoneally injected with ET 3.3 mg/kg,once a day,the 2.5 mg/kg group was intraperitoneally injected with ET 2.5 mg/kg,once a day;the 1.7 mg/kg group was intraperitoneally injected with ET 1.7 mg/kg,once a day The 1.0 mg/kg group was intraperitoneally injected with ET 1.0 mg/kg,once a day,and the control group was intraperitoneally injected with an equal volume of PBS every day.The mice were dissected on the 14th day,the lung tissue hemorrhage was observed visually,and the pathological changes of the lung tissue were analyzed pathologically by HE staining.(3)The effect of different treatments on the incidence of DAH in mice.Forty female C57BL/6 mice were randomly divided into 5 groups:DAH model group,ET treatment group,EPO treatment group,combined administration group,and control group.In addition to the control group,the mice in the other four groups were injected intraperitoneally with 0.5ml pristane,the ET treatment group continued intraperitoneal injection of ET(1.7mg/kg)from day 1 to day 14;the EPO treatment group continued intraperitoneal injection from day 1 to day 14 EPO(1000U/kg)was injected;the combined administration group was intraperitoneally injected with ET(1.7mg/kg)and.EPO(1000U/kg)every day for 14 days.The control group was intraperitoneally injected with 0.5 ml of normal saline,and an equal volume of PBS was injected every day.After 14 days,observe the treatment effect of different administration groups on DAH and conduct pathological analysis.(4)The effect of the whole course of ET treatment on the mortality of DAH mice.Fourty female C57BL/6 mice were randomly divided into ET treatment group,DAH model group.ET treatment group and dexamethasone group were given a one-time intraperitoneal injection of 0.5 ml Pristane to construct DAH on the day of the experiment.Model,the ET treatment group was intraperitoneally injected with ET(10mg/kg)from day 1 to day 14 for 14 consecutive days;For 14 consecutive days;the DAH model group was intraperitoneally injected with an equal volume of PBS every day.Observed for 30 days,the death of the two groups of mice was counted and the survival rate was analyzed.2.(1)The effect of ET on inflammatory cells and inflammatory factors in SLE-DAH mice.The experimental method is the same as 1(1).On the 14th day,the mice were dissected,the lung tissue hemorrhage was observed visually,the pathological changes of the lung tissue were analyzed by HE staining,and the IL-6,IL-10,TNF-α,and IL-6 in the lung tissue were detected by the QPCR method.The expression level of IFN-y,the expression levels of IL-6,IL-10,TNF-α and IFN-y in BALF were detected by enzyme-linked immunosorbent assay,and the macrophages in lung tissue were detected by immunofluorescence staining method(F4/80 Marker)and the number of neutrophils(Ly6G marker),M1 macrophages(iNOS marker)and M2 macrophages(CD 163 marker).(2)The effect of ET on the apoptotic protein in SLE-DAH mice,the experimental method is the same as 1(1),the mice were dissected on the 14th day,the lung tissue hemorrhage was observed visually,the pathological changes of the lung tissue were analyzed by HE staining pathology,TUNEL The number of apoptotic cells was detected by staining,the expression levels of Bcl-2,Bax and Caspase-3 in lung tissue were detected by QPCR,and the protein expression levels of Bcl-2,Bax and Caspase-3 were detected by Western blot.Results1.(1)The lung tissue of the ET treatment group and the control group was normal pink with no bleeding.Histopathology showed that the lung tissue was normal and there was no inflammatory cell infiltration.The lung tissue of the DAH model group was dark red with diffuse hemorrhage.Pathology showed that the alveolar cavity was filled with a large number of red blood cells and was accompanied by inflammatory cell infiltration.(2)The therapeutic effect of ET is related to the dose.Among the set concentration gradients,the 3.3 mg/kg group has the best therapeutic effect.(3)ET-centered combined treatment plan ET combined with EPO group has the best treatment effect.(4)Compared with the DAH group,the survival rate of DAH mice was significantly improved in ET treatment.2.Compared with the control group,immunofluorescence staining in the DAH model group showed that macrophages(F4/80 labeled)and neutrophils(Ly6G labeled)increased significantly.Compared with the DAH model group,the ET treatment group had macrophages Cells(marked by F4/80)and neutrophils(marked by Ly6G)were significantly reduced;pro-inflammatory factors(IL-6,TNF-α,IFN-γ)and M1 macrophages(marked by iNOS)in the ET treatment group Significantly reduced,but the anti-inflammatory factor(IL-10)and M2 type macrophages(CD 163 marker)did not change significantly.3.The results of Tunel staining of lung cell apoptosis in the 3 groups showed that the apoptotic cells in the DAH model group were significantly higher than those in the control group and the ET treatment group.There were statistically significant differences in the apoptosis rate of lung cells among the three groups.Compared with the model group,the ET treatment group had significantly reduced TUNEL staining positive cells and pro-apoptotic protein Bax,while the anti-apoptotic protein Bcl-2 significantly increased and the apoptotic protein caspase-3 significantly decreased.Compared with the control group,the DAH model group has significantly increased TUNEL staining positive cells and pro-apoptotic protein Bax,while the anti-apoptotic protein Bcl-2 significantly decreased and the apoptotic protein caspase-3 significantly increased.Conclusions1.ET has a significant therapeutic effect on Pristane-induced DAH.2.ET may inhibit cell apoptosis by regulating the mitochondrial pathway and reduce the recruitment of pro-inflammatory cells to play a therapeutic role.