| BackgroundDiffuse alveolar hemorrhage(DAH)is a rare and fatal complication of systemic lupus erythematosus(SLE).Due to the complexity of the pathogenesis of DAH,At present,the clinical treatment effect of DAH is relatively poor,so that the mortality rate of DAH is still as high as 90%.Therefore,Exploring safe and effective DAH treatment methods is a problem that needs to be solved urgently.Erythropoietin(EPO)is a glycoprotein hormone that is clinically used for the treatment of anemia.With the continuous in-depth understanding of the function of EPO,more and more studies have found that it has multiple extra-hematopoietic biological functions.In the model of cerebral ischemia-reperfusion injury,acute kidney injury and postoperative cognitive dysfunction,EPO plays the role of immunomodulation,apoptosis inhibition and organ protection.Studies have shown that EPO regulates its biological activity by binding to EPOR on macrophages,and macrophages play an important role in the pathogenesis of DAH.However,few studies have focused on the therapeutic role of EPO in DAH.This study evaluated the therapeutic effect of EPO on DAH by using Pristane-induced DAH mouse model,and explored the mechanism of EPO treatment of DAH through animal models and in vitro experiments.ObjectiveIntraperitoneal injection of Pristane induced DAH mouse model to explore the therapeutic effect and mechanism of EPO in DAH mouse model.MethodsEffect of EPO on the treatment of DAH mice.(1)Study on the therapeutic effect of EPO full treatment on DAH mice.30 female C57BL/6 mice were randomly divided into a control group,a DAH model group and an EPO treatment group of 10 each.On the day of the start of the experiment,mice in the DAH model group and the EPO treatment group were intraperitoneally injected with Pristane(0.5 mL),and the EPO treatment group was intraperitoneally injected with EPO(5 000 U/kg)once a day.On 14th 14,the mice were dissected,Visually observe the bleeding of lung tissue,histopathological analysis of lung tissue pathological changes.(2)Effects of different EPO treatment regimens on the incidence of DAH in mice40 female C57BL/6 mice were randomly divided into 10 groups each of DAH model group,D1-3 group,D7-14 group and D1-14 group.On the day of the experiment,4 groups of mice were intraperitoneally injected with Pristane(0.5 mL).Groups D1-3 received intraperitoneal injection of EPO(5000U/kg)from day 1 to day 3,and groups D7-14 received intraperitoneal injection of EPO(5 000U/kg)from day 7 to day 14.Times;the D1-14 group was intraperitoneally injected with EPO(5 000 U/kg)from day 1 to day 14;the model group was intraperitoneally injected with equal volume of PBS daily.On the 14th day,the mice were dissected,the bleeding of the lung tissue was observed with the naked eye,and the pathological changes of the lung tissue were analyzed by histopathology.(3)Effect of EPO full treatment on the mortality of DAH mice.Randomly divide 40 female C57BL/6 mice into EPO treatment group and 20 DAH model groups each:on the day of the experiment,one-time intraperitoneal injection of Pristane 0.5 mL was used to prepare the DAH model.EPO(5 000 U/kg)was intraperitoneally injected once a day for 14 days;the DAH model group was intraperitoneally injected with an equal volume of PBS for 14 days.After 30 days of observation,the deaths of the two groups of mice were counted and the survival rate was analyzed.Changes of macrophage polarization before and after treatment of DAH mice with EPO:The experimental method is the same as 1(1).The mice were dissected on the 14th day,the bleeding of the lung tissue was observed with the naked eye,and the pathological changes of the lung tissue were analyzed by histopathology,TUNEL staining to detect the number of apoptotic cells,QPCR method to detect miR-155-3p,miR-15-3p,miR-494-3p,IL-12,IL-6,IL-10 and TNF-? expression levels,Western blot method to detect p-JAK2,JAK2,p-STAT3,Bcl-2,Bcl-xl and Bax expression levels of lung tissue.The number of M1 type macrophages(iNOS labeled)and M2 type macrophages(CD 163 labeled)in lung tissue was detected by immunohistochemical method,and IL-12,IL-6,IL-10 and TNF-? in BALF were detected by ELISA.The expression level of M1 type macrophages(iNOS and CD86 markers)and M2 type macrophages(CD206 and Arg-1)in BALF were detected by Western blot.(2)Prepare DAH mouse model by Pritane induction,and evaluate the therapeutic effect of EPO treatment cycle on DAH mice.Visually observe the bleeding of lung tissue,histopathological analysis of pathological changes of lung tissue.(3)DAH mouse model was prepared by Pritane induction,and the effect of EPO treatment on the survival rate of DAH mice was evaluated.The mortality of thetwo groups of mice was counted and the survival rate was analyzed.EPO regulates macrophage polarization via miR494/JAK2/STAT3 axis.(1)Effect of EPO on the polarization of macrophage.LPS,IFN-? and IL-4 were added to RAW 264.7 cells for 24 h,and then stimulated with EPO,and cytokines(IL-12,IL-6,TNF-?)were detected by ELISA And IL-10)expression levels,flow cytometry was used to detect the number of M1 macrophages(iNOS and CD86 markers)and M2 macrophages(CD206 and Argl markers).(2)EPO regulates macrophage polarization through JAK2/STAT3 pathway.Using shRNA to silence JAK2,STAT3 and STAT6 in RAW 264.7 macrophages,and then adding LPS,IFN-y and IL-4 to RAW 264.7 cells for 24 h,and then further stimulated with EPO.The mRNA expression levels of JAK2,STAT3 and STAT6 were detected by QPCR method,and the number of M2 macrophages(CD206 and Argl labeled)was detected by flow cytometry.(3)MiR-494 regulates macrophage polarization through JAK2/STAT3 pathway.Add miR-494-3p inhibitor to RAW 264.7 cells,then add LPS,IFN-y and IL-4 to RAW 264.7 cells for 24 h,and then stimulate with EPO.The number of M2 macrophages(CD206 and Argl labeled)was detected by flow cytometry,the expression levels of miR-494-3p,JAK2,STAT3 and STAT6 were detected by QPCR,and JAK2,p-JAK2,STAT3,p-STAT3,STAT6 and P-STAT6 protein expression levels.Results(1)The lung tissue of the EPO treatment group and the control group was normal milky white,no bleeding,histopathology showed normal lung tissue,no inflammatory cell infiltration,DAH model group lung tissue was dark red,diffuse hemorrhage,histopathology It showed that the alveolar cavity was filled with a large number of red blood cells and accompanied by infiltration of inflammatory cells.(2)Only D1-14 group(from the first day to the 14th day of continuous intraperitoneal injection of EPO)has the best treatment effect.(3)The survival rate of DAH mice increased significantly after EPO treatment.Compared with the DAH model group,TUNEL staining positive cells and pro-apoptotic protein(Bax)in the EPO treatment group were significantly reduced,while anti-apoptotic proteins(Bcl-2 and Bcl-xl)were significantly increased;EPO treatment group was pro-inflammatory Factors(IL-6,IL-12 and TNF-?)and M1 type macrophages(iNOS and CD86 markers)were significantly reduced,while anti-inflammatory factors(IL-10)and M2 type macrophages(CD206,CD 163 and Arg-1 markers)were significantly increased;miR-494-3p,miR-155-3p,miR-15-3p were significantly reduced in the EPO treatment group,and p-JAK2 and p-STAT3 were significantly increased.Under the stimulation of LPS,IFN-y and IL-4 on macrophages,proinflammatory factors(IL-6,IL-12 and TNF-?)and M1 type macrophages were reduced after EPO treatment,p-JAK2,p-STAT3,anti-inflammatory factor(IL-10)and M2 type macrophages were all increased.The number of JAK2,STAT3 and STAT6 in gene-silencing macrophages,stimulated by LPS,IFN-y and IL-4 to macrophages,the number of M2 macrophages(CD206 and Arg-1 markers)after EPO treatment was not seen Significant increase.Under the stimulation of macrophages by LPS,IFN-y and IL-4,miR-494-3p inhibitor treatment promoted JAK2,STAT3 and STAT6 mRNA expression,co-treatment of miR-494-3p inhibitor and EPO significantly increased The number of M2 macrophages(CD206 and Arg-1 markers).Conclusions1.EPO has a significant therapeutic effect on Pristane-induced DAH.2.EPO may play a therapeutic role by regulating the miR-494/JAK2/STAT3 axis to promote the polarization of macrophages. |
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