The Preliminary Study Of The Therapeutic Effect Of Erythropoietin On Lupus Nephritis

Objective:Systemic lupus erythematosus(SLE)is an autoimmune disease with complicated etiologies,unpredictable disease course,various presentations.It mainly involves women of child-bearing age.Multiple target organs can be damaged by SLE,such as skin,joints,kidney,respiratory system,cardiovascular system and nervous system.Renal involvement is common in SLE,affecting about 40-70%percent of lupus patients.Nearly 30%lupus nephritis(LN)patients will develop into end-stage renal disease(ESRD)which significantly increases the mortality of lupus patients.With improvement of basic researches and clinical studies,much more therapeutic methods were discovered.However,the rate of ESRD caused by LN did not decrease in past ten years due to the different ethnic heredity,social economy and the complex pathological types of LN.The exploration of new treatments of LN is still the focus of scholars.In the process of adaptive immune,T cells can not only directly kill target cells,but also help or inbibit the antibodies production of B cells.CD4~+helper T cells(Th cells)are the vital subsets of T lymphocytes which mediate different types of hypersensitivity reactions and play a crucial role in the pathogenesis of LN.As the important subsets of Th cells,Th1、Th2、Th17 and Treg cells can regulate disease condition of LN via inducing different immune responses as well as locally infiltrating leading to direct injury.In addition,inflammatory reactions,dysfunction of pro-inflammatory and anti-inflammatory factors balance are also associated with the progress of LN.The course of LN accompanies mounts of proinflammatory cytokines production and the levels of these inflammatory factors are positively correlated with the development and prognosis of LN.Therefore,the key points of LN treatment include the regulation of Th cells subsets and inflammation control.Well-known for the regulation of erythrocytes production in bone marrow,erythropoietin(EPO)is widely used to treat anaemia in clinic.Nowadays,more and more studies found that EPO had multiple non-haematopoietic activities.The attentions of EPO were paid to the functions of organs protection,immunoregulation,anti-apoptosis and inflammatory inhibition.And studies have proved that EPO improved many diseases condition including ischemia reperfusion injury of brain,acute kidney injury,diabetic nephropathy and multiple sclerosis.However,there were less focus on the effects of EPO in LN treatment.The aims of this study were to discuss wheather EPO had therapeutic effects on LN and the possible mechanisms.Moreover,we also combined EPO injection with oral glucocorticoid(GC)to identify whether EPO could synergize GC and enhance the efficacy of GC in LN treatment.Methods:PartⅠ:20 8-week-old female MRL/lpr mice were randomly divided into rhEPO(3000u/kg)intraperitoneally injected group(EPO group,n=10)and saline intraperitoneally injected group(NS group,n=10).As normal control group(NS group),10female 8-week-old C57BL/6 mice were also given saline intraperitoneal injection.All the mice were injected every three days for 10 weeks.Urine samples were collected every week.10-week later,the mice were sacrificed and we measured the size and weight of spleens.Serum was separated to detect the concentration of anti-dsDNA antibody with ELISA kit.Paraffin embedded kidney sections were used to histopathological stain(HE,PAS)to calculate glomerulonephritis activic index(AI)and chronic index(CI).Immunonohistochemical stain was used to observe the deposition of glomerular IgG,C3.PartⅡ:The experimental mice were grouped and treated as that in PartⅠ.Cell suspension of fresh spleen was prepared after grinding and filtering.FITC-CD4,PE-CD25and Alexa Fluor?647-Foxp3 antibodies were used to detect Treg cells by flow cytometry.Th1、Th2 and Th17 cells were detected by using FITC-CD4,PE-IFNγ,APC-IL-4 and PE-IL17A antibodies after 6-hour stimulation.Protein expression of T-bet,GATA3,RORc,Foxp3,STAT1,STAT3,STAT5,I-κBα,pNF-κB,NF-κB in spleen and protein expression of I-κBα,pNF-κB,NF-κB in kedney were detected by western blot.RT-PCR was used to determine the mRNA expression of IL-1β,IL-4,IL-6,IL-10,IL-17,IL-23,TNFα,IFNγ,T-bet,GATA3,RORc,Foxp3,STAT1,STAT2,STAT3,EPOR,NF-κB in spleen and mRNA expression of IL-4,IL-6,IL-17,TNFα,IFNγ,EPOR,NF-κB in kidney.Immunonohistochemical staining was used to evaluate the exprssion of NF-κB in kidney.PartⅢ:16 8-week-old female MRL/lpr mice were divided into GE group(n=8)and GC group(n=8),randomly.Mice in GE group were intragastricly administrated prednisone(5mg/kg)every day accompanying rhEPO(3000u/kg)intraperitoneal injection every three days.Mice in GC group were only given prednisone(5mg/kg)intragastric administration every day.The treatment totally lasted for 10 weeks.The basic laboratory methods were in accordance with PartⅠand PartⅡ,as detail:detecting weekly urinary protein by BCA,serum anti-dsDNA antibody concerntration by ELISA,local IgG,C3deposition within glomerulus and expreesion of NF-κB by immunonohistochemical staining,spleenic Th cells subsets by flow cytometry,protein expression of I-κBα,pNF-κB,NF-κB by western blot,mRNA expression of inflammatory factors by RT-PCR.Results:PartⅠ:1.Compared to NS group,MRL/lpr mice in EPO group showed smaller spleen hyperplasia,less urinary protein,lower concentration of serum anti-dsDNA antibody.2.For the pathological features of kidney,the main manifestations of mice in EPO group were segmental mesangial cell proliferation,occasional glomerular sclerosis and the interstitial fibrosis and infiltration were relatively mild.The main manifestations of mice in NS group were diffuse mesangial hyperplasia and basement membrane thickening,accompanying glomerular secrosis,crescent formation and had much severe inflammatory infiltration and interstitial fibrosis.3.The scores of AI,CI and the depositon of IgG,C3 in glomerulus of EPO group were all less than NS group.PartⅡ:1.Compared to NS group,the level of Th1,Th17 cells decreased while the level of Th2,Treg cells increased in spleen of EPO group.2.In EPO group,both mRNA and protein expressions of T-bet,RORc were lower,but GATA3,Foxp3 were higher than NS group.3.Comparing with NS group,the expression of STAT1,STAT3 decreased while the expression of STAT5 increased in spleen of EPO group.4.Production of pro-inflammatory factors in both spleen and kidney of EPO group,such as:IL-6,IL-17,TNFα,IFNγ.etc,were less than that of NS group.5.Expressions of NF-κB in spleen and kidney between EPO and NS group had no statistic significance,but the expression of I-κBαincreased,the expression of pNF-κB decreased after EPO treatment.PartⅢ:1.MRL/lpr mice in GE group had less urinary protein and lower concentration of anti-ds DNA antibody.2.Renal mesangial cell proliferation,mesangial and basement membrane thickening and interstitial infiltration were relatively mild and AI scores were relatively low in EPO group.3.The proportion of Th17,Th1 cells decreased while the proportion of Th2,Treg cells increased in spleen of GE group.Moreover,the expression of T-bet,RORc decreased but the expression of GATA3、Foxp3 increased in EPO group.4.Production of pro-inflammatory factors in both spleen and kidney of GE group was lower than GC group.In addition,the expression of I-κBαincreased while the expression of pNF-κB decreased in GE group comparing with GC group.Conclusions:1.EPO amilerated the LN disease manifestations of MRL/lpr mice and effectivly improved the renal pahological injury and deposition.2.EPO treatment decreased the Th1,Th17 cells but increased Th2,Treg cells via inhibiting production of STAT1,STAT3 and inducing production of STAT5,then corrected the balances of Th1/Th2 and Th17/Treg cells,thus amilerating the LN of MRL/lpr mice.3.EPO inhibited the expression of proinflammatory cytokines in spleen and kidney via inhibiting the activation of NF-κB,further alleviating lupus nephritis of MRL/lpr mice.4.Compared to glucocorticoid administration alone,EPO combined with glucocorticoid had better efficacy on lupus nephritis in MRL/LPR mice.5.EPO enhanced the therapeutic effect of glucocorticoid on lupus nephritis via synergistically regulating the Th1、Th2、Th17和Treg lymphocytes subsets and further inhibiting the activation of NF-κB.