Effect Of AG490 On Prolactin-mediated JAK/STAT Signal Transduction Pathway In The Systemic Lupus Erythematosus Patients

Systemic lupus erythematosus(SLE) is a common immunity disease which can be found in women.It has diversity and non-specifice clinical manifestations involved in different visceras.There are main reasons that the abnormity of immunoregration involved in many immunity cells can induce SLE.The incidence of SLE is from 0.13%o to 0.39%0.But its incidence can reach up to 3.72%0 in women 18 and older.In our country,the incidence of SLE is about 0.7%0,and it increases annually.The pathological mechanism of SLE involved in many factors such as inheritance,environment,sex hormone.But it is not clear now.Prolactin(PRL),a versatile hormone with more than 300 separate functions,is produced in the anterior pituitary gland and affects mammary growth and differentiation and its secretion is mediated via the dopaminergic pathway.As an important factor,PRL can provoke cell-mediated immune response and humoral immune response in vivo.Furthermore,PRL has widespread influences on proliferation and differentiation of a variety of cells in the immune system.Its classical function is to regulate the growth and differentiation of mammary glands, ovary and men's affiliated generative organ and increase milk secretion.PRL involved many functions such as attending T cells differentiation,activating PKC and guanyliccyclase,impromting mitogen differentiation,inducing the express of IL-12 receptor and many interrelated grouth factor genes,increasing the express of interferon-γby provoking interferon regulatory factor(IRF),provoking the production of autoantibody.Vera-Lastra's group demonstrated that more than 20-30%of SLE patients have mild and moderate hyperprolactinemia(HPRL) which associated with active lupus. HPRL can stimulate the production of many autoantibodies.Some clinical researches indicated that the increasing of serum PRL in SLE patients is positive correlation to ANA's titre and some activity indexes of clinical disease.Some sciestists demonstrated that injected-Prolactin(PRL) can induce SLE mouse dead. Furtheremore the mortality rate of SLE mouse was reduced as soon as inhibited the synthesis and secretion of PRL.These evidences show that PRL act as an important role in the pathological mechanism of SLE.From Yang's research,PRL could induce fast phosphorylation happened in multiplex TCR/CD3 complex protein and affect the antigen function latently.From our research the second signal passway about PRL in cell-activation,we found that PRL could up-regulate the express of CD154 located in the surface of PBMC.The intracellular signalling pathways of PRL becomes a hot research field in recent years.It is identify that the trimer of PRL-PRLR composite induce the JAK/STAT transmembrane signalling pathways.After being activated,the PRL-R can introduct many massages into nucleolus and induce the expression of IRF-1 and IL-6 through JAK/STAT signal passage.The message can be send into nucleolus and cause the expression of IL-6.IL-6 is a cytokine secreted from monocyte,T lymphocyte and B lymphocyte.It can provoke T and B lymphocyte differentiation and induce B cell to produce many autoantibodies.It develops its biological function through JAK/STAT signaling pathways in vivo.It is clarified that IL-6 is positive correlation with SLEDIA closely related with SLE.But it is unclear how PRL can activate the JAK/STAT5 signalling pathways in vivo and whether HPRL is related with IL-6 in SLE patients.It is also unknown whether JAK/STAT singalling pathway acts as a link between HPRL and IL-6.In this study we will do some work on the influence of serum prolactin on JAK/STAT5 signalling pathways in the peripheral blood mononuclear cells of the patients with systemic lupus erythematosus and its inhibitor,AG490,can affect this signal passway.AG490(MW:294.3) was an artificiality-synthesized compound from 2-Cyano-3-(3,4-dihydroxyphenyl)-N-(benzyl)-2-propenamide.It can compete with receptor tyrosine kinases because of its tyrosine-likely structure.So it can inhibit the signal passways of JAK/STAT5 and inhibit the production of inflammatory factors further.In this study,we try to clarify the pathogenesis machine of SLE and look forward to find a new method for SLE treating.Obeetive1.To detect the level of serum PRL in SLE patients,analysis its relationship among SLE activity,clinical manifestations and laboratory indexes.2.To research the affection of PRL and rhPRL to intracellular JAK/STAT5 signal passways in SLE patient's PBMC.Try to clarify the pathogenesis machine of SLE by detecting the expression of STAT5.3.To detect the affection of PRL and rhPRL to SLE patients' PBMC secreting IL-6. Try to clarify the relationship between PRL-mediated JAK/STAT5 signal passway.4.To detect the effect of AG490 on prolactin-mediated JAK/STAT5 signal transduction pathway in the systemic lupus erythematosus patients.Try to clarify the mechanism of PRL-mediated PBMC secreting IL-6 so that we can confirm a new method to treat SLE through biology. Method1.The content of the serus PRL in standard basal state was examined by electrochemilumescence-meter in 40 cases of SLE patients and 20 cases of healthy controls.According to a criterion supported by a PRL-kit,we define the value of serum PRL over 15.2ng/ml as HPRL(man) and over 23.3ng/ml as HPRL(woman).2.To divide samples as follows:â‘ SLE group with high PRL(N=22);â‘¡SLE group with high PRL+AG490(N=22);â‘¢SLE group with high PRL+AG490+rhPRL(N=22);â‘£Normal control groups+rhPRL(N=20);⑤SLE group with normal PRL(N=18);â‘¥SLE group with normal PRL+rhPRL(N=18);⑦SLE group with normal PRL+rhPRL+AG490(N=18).Take 6 bore planks,and to divide cells in six bores equally,make the each bore cells to attain 1×10⁶/ml.The concentration of rhPRL is 10ng/ml finally,and the concentration of AG490 is 1μmol/ml finally.3.PBMC were separated by density gradient centrifugation as follow steps:â‘ In the calibration(15ml) of sterile centrifuge tube.Each tube was joined 5ml Ficoll lymphocytes isolated liquid-take the peripheral blood 10ml heparin(including 40 SLE patients and 20 normal controls) with the same blending PBS fully diluted.With the pasteur dropper drawed with 5ml anticoagulant blood(anti-clotting diluted with PBS equivalent),along slowly superimposed on the wall of lymphocyte Liquid cell separation(by 4 per sample tube),20℃,20min 2000rpm level centrifugal;â‘¡using the suction to reache gray hair cell layer,and drawing the mononuclear cells placed in a separate centrifuge tube,adding more than five times the size of PBS,20℃,10min 1500rpm level centrifuge,washing the cells one times.â‘¢Everygroup sample was sent to FCM,after incubation in 37℃,5%CO₂ incubator within 48 hours.4.ELISATo according ELISA kit instruction to do the following experiment.First the serum from PBMC and standard sample were put into coated 96-well polystyrene plates after 90min,no wash;second,add bioti-labeled antibody into the mixture,60mins, wash 3 times.Third,add avidin-peroxidase complex,30rains,wash 5 times;last,add TMB,10-15mins and use 2NH₂SO₄ stop the reaction.The absorbance at 450nm (A450) was recorded with an ELISA reader.5.Protein isolation and Western blottingThe proteins were isolated from PBMC.The total proteins(10-50mg/lane) were electrophoresed,separated on 10%SDS-PAGE,and transferred to a polyvinylidene difluoride membrane(Millipore Corporation,Billerica,MA,USA),which was blocked in 5%non-fat dry milk in Tris-buffered saline Tween(TBST;pH 7.6).The membrane was incubated overnight with a rabbit polyclonal antibody to rat pSTAT5 or a monoclonal antibody to mouse STAT5(Calbiochem USA) at a dilution of 1:1000 on a rotating platform at 4℃.Subsequently,the membrane was rinsed in TBST(pH 7.6) for 40 min and incubated with horseradish peroxidase(HRP)-conjugated antirabbit immunoglobulin G(IgG) antibodies(Boster,Wuhan,China) diluted in TBST(1:4000) for 1h on a rotating platform at 37℃.Bands were visualized using a HRP developer,and background-subtracted signals were quantified on a laser densitometer(Bio-Rad,Hercules,California,USA).Blots were probed with a mouse anti-(?)-actin monoclonal antibody(Boster,China) to ensure equal protein loading.All protein levels were assessed by densitometry,withβ-actin used as a control.6.Statistical treatmentThe statistical treatment consisted of analyzing variance and multiple comparision of group means by using of SPSS13.0.The study makes a comparison among the groups of sample through independent sample T-test.If our dates were fit for test of homogeneity of variances,we dealed them with LSD.If our dates were not fit for it, we analyzed all datas through Welch and Dunnett's T3,crosstabs x² test.We analyse comparison of many sample rates,the relationship of bi-variance and multi-variance by using of crosstabs x² test,bivariate correlation and Curve Expert respectively.We get Standard curve and selete curve fitting equation through Curve Expert1.3.It is significance when P value less than 0.05.All datas are showed by(?)±SD.Result1.The level of serum PRL in SLE patients are significient higher than control,and it is obvious increasing in active stage.It is positive correlation to SLEDIA.(r=0.568, P=0.000)2.The incidence of central nervous system symptoms in HPRL SLE patients such as headache,falling sickness,SBP-RI,IF-anti-ds-DNA,tetter,ulcer of oral are obvious higher than those of NPRL.On the country the incidence of arthritis and ttematologic system diseases are common in NPRL SLE patients.There is no obvious discrepancy in decreasing C3 and C4,fever,oromeningitis.3.â‘ The level of IL-6,STAT-5 and p-STAT-5 in HPRL patients are higher than those of in NPRL patients and normal people respectively(P＜0.05)â‘¡The level of IL-6,STAT-5 and p-STAT-5 are reduced in HPRL-SLE patients which have been pretreated by using of AG-490(P＜0.05)â‘¢The level of IL-6,STAT-5 and p-STAT-5 are obvious reduced in HPRL-SLE patients that have been pretreated by using of rhPRL and AG-490.Compared to only use AG490,we found the expression of STAT-5 and IL-6 increased after treated by rhPRL and AG490(P=0.000).4.â‘ The level of IL-6,STAT-5 and p-STAT-5 are increased in NPRL-SLE patients which have been pretreated by using of rhPRL(P＜0.05).â‘¡Compared to negative control,the level of IL-6 and STAT5 are obvious increased in NPRL-SLE patients after treating by rhPRL(P=0.000).But the expression of p-STAT5 does not change.(P=0.135)â‘¢The level of IL-6,STAT-5 have obvious reduced in NPRL-SLE(P＜0.05) which have been pretreated by using of rhPRL and AG490.But p-STAT-5 have been no changed(P=0.067).Conclusion1.The increasing of serum PRL in SLE patients is related with its pathogenetic condition.It can be regarded as an index of SLE pathogenetic condition2.Both endogenic and exogenous PRL can increase the expression of total STAT5, p-STAT5 and IL-6 in the secretion of IL-6 in SLE patients' PBMCs.The up-regulation will be inhibited obviously by AG490.3.AG490 can obvious inhibit the secretion of IL-6 which was induced by endogenic and exogenous PRL in PBMCs through down-regulated the expression of total STAT5 and p-STAT5.It suggests that JAK/STAT singalling pathway acts as a link between HPRL and IL-6 in SLE patients.4.Compared to normal group,the expression of p-STAT5 has no significant deviationin in NPRL SLE patients.This phenomenon does not change after being stimulated by rhPRL.It is further confirmed that PRL makes its function in the pathogenesis of SLE through JAK/STAT signalling pathway.