来氟米特对狼疮小鼠治疗作用机制的探讨

目的:探讨来氟米特(LEF)对MRL/lpr自发狼疮小鼠的治疗作用机制。方法:用LEF35mg·kg-1·d-1治疗12周龄雌性MRL/lpr自发狼疮小鼠,并与未干预组对照。12周后用放射免疫方法检测小鼠血清抗dsDNA抗体结合率、血清和尿中肿瘤坏死因子(tumornecrosisfactor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)的变化;并观察小鼠尿蛋白、血肌酐及肾组织CD14 细胞数的变化。结果:治疗12周后,LEF组与对照组比较,抗dsDNA抗体结合率、血清和尿中TNF-α、IL-6水平降低(P<0.05);24h尿蛋白排泄量、血肌酐水平及肾组织CD14 细胞数差异均有统计学意义(P<0.05)。结论:细胞因子产生异常参与了LN的发生、发展过程,检测血清和尿中TNF-α、IL-6水平有助于监测肾损害的程度,来氟米特治疗MRL/lpr自发狼疮小鼠,可能通过抑制单核/巨噬细胞和T淋巴细胞所产生的TNF-α、IL-6水平,减轻了MRL/lpr小鼠肾损害,对LN有较好的治疗作用。     

TACI-Ig对MRL/lpr小鼠的治疗作用及其对小鼠肾组织JAK1-STAT1信号通路的影响

目的研究TACI-Ig对MRL/lpr小鼠的治疗作用以及TACI-Ig对狼疮性肾炎肾组织JAK1-STAT1信号通路活化的影响。方法将MRL/lpr红斑狼疮转基因小鼠随机分成6组,即模型组、TACI-Ig 3个剂量治疗组(3.75、7.5、15 mg.kg-1)组、强的松阳性对照组和IgG-Fc阴性对照组,另选用BALB/c小鼠作为正常对照组。除强的松组每日灌胃给药外,其余各组隔日皮下注射给药,共计8周,模型组和正常组给予生理盐水。观察TACI-Ig对MRL/lpr小鼠一般体征、损伤指数的影响,目测半定量尿蛋白试纸法检测动物的尿蛋白变化,HE染色法观察肾组织病理学改变情况,全自动生化分析仪检测血清中肌酐和尿素氮的水平,ELISA法检测血清中BLyS、IL-10和IFN-γ的水平,免疫组化法检测肾脏磷酸化的JAK1和STAT1的表达分布情况。结果 TACI-Ig(7.5、15mg.kg-1)皮下注射给药8周可明显降低模型小鼠的尿蛋白水平和损伤指数;有效改善肾小球系膜细胞增生和肾小球纤维化,明显减轻炎性细胞浸润;TACI-Ig给药可明显降低小鼠血清中的肌酐和尿素氮水平以及血清中BLyS、IL-10、IFN-γ等细胞因子水平。免疫组化结果显示模型组小鼠的肾脏磷酸化的JAK1和STAT1蛋白的表达明显升高,在肾小球、肾间质中呈强阳性表达,TACI-Ig给药后可使其表达明显降低。结论 TACI-Ig可明显改善MRL/lpr小鼠红斑狼疮样的临床表现和肾脏病理特征,降低血清中细胞因子和肾组织中磷酸化JAK1、STAT1蛋白的表达水平,这可能是TACI-Ig治疗狼疮性肾炎的机制之一。     

雷公藤内酯通过CCAAT/增强子结合蛋白α抑制狼疮样肾炎小鼠IL-12/IL-23的表达

目的探究雷公藤内酯通过CCAAT/增强子结合蛋白α(CCAAT/enhancerbinding proteinα,C/EBPα)抑制狼疮样肾炎小鼠IL-12/IL-23表达的机制。方法实验分为3组:对照组(健康小鼠,n=15)、MRL/lpr组(狼疮样肾炎小鼠,n=15)和雷公藤内酯组(狼疮样肾炎小鼠经雷公藤内酯治疗,0. 125 mg/kg/2 d,n=15);使用HITACHI-7 080自动生化分析仪检测血清尿素氮和肌酸酐水平;用苏木素-伊红染色进行肾组织病理学测定,评估肾小球肾炎,间质性肾炎和血管病变的组织学评分;通过逆转录-实时定量PCR(RT-qPCR)和蛋白质印记分析了肾脏中炎性细胞因子(TNF-α、IL-6、IL-12和IL-23) mRNA和蛋白质表达;通过定点诱变将碱基取代检测IL-12/IL-23启动子活性;通过Ch IP测定小鼠C/EBP结合活性。结果与对照组(8. 47±0. 85,15. 38±1. 06)相比,MRL/lpr组尿素氮(29. 14±2. 05)和肌酸酐(40. 18±3. 72)水平增加(P0. 05),雷公藤内酯(11. 36±1. 23,20. 17±2. 45)治疗降低MRL/lpr小鼠尿素氮和肌酸酐水平(P0. 05); MRL/lpr组中的小鼠表现出肾损伤,组织学评分均增加(P0. 05),其特征在于系膜基质增加,管状铸型沉积和间质细胞浸润。相反,在雷公藤内酯处理的小鼠中这些病理特征改善; MRL/lpr组较对照组炎性细胞因子的mRNA和蛋白质表达增加(P0. 05)。雷公藤内酯降低了MRL/lpr小鼠中TNF-α、IL-6、IL-12和IL-23 mRNA和蛋白质表达(P0. 05); C/EBP的突变缓解雷公藤内酯对小鼠IL-12/IL-23启动子活性的抑制。结论雷公藤内酯通过C/EBPα抑制IL-12/IL-23的表达来改善MRL/lpr小鼠中的狼疮样肾炎。     

基于HPV16的新型伪病毒治疗MRL/lpr小鼠狼疮性肾炎的实验研究

目的 构建基于人乳头状病毒(human papilloma virus,HPV)16型的新型伪病毒,探讨对MRL/Ipr,小鼠狼疮性肾炎的治疗作用,为临床治疗系统性红斑狼疮提供实验依据.方法 将12只3月龄MRL/Ipr狼疮小鼠随机均分为治疗组和对照组,治疗前后测定尿蛋白、血清尿素氮、肌酐及抗ds-DNA抗体滴度.结果 治疗组小鼠治疗后的尿蛋白、血清尿素氮、肌酐及抗ds-DNA抗体均下降,与治疗前有显著性差异(P＜0.05),而对照组治疗前后无显著性差异(P＞0.05),并且治疗组小鼠的平均生存期比对照组明显延长(p＜0.05).结论 基于人乳头状病毒16型的新型伪病毒可显著改善MRL/Ipr小鼠的免疫状况和肾脏功能,提高平均生存期.该研究结果为临床治疗系统性红斑狼疮提供了新的启示.     

商陆皂苷甲对大鼠Heymann肾炎的治疗作用及对细胞因子的影响

目的： 商陆皂苷甲(EsA)对大鼠Heymann肾炎(passive Heymann nephritis,PHN)的治疗作用及机理。方法： 大鼠sc Fx1A抗原的抗体制成自身免疫性肾小球肾炎,EsA 5,10和20 mg.kg^-1.d^-1 ip,连续14 d。结果： EsA可以显著减少肾炎大鼠尿蛋白的产生,电镜和免疫荧光检查发现EsA治疗后肾炎大鼠病理情况明显好转。EsA治疗对血清中炎性细胞因子肿瘤坏死因子（TNF）、白细胞介素1（IL-1）和白细胞介素6（IL-6）的产生具有显著的抑制作用。结论： EsA对大鼠Heymann肾炎具有显著治疗作用,抑制细胞因子产生可能参与EsA抗炎机制。     

芦丁通过抑制JAK2-STAT3信号通路活性改善MRL/lpr狼疮小鼠的肾损伤

目的 研究芦丁对小鼠系统性红斑狼疮肾损伤的保护作用。方法 10只正常雌性Balb/c小鼠为正常对照组，50只雌性MRL/lpr小鼠随机分为5组，模型对照组、醋酸泼尼松组(给予醋酸泼尼松0.1 mg·kg^-1)及芦丁小、中、大剂量组(给予芦丁25,50,100 mg·kg^-1)。8周龄时开始连续灌胃给药8周。检测尿蛋白浓度、血清Anti-dsDNA IgG和IgM、白细胞介素(IL)-6、干扰素(IFN)-γ、血肌酐(CRE)、血尿素氮(BUN),采用苏木精-伊红(HE)染色法光镜下观察各组小鼠肾脏组织的病理变化。免疫印迹法检测p-JAK2、JAK2、p-STAT3、STAT3的水平。结果 与模型对照组比较，芦丁各剂量组尿蛋白含量、血清BUN、CRE浓度、Anti-dsDNA IgG、IgM的含量均显著下降，肾脏病理性损伤减轻，血清中IL-6和IFN-γ的含量显著减少，p-JAK2、p-STAT3水平有所下调，且呈剂量依赖性。结论 芦丁对MRL/lpr自发系统性红斑狼疮小鼠肾损伤有一定保护作用，其机制可能与抑制JAK2-STAT3信号通路活化有...     

清化狼疮汤对MRL/lpr小鼠血清ANA、SIL-2R的影响

目的:研究中药清化狼疮汤对MRL/lpr小鼠血清抗核抗体(ANA)、SIL-2R的影响.方法:取MRL/lpr♀小鼠40只和♂小鼠8只,随机分成4组,分别予以胃饲生理盐水(模型组)、清化狼疮汤混悬液(中药组)、泼尼松(西药组)和清化狼疮汤混悬液加泼尼松(中西药组).30 d后,免疫荧光法检测小鼠血清ANA滴度,单克隆抗体技术酶联免疫吸附(ELISA)双抗体夹心法检测小鼠血清SIL-2R表达水平.结果:中药组、西药组和中西药组MRL/lpr小鼠的血清ANA滴度和SIL-2R表达水平,均明显低于生理盐水组(P＜0.01或P＜0.05).结论:清化狼疮汤可以降低MRL/lpr小鼠血清ANA滴度和SIL-2R的表达水平.     

IL-23／IL-17炎症轴在实验性自身免疫性肝炎中的作用

目的通过建立实验性自身免疫性肝炎小鼠模型,观察外源性IL-23对实验性自身免疫性肝炎模型小鼠中IL-17的影响,探讨其可能的作用机制。方法以肝抗原S-100免疫C57BL／6小鼠制作自身免疫性肝炎动物模型,提取出脾淋巴细胞,将IL-23加入到抗CD,抗体活化后的脾淋巴细胞中进行培养,免疫组化观察IL-17在肝细胞中的分布及表达水平,RT-PCR和ELISA检测IL-17在脾淋巴细胞中的表达。结果与对照组相比,模型组肝细胞中IL-17表达升高;IL-23作用于模型组活化的脾淋巴细胞后IL-17的表达水平明显高于对照组。结论IL-23／IL-17炎症轴在EAH的病理机制中发挥着重要的作用,IL-23可能通过上调IL-17的表迭使AIH模型小鼠症状加重,并为AIH的治疗提供了新的靶点。     

三氧化二砷对MRL/lpr小鼠免疫功能和肾脏组织病理变化的影响(英文)

目的研究三氧化二砷(As2O3)对MRL/lpr小鼠免疫功能和肾脏组织病理变化的影响。方法45只MRL/lpr狼疮小鼠ip给予环磷酰胺50 mg·kg-1(每周1次)和As2O30.8 mg·kg-1,每天1次,共2个月。用ELISA法检测血清抗双链DNA(dsDNA)抗体、干扰素γ(IFN-γ)和白细胞介素12(IL-12)浓度;用流式细胞术测定脾CD3+,CD19+,CD3+CD4+和CD3+CD8+细胞亚群的百分比;用PAS染色法观察肾组织病理变化;用免疫荧光方法检测肾组织IgG和补体C3的表达。结果与给药前比较,给药2个月后,正常对照组血清抗dsDNA抗体水平升高,由给药前1.18±0.26升高至1.80±0.26(P<0.01),As2O3和环磷酰胺组该抗体水平明显降低,分别由给药前1.14±0.58和1.09±0.22降低至0.92±0.06和0.67±0.14(P<0.05,P<0.01)。与正常对照组比较:①As2O3和环磷酰胺组血清抗ds-DNA抗体、IFN-γ和IL-12浓度明显降低(P<0.05),环磷酰胺组抗ds-DNA抗体比As2O3组显著降低(P<0.01);②As2O3组CD3+,CD3+CD4+和CD19+细胞百分率明显降低(P<0.01),环磷酰胺组CD3+,CD3+CD8+和CD19+细胞百分率明显降低(P<0.01);As2O3组CD3+CD4+细胞百分率明显降低(P<0.01);③As2O3和环磷酰胺组小鼠肾小球细胞计数和活动度积分明显降低(P<0.05,P<0.01),As2O3和环磷酰胺组无显著差异;④As2O3和环磷酰胺组肾IgG表达明显降低(P<0.05),补体C3表达无明显差异,As2O3和环磷酰胺组之间无显著性差异。结论As2O3能降低MRL/lpr狼疮小鼠血清抗ds-DNA抗体水平,抑制T,B和Th细胞活化和增殖,降低血清IFN-γ和IL-12水平,从而缓解狼疮肾炎的病理变化。     

来氟米特对MRL/1pr小鼠肾脏JAK/STAT信号转导途径的影响

目的 探讨来氟米特对MRL/1pr狼疮小鼠肾脏JAK/STAT 1信号转导途径的影响.方法 14只12周龄雌性MRL/1pr狼疮小鼠随机分为空白对照组和治疗组(来氟米特35 mg·kg-1·d-1×8周).检测小鼠肾脏磷酸化SrAT 1(p-STAT 1)蛋白及细胞信号传导抑制因子(SOCS)-1 mRNA表达情况,并观察小鼠体重、24-h尿蛋白、血浆抗双链DNA(ds-DNA)抗体浓度、肾组织病理改变及肾小球免疫荧光变化.结果 治疗组小鼠24-h尿蛋白及血浆抗ds-DNA抗体浓度均明显低于对照组(P<0.01),肾脏p-STAT 1蛋白及SCCS-1 mRNA表达均低于对照组(P<0.01);肾组织病理明显改善.结论 来氟米特能抑制肾脏JAK/STAT 1信号转导途径,对MRL/1pr狼疮小鼠有治疗作用.     

Microarray analysis of glomerular gene expression in murine lupus nephritis

To elucidate the molecular mechanism of glomerular events in lupus nephritis, we performed genome-wide mRNA expression analysis of glomeruli microdissected from lupus mice. MRL/lpr mice (12-week-old) were orally given vehicle or prednisolone (10 mg/kg per day) for 4 weeks. Renal histology of MRL/lpr mice revealed mesangial proliferative glomerulonephritis with cellular infiltration of macrophages, T cells, and neutrophils. We identified 567 up-regulated genes in MRL/lpr glomeruli compared to control congenic mice. Those included complement components, adhesion molecules, chemokines and their receptors, and molecules related to antigen presentation. Over 130 genes were considered preferentially or exclusively expressed in hematopoietic cell lineages possibly reflecting leukocytes accumulation. Of note is the finding that chemokines and chemokine receptors (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16, CCR5, CXCR3, and CXCR6) that are related to T helper 1 (Th1) cells accumulation were up-regulated concomitantly with increased expression of Ebi3, a subunit of IL-27 that plays a role in Th1 predominance. These changes were accompanied by increased mRNA expression of many genes that were inducible by Th1 cytokine interferon-gamma. Prednisolone markedly attenuated glomerular lesion and leukocyte influx parallel with the reduction of enhanced gene expression. The present study shows additional evidence supporting glomerular Th1 cells accumulation and their role. Our data also provide an important resource in seeking new therapeutic targets to lupus nephritis. Supplemental table: available only at http://dx.doi.org/10.1254/jphs.FP0071337.     

Immunomodulating effect of a traditional Japanese medicine, hachimi-jio-gan (ba-wei-di-huang-wan), on Th1 predominance in autoimmune MRL/MP-lpr/lpr mice

Hachimi-jio-gan (Ba-Wei-Di-Huang-Wan, HMG), a traditional Japanese herbal medicine, has been used for disorders accompanying aging. Oral administration of HMG from 8 to 16 weeks of age to MRL/lpr mice as a lupus-like autoimmune model ameliorated significantly some nephritis parameters, proteinuria and immune complex deposition in the kidney. Further, HMG reduced significantly the degree of lymphadenopathy and the serum level of immunoglobulin (Ig) G2a anti-dsDNA specific auto-antibody, even at 12 weeks of age. Simultaneously, interferon (IFN)-gamma production from anti-CD3 stimulated B220- T cells was suppressed by HMG, whereas interleukin (IL)-4 production was promoted. Examination of cytokine mRNA expressions in CD4 positive cells showed clearly that T cell differentiation was shifted from T helper (Th)1 to Th2 predominance by HMG. Furthermore, we demonstrated that HMG suppressed IL-12 mRNA expression in spleen cells which is a marker of Th1 predominance in MRL/lpr mice. These results suggested that HMG modulated an imbalance toward Th1 predominance in MRL/lpr mice through inhibition of IL-12 production and ameliorated autoimmune disorders.     

Clinical efficacy of buprenorphine to minimize distress in MRL/lpr mice

MRL/MpJ-Fas(lpr) (MRL/lpr) mice are an accepted animal model to study human systemic lupus erythematosus. We tested if a commonly used analgesic (buprenorphine hydrochloride) would reduce pain and distress in these mice without impacting the progression of autoimmune disease. Female MRL/lpr mice were randomly separated into four groups. Experimental groups received cyclophosphamide (25 mg/kg i.p. weekly), buprenorphine (0.09 mg/kg/mouse/day via drinking water), or cyclophosphamide+buprenorphine from 11 to 21 weeks of age. Controls received no treatments. Mice were monitored daily by a licensed veterinarian (blinded observer) and assigned a score weekly on parameters associated with pain and distress as well as progression of disease. Proteinuria was measured weekly, and serum anti-dsDNA antibody levels were determined at 11, 15, and 18 weeks of age. At 21 weeks of age, the animals were euthanized and the kidneys and spleens were removed for evaluation. Regardless of the parameter observed, buprenorphine did not significantly decrease distress when compared to the controls. Buprenorphine did not alter the progression of autoimmune disease, based on characteristics of splenic architecture and splenocyte cell profiles, development of lymphadenopathy, or kidney histology as compared to controls. This study indicates that buprenorphine at this dose and route of administration was ineffective in reducing distress associated with disease progression in the MRL/lpr strain. More studies are needed to determine if, at a different dose or route, buprenorphine would be useful as adjunctive therapy in reducing distress in MRL/lpr mice.     

CD4+ B220+ TCRγδ+ T cells produce IL-17 in lupus-prone MRL/lpr mice

Systemic lupus erythematosus is an autoimmune disease with comprehensive immune cell disorders. Recent studies suggested that pro-inflammatory cytokine IL-17 plays important role in lupus, leaving the cellular sources and their pathogenic and physiologic characters largely unknown. In the current study, by using lupus-prone MRL/lpr mice, we demonstrated that Th17 response prevails in lupus disease regarding significantly accumulated serum IL-17, increased IL-17-producing splenocytes, and elevated phospho-STAT3 in CD4⁺ T cells. Intracellular staining revealed that unusual CD4⁺ B220⁺ T cells are major IL-17-producing cells, whereas conventional CD4⁺ B220⁻ T cells are major IFN-γ-producing cells. Subsequent studies showed that CD4⁺ B220⁺ cells contains both αβ and γδ T cells in the spleen and thymus of MRL/lpr mice. Further study showed that around 60% of γδ T cells in MRL/lpr mice co-express both B220 and CD4 on their surface, and are the major RORγt⁺ cells in MRL/lpr mice. Finally, CD4⁺ B220⁺ T cells alone do not proliferate, but could enhance the proliferation and IFN-γ-production of conventional CD4⁺ B220⁻ T cells. Our findings suggest the pathogenic role of unusual CD4⁺ B220⁺ T cells in lupus disease in MRL/lpr mice according to their IL-17-producing ability and stimulatory function for conventional CD4⁺ B220⁻ T cells.     

The selective p38 mitogen-activated protein kinase inhibitor, SB203580, improves renal disease in MRL/lpr mouse model of systemic lupus

Systemic lupus erythematosus (SLE) is an autoimmune disease accompanying excessive inflammatory responses in a wide range of organs. Abnormal activation of p38 MAPK has been postulated to contribute to the inflammation of SLE, leading to progressive tissue and organ damages to develop lupus nephritis and autoimmune hepatitis. In order to determine whether p38 MAPK inhibitor is effective in mouse model of SLE, a specific inhibitor of p38 MAPK SB203580 was orally administrated to MRL/lpr mice aged from 14 to 22 weeks. Renal and hepatic functions, as well as pathologic changes of important organs including kidney, liver and spleen of MRL/lpr mice were evaluated. As a result, we showed that SB203580 improved renal function by decreasing the levels of proteinuria and serum BUN, ameliorating the pathologic changes of kidney and reducing Ig and C(3) depositions in the kidney. Hepatocytes necrosis, recruitment and proliferation of leucocytes in liver and spleen were found to be inhibited by administration of SB203580. Therefore, p38 MAPK activation may be partially responsible for escalating autoimmune renal, hepatic and splenic destruction, and its inhibitor may lighten the autoimmune attack in these important organs and improve renal function. Our study reveals that the selective blockade of p38 MAPK is effective to prevent and treat the disease in this model of SLE.     

A soy diet accelerates renal damage in autoimmune MRL/Mp-lpr/lpr mice

Isoflavones, which are phytoestrogens present in large quantities in soy and soy-derived products, have estrogenic activity, inhibit protein tyrosine kinase, and exert other effects in the human body. Thus, the recent spread of soy consumption in Western populations emphasizes the need to more fully understand the potential effects in the body, especially in abnormal immune conditions. In the present study, the influence of a soy diet on lupus disease in MRL/Mp-lpr/lpr (MRL/lpr) mice was investigated. Weanling female MRL/lpr mice (4 weeks) were fed a soy diet (20% soybean protein and 5% soybean oil). The soy diet exacerbated renal damage; findings in this mouse strain included accelerated proteinuria, elevated serum creatinine concentrations, and reduced creatinine clearance. No effects were detected, however, in C3H/HeN mice, which have the same H-2(k) genetic background as MRL/lpr mice do. A tendency toward an increase in thymus weight and proliferation of T cells in spleen and B cells in lymph nodes were found at the age of 16 weeks. These findings indicate that a soy diet, in comparison with a casein diet, significantly exacerbates the clinical course of this autoimmune disease. Further research on the mechanism of this effect of soy-rich diets is needed, and isoflavone supplementation for systemic lupus erythematosus patients should be carefully reevaluated.     

Biological activities of deoxyspergualin in autoimmune disease mice

An assessment of the prophylactic and ameliorative effects of deoxyspergualin (NKT-01), an immunosuppressive agent, was carried out in male MRL/MpJ-lpr/lpr (MRL/1) mice which spontaneously develop lupus-like lesions. When NKT-01 was administered ip daily from the age of either 8 or 19 weeks, diseases such as massive lymphadenopathy, circulating anti-DNA antibody and lupus nephritis were markedly suppressed. The primary response to lipopolysaccharide was significantly reduced in MRL/1 mice administered NKT-01 but the response to sheep red blood cells was not affected. The ability of spleen cells to release interleukins 2 and 3 with or without mitogen was significantly enhanced in mice receiving NKT-01. These findings demonstrate that NKT-01 has therapeutic activity against the development of spontaneous disease in MRL/1 mice.