青蒿琥酯治疗MRL／lpr狼疮鼠肾炎的病理变化及机制

目的研究青蒿琥酯对MRL／lpr狼疮鼠肾炎的疗效，探讨青蒿琥酯治疗狼疮鼠肾炎的病理变化及机制，为临床用于治疗狼疮患者提供依据。方法MRL／lpr鼠随机分为青蒿琥酯治疗组、环磷酰胺（CTX）治疗组和对照组。16周龄时青蒿琥酯组给予青蒿琥酯125mg／（kg·d）治疗16周，CTX组给予CTX100mg／kg×2d腹腔注射。PAS染色观察病理改变，免疫荧光检测补体G沉积，运用反转录-聚合酶链反应（RT-PCR）检测小鼠肾脏血管内皮生长因子（VEGF）的mRNA表达水平，用免疫组化法检测肾脏中VEGF的蛋白表达。结果①青蒿琥酯组和CTX组肾脏病理损伤较对照组明显减轻；②青蒿琥酯组和CFX组肾脏内补体C3沉积较对照组明显减少；③青蒿琥酯组肾脏VEGFmRNA表达（0．72±0．11）和CFX组肾脏VEGFmRNA表达（066±0．19）均低于对照组（0．92±0．06）（P〈0．05）；④青蒿琥酯组和CTX组肾脏VEGF表达比对照组明显减少。结论青蒿琥酯治疗MRL／lpr狼疮鼠可以改善。肾脏病理损伤。抑制C3的沉积及VEGF的产生是青蒿琥酯治疗MRL／lpr狼疮鼠肾炎的有效的可能机制。     

青蒿琥酯通过抑制ICAM-1治疗鼠狼疮性肾炎的研究

目的探索青蒿琥酯对狼疮模型鼠-MRL/lpr鼠狼疮性肾炎(LN)的疗效及作用机制。方法分别以青蒿琥酯50mg/(kg.d)和相同体积生理盐水灌胃治疗MRL/lpr鼠,治疗16周后检测小鼠24 h蛋白尿、血肌酐水平,检测小鼠外周血血清中ICAM-1浓度及淋巴细胞表面CD54阳性率,检测小鼠肾组织中ICAM-1表达。结果青蒿琥酯能够显著减少狼疮鼠的蛋白尿(P0.01)及血肌酐水平(P0.05),显著降低狼疮鼠的血清中可溶性ICAM-1浓度(P0.05)及淋巴细胞表面CD54的阳性率(P0.05),导致肾组织中ICAM-1的表达明显下降。结论青蒿琥酯显著缓解MRL/lpr鼠LN病情,此作用可能与抑制ICAM-1的表达有关。     

青蒿琥酯通过抑制ICAM-1治疗鼠狼疮性肾炎的研究

目的探索青蒿琥酯对狼疮模型鼠-MRL/lpr鼠狼疮性肾炎（LN）的疗效及作用机制。方法分别以青蒿琥酯50mg/（kg.d）和相同体积生理盐水灌胃治疗MRL/lpr鼠,治疗16周后检测小鼠24 h蛋白尿、血肌酐水平,检测小鼠外周血血清中ICAM-1浓度及淋巴细胞表面CD54阳性率,检测小鼠肾组织中ICAM-1表达。结果青蒿琥酯能够显著减少狼疮鼠的蛋白尿（P〈0.01）及血肌酐水平（P〈0.05）,显著降低狼疮鼠的血清中可溶性ICAM-1浓度（P〈0.05）及淋巴细胞表面CD54的阳性率（P〈0.05）,导致肾组织中ICAM-1的表达明显下降。结论青蒿琥酯显著缓解MRL/lpr鼠LN病情,此作用可能与抑制ICAM-1的表达有关。     

双氢青蒿素对MRL/lpr狼疮鼠的治疗作用及其对滤泡辅助性T细胞的调控作用

目的：探究双氢青蒿素对MRL/lpr狼疮鼠的治疗作用及其对滤泡辅助性T细胞（TFH）的调控作用。方法：将12只雌性狼疮鼠（12周龄）随机分为：双氢青蒿素治疗组和对照组，治疗组双氢青蒿素（150mg/kg）灌胃给药，对照组给予PBS溶液，均每天1次，持续给药4周至16周龄时，考马斯亮蓝法检测24小时尿蛋白，其后处死狼疮鼠。取肾脏行苏木精-伊红染色，肾脏病理评分评估肾脏损伤情况；收集血清，ELISA检测抗ds-DNA抗体及ANA抗体含量；取脾脏称重并分离单个核细胞，流式细胞仪检测滤泡辅助性T细胞百分比。体外实验中，免疫磁珠分选MRL/lpr狼疮鼠中初始CD4+ T细胞，根据双氢青蒿素干预的不同浓度分为4组（0μM、0.1μM、1.0μM、10μM），每组加入10ng/mL IL-21、20ng/mL IL-6和T细胞活化/扩增试剂盒中的试剂诱导分化5天，流式细胞仪检测TFH细胞比例；收集TFH细胞培养上清，ELISA检测TFH细胞分泌的IL-21含量。采用GraphPad prism 7.0软件进行统计学分析，数据采用均数±标准差，定量资料通过student-t检验和方差分析组间比较，P <0.05具有统计学意义。结果：双氢青蒿素对MRL/lpr鼠具有治疗作用：可有效改善肾脏损伤，双氢青蒿素治疗组尿蛋白（1536±150.1）μg/24h显著小于对照组（2548±93.30）μg/24h，P <0.01；治疗组可显著降低肾脏病理评分（2.410±0.535 vs. 5.713±0.662，P <0.01）；可有效降低狼疮鼠体内自身抗体滴度，抗ds-DNA抗体治疗组vs对照组（345.3±29.00 vs. 477.8±20.07，t=7.514，P <0.01）和ANA治疗组vs对照组（514.5±19.64 vs. 726.3±16.27，t=8.302，P <0.01）；可有效降低脾脏TFH细胞比例，治疗组（12.40%±2.726%）低于对照组（36.33%±2.750%），P <0.01。体外双氢青蒿素可抑制TFH细胞分化，0μM、0.1μM、1.0μM、10μM双氢青蒿素剂量依赖性抑制TFH细胞百分比（分别为29.43%±1.94%、21.07%±1.19%、11.27%±1.40%、6.28%±1.02%，P <0.01），且抑制细胞因子IL-21 的分泌（分别为87.07±5.49、63.63±3.70、47.67±4.02、34.37±5.10）pg/mL，P <0.01。结论：双氢青蒿素对MRL/lpr狼疮鼠具有治疗作用，可有效改善狼疮肾炎、降低抗体滴度，其机制可能与抑制TFH细胞分化有关。     

脐带间充质干细胞移植对系统性红斑狼疮免疫系统的影响

背景:目前有关脐带间充质干细胞对系统性红斑狼疮的免疫抑制作用及对各个脏器的影响报道较少。目的:探讨脐带间充质干细胞治疗MRL/lpr狼疮鼠的疗效及对免疫系统和各个脏器的影响。方法:MRL/lpr狼疮小鼠随机分为对照组、环磷酰胺组、环磷酰胺+脐带间充质干细胞组、脐带间充质干细胞组。对照组给以生理盐水,其他各组分别给予相应的治疗。采用考马斯亮蓝法检测24h尿蛋白定量;酶联免疫吸附法检测白细胞介素10、白细胞介素17、干扰素γ、PDGF、抗ds-DNA抗体;间接免疫荧光法检测抗核抗体水平;常规病理苏木精-伊红染色观察小鼠的肾脏病理改变肾脏病理改变;免疫组织化学法检测间充质干细胞表面标志CD44、CD105在肾脏、肺脏、脾脏中的表达。结果与结论:①环磷酰胺+脐带间充质干细胞组干扰素γ、白细胞介素17、白细胞介素10水平与对照组相比明显降低(P<0.05)。②32周时环磷酰胺+脐带间充质干细胞组和脐带间充质干细胞组尿蛋白定量低于环磷酰胺组和对照组(P<0.01)。③32周时环磷酰胺+脐带间充质干细胞组抗ds-DNA抗体水平与对照组相比明显降低。④脐带间充质干细胞组肾小球硬化及炎性细胞浸润程度均较对照组为轻。⑤32周时肾脏、肺脏、脾脏中间充质干细胞表面标志CD44、CD105表达为阴性。提示,应用脐带间充质干细胞移植治疗MRL/lpr狼疮鼠可以显著降低狼疮活动指标,对狼疮肾炎具有治疗作用,同时可以通过降低促炎因子的水平发挥免疫抑制作用。     

脐带间充质干细胞移植对系统性红斑狼疮免疫系统的影响

背景：目前有关脐带间充质干细胞对系统性红斑狼疮的免疫抑制作用及对各个脏器的影响报道较少。目的：探讨脐带间充质干细胞治疗MRL/lpr狼疮鼠的疗效及对免疫系统和各个脏器的影响。方法：MRL/lpr狼疮小鼠随机分为对照组、环磷酰胺组、环磷酰胺＋脐带间充质干细胞组、脐带间充质干细胞组。对照组给以生理盐水,其他各组分别给予相应的治疗。采用考马斯亮蓝法检测24h尿蛋白定量;酶联免疫吸附法检测白细胞介素10、白细胞介素17、干扰素γ、PDGF、抗ds-DNA抗体;间接免疫荧光法检测抗核抗体水平;常规病理苏木精-伊红染色观察小鼠的肾脏病理改变肾脏病理改变;免疫组织化学法检测间充质干细胞表面标志CD44、CD105在肾脏、肺脏、脾脏中的表达。结果与结论：①环磷酰胺＋脐带间充质干细胞组干扰素γ、白细胞介素17、白细胞介素10水平与对照组相比明显降低（P〈0.05）。②32周时环磷酰胺＋脐带间充质干细胞组和脐带间充质干细胞组尿蛋白定量低于环磷酰胺组和对照组（P〈0.01）。③32周时环磷酰胺＋脐带间充质干细胞组抗ds-DNA抗体水平与对照组相比明显降低。④脐带间充质干细胞组肾小球硬化及炎性细胞浸润程度均较对照组为轻。⑤32周时肾脏、肺脏、脾脏中间充质干细胞表面标志CD44、CD105表达为阴性。提示,应用脐带间充质干细胞移植治疗MRL/lpr狼疮鼠可以显著降低狼疮活动指标,对狼疮肾炎具有治疗作用,同时可以通过降低促炎因子的水平发挥免疫抑制作用。     

MRL/lpr狼疮鼠骨髓间充质干细胞表面粘附分子N-钙粘附蛋白表达增高并伴有骨髓中B淋巴细胞生成障碍

目的:观察MRL/lpr狼疮鼠骨髓的血液生成情况及骨髓微环境中骨髓间充质干细胞(bone marrow masenchymal stem cell,BMMSC)表面的粘附分子N-钙粘附蛋白(N-cadherin)的表达水平。方法:采集MRL/lpr狼疮鼠外周血进行血细胞计数;流式细胞术检测和分析狼疮鼠骨髓各系血细胞的比例;甲基纤维素半固体集落形成试验(CFU)检测狼疮鼠骨髓中CFU-pre-B、BFU-E、CFU-GM数目。分离培养狼疮鼠的BMMSC,Western blot检测其N-cadherin的表达。最后,分别用BMP/Smad通路的激动剂BMP-2和抑制剂Noggin处理BMMSC,尔后观察狼疮鼠BMMSC的N-cadherin的表达变化。结果:与C57BL/6小鼠比较,狼疮鼠的血红蛋白、红细胞计数和血细胞比容显著降低(P0.01;n=7);狼疮鼠骨髓中B220+细胞比例和CFU-pre-B数目均明显下降(P0.05;n=5)。狼疮鼠BMMSC的粘附分子N-cadherin的表达水平明显高于对照组(P0.05;n=3)。BMP-2处理狼疮鼠BMMSC后N-cadherin的水平下降,而在Noggin处理后N-cadherin表达则上调(P0.05;n=3)。结论:MRL/lpr狼疮小鼠骨髓中B淋巴细胞生成障碍。此外,小鼠骨髓微环境中BMMSC表面的粘附分子N-cadherin水平升高,其可能参与了MRL/lpr狼疮小鼠的骨髓血液生成异常。     

凉血化瘀解毒法对MRL/lpr 鼠相关细胞因子表达的影响

目的 观察凉血化瘀解毒法对MRL /lpr 鼠相关细胞因子表达的影响,探讨系统性红斑狼疮(SLE)发病中瘀热将MRL /lpr 鼠分为模型组、阴性组、中药组、西药组、中西药组,中药组予凉血化瘀解毒复方,西药组给予强的松加环磷酰胺,观察MRL /lpr 鼠血清中抗ds-DNA 、抗核小体抗体变化及肾组织中金属基质蛋白酶-9 (MMP-9 )、转移生长因子-β(TGF-β)、肿瘤坏死因子-α(TNF-α)、血管内皮生长因子(VEGF)、血栓调节蛋白(TM)表达的变化.结果 抗dsDNA 在模型组含量最高,西药能降低抗dsDNA,中药作用较弱.抗核小体抗体在模型组含量最高,中、西药不能明显降低抗核小体抗体.MMP-9 、TNF-α、TGF-β、VEGF 、TM 在模型组表达较高,中药组及中西药组能下调MMP-9 、TNF-α、VEGF 表达,西药组能下调MMP-9 、VEGF 的表达,而对TNF-α表达影响较小,对TM 中药组、西药组、中西药组表达有所下调,但没有统计学意义.结论 凉血化瘀解毒法对MRL /lpr 鼠相关细胞因子表达的影响与现代医学基础治疗基本一致,并协调其部分作用,提示瘀热毒在SLE 发病中具有重要作用.     

蛤士蟆对MRL/lpr狼疮鼠体内雌激素与抗ds-DNA的影响

目的 探讨蛤士蟆能否刺激狼疮鼠的免疫反应,以证实蛤士蟆是否引起或加重系统性红斑狼疮病情活动.方法 取12只MRL/lpr雌性狼疮小鼠,随机分为蛤士蟆组和生理盐水组各6只,分别用蛤士蟆浸出液和生理盐水给MRL/lpr狼疮鼠灌胃,用ELISA法检测2组尾静脉血中雌激素水平和抗ds-DNA水平.结果 灌胃后,蛤士蟆组狼疮鼠体内雌激素水平较生理盐水组显著增高;蛤士蟆组狼疮鼠体内抗ds-DNA水平也显著高于生理盐水组.结论 蛤士蟆能增加MRL/lpr狼疮鼠体内雌激素水平与抗ds-DNA水平.     

脐带间充质干细胞对MRL/lpr小鼠免疫炎性易栓状态的调节作用

目的:通过检测MRL/lpr狼疮小鼠的外周血CD4~+CD25~+T细胞表达、血浆炎症因子TNF-α和IL-6的表达以及易栓指标TF和FIB的水平,探讨脐带间充质干细胞(UC-MSC)免疫调节作用对MRL/lpr小鼠免疫炎性紊乱及易栓状态的修复影响。方法:将25只MRL/lpr小鼠分为对照(C)组、UC-MSC 1次治疗(UT1)组和UC-MSC 3次治疗(UT3)组,给予UT1和UT3组小鼠经尾静脉注射UC-MSC悬液。在SPF环境中饲养,16周后每2周取血分离血浆,采用流式细胞术法检测外周血CD4~+CD25~+T细胞表达水平;ELISA法检测血浆炎症因子TNF-α、IL-6和易栓指标TF、FIB水平。结果:治疗组第16-18周外周血CD4~+CD25~+T细胞的表达水平上调,至20周后下降并趋于稳定,至第30周时低于对照组;治疗组小鼠16周后血浆TNF-α和IL-6水平下降,至30周时明显低于对照组(P0.05);治疗组小鼠16周后血浆TF和FIB水平下降,TF水平自18周起明显低于C组(P0.05~0.01)。结论:UC-MSC可通过其免疫调节作用,修复MRL/lpr小鼠免疫炎性微环境紊乱;UC-MSC有利于MRL/lpr小鼠的免疫炎性易栓状态的修复。     

Distinct clonotypes of anti-DNA antibodies in mice with lupus nephritis.

Clonotypes of IgG anti-DNA antibodies were studied by isoelectric focusing in various autoimmune mice with or without lethal lupus nephritis. MRL/MpJ-lpr/lpr mice exhibited the most heterogeneous spectrotypes of anti-DNA antibodies in the pH range from 6.5 to 8.5, with marked variation in individual mice. Female (NZB X NZW)F1 mice expressed rather uniform DNA-binding bands composed of at least five to six distinct subgroups, having isoelectric points from 6.5 to 8.0. Male BXSB mice showed major characteristic bands confined to alkaline pH range from 7.8 to 8.5, similar to C57BL/6J-lpr/lpr mice, which showed markedly restricted bands in this region. Both AKR/J-lpr/lpr and C3H/HeJ-lpr/lpr mice expressed DNA-binding bands mostly focused between pH 6.5 and 8.2. The aging study indicated that three autoimmune mice (MRL/MpJ-lpr/lpr, [NZB X NZW]F1, and male BXSB) that developed fatal glomerulonephritis showed clonal expansion of anti-DNA antibodies throughout their life. In contrast, such age-dependent expansion of anti-DNA clonotypes was not evident in three lpr cogenic mice (C57BL/6J-lpr/lpr, AKR/J-lpr/lpr, and C3H/HeJ-lpr/lpr) that developed only mild glomerulonephritis; rather, their expression of anti-DNA spectrotypes diminished as they aged. Anti-DNA activities in renal eluates from nephritic autoimmune mice were mostly distributed in the pH range from 6.5 to 8.0, without significant concentrations in the high alkaline range of more than pH 8.0. These results suggest that there exist distinct anti-DNA clonotypes in each mouse strain and that the development of lupus nephritis does not appear to be associated with particular spectrotypes of anti-DNA antibodies. Rather, the age-dependent expansion of anti-DNA clonotypes may be a feature more characteristic of mice developing lethal lupus nephritis.     

间歇大剂量环磷酰胺冲击治疗重症狼疮性肾炎临床研究

目的：探讨间歇静脉注射大剂量环磷酰胺（IV-CTX）治疗重症狼疮性肾炎（LN）的疗效。方法;将146例重症LN患者随机分为3组.A组56例,采用IV-CYX0.6～1.0g加强的松1mg.kg-1／d口服。B组46例,口服强的松1.5mg.kg-1／d）,明显狼疮活动时给予静脉注射甲基强的松龙1mg／d,共3d。间歇4d,间歇期口服强的松（剂量同前）,7d为1疗程,可连用2～3个疗程。C组44例.口眼强的松1～1.5mg加CTX2mg.kg-1／d）。整个观察时间为2个月。结果：3组治疗结束后免疫学指标均有改善,尿蛋白减少、肾功能好转。组间比较无统计学意义。但3组副反应比较：A组最轻,仅出现轻微而短暂的消化道症状。B组感染率最高。C组以肝功能损害及白细胞减少明显。结论：IV-CTX加中、小剂量强的松龙治疗重症LN,能减少蛋白尿,改善肾功能。与口服大剂量强的松或口服CTX相比疗效等同.且副反应轻微。     

Ultrasonographic Evaluation of Renal Resistive Index in Patients with Lupus Nephritis: Correlation with Histologic Findings

We analyzed the association between the renal arterial resistive index (RI) and the histologic features of lupus nephritis. All consecutive patients with systemic lupus erythematosus (SLE) who required a kidney biopsy were enrolled. The study protocol included ultrasonographic assessment to measure the RI and kidney biopsy (International Society of Nephrology/Renal Pathology Society classification). A RI > 0.7 was considered pathologic. Patients with non-renal SLE and healthy patients were studied as control groups. We enrolled 42 patients with renal SLE, 10 with non-renal SLE and 14 healthy patients: their mean (±standard deviation) RI values were 0.64 ± 0.08, 0.60 ± 0.04 and 0.59 ± 0.01, respectively (p = not significant). RIs > 0.7 were recorded only in patients with renal SLE (5/42, 11.9%). The percentage of patients with a pathologic RI was significantly higher in class IV nephritis in comparison with other classes (p < 0.009). In conclusion, we found a significant correlation between pathologic RI and class IV nephritis, suggesting a role for RI as a severity marker.     

Efficacy of a pure compound H1-A extracted from Cordyceps sinensis on autoimmune disease of MRL lpr/lpr mice

Cordyceps sinensis (CS) is a traditional Chinese medicine with immunomodulatory effect and is effective in improving the survival of lupus mice. In the present study we isolated a pure compound (H1-A) from CS and investigated its effect on inhibiting autoimmune disease progression in MRL Ipr/Ipr mice. Our results demonstrated that MRL Ipr/Ipr mice treated daily with H1-A (40 microg/kg/d orally) for 8 weeks had a progressive reduction in anti-ds-DNA production (optical density value decreased from 0.172 +/- 0.009 to 0.112 +/- 0.015) when compared with the control group (optical density value increased from 0.141 +/- 0.036 to 0.198 +/- 0.047). In clinical presentation, the treated group had a reduction in lymphadenopathy, a delayed progression of proteinuria, and an improvement in kidney function. Histologic analysis of kidney tissue indicated that H 1-A could inhibit the mesangial proliferation that was evident in lupus nephritis. However, there was no significant change in immune complex deposition. The studies reveal that the pure compound (H1-A) may be potentially useful for treating systemic lupus erythematosus in human patients, and they provide some questions for further investigation of the pathogenesis of systemic lupus erythematosus and lupus nephritis.     

Somatic mutation and light chain rearrangement generate autoimmunity in anti-single-stranded DNA transgenic MRL/lpr mice.

Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp-lpr/lpr (MRL/lpr) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo.