【作者】 王志强；

【导师】 李振彬；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种具有多系统损害及多种自身抗体的自身免疫性疾病,可累及全身各组织器官。狼疮肾炎(lupus nephritis,LN)是SLE最重要的临床表现之一,肾脏病变的严重程度是直接影响SLE预后的重要因素。近年研究表明,转化生长因子β1(transform growth factorβ1,TGF-β1)与多种肾病的发生、发展有明显相关性,可促进肾小球系膜细胞和内皮细胞增生以及细胞外基质(extracellular matrix,ECM)的合成和积聚,从而加重肾小球硬化和间质纤维化。新近发现骨形成蛋白7(bone morphogenetic protein 7,BMP-7)是TGF-β1发挥生物学作用的重要的下游调控介质。作为纤维化的负性调节因子,BMP-7通过维持上皮细胞表型、抑制肾脏上皮细胞的凋亡、激活细胞外基质(extracellular matrix,ECM)的降解途径、减少多种促炎症因子表达、影响TGF-β/Smads传导途径以及与TGF-β1的互逆作用,对包括肾纤维化在内的各种纤维化病变起到预防及逆转作用。雷公藤多苷(tripterygium glycosides,TG)广泛用于治疗风湿病、肾病等免疫相关性疾病,但具体机制仍在进一步研究中,同时其毒副作用明显。如何增强TG的疗效并防治其毒性反应是目前研究的重点之一。研究证实,白芍总苷(total glucosides of paeony,TGP)具有良好的抗炎、镇痛以及免疫调节作用,同时可明显改善不同的肝脏损伤,且不良反应少,患者耐受性良好。本课题拟在建立慢性移植物抗宿主病(chronic graft-versus-host disease,cGVHD)狼疮小鼠模型的基础上,观察其肾组织中BMP-7及TGF-β1的表达情况及TG联合TGP的干预作用,探讨BMP-7、TGF-β1在LN病理过程中的可能作用和TG联合TGP治疗LN的可能机制以及TGP联合TG的增效减毒作用,为临床中药有效成分配伍治疗LN提供实验依据。方法:1.(C57BL/6J×DBA/2)F1(即B6D2F1)雌性杂交鼠,建立慢性移植物抗宿主病(cGVHD)狼疮模型。将成模小鼠随机分6组:模型组(LN group)、泼尼松组(Pred group)、来氟米特组(LEF group)、雷公藤多甙组(TG group)、白芍总苷组(TGP group)、雷公藤多苷+白芍总苷组(TG+TGP group)。另设正常对照组(Control group)。2.在造模后第9周,正常对照组、模型组给予生理盐水灌胃,其他各组分别给予泼尼松、来氟米特、雷公藤多苷、白芍总苷、雷公藤多苷和白芍总苷灌胃治疗,共4周。3.在造模及用药前后观察小鼠的一般情况。4.在治疗第4周末,各组小鼠全部处死。处死前1d置于代谢笼内收集24小时尿标本,检测24小时尿蛋白排泄率(UPE)。5.从心脏取血,离心,-50℃保存,检测尿素(BUN)、肌酐(SCr)、丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)。6.摘取肝脏制成组织匀浆,检测超氧化物歧化酶(SOD)、丙二醛(MDA)、还原型谷胱甘肽(GSH)。7.取肾脏组织进行固定、石蜡包埋,经HE染色、PAS染色和Masson染色,光镜下观察肾脏组织病理变化并进行对比。8.采用免疫组织化学染色法检测BMP-7、TGF-β1在肾组织的表达,并采用彩色病理图文报告分析系统对其表达量进行半定量分析。9.所有实验数据均采用SPSS 13.0软件包进行统计学处理,所得结果以均数±标准差(±s)表示。各组间均数比较采用单因素方差分析(one-way ANOVA)。方差分析前进行方差齐性检验,采用LSD法进行组间两两比较。相关性分析采用Pearson相关检验,以P<0.05认为有统计学意义。结果:1.与正常对照组相比,LN组小鼠的UPE、BUN、SCr均显著增高(P<0.01)。与模型组相比,Pred组、LEF组、TG组、TGP组、TG+TGP组均可有效降低LN小鼠的UPE、BUN、SCr,差异有统计学意义(P<0.05或P<0.01)。同时TG+TGP组与TG组比较,上述指标均有显著性差异(P<0.05或P<0.01)。2.与正常对照组、LN组相比,LEF组、TG组小鼠的ALT、AST均显著增高(P<0.01)。与TG组相比,TG+TGP组的ALT、AST均显著降低(P<0.01)。3.与正常对照组、LN组相比,LEF组、TG组小鼠的SOD、GSH均显著降低(P<0.05或P<0.01)。与TG组相比,TG+TGP组的SOD、GSH均显著增高(P<0.01)。与正常对照组、LN组相比,LEF组、TG组小鼠的MDA均显著增高(P<0.01)。与TG组相比,TG+TGP组的MDA显著降低(P<0.01)。4.病理结果显示,正常对照组小鼠肾脏未见明显病变。LN组小鼠肾小球系膜增宽、基底膜增厚、肾间质淋巴细胞浸润。各治疗组小鼠肾脏病变均较LN组有所减轻,其中以TG+TGP组病变最轻。5. BMP-7在正常对照组小鼠肾组织中高度表达,主要分布于肾小管及肾间质,而在肾小球内基本无表达。LN组小鼠肾小管及肾间质BMP-7的表达明显减少,各治疗组较LN组小鼠肾小管及肾间质BMP-7的表达增强。图像分析结果显示:LN组小鼠肾脏组织中BMP-7的表达与正常对照组比较明显减弱(P<0.01);除TGP组以外的各治疗组较LN组小鼠肾脏组织中BMP-7的表达明显增强(P<0.05或P<0.01);TG+TGP组较TG组小鼠肾小管及肾间质中BMP-7的表达更强(P<0.01)。6.正常对照组小鼠仅在少数肾小管有微弱TGF-β1表达。LN组小鼠肾小球系膜细胞、肾小管上皮细胞、血管内皮细胞、肾小球和肾间质浸润的炎症细胞胞质均有TGF-β1显著表达。各治疗组TGF-β1的表达均减弱。图像分析结果显示:LN组小鼠肾脏组织中TGF-β1的表达与正常对照组比较明显增强(P<0.01);各治疗组较LN组小鼠肾脏组织中TGF-β1的表达明显减弱(P<0.05或P<0.01);TG+TGP组较TG组小鼠肾脏组织中TGF-β1的表达更弱(P<0.05)。7.各组小鼠肾组织中BMP-7表达与UPE、BUN、SCr水平均呈显著负相关,r分别为-0.814、-0.555、-0.766(P均<0.01)。各组小鼠肾组织中TGF-β1表达与UPE、BUN、SCr水平均呈显著正相关,r=0.800、0.684、0.740(P均<0.01)。8.各组小鼠肾组织中BMP-7与TGF-β1的表达呈显著负相关,r=-0.665(P<0.01)。结论:1. BMP-7是维持肾脏功能稳定的一个重要因子,BMP-7在狼疮肾炎中具有与TGF-β1相反的作用,其表达水平下降可能参与了狼疮肾炎的发生、发展。2.雷公藤多苷、雷公藤多苷联合白芍总苷对cGVHD狼疮肾炎小鼠有显著的治疗作用;上调BMP-7表达、下调TGF-β1表达可能是其肾脏保护作用的重要机制。3.雷公藤多苷联合白芍总苷对cGVHD狼疮肾炎小鼠的治疗作用以及对BMP-7、TGF-β1表达的干预作用优于单用雷公藤多苷,提示白芍总苷与雷公藤多苷有明显的协同/增效作用。4.雷公藤多苷可导致肝脏损伤,其机制与氧化损伤有关;白芍总苷对雷公藤多苷所致的肝损伤具有保护作用,抗氧化损伤可能是其作用机制之一。5.中药有效成分雷公藤多苷与白芍总苷配伍治疗cGVHD狼疮肾炎具有增效减毒作用,该用药模式提示了中药现代化的新思路和新途径。更多还原

【Abstract】 Objective: Systemic lupus erythematosus (SLE) is a serious multi-system autoimmune disease characterized by the formation of antibodies. Lupus nephritis (LN) is one of the most important manifestation of SLE and is relevant to prognosis of SLE tightly. Research have showed that TGF-β1 (transform growth factor-β1) could aggravate glomerulosclerosis and renal interstitial fibrosis through enhancing mesangial cell and endotheliocyte proliferation and exacerbating extracellular matrix (ECM) synthesis and accumulation. Recent research suggested bone morphogenetic protein-7 (BMP-7) can reduce glomerulosclerosis and renal interstitial fibrosis through maintains epithelial cells phenotype, inhibit apoptosis of epithelial cells, activate degradation of extracellular matrix (ECM), decrease expression of proinflammatory cytokine, interfere with signal transduction pathway of TGF-β/Smads, and reverse effects of TGF-β1. Tripterygium glycosides (TG) is used for immune-associated disease such as rheumatism and nephrosis, but the toxic and side-effect of TG are apparent. The research of Action-Enhancing and Toxicity-Reducing effect on TG is necessary. Total glucosides of paeony (TGP) has favourable effects of anti-inflammatory, analgesia and immunoregulation, and satisfactory tolerance. In addition, TGP can amelioration various liver injurys obviously. The aim of this study based on murine model of chronic graft-versus-host disease(cGVHD) is to investgate the effect of BMP-7 on LN, the treatment mechanism of TG combine with TGP in LN and the action-enhancing and toxicity-reducing effect of TGP on TG through observe the effect of TGP combine with TG on the renal expression of BMP-7, renal function and hepatic function of cGVHD mice for the evidence of rational administration of TG and compatibility of effective component of the traditional Chinese medicine (TCM).Methods:(1) The murine LN model of cGVHD was employed, which was established by injecting with lymphocytes from female DBA/2 mice to Female (C57BL/6J×DBA/2) F1 (B6D2F1) hybrids aged 8-10 weeks intravenously 4 times at 3 days interval. The B6D2F1 hybrids were then randomly divided into lupus nephritis group, prednisone group, leflunomide group, TG group, TGP group, and TG+TGP group, with age-matched normal mice as the control group injected with physiological saline. (2) The medicines were given from the 9th week to the 12th week once a day. The general status of mice was observed before and after model and administration. After 4 weeks administration, 24hrs urinary protein excretion (UPE) and the levels of serum BUN, SCr, ALT, AST and liver tissue homo- genate SOD, MDA, GSH were detected. The pathologic change of kidneys of each group were observed by light microscope. The renal expression levels of BMP-7 and TGF-β1 were examined by immunohistochemical technique. (3) All statistical analyses of data were computed using Statistical Package for Social Sciences (version 13.0). Results are expressed as the mean±SD for the number of animals indicated. The sources of variation for multiple comparisons were assessed by one-way analysis of variance (ANOVA). Pearson correlation analysis was adopted. The differences were considered statistically significant at P<0.05.Results: (1) The levels of UPE, BUN and SCr of lupus nephritis group were increased significantly compared to control group (P<0.01). Compared with lupus nephritis group, the levels of UPE, BUN and SCr of prednisone group, leflunomide group, TG group, TGP group, and TG+TGP group were decreased notablely (P<0.05 or 0.01), and the TG+TGP group were the lowest (P<0.05 or 0.01). (2) The levels of ALT, AST of leflunomide group and TG group were increased significantly compared to control group and lupus nephritis group (P<0.01), and the levels of ALT, AST of TG+TGP group were decreased remarkably compared to TG group (P<0.01). (3) Compared with control group and lupus nephritis group, the levels of liver tissue homogenate SOD, GSH in the TG group were decreased significantly (P<0.05 or 0.01). The levels of liver tissue homogenate SOD, GSH in the TG+TGP group were signify- cantly higher than the TG group (P<0.01). Each group’s MDA level was converse to SOD, GSH. (4) The pathology of kidney tissue was no changes in control group mice. There was glomerular mesangial proliferation, basilar membrane thickening, extracellular matrix expansion and interstitial cellular infiltration in lupus nephritis group. The kidney pathological changes were all alleviated in each treatment group, and there was significant difference between TG group and TG+TGP group. (5) The expression of BMP-7 in renal tubular epithelial cells and interstitial tissue was shown to be lower in the lupus nephritis group than in the control, though it was not found in renal glomerular cells. The levels of BMP-7 expression in each treatment group were increased obviously (P<0.05 or 0.01). The expression of BMP-7 was higher in TG+TGP group than TG group remarkably (P<0.01). (6) There was only little TGF-β1 expression in kidney tubules of control groups rats. The expression of TGF-β1 in mesangial cells, renal tubular epithelial cells, vascular endothelial cells, and inflammatory cells was shown to be higher in the lupus nephritis group than in the control group (P<0.01). The levels of TGF-β1 expression in each treatment group were decreased obviously (P<0.05 or 0.01). The difference of kidney TGF-β1 expression between TG+TGP group and TG group was noticeable (P<0.05). (7) The levels of UPE、BUN、SCr were relevant to BMP-7 expression in kidney negatively (r=-0.814, -0.555, -0.766 respectively, all P<0.01) and TGF-β1 positively (r=0.800, 0.684, 0.740 respectively, all P < 0.01). (8) The correlation coefficient between the level of BMP-7 and TGF-β1 expression in kdney of mice was -0.665 (P<0.01).Conclusions:(1) BMP-7 is an important cytokine to protect renal function, and has adverse effect to TGF-β1. The down-regulation of BMP-7 is suggested to be related to developing and progress of lupus nephritis. (2) Tripterygium glycosides and tripterygium glycosides combined with total glucosides of paeony have prominent therapeutic effect on cGVHD lupus-like mice. The renoprotective effect of tripterygium glycosides and tripterygium glycosides combined with total glucosides of paeony maybe through up-regulation of BMP-7 and down-regulation of TGF-β1. (3) The therapeutical effect on UPE, BUN, Scr, BMP-7 and TGF-β1 in cGVHD lupus nephritis mice of tripterygium glycosides combined with total glucosides of paeony is better than tripterygium glycosides alone, which suggested that tripterygium glycosides and total glucosides of paeony have synergistic effect. (4) Tripterygium glycosides has noticeable hepatotoxicity, which is related to oxidative damage, and total glucosides of paeony has hepatoprotective effect, which is related to anti-oxidation.(5) Compatibility of tripterygium glycosides and total glucosides of paeony has action-enhancing and toxicity-reducing effect to cGVHD lupus-like mice, which suggest that the medication model of the effective component of the traditional Chinese medicine (TCM) provide a significant information for moderni- zation of TCM.更多还原