雷公藤内酯醇滴眼液防治角膜移植免疫排斥反应的实验研究

目的探讨雷公藤内酯醇(T10)滴眼液局部应用对角膜移植免疫排斥反应的影响.方法建立封闭群大鼠角膜移植模型,随机分组A、B、C组为SD-Wistar组间同种异体角膜移植,SD为受体,Wistar为供体,其中A组为空白对照组,B组为o.5mg@L1T10滴眼液组,C组为1mg@L-1T10滴眼液组,D组为SD-SD对照组,即SD大鼠间同种异体移植组和E组SD自体角膜移植对照组.用裂隙灯显微镜记录及比较各组角膜透明度、水肿度、新生血管度、移植排斥指数(RI)以及角膜排斥发生时间.结果术后各组角膜植片透明度、水肿度、新生血管程度以及发生角膜移植排斥时间,A组与C组比较有显著性差异(P＜0.05);D组和E组比较无显著性差异(P＞0.05).结论T10滴眼液可有效防治角膜移植免疫排斥反应.     

Effects of triptolide on proliferation and apoptosis in rat mesangial cells induced by transforming growth factor β1 and related mechanisms

Objective To investigate the effects of triptolide on proliferation, apoptosis and the changes of Ski, Smad3, Smad7 and collagen type Ι (ColΙ) in cultured rat mesangial cells induced by transforming growth factor (TGF)?β1. Methods Cultured HBZY?1 rat mesangial cells were divided into 5 groups: (1)normal control group; (2)TGF?β1 group (10 μg/L); (3)-(5)triptolide (0.4, 2, 10 μg/L)+TGF?β1 (10 μg/L) groups. The cell proliferation was detected by MTT. Apoptosis of mesangial cells was detected by TUNEL assay. The expressions of Ski, Smad3, Smad7 mRNA were examined by real?time quantitative PCR. The expressions of Ski, Smad3, Smad7 and ColΙ protein were detected by Western blotting. The localizations of Ski and Smad3 protein were detected by laser confocal fluorescence microscope. Results Compared with the normal control, TGF?β1 (10 μg/L) significantly stimulated mesangial cells proliferation, while decreased apoptosis. The mRNA and protein expressions of Ski, Smad7, Smad3 and ColΙ protein expression in TGF?β1 group were increased (P＞0.05). In comparison with TGF?β1 group, triptolide could significantly inhibit TGF?β1?induced mesangial cells proliferation in dose?dependent manner, and promote the apoptosis of mesangial cells. In TGF?β1 group, mRNA and protein expresscons of Ski and Smad7 were increased (P<0.05), Smad3 mRNA and protein were decreased (P＞0.05), and ColΙ protein was decreased (P<0.01). In comparison with TGF?β1 group, fluorescence intensity of Ski, Smad3 proteins was significantly increased in cytoplasm, while decreased in nucleus. Conclusions Triptolide can inhibit TGF?β1?induced mesangial cells proliferation through regulating the expressions of Ski, Smad7 mRNA and protein, inhibiting Ski. Smad7 translocation to the nucleus, and down?regulating Smad3 mRNA and protein expression. Triptolide can promote apoptosis of mesangial cells.     

Molecular cloning and functional identification of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Tripterygium wilfordii

The 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the last step key enzyme of the methylerythritol phosphate (MEP) pathway, synthesizing isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which is important for regulation of isoprenoid biosynthesis. Here the full-length cDNA of HDR, designated TwHDR (GenBank Accession No. KJ933412.1), was isolated from Tripterygium wilfordii for the first time. TwHDR has an open reading frame (ORF) of 1386 bp encoding 461 amino acids. TwHDR exhibits high homology with HDRs of other plants, with an N-terminal conserved domain and three conserved cysteine residues. TwHDR cDNA was cloned into an expression vector and transformed into an Escherichia coli hdr mutant. Since loss-of-function E.coli hdr mutant is lethal, the result showed that transformation of TwHDR cDNA rescued the E.coli hdr mutant. This complementation assay suggests that the TwHDR cDNA encodes a functional HDR enzyme. The expression of TwHDR was induced by methyl-jasmonate (MJ) in T. wilfordii suspension cells. The expression of TwHDR reached the highest level after 1 h of MJ treatment. These results indicate that we have identified a functional TwHDR enzyme, which may play a pivotal role in the biosynthesis of diterpenoid triptolide in T. wilfordii.     

雷公藤甲素对Heymann肾炎模型足细胞病变的影响

目的利用被动型Heymann肾炎模型(passive Heymann nephritis, PHN)研究雷公藤甲素(triptolide)对膜性肾病的疗效及其对足细胞病变的影响. 方法实验动物分为正常对照组、PHN组和雷公藤甲素治疗组.雷公藤甲素(200 μg/kg·d)灌胃治疗,分别在观察7天、14天、21天和28天时宰杀大鼠,留取血清和尿标本,观察尿蛋白、血清白蛋白、肝酶、肌酐和外周血白细胞等指标的变化;留取肾组织标本,行光镜、免疫病理、电镜和免疫电镜检查,并对肾组织IgG、C5b-9沉积以及Nephrin和Podocin的分布进行观察. 结果(1)尿蛋白雷公藤甲素治疗7天即可显著降低PHN大鼠的蛋白尿,至14天、21天和28天时,治疗组大鼠的尿蛋白进一步降低,与Heymann肾炎大鼠之间差异有统计学意义(P＜0.01).在尿蛋白显著降低的同时,血浆白蛋白显著增加.(2)肾小球上皮侧免疫沉积物电镜观察结果显示,雷公藤甲素治疗组上皮侧免疫复合物、钉突以及基膜反应性增殖均较Heymann肾炎组似有所改善,治疗后上皮侧电子致密物减少,基膜反应减轻,但在尿蛋白明显减少的同时,上皮侧电子致密物始终存在.(3)肾小球IgG和C5b-9的沉积经雷公藤甲素治疗后肾小球IgG和C5b-9的沉积与Heymann肾炎大鼠相比,荧光强度有所减少,尤其在治疗后的第7天,减少较为明显.(4)足细胞病变经雷公藤甲素治疗7天、14天、21天和28天后,均可见足细胞的足突损伤比对照组明显减轻,足突融合显著改善,足突宽度显著降低,观察至第28天时,治疗组可见大部分足细胞的足突形态基本恢复正常.(5)足细胞裂孔膜蛋白的变化经雷公藤甲素治疗7天后,足细胞表面Nephrin和Podocin的表达比Heymann肾炎大鼠有明显增加,分布上的异常开始得到纠正,至第21天基本恢复成连续的线样分布.免疫电镜的结果再次显示了治疗后Nephrin的上述修复过程. 结论雷公藤甲素对被动型Heymann肾炎具有显著的治疗作用,能有效地减少蛋白尿,减轻肾组织免疫损伤,促进足细胞病变和裂孔膜蛋白结构的修复.雷公藤甲素疗效机制除了其免疫抑制和抗炎作用外,还与它能显著地改善和修复足细胞病变有关.     

雷公藤内酯醇在小鼠同种异体胰岛移植中的抗排斥作用

目的探讨雷公藤内酯醇（triptolide,TPT）在小鼠同种异体胰岛移植中的抗排斥作用及其机理。方法采用BALB/c小鼠作为供体,进行胰岛分离,以链脲佐菌素（streptozotocin,STZ）诱导的C57BL/6糖尿病小鼠作为受体,行左肾被膜下胰岛移植。将移植后糖尿病小鼠随机（随机数字表法）分为3组,每组8只。于胰岛移植术后前5 d分别给予腹腔注射1%吐温80溶剂（对照组）、TPT 50μg/kg（L-TPT组）和100μg/kg（H-TPT组）,之后隔天注射1次,至术后第14天结束。术后监测受体血糖水平变化;并于术后第10天每组随机（随机数字表法）选取3只小鼠,切取左侧肾脏行病理学检查,流式细胞术检测脾淋巴细胞中CD4＋CD25＋Foxp3＋调节性T细胞比例。结果对照组、L-TPT组和H-TPT组移植胰岛的中位存活时间分别为12.6 d（9～16 d）、21.4 d（14～27 d）和27.6 d（19～34 d）;脾淋巴细胞中CD4＋CD25＋Foxp3＋调节性T细胞比例分别为（5.2±0.6）%、（12.0±1.3）%和（15.7±1.8）%。与对照组相比,L-TPT组和H-TPT组小鼠移植胰岛的中位存活时间明显延长（P〈0.05）,CD4＋CD25＋Foxp3＋调节性T细胞比例显著增高（P〈0.05）。结论 TPT通过上调移植受体CD4＋CD25＋Foxp3＋调节性T细胞比例,减轻了胰岛移植后的排斥反应,显著延长了移植胰岛的存活时间,其免疫抑制作用呈剂量依赖性。     

雷公藤甲素配伍芍药苷对L-02细胞毒性的影响

目的:研究芍药苷配伍雷公藤甲素对L-02细胞毒性的影响。方法:采用MTT法比较雷公藤甲素单用与雷公藤甲素配伍芍药苷对人肝细胞L-02细胞存活率的影响,以研究芍药苷对雷公藤甲素的减毒作用。结果:雷公藤甲素对L-02细胞的无毒范围在6.250 0 ng/ml以下,芍药苷对L-02细胞的无毒范围在1 000.0μg/ml以下。芍药苷与雷公藤甲素以1 000:1的比例配伍可降低雷公藤甲素对L-02细胞的毒性作用,减少雷公藤甲素对L-02细胞损伤,提高细胞存活率。结论:配伍芍药苷可降低雷公藤甲素的细胞毒性,这为后期研究和药物比例选择提供了依据。     

Prediction of triptolide targets in rheumatoid arthritis using network pharmacology and molecular docking

Network pharmacology is a novel approach that uses bioinformatics to predict and identify multiple drug targets and interactions in disease. Here, we used network pharmacology to investigate the mechanism by which triptolide acts in rheumatoid arthritis (RA). We first searched public databases for genes and proteins known to be associated with RA, as well as those predicted to be targets of triptolide, and then used Ingenuity Pathway Analysis (IPA) to identify enriched gene pathways and networks. Networks and pathways that overlapped between RA-associated proteins and triptolide target proteins were then used to predict candidate protein targets of triptolide in RA. The following proteins were found to occur in both RA-associated networks and triptolide target networks: CD274, RELA, MCL1, MAPK8, CXCL8, STAT1, STAT3, c-JUN, JNK, c-Fos, NF-κB, and TNF-α. Docking studies suggested that triptolide can fit in the binding pocket of the six top candidate triptolide target proteins (CD274, RELA, MCL1, MAPK8, CXCL8 and STAT1). The overlapping pathways were activation of Th1 and Th2 cells, macrophages, fibroblasts and endothelial cells in RA, while the overlapping networks were involved in cellular movement, hematological system development and function, immune cell trafficking, cell-to-cell signaling and interaction, inflammatory response, cellular function and maintenance, and cell death and survival. These results show that network pharmacology can be used to generate hypotheses about how triptolide exerts therapeutic effects in RA. Network pharmacology may be a useful method for characterizing multi-target drugs in complex diseases.     

双波长薄层扫描法测定优力藤片中雷藤素甲的含量

用双波长薄层扫描法测定优力藤片（ＹＯＬＩＴＥＮ）中雷藤素甲（ｔｒｉｐｔｏｌｉｄｅ）的含量。采用硅胶Ｇ－ＣＭＣＮａ薄板，样品经氧化铝吸附、氯仿回流提取、浓缩后点样。展开剂为乙酸乙酯－氯仿（１．３：２．０），用岛津ＣＳ－９０００型扫描仪λ＿ｓ＝５１０ｎｍ，λ＿Ｒ＝６１０ｎｍ波长处扫描。随行外标两点法计算含量。线性范围为０．１２３１～０．７３８６μｇ，回归方程Ａ＝６１８０３．２６Ｃ＋５５７６．８５，ｒ＝０．９９９８。回收率为１０４．１％，ＲＳＤ＝２．７３％（ｎ＝６）。     

雷公藤内酯醇的结构修饰

为降低雷公藤内酯醇的毒性,寻找高效低毒的抗炎免疫化合物,对雷公藤内酯醇(trip-tolide,1)的结构进行了修饰,合成了九个雷公藤内酯醇衍生物。初步活性测定显示雷公藤氯内酯(tri-pchlorolide,2)和雷公藤溴内酯醇(tripbromolide,3)的免疫抑制活性与雷公藤内酯醇近似,但毒性有所降低。其他化合物的活性大大低于雷公藤内酯醇。     

雷公藤内酯醇对哮喘豚鼠血浆和肺组织内皮素1的影响

目的探讨雷公藤内酯醇(TP)干预对哮喘脉鼠血浆和肺组织中内皮素1(ET—1)水平的影响。方法42只脉鼠随机分为对照组(C)6只、哮喘组(A)18只，干预组(T)18只。后两组依次分为4小时组(A4、T4)、8小时组(A8、T8)及慢性组(A2w、T2w)，每组6只。建立哮喘模型，放免法测定血浆和肺组织ET—1。结果A4、T4、A8组肺组织ET—1显高于血浆，C、T2w组血浆ET—1显高于肺组织。较C组，A4、T4、A8、T8、A2w组肺组织及A8、T8组血浆ET—1显增加。各干预组肺组织ET—1显低于哮喘组。结论推测抑制肺组织ET—1的合成与分泌及其生物学效应等，可能是雷公藤治疗哮喘的机制之一。