雷公藤多甙对恶唑酮诱导小鼠肠炎模型脾淋巴细胞因子的作用及其机制

目的观察小鼠肠炎模型体外实验中,雷公藤多甙(MGT)对唑酮(oxazolone,OXZ)诱导的小鼠肠炎模型中脾脏淋巴细胞细胞因子分泌类型的影响,从免疫学、分子生物学角度探讨MGT治疗炎症性肠病(IBD)的作用机制.方法采用SJL/J小鼠,经直肠注入OXZ制成IBD模型;3d后将小鼠处死,即刻取出脾脏,收集脾细胞,将MGT(0.1和0.01 mg/ml两个浓度)分别加入培养的淋巴细胞液中,行FILISA检测白介素-4(IL-4)和γ-干扰素(IFN-γ).结果1.MGT对IFN-γ生成的影响(1)正常对照组未加入MGT组(空白对照)的IFN-γ为(1 24±0 13)pg/ml;0.01mg MGT组为(0.97±0 26)pg/ml;0.1mg MGT组为(0 87±0 18)pg/ml;(P＜0.02);(2)OXZ肠炎模型组与正常对照组比较,未加入MGT组(空白对照)IFN-γ显著降低[(0 65±0 08)pg/ml比(1.24±0.13)pg/ml,P＜0.01],0.01mg MGT组和0.1mg MGT组IFN-γ均低于空白对照[分别为(0.47±0.05)pg/ml;(0 46±0 09)pg/ml],但差异无显著性.2.MGT对IL-4生成的影响(1)正常对照组未加入MGT组(空白对照)的IL4为(5.65±0.48)pg/ml;MGT有显著抑制作用[0.01 mg/ml MGT组为(4.97±0.38)pg/ml;0.1 mgMGT组为(3.98±0.32)pg/ml;P＜0.01];(2)0XZ肠炎模型组与对照组比较,未加入MGT组(空白对照)IL-4显著升高[(7.83±0.69)pg/ml比(5.65±0.48)pg/ml,P<0.01];0.01mg MGT组(7.07±0.47)pg/ml和0.1 mg MGT组(6.35±0.48)pg/m1与空白对照组比较,差异有显著性(P<0.01).结论雷公藤既可抑制IFN-γ(Th1)生成,亦可抑制IL-4(Th2)分泌.由于OXZ诱导的小鼠实验性肠炎模型系以1L-4过量生成为主的Th2型免疫反应,由此可以推测MGT治疗溃疡性结肠炎的机制可能与其抑制IL-4(Th2)生成有关.     

长期应用尼古丁对恶唑酮诱导的实验性小鼠肠炎模型的影响及其机制探讨

目的分析和探讨长期应用尼古丁对恶唑酮(OXZ)诱导的实验性小鼠肠炎的影响以及肠黏膜和脾细胞生成细胞因子的类型.方法采用BALB/c小鼠,经直肠注入OXZ制成炎症性肠病(IBD)模型;皮下注射尼古丁0.5mg@kg.@d,连续3周.将小鼠处死后,取出大肠及脾脏.病理组织学观察大肠黏膜改变.采用ELISA法测定大肠黏膜及脾细胞产生的干扰素γ(IFN-γ)和白介素-4(IL-4).采用流式细胞仪检测技术(FACScan)分析脾细胞内细胞因子IFN-γ和IL-4的表达.结果尼古丁组的病理组织学计分显著低于OXZ组(19.8比23.7,P＜0.02).与对照组比较,OXZ组大肠黏膜[(185±47)pg/g比(94±14)pg/g]和脾细胞生成的IL-4明显高于对照组[(59±12)pg/ml比(10±1)pg/ml];与OXZ组比较,尼古丁组IL-4生成量更趋降低[肠黏膜(157±38)pg/g比(185±47)pg/g,P＜0.05;脾细胞(50±13)pg/ml比(59±12)pg/ml,P＜0.05].OXZ组IFN-γ/IL-4比值明显低于对照组(黏膜1.10±0.37比3.40±0.35,P＜0.02;脾细胞275±1.90比30.70±3.90,P＜0.01),与尼古丁组差异无显著性(黏膜110±0.37比0.78±0.14;脾细胞2.75±1.90比0.78±0.40).OXZ组,表达IL-4的CD+4细胞数较表达IFN-γ的CD+4细胞数高13.6倍.尼古丁组表达IL-4及IFN-γ的CD44+细胞数仅为OXZ组的32%和39%.结论OXZ诱导的小鼠IBD肠炎模型是Th2亚型(IL-4升高)为主的免疫应答反应;长期应用尼古丁,通过抑制IL-4生成,从而减轻OXZ诱导的实验性小鼠肠炎的组织学损伤.     

雷公藤多甙体外对成骨细胞功能表达的影响

目的　观察雷公藤多甙 (MT)体外对成骨细胞 (OB)功能表达的影响 ,以进一步探讨雷公藤 (TW)致女性骨质疏松的机制。方法　分离新生SD大鼠头盖骨OB进行原代培养 ,经 5～ 6d至汇合 ,传代后加入不同浓度MT ,用对硝基苯磷酸法和考马斯亮蓝法作碱性磷酸酶 (ALP)比活性测定 ,茜素红染色法作钙化结节计数测定OB矿化能力。结果　培养 6d ,MT 1μg/ml起显著抑制OBALP比活性 (P <0 0 5 ) ;培养 2周 ,MT 10 -4 μg/ml起显著抑制OB矿化能力 (P <0 0 5 ) ;药物对OB功能表达的抑制程度与其剂量密切相关 (P <0 0 5 ,P <0 0 1)。结论　MT显著抑制OB功能表达且呈剂量依赖性。TW对OB的直接抑制作用 ,是其导致药物性骨质疏松的一个重要原因。     

雷公藤多甙对胶原诱导关节炎大鼠的治疗作用及其作用机制

目的探讨雷公藤多甙对类风湿关节炎(rheumatoid arthritis,RA)动物模型,胶原诱导关节炎(collagen induced arthritis,CIA)大鼠血清炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)、血管内皮生长因子(vascuoar endothelial cell growth factor,VEGF)、基质金属蛋白酶(matrix metalloproteinases,MMP)-1、2、9表达的影响,及对成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)ERK信号转导通路的影响,研究雷公藤多甙治疗RA的可能机制。方法采用Ⅱ型胶原建立CIA大鼠模型;观察雷公藤多甙治疗后模型大鼠关节肿胀情况,用免疫组化法观察大鼠关节炎症及滑膜微血管密度;用Western blot法检测CIA大鼠血清IL-1β、TNF-α、VEGF、MMP-1、2、9的表达,并用胶原酶消化法分离培养正常大鼠及CIA大鼠原代FLS;用Western blot法检测不同浓度雷公藤多甙治疗后CIA大鼠FLS中ERK和p-ERK的表达。结果 CIA大鼠造模成功,雷公藤多甙治疗能降低CIA大鼠血清炎症因子及血管新生相关因子表达(P0.05);免疫组化结果显示雷公藤多甙能抑制CIA大鼠关节滑膜血管新生(P0.05);不同浓度雷公藤多甙对FLS中ERK蛋白表达无明显影响,但可降低FLS的p-ERK表达(P0.05)。结论雷公藤多甙对CIA大鼠抗炎及抑制血管新生的作用可能与调节ERK信号转导通路有关。     

吡格列酮对噁唑酮诱导的小鼠实验性结肠炎模型的影响

目的　观察吡格列酮对唑酮结肠炎的干预 ,探讨其是否通过Fas Fas配体 (FasL)途径促进细胞凋亡参与免疫调节作用。方法　经唑酮诱导成功的BALb/c小鼠 ,每组 6只 ,第 1组于灌肠后3d分离结肠固有层单个核细胞 (LPMC) ,并在体外用吡格列酮 (40 μmol/L)处理 2 4h ,采用annexin V FITC试验法分析凋亡细胞的百分率 ,采用流式细胞术检测Fas及FasL表达 ;并没未处理的正常小鼠组。第 2组 (对照组 )和第 3组 (实验组 )于灌肠后 3d分别给予二甲基纤维素和吡格列酮 (2 0mg·kg-1·d-1)连续 7d ,结肠炎症的评价包括炎症活动指数 (DAI)、大体形态损伤、组织学改变及肠黏膜髓过氧化物酶 (MPO)活性 ,采用ELISA法检测白细胞介素 (IL) 4和IL 5。结果　正常小鼠和结肠炎小鼠结肠组织LPMC凋亡率、Fas、FasL表达分别为 12 .89± 1.2 3、70 .6 3± 6 .2 4、8.5 9± 5 .4 7和 4 .2 5± 0 .84、6 2 .6 0±5 .85、2 3.75± 10 .2 3;经吡格列酮处理后 ,分别为 4 0 .5 8± 10 .32、83.98± 11.38、10 .0 4± 5 .2 1。对照组和实验组大体形态、组织学损伤、MPO值、IL 4、IL 5分别为 2 .5 0± 0 .5 5、8.83± 0 .75、3.81± 0 .17、2 16 .4 6± 34.32、10 2 .2 8± 2 5 .74和 0 .33± 0 .5 2、4 .0 0± 0 .6 3、1.2 5± 0 .16、1     

雷公藤多甙对糖尿病肾病蛋白尿的影响

目的 观察雷公藤多甙对糖尿病肾病患者蛋白尿的作用及安全性.方法 选取天津医科大学代谢病医院2008年4月至12月肾病科住院及门诊的81例2型糖尿病患者为研究对象,24 h尿蛋白定量≥1.0 g,血清肌酐(Scr)<265.2 μmol/L.随机分为4组,对照组(21例)应用常规荆量血管紧张素转换酶抑制剂(ACEI)或血管紧张素受体拮抗剂(ARB),治疗组在应用常规剂量ACEI或ARB基础上,分别给予雷公藤多甙20 mg/d(低剂量组)、30 mg/d(中剂量组)、40 mg/d(高剂量组)(每组各20例),疗程3个月,观察治疗前后24 h尿蛋白定量、肝肾功能及血常规变化.结果 治疗组与对照组24 h尿蛋白定量均减少.雷公藤多甙低剂量、中剂量、高剂量治疗组24 h尿蛋白分别由治疗前(2.7±0.9)g、(2.7±0.9)g、(2.8±0.9)g减少到治疗后(2.2±1.0)g、(2.1±0.9)g、(1.9±1.0)g(P均<0.01),下降率分别为17.8%、22.8%、30.4%;对照组24 h尿蛋白由治疗前(2.4±1.2)g减少为治疗后(2.0±0.9)g(P>0.05),下降率为16.3%.雷公藤多甙治疗前后肝肾功能及外周血象比较差异均无统计学意义(P>0.05).结论 在常规治疗基础上,加用雷公藤多甙可在一定程度上降低糖尿病肾病患者蛋白尿水平,且随雷公藤多甙用药剂量在一定范围内增加,24 h尿蛋白定量下降率增大,趋势检验有统计学意义(P<0.05),对患者的肝、肾功能及外周血象未见明显不良影响.     

雷公藤多甙对大鼠肾移植急性排异反应治疗作用的观察

利用大鼠肾移植模型,观察雷公藤多甙(GTW)、雷公藤甲素及小剂量环孢霉素A(CsA)对大鼠肾移植急性排异反应的影响。结果证实,应用GTW或CsA后大鼠移植肾肾长明显减少,肾重明显减轻,肾小球基底膜增厚,肾小管空泡变性、坏死及间质水肿明显减轻;利用微机肾组织切片定量分析示移植术后第四、八天,肾小管面积、数量也明量增加,而间质面积明显减少。雷公藤甲素来证实有上述作用.另外,观察应用GTW治疗者,移植术后第九～第11天存活率明显增加.     

三硝基苯磺酸和噁唑酮诱导肠炎小鼠脾脏中Th1/Th2类细胞因子基因表达的动态研究

目的 对比研究三硝基苯磺酸（TNBS）和 唑酮（OXZ）诱导的实验性小鼠肠炎模型脾脏中Th1／Th2类细胞因子基因表达的动态变化。方法 雌性BALB／c小鼠，随机分为50％乙醇对照组，OXZ诱导肠炎模型组，TNBS诱导肠炎模型组（保证每组小鼠取材时6只以上）。采用RT—PCR方法观察OXZ和TNBS诱导肠炎后小鼠脾脏于3及7d IFN-γ、IL-4 mRNA的表达变化。结果 TNBS模型组小鼠在造模后3及7d脾脏中IFN-γ mRNA的表达量均高于OXZ模型组（P〈0．05），IL-4 mRNA的表达量低于OXZ模型组（P〈0．05）。结论 在造模后3和7d TNBS模型小鼠的外周免疫皆以Th1型为主，OXZ模型小鼠的外周免疫以Th2型为主，可代表肠道局部免疫类型。     

雷公藤多甙对气道变应性炎症作用的实验研究

采用超声雾化吸入1％卵蛋白生理盐水致敏，并以同法诱喘制备豚鼠哮喘模型45只，随机分成3组（治疗前组、治疗组、对照组），并设正常组15只．治疗组每天灌服GTT混悬液2ml（含GTT5mg），连续灌21d．治疗前组及对照组豚鼠气管及肺组织病理切片可见气管、支气管粘膜上皮细胞之间及粘膜下层，有多量嗜酸细胞及淋巴细胞浸润，粘膜上皮细胞脱落，杯状细胞脱颗粒等变应性炎症的表现．治疗组治疗后诱喘潜伏期显著延长，发作程度也显著减轻，诱喘的卵蛋白阈浓度有所增高，提示GTT对豚鼠哮喘有明显的平喘作用．治疗组治疗后气管。支气管各层组织中嗜酸细胞和淋巴细胞浸润减轻，粘膜上皮细胞脱落亦有改善，提示GTT对豚鼠气道变应性炎症有一定的治疗作用．     

Insulin-modulated interleukin-2 production by murine splenocytes and a T-cell hybridoma

Murine interleukin-2 (IL-2)-producing AOFS 21 T-cell hybridoma cells and normal murine splenocytes were stimulated in serum-free media by 16 potential mitogens/growth factors. Only insulin, concanavalin A (Con A), peptidoglycan monomer and a tumour-derived insulinoid stimulated [3H]thymidine incorporation by AOFS 21 cells and splenocytes. Supernatants of these stimulated cultures were tested for IL-2 activity which generally followed the pattern of growth stimulation. Both the mitogenicity and stimulation of IL-2 secretion by insulin were second only to the effects of Con A.     

Tripterygium glycosides suppress production of interleukin‐4 by splenocytes in oxazolone‐induced murine colitis

OBJECTIVE: To investigate the in vitro effect of Tripterygium glycosides (TG) on cytokine production by splenocytes in oxazolone (OXZ)‐induced colitis in mice. METHODS: Oxazolone (6 mg in 50% ethanol) was administered to male SJL/J mice intrarectally to induce colitis and the mice were killed 3 days later. Isolated splenocytes were cultured for 24 h in the presence of phorbol myristate acetate and ionomycin. A preparation of Tripterygium glycosides at a concentration of either 0.1 mg/mL or 0.01 mg/mL was added to the culture medium of splenocytes. Production of interferon gamma (IFN‐γ) and interleukin‐4 (IL‐4) in the supernatant were measured by ELISA. RESULTS: Production of IFN‐γ in the normal control group was suppressed by TG at both concentrations (0.01 and 0.1 mg/mL; 1.24 ± 0.13 pg/mL (samples without TG) → 0.97 ± 0.26 pg/mL (0.01 mg/mL TG) → 0.87 ± 0.18 pg/mL (0.1 mg/mL TG); P < 0.02) in a dose dependent manner. In the OXZ‐induced colitis group, the basic level of IFN‐γ was significantly lower than that of the normal control group (1.24 ± 0.13 pg/mL vs 0.65 ± 0.08 pg/mL; P < 0.01); but IL‐4 production was significantly increased in the OXZ‐induced colitis without TG group (7.83 ± 0.69 pg/mL vs 5.65 ± 0.48 pg/mL, P < 0.01). In both groups, TG suppressed IL‐4 production in a dose‐dependent manner (normal control group: 5.65 ± 0.48 pg/mL (samples without TG) → 4.97 ± 0.38 pg/mL (0.01 mg/mL TG) → 3.98 ± 0.32 pg/mL (0.1 mg/mL TG), P < 0.01; OXZ group: 7.83 ± 0.69 pg/mL (samples without TG) → 7.07 ± 0.47 pg/mL (0.01 mg/mL TG) → 6.35 ± 0.48 pg/mL (0.1 mg/mL TG), P < 0.01). CONCLUSION: Oxazalone‐induced IL‐4 overproduction by splenocytes was significantly suppressed by TG in a dose dependent manner and the beneficial effects of TG on ulcerative colitis might be related to the suppression of the Th2 type (e.g. IL‐4) mediated immunological response of splenocytes.     

阿泰宁对噁唑酮诱导的大鼠结肠炎模型的治疗作用

目的:建立嗯唑酮诱导的大鼠结肠炎模型.观察阿泰宁对噁唑酮诱导的大鼠结肠炎的治疗作用及机制.方法:经直肠注入嗯唑酮建立大鼠溃疡性结肠炎模型.将大鼠随机分为:空白对照组(正常组,n=8),模型组(n=10),美沙拉秦组(正常组,n=10),阿泰宁组(n=10),治疗21d后处死动物,肉眼观察结肠病变,分别测体质量、结肠湿质量、脾脏质量和结肠组织病理学变化,ELISA法测定大鼠血清IL-1β、IL-10、TNF-α以及结肠黏液内容物sIgA含量,考马斯亮兰法测定血清总蛋白,溴甲酚绿比色法测定白蛋白以及肠道菌群培养.结果:模型组、阿泰宁组和美沙拉秦组大鼠体质量均比正常组显著降低(均P＜0.01).美沙拉秦组和阿泰宁组血清球蛋白含量、结肠湿质量指数较模型组差异显著(29.9±5.7,29.1±5.4vs23.7±9.5:6.0±0.9.6.2±0.4vs7.4±1.6,均P<0.05).模型组大鼠血清IL-1β和TNF-α含量较正常组、美沙拉秦组和阿泰宁组差异显著(44.6±17.2vs8.8±7.9,14.5±4.7,8.6±3.4,均P<0.01;33.5±7.2 vs 22.6±6.7,22.3±9.2,24.4±10.8,均P＜0.05).模型组大鼠血清IL-10含量较正常组、阿泰宁组差异显著(101.5±35.8vs280.5±36.1,271.3±33.8,P<0.01).阿泰宁组脾脏指数、sIgA含量比正常组显著升高(3.4±0.8vs2.7±0.3;46.0±20.3vs23.4±18.5,均P<0.05),而美沙拉秦组差异不显著.较模型组双歧杆菌数量,阿泰宁治疗后明显上升,梭杆菌数量明显下降(9.7±0.1vs9.3±0.2:3.7±0.3vs5.8±0.7,均P<0.01).结论:阿泰宁能够有效治疗噁唑酮诱导的大鼠结肠炎.     

Characterization of murine hematopoietic progenitor subsets involved in interleukin-3-induced interleukin-6 production

Various murine cell populations were tested for their ability to generate interleukin-6 (IL-6) in response to IL-3. Among these, bone marrow cells exhibit the most prominent IL-6 production. The responder cells in this organ have been further characterized by cell fractionation on a discontinuous Ficoll gradient, fluorescence-activated cell sorting, and in situ hybridization. These procedures have allowed us to ascribe the following features to the cells mainly responsible for IL-3-induced IL-6 production: (1) they possess a low density and a relatively high forward and perpendicular light scatter (FLS/PLS); (2) they are characterized by a high rhodamine (Rh) retention; and (3) their enrichment in various subpopulations is similar to that obtained for progenitors forming colonies in the methylcellulose assay colony-forming units (CFU-C). In contrast, IL-3 target cells in terms of IL-6 production are absent both in the mature and in the most immature bone marrow compartment. Indeed, the Rh-dull population that is enriched for cells with marrow repopulating activity does not respond to the growth factor and mature cells cannot be induced to express IL-6 as assessed by (1) FLS/PLS characteristics, (2) the monoclonal antibody ER-MP 20 recognizing monocytes and granulocytic cells, and (3) in situ hybridization. Taken together, our data support the conclusion that the bone marrow cells generating IL-6 in response to IL-3 belong to a progenitor population with enhanced mitochondrial activity, comprising probably several types of immature cells of the myeloid lineage including macrophage/granulocyte precursors.