【作者】 李俊；

【导师】 凌昌全；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2012， 硕士

【摘要】 背景：糖皮质激素（Glucocorticoids，GC）是目前临床应用较多的一类药物，广泛用于炎症及免疫相关疾病的治疗，但是临床上有相当一部分病人的激素疗效并不理想。由于GC的药理作用主要是通过糖皮质激素受体（glococorticoid receptors,GR）介导发挥的，因此有研究认为GR的表达及其结和力是GC疗效的决定因素之一。如果GR表达或结合力处于一种较低的状态，即使体内GC水平很高，实际能与GR结合形成配体受体复合物的GC数量却有限，故激素的生物效应仍然较低。我课题组研究发现，出现激素抵抗的系统性红斑狼疮病人外周血单个核细胞GR的表达及结合力就明显低于激素敏感的病人。对于这部分病人，通常采用补充大剂量糖皮质激素，或者给予免疫抑制剂联合治疗的方法提高其疗效。然而，大剂量GC和免疫抑制剂的使用可以引起各种副作用的发生。而且长期大剂量应用激素又会进一步导致GR表达减少和结合力降低，从而加大后续治疗的难度。因此，寻找一种有效调节GR的药物一直是近年来研究的热点。近年来，中药在临床治疗中的地位越来越受到重视，我科室在前期研究中发现人参中主要有效成分人参总皂苷（ginsenosides，GSS）在体内外具有上调GR，并增强激素效应的作用。随后的临床研究也证实，GSS可以增强系统性红斑狼疮的糖皮质激素疗效，在不影响疗效的前提下有助于GC的减量。但是总皂苷成份复杂，包含了Rb1、Rb2、Rb3、Rc、Rd、Re、Ro、Rf、Rg1、Rg2、Rg3、Rh1、20(S)-Rh2、20(R)-Rh2、F1、F2等40余种单体皂苷，不利于临床推广和机制研究。因此，本研究将从总皂苷中筛选出一种或几种具有上调GR作用的单体，观察其在体内外对糖皮质激素抵抗的改善作用，并做初步机制探讨。本科题研究目的：1、从成分复杂的GSS中筛选能够上调GR表达和结合力的一种或几种单体，并筛选出上调效果最强的单体；2、以地塞米松(dexamethasone,Dex)作为阳性对照，观察该单体在体内外对糖皮质激素抵抗的炎症模型的作用，并对其机制做初步探讨。本课题采用的方法：1.采用Western-Blot、定量RT-PCR法筛选具有上调GR表达的单体，并运用放射性配基结合实验检测其对GR结合力的上调作用。2.体外观察Dex联合人参皂苷单体的抗炎作用，并初步探讨其分子机制。（1）我们先用Dex（1uM）联合人参皂苷单体(10uM)及单用Dex（1uM）预处理细胞2h（短期作用组，非激素抵抗模型）或者24h （长期作用组，激素抵模型），再用TNF-α干预8h，然后运用定量RT-PCR技术检测炎症因子IL-6、IL-17、MMP-1、TNF-α mRNA的表达，对比Dex联合人参皂苷单体与单用Dex在不同条件下的抗炎效果。（2）为了探讨Dex联合人参皂苷单体优于单用Dex的抗炎机制是否与NF-κB通路有关，我们设立短期作用组（2h）及长期作用组（24h），TNF-α干预，相同条件下，采用Western-Blot技术观察其抗炎作用是否通过影响P65通路发挥。（3）因为人参皂苷单体能够抑制GR蛋白表达及结合力的下调，我们运用双荧光素酶报告基因法观察其是否增强GRE的转录激活。同时用Dex联合人参皂苷单体及单用Dex处理肝原代细胞，观察GR的数量及结合力的提高是否会激活与高血糖相关的酶，因此我们运用定量RT-PCR法检测糖异生相关基因G-6-P、PEPCK，观察其是否处于高表达状态。3.DBA-1关节炎症模型小鼠验证所选单体的体内抗炎效果。牛Ⅱ型胶原与完全弗氏佐剂混合物诱导关节炎模型，将模型小鼠随机分组分成生理盐水（Nerative）组、Dex（1.5mg/kg·d）组、Dex（1.5mg/kg·d）+Rh1（25mg/kg·d）组，连续给药14天。每天观察小鼠关节炎症反应情况，并给予评分，治疗结束后观察血糖，定量RT-PCR法检测GR、G-6-P、PEPCK的表达。结果表明：1.人参总皂苷中三个单体Rh1，Rb1，Rb3具有抑制GR表达和结合力下调的作用，其中Rh1作用最为明显；2.体外研究显示，人参皂苷Rh1与Dex联合应用对TNF-α诱导生成的炎症因子IL-6、IL-17、MMP-1、TNF-α mRNA表达具有明显的抑制作用（P<0.01），即使对Dex处理24h后的细胞（激素抵抗模型），该联合方案也具有明显的抑制作用，而单独使用Dex则抑制炎症作用减弱。进一步研究发现，无论是在激素抵抗还是非激素抵抗模型中，人参皂苷Rh1联合Dex均可以抑制P65核转位，抑制IkB磷酸化,而单独使用Dex对激素抵抗的P65通路则无抑制作用。以上作用与Rh1抑制GR表达和结合力下调密切相关。由于Rh1具有抑制GR的下调作用，双荧光素酶报告基因结果也显示与Dex组相比，Rh1联合Dex可以轻微增强GRE的转录激活，但有趣的是，对GRE激活的糖异生相关基因G-6-P、PEPCK，Rh1联合Dex却没有激活作用。体内研究采用DBA-1关节炎模型小鼠，研究证实，与单独给予Dex比较，Rh1联合Dex能够更有效地抑制小鼠关节炎症反应（P<0.01），且不会引起GR下调，同时也不会产生血糖升高的副作用。结论:人参皂苷单体Rh1通过抑制GR下调明显改善激素抵抗，同时抑制高血糖的发生，因此可以作为激素辅助用药增强疗效，减轻副作用，值得临床进一步验证。更多还原

【Abstract】 BackGRound：Glucocorticoids (GCs) are widely used in treating inflammatoryand immune related diseases. However, although the first impressions were positive, itsoon became clear that the frequent and prolonged administration of GCs influence adiverse set of key biological functions and cause severe, sometimes irreversible,side-effects. The physiological and pharmacologic effects of GC are mainly mediated byglucocorticoid receptors (GRs). It is reported that the quantity and binding capacity of GRsare important in determinanting the curative effects of GCs. Even if the level of GCs arehigh in vivo, the effects of GCs are still poor for few quantities and binding of GR, whichcould binding with ligand are very small. In our prior studies, the GRs expression andbinding capacity in peripheral blood mononuclear cells (PBMC) from systemic lupuserythematosus patients with GRs resistance are lower than those in GCs sensitive patiens.For good curative effects, we often treat the patients with high-dose GCs, or combinedwith immunosuppressive agents, whih give rise to various of side effects. Moreover,high-dose GCs used in a long time is account for further reduced expresson and bindingcapacity of GRs, which will make the subsequent therapy more difficult. As a result, a newmedicine which can regulate GCs effectively is still the hot ports.In recent years, Chinese herbs exert an increasingly effect in clinical treatment. Inour previous research, we found Ginsenosides (GSS) could induce a homologousupregulation of GRs and enhance the effects of GCs. In the subsequent clinical studies, it isrevealed that GSS could enhance the effects of GCs in treating systemic lupuserythematosus patients, and reduce the dose of GCs without reduced the clinical effects.The GSS includes about40Ginsenosides,such as Rb1、Rb2、Rb3、Rc、Rd、Re、Ro、Rf、Rg1、Rg2、Rg3、Rh1、20(S)-Rh2、20(R)-Rh2、F1、F2,ect.Because of its complicatedcompositions,GSS can not be used in clinical treatment widely,and it is difficult to clarifyits possible mechanism in clinic.OBJECTIVE:1.Pick out one or several Ginsenoside(s),which can upregulate thelevel of the expression and binding capacity of GRs. Then find out the most effectivecomponent on GRs from them.2.Study the selected-Ginsenoside’s effects on inflammationmodels on GCs resistance in vivo and in vitro, and exlpore its possible mechanism.METHODS:1.We first pick out the Ginsenoside(s) which can upregulate the GRsprotein levels by Western-Blot and quantity RT-PCR methods. Radioligand binding analysis is used for GR binding capacity.2.Observe the anti-inflammatory effects of Dex combined with the selected-Ginsenoside in vitro and vivo and explore its possible mechanism.（1）We compared the therapeutic potential of Dex combined with the selected-Ginsenoside&Dex alone treatment protocols in two different experimental settings. FirstRAW264.7cells were pretreated with solvent, Dex (1μM) or the selected-Ginsenoside (10μM) combined with Dex (1μM) for2h (short-term treatment protocol) or24h (prolongedtreatment protocol).Then we use quantity RT-PCR method to test the pro-inflammatorymediators IL-6, IL-17, MMP-1and TNFα.（2）We tested whether the enhancement of the inhibition of Dex combined with theselected-Ginsenoside on pro-infalmmatroy cytokines is in a NF-κB dependent way. Theshort-term and the long-time treatment protocol was performed as above described.First,weuse Western-Blot method to test whether Dex combined with the selected-Ginsenosideinhibit the translocation of P65more efficiently compared with Dex alone in both theshort-term and the long-time treatment protocols.And in order to investigate the molecularbasis of the altered subcellular localization of P65, we detect phospho-IκBα and total IκBαprotein still by Western-Blot method.（3）Because the selected Ginsenoside can upregulate GRs in quantity and bindingcapacity, we tset whether this upregulation would result in enhancement of GRE promotoractivity by dual-luciferase reporter gene assay. We use RAW264.7cells,which werepretreated with solvent, Dex (1μM) or Dex (1μM) combined with the selected-Ginsenoside (10μM) for12h. Meanwhile we use primary hepatocytes which were treatedwith solvent, Dex alone or Dex combined with the selected-Ginsenoside for12h toobserve whether the upregulation of GRs would cause a hyperglycemic side effect.（4）We also use bovine type II collagen-induced arthritis (CIA) mouse model ofDBA-1mouse to anti-inflammation properties in vivo.The arthritis (CIA) mice wererandomly divided into3groups according to the scores of inflammation as follows: normalsaline (NS) group,Dex (1.5mg/kg·d) group, Dex（1.5mg/kg·d）+Rh1(25mg/kg·d)group.They were given treatment one time every day,in continuous10days.We observedtheir inflammatory symptoms and scored them everyday.The arthritis mice weresacrificed after10days treatment.Then we detect glycemic levels,and tested the level ofGR、G-6-P、PEPCK by quantity RT-PCR method.RESULTS:1. Ginsenoside Rb1、Rb3、Rh1can upregulate the level of GR in quantity and binding capacity. Ginsenoside Rh1is the most effective in upregulating GR amongthem in the future study.2. In vitro study, Dex and Dex combined with Rh1both can repress the mRNAexpression of IL-6, IL-17, MMP-1and TNFα efficiently, which induced by TNF-αin theshort-term treatment protocol (P<0.01). However, Dex combined with Rh1could represspro-inflammatory cytokine mRNA expression efficiently even in the long-time treatmentprotocol, while Dex couldn’t. The parallel western blot experiment and radioligand bindinganalysis confirmed that, in the prolonged treatment protocol, the effect of Dex on GRexpression and binding capacity became more pronounced than in the short-term treatmentprotocol, thereby explaining the abolishment of its anti-inflammatory effect. The sameresults were also found in P65pathway.3. For up-regulating GRs expression and binding of Rh1, GRE promotor activity wasdetermined. Interstingly, the results of the dual-luciferase reporter gene assay showedthat compared to Dex alone,Dex combined with Rh1enhance the Dex-induced GREpromotor activity slightly in RAW264.7cells, but could not improve the expression ofPEPCK or G6P like Dex alone.4. In vivo investigations in the arthritis (CIA) mouse model of DBA-1mouse showedthat Dex combined with Rh1could inhibit the inflammatory symptoms of arthritis moreefficiently, compared to Dex alone(P<0.01). However it did not affect blood glucose leveland downregulated the express of GRs, while Dex group had a significant increase inblood glucose levels and lower GRs expression(P<0.01).CONCLUSIONS: Ginsenoside Rh1could ameliorate GCs resistance in vivo and invitro without inducing side effects, such as hyperglycemia.更多还原