黄芩苷和黄芩素的抗炎和免疫调控作用研究
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摘要
研究背景
黄芩苷(Baicalin)及其苷元黄芩素(Baicalein)是从黄芩根中分离的黄酮类化合物，具有广泛的药理作用，其中以抗炎和免疫调控作用尤为突出。在多种炎症和自身免疫性动物模型上黄芩苷显示明确的抗炎和免疫调控作用，这些模型包括：急性胰腺炎，哮喘，急性肺损伤，急性肝损伤，肝纤维化，实验性牙周炎，实验性自身免疫性脑脊髓炎等。黄芩苷体内给药一般可以减轻组织炎症性损伤，下调模型动物血浆促炎细胞因子水平（TNF-α、IL-1、IL-6等），并可以调节某些免疫细胞分泌细胞因子，如抑制哮喘模型动物的脾细胞分泌IL-4和IL-5，增加IL-10；抑制实验性自身免疫性脑脊髓炎动物淋巴结细胞分泌IFN-γ，但增加IL-4分泌。体外实验表明：黄芩苷对刀豆蛋白(Con A)、佛波醇酯（PMA）/离子霉素(ionomycin)、或内毒素（LPS）等多种丝裂原诱导的淋巴细胞增殖均有明显的抑制作用，进一步提示淋巴细胞可能是黄芩苷免疫调控的重要靶细胞。来自肿瘤细胞的研究表明黄芩苷和黄芩素不仅调控细胞的增殖而且具有促凋亡活性，但目前对黄芩苷和黄芩素是否也调控免疫细胞的凋亡尚未进行充分研究。
免疫细胞的正常凋亡是机体建立自身免疫耐受、维持免疫系统自稳、以及避免自身免疫应答发展的的重要机制。而该调控机制出现异常则与各种自身免疫性疾病的发生和发展密切相关。转基因动物实验表明，选择性的敲除免疫细胞的促凋亡基因，导致其不能正常凋亡、大量累积，从而可通过慢性淋巴细胞激活反应加速自身免疫反应的发展。在多种自身免疫性疾病动物模型或临床病人体内也发现免疫细胞的凋亡机制发生异常，即相关炎症组织或免疫器官内免疫细胞凋亡减少，使大量的炎性细胞和活化的淋巴细胞蓄积，导致炎症持续反复的发作，这可能是自身免疫性疾病患者自身组织炎性损伤和器官功能受损的主要原因之一。许多对类风湿性关节炎等自身免疫性疾病有明显的防治作用的免疫抑制剂，包括糖皮质激素、环孢霉素(cyclosporine A，CsA)、FK506、雷帕霉素(rapamycin，RAPA)1α,25(OH)2D3等，不仅抑制T细胞的活化增殖，同时也能诱导激活的T细胞凋亡来发挥免疫抑制作用，这是免疫抑制剂治疗自身免疫性疾病的重要作用机制之一。目前对涉及淋巴细胞死亡的活化诱导的细胞死亡(activation-induced cell death，AICD)调控机制已经有了较深入的研究，普遍认为，AICD是机体清除活化的淋巴细胞，避免过强免疫应答和维持外周免疫耐受的一种重要的负反馈机制，因而通过调节免疫细胞的凋亡机制促进其凋亡是进行免疫抑制和防治自身免疫性疾病的有效手段。
基于上述研究进展，本项目首先重点探讨了黄芩苷和黄芩素在体外细胞模型上对不同种类和活化状态的免疫细胞凋亡的影响，进而采用胶原诱导的关节炎和Con A诱导的免疫性肝炎小鼠模型，探讨了黄芩苷和黄芩素防治自身免疫性疾病的效应及其免疫抑制和体内诱导T和B淋巴细胞凋亡的作用。
研究目的
进一步了解黄芩苷和黄芩素的抗炎和免疫调控作用机制及其应用潜力，为它们的临床应用提供研究基础。
研究方法
1、采用Con A和LPS刺激的小鼠脾细胞、抗CD3/CD28抗体刺激的免疫磁珠纯化的CD3~+T淋巴细胞、LPS刺激的免疫磁珠纯化的CD19~+B淋巴细胞、小鼠腹腔渗出的巨噬细胞、胸腺细胞、骨髓细胞、骨髓衍生的树突状细胞、PMA/离子霉素刺激的Jurkat T细胞、RAW264.7巨噬细胞、bEnd.3小鼠内皮细胞、以及L929小鼠成纤维细胞等为细胞模型，分析黄芩苷和黄芩素对不同种类及不同活化状态下的免疫细胞的凋亡的影响。免疫细胞的凋亡检测采用荧光标记的免疫细胞特性表面标志抗体（CD3、CD19、CD69等）标记细胞后再行annexin V/propidium iodide(PI)染色，或用抗活化的Caspase3抗体标记，用流式细胞仪分析不同种类和不同活化状态免疫细胞的凋亡率；通过流式分析细胞表面Fas和FasL的表达水平和细胞内线粒体膜电位改变，初步分析黄芩苷和黄芩素的促凋亡机制。
2、体内实验采用胶原Ⅱ（CⅡ）诱导的类风湿性关节炎小鼠（CIA）为自身免疫性疾病模型，探讨黄芩苷的抗炎和免疫抑制效应及其体内诱导淋巴细胞凋亡的作用。DBA/1小鼠于尾根部皮内接种乳化的鸡Ⅱ型胶原，21天后再用鸡Ⅱ型胶原加强免疫，诱发关节炎，于第二次免疫接种后第1天开始每天腹腔注射黄芩苷（50mg/kg体重），直至第14天。通过小鼠关节炎病理评分、关节组织病理分析、小鼠后肢计算机断层摄影（Micro-CT）分析等方法检测黄芩苷对CIA小鼠关节炎的治疗作用，采用ELISA法检测各实验组（正常对照、模型组和给药组）小鼠血清抗体水平以及小鼠脾细胞体外对CⅡ在刺激诱导的Th1和Th2型细胞因子的分泌水平分析黄芩苷的免疫抑制作用。采用TUNEL染色和荧光标记的抗Caspase3抗体进行脾脏的免疫组化分析，以及分离制备脾细胞悬液后流式分析各类免疫细胞的凋亡率，分析药物的体内促凋亡作用。
3、采用ConA诱导的免疫性肝炎小鼠模型检测黄芩素的抗炎和体内促凋亡作用。BALB/c小鼠尾静脉注射ConA（20mg/kg）诱导急性肝损伤，黄芩素（100mg/kg）于ConA注射前2h腹腔注射给药。ConA注射8h后采集外周血，检测血清ALT水平，24h取小鼠肝脏和脾脏组织，切片后进行H&E染色和免疫荧光化学分析，检测肝脏炎症和损伤程度以及各类免疫细胞凋亡情况，部分脾脏用以制备脾细胞悬液，流式分析各类免疫细胞的凋亡率，分析药物的体内促凋亡作用。
研究结果
1、黄芩苷和黄芩素在5-40μM剂量范围内能剂量依赖性地抑制ConA（5μg/ml）或LPS（10μg/ml）刺激的小鼠脾细胞增殖，表明黄芩苷和黄芩素对T和B淋巴细胞的功能具有显著的抑制作用。
2、在5-20μM剂量范围内黄芩苷和黄芩素能剂量依赖性地诱导Con A刺激的脾细胞发生凋亡，进一步分析脾细胞中CD3阳性的T细胞、CD19阳性的B细胞以及CD11b阳性的巨噬细胞凋亡的情况，结果发现黄芩苷和黄芩素对脾细胞中CD3~+T细胞和CD19~+B细胞均有有显著的促凋亡作用，但对CD11b+巨噬细胞的促凋亡作用极弱。
3、比较了黄芩苷和黄芩素（剂量为10μM，作用时间为24小时）对不同种类和不同活化状态的细胞存活的影响，发现黄芩苷对脾细胞、ConA刺激活化的脾细胞中的T细胞、LPS刺激的脾细胞中的B细胞、骨髓细胞、以及PAM/ionomicin刺激活化的Jurkat T细胞具有较强的促凋亡作用，对胸腺细胞和骨髓衍生的树突状细胞仅有较弱的促凋亡作用，而对未活化的Jurkat T细胞、腹腔渗出的巨噬细胞、RAW264.7巨噬细胞、bEnd.3小鼠内皮细胞、以及L929小鼠成纤维细胞的凋亡没有影响。说明黄芩苷并非无选择地促进所有细胞的凋亡，而是对B细胞和T细胞作用较强。尤其值得注意的是，ConA刺激活化的脾细胞中的CD3~+T细胞比na ve脾细胞中的CD3~+T细胞对黄芩苷的促凋亡作用更加敏感，PAM/ionomicin刺激活化的Jurkat T细胞比未刺激的Jurkat T对黄芩苷的促凋亡作用更加敏感，LPS刺激活化的脾细胞中的CD19~+B细胞比na ve脾细胞中的CD19~+B细胞对黄芩苷的促凋亡作用更加敏感，这些结果强烈提示：黄芩苷对活化的T和B细胞具有选择性的促凋亡作用。黄芩素的作用与黄芩苷基本类似。上述结果表明黄芩苷和黄芩素主要是促进活化的T细胞和B细胞发生凋亡。
4、采用免疫磁珠纯化的CD3~+T细胞进一步分析黄芩素和黄芩苷对T淋巴细胞的促凋亡作用，发现黄芩苷在5-20μM剂量范围内对未活化的CD3~+T细胞几乎没有促凋亡作用，但对抗CD3/CD28抗体联合刺激活化的CD3~+T细胞则有显著的促凋亡作用；黄芩素在5-20μM剂量范围内对未活化的和抗CD3/CD28抗体联合刺激活化的CD3~+T细胞均有显著的剂量依赖性的促凋亡作用，但对活化的CD3~+T细胞的促凋亡作用更强。
5、采用免疫磁珠纯化的CD19~+B细胞分析黄芩苷和黄芩素对B细胞的促凋亡作用，发现黄芩苷在5-20μM剂量范围内，作用24h，可以剂量依赖性地诱导CD19~+B细胞发生凋亡，而且LPS刺激活化的B细胞比Na ve B细胞对黄芩苷的促凋亡作用更加敏感；黄芩素促进CD19~+B细胞凋亡的作用更强，在5-20μM剂量范围内，黄芩素作用24h即能依赖性地诱导CD19~+B细胞发生凋亡（黄芩苷作用24h时对B细胞凋亡的影响极弱），而且对LPS刺激活化的B细胞的促凋亡作用明显强于Na ve B细胞。
6、脾细胞在不同浓度（0-20μM）的黄芩苷和黄芩素存在下培养24h，流式检测线粒体膜电位的变化以及脾细胞表达Fas蛋白及Fas配体的变化（Fas和FasL阳性细胞百分率）。结果发现：在5-20μM剂量范围内黄芩苷和黄芩素以剂量依赖的方式诱导脾细胞内线粒体膜电位下降，但在同样剂量范围内，脾细胞表面Fas及FasL表达的百分率未发生明显增加，表明黄芩苷和黄芩素主要通过激活线粒体介导的内源性的凋亡途径促进脾细胞的凋亡，而对死亡受体Fas介导的外源性凋亡途径无影响。
7、采用CIA小鼠为自身免疫性疾病模型，探讨黄芩苷的抗炎和免疫抑制效应及其体内诱导淋巴细胞凋亡的作用，发现黄芩苷治疗组小鼠的关节炎病理评分显著低于模型对照组，关节组织病理切片检查显示CIA模型小鼠关节组织有明显的炎性细胞浸润，滑膜增殖，关节软骨的破坏和骨质侵蚀等症状，给予黄芩苷治疗后，上述症状明显减轻或消失，说明黄芩苷有显著的抗关节炎效应；黄芩苷治疗组小鼠脾细胞在CⅡ体外再刺激下分泌IFN-γ、IL-17和IL-2的能力显著降低，血清抗CⅡ抗体水平（包括抗体总水平和IgG1、IgG2a亚型抗体水平）亦明显降低，表明黄芩苷治疗后，体内的细胞和体液免疫水平均受到抑制；脾脏冰冻切片免疫荧光分析表明黄芩苷治疗组小鼠脾细胞的凋亡明显增加，同时流式分析表明脾细胞中T和B细胞的百分率减少，T和B细胞的凋亡率增加，表明黄芩苷体内给药亦具有促进T和B细胞凋亡的效应。上述体内实验的结果表明：黄芩苷具有体内促进淋巴细胞凋亡和免疫抑制及抗关节炎效应。
8、采用ConA诱导的免疫性肝炎小鼠模型检测黄芩素的抗炎和体内促凋亡作用，发现黄芩素（100mg/kg）于ConA注射前2h腹腔注射给药可以降低ConA刺激所致的血清转氨酶升高，减轻肝组织的损伤和炎性细胞的浸润，同时增加脾脏内T、B淋巴细胞的凋亡，表明黄芩素具有体内促进淋巴细胞凋亡和防治免疫性肝炎的作用。
结论
黄芩苷和黄芩素体外对活化的T和B细胞具有选择性的促凋亡作用，其促凋亡机制与线粒体相关的内源性凋亡途径的激活有关。在CⅡ诱导的关节炎和ConA诱导的免疫性肝炎小鼠模型上，黄芩苷和黄芩素分别显示抗炎、免疫抑制和促进淋巴细胞凋亡的体内效应。这些发现拓展了对黄芩苷和黄芩素抗炎和免疫抑制作用机制的认识，进一步揭示了它们在自身免疫性疾病防治方面的应用潜力，为它们的临床应用提供了研究基础。
Background
Baicalein and its glycoside baicalin are major flavonoid constituents found in thetraditional Chinese medicinal herb Baikal skullcap (Scutellaria baicalensis Georgi), knownas “Huang qin” in China, which has been widely used for the treatment of various diseasessuch as pneumonia, hepatitis, and diarrhea. Previous studies have demonstrated thatbaicalin possess a wide range of pharmacological and biological activities such asanti-inflammatory, anti-tumor properties, anti-allergic, antimicrobial, antioxidant activities.Its anti-inflammatory activity has been estimated in various animal models of acute andchronic inflammation, including acute pancreatitis, asthma, ligature-induced periodontitis,embolism-induced acute lung injury, liver injury, hepatic fibrosis, and experimentalautoimmune encephalomyelitis. Baicalin administration generally could alleviateinflammatory tissue injury, reduce serum pro-inflammatory cytokine levels, and modulatecytokine production from immune cells, such as suppressing IL-4and IL-5but enhancingIL-10production in splenocytes in asthma model, suppressing IFN-γ but enhancing IL-4release from lymphocytes in experimental autoimmune encephalomyelitis model. In vitrostudy showed that baicalin inhibited lymphocyte proliferation stimulated by variousmotigens such as Con A, PMA/ionomycin, and LPS, further suggesting that lymphocytesmight be one of the important targets for baicalin. Evidence from studies using tumor celllines revealed that, in addition to anti-proliferating activity, baicalin also processpro-apoptotic activity. However, the influence of baicalin and baicalein on lymphocyteapoptosis has not been explored yet.
Apoptosis of immune cells represents one of the most important mechanisms ensuresthe development of protective immunity, maintenance of self-tolerance and prevention ofautoimmunity. Defects in immune cell apoptosis are closely associated with thedevelopment of various autoimmune diseases. Evidence from transgenic mice revealed thatselective knockdown of pro-apoptotic gene in immune cells resulted in their accumulationand, in turn, chronic lymphocyte activation and development of autoimmunity. Inautoimmune animal models, as well as patients with autoimmune diseases, various defectsof apoptosis in immune cells have been detected, which result in an overabundance oflymphocytes and repeat occurrence of inflammation. This might be a major cause ofinflammatory tissue injury and dysfunction of organs. Many classical immunosuppressive agents, such as cyclosporine A, rapamycin, dexamethasone, and1,25(OH)2D3, not onlyinhibit activation and proliferation of lymphocytes, but also promote their apoptosis, whichis attributed to their immunosuppressive effects. So far, the mechanisms ofactivation-induced cell death (AICD) that related to lymphocytes death have been widelyexplored, and it appears to serve as a negative immuno-regulatory mechanism that iscritical to ensure protective immunity and avoid autoimmunity. Therefore, interferencewith regulatory mechanisms of lymphocyte apoptosis represents a promising approach forthe development of new immune-modulatory therapies for autoimmune diseases.
Based on above background, the present study at first focused on the in vitro influenceof baicalin and baicalein on apoptosis of immune cells of different types and differentactivating status, and then further explored their in vivo anti-inflammatory andimmune-modulating activities, as well as their pro-apoptotic activities on lymphocytes, incollagen Ⅱ (CⅡ)-induced arthritic mice and Con A-induced immune-mediated hepatiticmice.
Aim
The present study is aimed to further understand the mechanisms underlying theanti-inflammatory and immune-modulating activities of baicalin and baicalein, and toexplore their therapeutic potential in treatment of autoimmune diseases, thus providingresearch basis for their clinical use.
Method
1. The in vitro effects of baicalin and baicalein on apoptosis of immune cells wereassayed using Con A-or LPS-stimulated mouse splenocytes, anti-CD3/CD28antibody-stimulated CD3~+T cell purified from mouse splenocytes with immunalmicrobeads, LPS-stimulated CD19~+B cells purified from mouse splenocytes with immunalmicrobeads, mouse thymocytes, bone-marrow cells, bone marrow-derived dendritic cells,PMA/ionomycin-stimulated Jurkat T cells, RAW264.7macrophages, bEnd.3endothelialcells, and L929mouse fibroblast cells. Cellular apoptosis was analyzed by flow cytometryafter stained with fluorescence-labeled antibodies against surface markers (such as CD3,CD19, and CD69) and with annexin V/propidium iodide (PI) or anti-active caspase3antibody. The effects of baicalin and baicalein on mitochondrial membrane potential insplenocyte and surface expression of Fas and FasL were assayed with flow cytometry to analyze the intracellular mechanisms of their pro-apoptotic activities.
2. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CⅡ-induced arthritic (CIA) mice. DBA/1mice were injectedintradermally at the base of the tail with0.1ml of the emulsion containing200μg of CⅡ,and then intravenously injected with0.1ml of PBS containing200ug of CⅡ at an intervalof21days to induced arthritis. Baicalin (50mg/kg) was administrated intraperitoneallydaily from days1to14after second CⅡ immunization. Arthritis symptoms were assesseddaily after drug administration by using a scoring system (0=no signs of arthritis;1=swelling and/or redness of the paw or one digit;2=two joints involved;3=more than twojoints involved; and4=severe arthritis of the entire paw and digits). The hind limbs of themice were removed postmortem, and histochemistry study with hematoxylin and eosinstaining, Micro-CT screening were performed to evaluate the anti-arthritic effects ofbaicalin. Levels of anti-CⅡ antibody in serum samples collected on day34weredetermined by ELISA. Splenocytes were prepared and cultured in medium containing CⅡ(50μg/ml) for48h, and Th1/Th2cytokines in the culture supernatants were measured byELISA. Apoptotic cells in spleen were identified by the TUNEL assay orimmunohistochemistry assay using fluorescence-labeled antibodies against CD3, CD19,and active caspase3. Lymphocyte apoptosis in splenocytes was also analyzed with flowcytometry after stained with FITC-annexin V and fluorescence-labeled antibodies againstCD3and CD19.
3. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. BALB/c mice wereintravenously injected with ConA（20mg/kg）to induced acute liver injury. Baicalein (100mg/kg) was administrated intraperitoneally2hours before ConA injection. Serum sampleswere collected8hours after ConA injection and measure for ALT levels. Liver and spleentissues were collected24hours after ConA injection, and histochemistry studies wereperformed to detect liver injury and apoptotic cells. Lymphocyte apoptosis in splenocyteswas also analyzed with flow cytometry after stained with FITC-annexin V andfluorescence-labeled antibodies against CD3and CD19.
Results
1. Baicalin and baicalein inhibited Con A or LPS-stimulated splenocytes proliferation dose-dependently in the dose range of5-40μM, indicating the inhibitory effects of baicalinand baicalein on lymphocytes function.
2. Baicalin and baicalein induced apoptosis of ConA-stimulated splenocytesdose-dependently in the dose range of5-20μM. Further analysis of cellular composition ofapoptotic cell showed that baicalin and baicalein mainly promote apoptosis in CD3~+T cellsand CD19~+B cells, but only mildly in CD11b+macrophages.
3. Comparison of pro-apoptotic activities of baicalin and baicalein (at10μM,exposure for24hours) on immune cells of different types and different activating statusrevealed that baicalin potently promote apoptosis in splenocytes, CD3~+T cells inConA-stimulated splenocytes, CD19~+B cells in LPS-stimulated splenocytes, bone-marrowcells, and PMA/ionomycin-stimulated Jurkat T cells, while only mildly in bonemarrow-derived dendritic cells, but showed almost no influence in non-stimulated Jurkat Tcells, RAW264.7macrophages, bEnd.3endothelial cells, and L929mouse fibroblast cells.These results demonstrated that baicalin does not unselectively promote apoptosis in allcells but selectively in T and B cells. It is noteworthy that Con A-activated CD3~+T cellswere more sensitive than na ve CD3~+T cells to the pro-apoptotic activity of baicalin.Similarly, PMA/ionomycin-stimulated Jurkat T cells were more sensitive thannon-stimulated Jurkat T cells, and LPS-activated CD19~+B cells were more sensitive thanna ve CD19~+B cells to the pro-apoptotic activity of baicalin. These results strongly suggestthat baicalin selectively promotes apoptosis in activated T and B cells. The action modes ofbaicalein are almost totally same as baicalin. These results suggest that baicalin andbaicalein mainly promote apoptosis in activated T and B cells.
4. The pro-apoptotic activities of baicalin and baicalein on T cell were further studiedusing CD3~+T cells purified from mouse splenocytes with immunal microbeads. The resultsshowed that, in dose range of5-20μM, baicalin had no influence on apoptosis of na veCD3~+T cells but dose-dependently promoted apoptosis in anti-CD3/CD28Ab-activatedones. Baicalein promote apoptosis in both na ve and anti-CD3/CD28Ab-activated T cellsdose-dependently in dose range of5-20μM, and anti-CD3/CD28Ab-activated T cells weremuch more sensitive to baicalein than na ve CD3~+T cells.
5. The pro-apoptotic activities of baicalin and baicalein on B cell were further studiedusing CD19~+B cells purified from mouse splenocytes with immunal microbeads. The results showed that, baicalin promote apoptosis in both na ve and LPS-activated CD19~+Bcells dose-dependently in dose range of5-20μM within24hours of exposure time, andLPS-activated CD19~+B cells were much more sensitive to baicalein than na ve ones.Baicalein had a more potent pro-apoptotic activity than baicalin. Within24hours ofexposure time, baicalein already significantly promote apoptosis in both na ve andLPS-activated B cells dose-dependently in dose range of5-20μM, and LPS-activated Bcells were much more sensitive to baicalein than na ve CD19~+B cells.
6. Exposure of baicalin or baicalein (5-20μM) for24hours resulted indose-dependent increase in the proportion of green fluorescence-positive cells (indicatingchange in mitochondrial membrane potential, that is low△φ) in JC-1stained splenocytes,indicating that baicalin and baicalein induced the loss of mitochondrial membrane potentialin splenocytes. However, within the same dose range, baicalin and baicalein did notenhance the expressions of Fas and FasL on cell surface of splenocytes. These resultssuggest that baicalin and baicalein promote lymphocyte apoptosis through activation ofmitochondrial-dependent endogenous pathway but not Fas-dependent exogenous pathway.
7. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CIA mice. The results showed that intraperitoneal administrationof baicalin (50mg/kg) decreased the severity of CⅡ-induced arthritis in mice. Thehistology of ankle joints from CIA mice showed severe proliferation of the synovium withsignificant infiltration of inflammatory cells, pannus formation, cartilages damage andbone erosion. Treatment with baicalin suppressed these pathologic changes, and the jointsshowed mild hyperplasia with reduced inflammatory cell infiltration, no pannus formation,and intact cartilage and bone. Baicalin treatment resulted in a significant decrease in serumlevel of anti-CⅡ IgG Abs. Production of IFN-γ, IL-2, and IL-17from collagen-stimulatedspleen cells was also reduced by baicalin treatment. These results indicated that bothhumoral and cellular immune responses against CⅡ in CIA mice were suppressed bybaicalin treatment. Immunohistochemistry and TUNEL staining of spleen sections showedthat baicalin treatment remarkably enhanced the apoptosis in lymphocytes. Flow cytometryanalysis of splenocytes revealed that baicalin treatment significantly reduced thepercentage of T and B cells in spleens, and increased their apoptosis at the same time.These results demonstrated that baicalin promotes the apoptosis of T and B lymphocytes invivo, thus exerts anti-inflammatory and immune-suppressing effects in CIA mice.
8. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. The results showed thatintraperitoneal administration of baicalein (100mg/kg) reduced serum ALT levels,alleviated Con A-induced liver injury and inflammatory infiltration, and enhancedapoptosis of T and B cells in spleens. These results demonstrated that baicalein promotesthe apoptosis of T and B lymphocytes in vivo, thus exerts anti-inflammatory inConA-induced hepatitis mice.
Conclusion
Baicalin and baicalein can selectively promote apoptosis in activated T and Blymphocytes possibly through modulation of mitochondrial-dependent endogenouspathway. Baicalin and baicalein exerts anti-inflammatory and immune-suppressing effectsthrough promote apoptosis of T and B lymphocytes in vivo, at least in part, in CⅡ-inducedarthritic mice and Con A-induced hepatitic mice, respectively. These findings extend ourunderstanding on the mechanism underlying the anti-inflammatory andimmune-suppressing effects of Baicalin and baicalein, and further reveal their therapeuticpotential in treatment of autoimmune diseases, thus providing research basis for theirclinical uses.
黄芩苷(Baicalin)及其苷元黄芩素(Baicalein)是从黄芩根中分离的黄酮类化合物，具有广泛的药理作用，其中以抗炎和免疫调控作用尤为突出。在多种炎症和自身免疫性动物模型上黄芩苷显示明确的抗炎和免疫调控作用，这些模型包括：急性胰腺炎，哮喘，急性肺损伤，急性肝损伤，肝纤维化，实验性牙周炎，实验性自身免疫性脑脊髓炎等。黄芩苷体内给药一般可以减轻组织炎症性损伤，下调模型动物血浆促炎细胞因子水平（TNF-α、IL-1、IL-6等），并可以调节某些免疫细胞分泌细胞因子，如抑制哮喘模型动物的脾细胞分泌IL-4和IL-5，增加IL-10；抑制实验性自身免疫性脑脊髓炎动物淋巴结细胞分泌IFN-γ，但增加IL-4分泌。体外实验表明：黄芩苷对刀豆蛋白(Con A)、佛波醇酯（PMA）/离子霉素(ionomycin)、或内毒素（LPS）等多种丝裂原诱导的淋巴细胞增殖均有明显的抑制作用，进一步提示淋巴细胞可能是黄芩苷免疫调控的重要靶细胞。来自肿瘤细胞的研究表明黄芩苷和黄芩素不仅调控细胞的增殖而且具有促凋亡活性，但目前对黄芩苷和黄芩素是否也调控免疫细胞的凋亡尚未进行充分研究。
免疫细胞的正常凋亡是机体建立自身免疫耐受、维持免疫系统自稳、以及避免自身免疫应答发展的的重要机制。而该调控机制出现异常则与各种自身免疫性疾病的发生和发展密切相关。转基因动物实验表明，选择性的敲除免疫细胞的促凋亡基因，导致其不能正常凋亡、大量累积，从而可通过慢性淋巴细胞激活反应加速自身免疫反应的发展。在多种自身免疫性疾病动物模型或临床病人体内也发现免疫细胞的凋亡机制发生异常，即相关炎症组织或免疫器官内免疫细胞凋亡减少，使大量的炎性细胞和活化的淋巴细胞蓄积，导致炎症持续反复的发作，这可能是自身免疫性疾病患者自身组织炎性损伤和器官功能受损的主要原因之一。许多对类风湿性关节炎等自身免疫性疾病有明显的防治作用的免疫抑制剂，包括糖皮质激素、环孢霉素(cyclosporine A，CsA)、FK506、雷帕霉素(rapamycin，RAPA)1α,25(OH)2D3等，不仅抑制T细胞的活化增殖，同时也能诱导激活的T细胞凋亡来发挥免疫抑制作用，这是免疫抑制剂治疗自身免疫性疾病的重要作用机制之一。目前对涉及淋巴细胞死亡的活化诱导的细胞死亡(activation-induced cell death，AICD)调控机制已经有了较深入的研究，普遍认为，AICD是机体清除活化的淋巴细胞，避免过强免疫应答和维持外周免疫耐受的一种重要的负反馈机制，因而通过调节免疫细胞的凋亡机制促进其凋亡是进行免疫抑制和防治自身免疫性疾病的有效手段。
基于上述研究进展，本项目首先重点探讨了黄芩苷和黄芩素在体外细胞模型上对不同种类和活化状态的免疫细胞凋亡的影响，进而采用胶原诱导的关节炎和Con A诱导的免疫性肝炎小鼠模型，探讨了黄芩苷和黄芩素防治自身免疫性疾病的效应及其免疫抑制和体内诱导T和B淋巴细胞凋亡的作用。
研究目的
进一步了解黄芩苷和黄芩素的抗炎和免疫调控作用机制及其应用潜力，为它们的临床应用提供研究基础。
研究方法
1、采用Con A和LPS刺激的小鼠脾细胞、抗CD3/CD28抗体刺激的免疫磁珠纯化的CD3~+T淋巴细胞、LPS刺激的免疫磁珠纯化的CD19~+B淋巴细胞、小鼠腹腔渗出的巨噬细胞、胸腺细胞、骨髓细胞、骨髓衍生的树突状细胞、PMA/离子霉素刺激的Jurkat T细胞、RAW264.7巨噬细胞、bEnd.3小鼠内皮细胞、以及L929小鼠成纤维细胞等为细胞模型，分析黄芩苷和黄芩素对不同种类及不同活化状态下的免疫细胞的凋亡的影响。免疫细胞的凋亡检测采用荧光标记的免疫细胞特性表面标志抗体（CD3、CD19、CD69等）标记细胞后再行annexin V/propidium iodide(PI)染色，或用抗活化的Caspase3抗体标记，用流式细胞仪分析不同种类和不同活化状态免疫细胞的凋亡率；通过流式分析细胞表面Fas和FasL的表达水平和细胞内线粒体膜电位改变，初步分析黄芩苷和黄芩素的促凋亡机制。
2、体内实验采用胶原Ⅱ（CⅡ）诱导的类风湿性关节炎小鼠（CIA）为自身免疫性疾病模型，探讨黄芩苷的抗炎和免疫抑制效应及其体内诱导淋巴细胞凋亡的作用。DBA/1小鼠于尾根部皮内接种乳化的鸡Ⅱ型胶原，21天后再用鸡Ⅱ型胶原加强免疫，诱发关节炎，于第二次免疫接种后第1天开始每天腹腔注射黄芩苷（50mg/kg体重），直至第14天。通过小鼠关节炎病理评分、关节组织病理分析、小鼠后肢计算机断层摄影（Micro-CT）分析等方法检测黄芩苷对CIA小鼠关节炎的治疗作用，采用ELISA法检测各实验组（正常对照、模型组和给药组）小鼠血清抗体水平以及小鼠脾细胞体外对CⅡ在刺激诱导的Th1和Th2型细胞因子的分泌水平分析黄芩苷的免疫抑制作用。采用TUNEL染色和荧光标记的抗Caspase3抗体进行脾脏的免疫组化分析，以及分离制备脾细胞悬液后流式分析各类免疫细胞的凋亡率，分析药物的体内促凋亡作用。
3、采用ConA诱导的免疫性肝炎小鼠模型检测黄芩素的抗炎和体内促凋亡作用。BALB/c小鼠尾静脉注射ConA（20mg/kg）诱导急性肝损伤，黄芩素（100mg/kg）于ConA注射前2h腹腔注射给药。ConA注射8h后采集外周血，检测血清ALT水平，24h取小鼠肝脏和脾脏组织，切片后进行H&E染色和免疫荧光化学分析，检测肝脏炎症和损伤程度以及各类免疫细胞凋亡情况，部分脾脏用以制备脾细胞悬液，流式分析各类免疫细胞的凋亡率，分析药物的体内促凋亡作用。
研究结果
1、黄芩苷和黄芩素在5-40μM剂量范围内能剂量依赖性地抑制ConA（5μg/ml）或LPS（10μg/ml）刺激的小鼠脾细胞增殖，表明黄芩苷和黄芩素对T和B淋巴细胞的功能具有显著的抑制作用。
2、在5-20μM剂量范围内黄芩苷和黄芩素能剂量依赖性地诱导Con A刺激的脾细胞发生凋亡，进一步分析脾细胞中CD3阳性的T细胞、CD19阳性的B细胞以及CD11b阳性的巨噬细胞凋亡的情况，结果发现黄芩苷和黄芩素对脾细胞中CD3~+T细胞和CD19~+B细胞均有有显著的促凋亡作用，但对CD11b+巨噬细胞的促凋亡作用极弱。
3、比较了黄芩苷和黄芩素（剂量为10μM，作用时间为24小时）对不同种类和不同活化状态的细胞存活的影响，发现黄芩苷对脾细胞、ConA刺激活化的脾细胞中的T细胞、LPS刺激的脾细胞中的B细胞、骨髓细胞、以及PAM/ionomicin刺激活化的Jurkat T细胞具有较强的促凋亡作用，对胸腺细胞和骨髓衍生的树突状细胞仅有较弱的促凋亡作用，而对未活化的Jurkat T细胞、腹腔渗出的巨噬细胞、RAW264.7巨噬细胞、bEnd.3小鼠内皮细胞、以及L929小鼠成纤维细胞的凋亡没有影响。说明黄芩苷并非无选择地促进所有细胞的凋亡，而是对B细胞和T细胞作用较强。尤其值得注意的是，ConA刺激活化的脾细胞中的CD3~+T细胞比na ve脾细胞中的CD3~+T细胞对黄芩苷的促凋亡作用更加敏感，PAM/ionomicin刺激活化的Jurkat T细胞比未刺激的Jurkat T对黄芩苷的促凋亡作用更加敏感，LPS刺激活化的脾细胞中的CD19~+B细胞比na ve脾细胞中的CD19~+B细胞对黄芩苷的促凋亡作用更加敏感，这些结果强烈提示：黄芩苷对活化的T和B细胞具有选择性的促凋亡作用。黄芩素的作用与黄芩苷基本类似。上述结果表明黄芩苷和黄芩素主要是促进活化的T细胞和B细胞发生凋亡。
4、采用免疫磁珠纯化的CD3~+T细胞进一步分析黄芩素和黄芩苷对T淋巴细胞的促凋亡作用，发现黄芩苷在5-20μM剂量范围内对未活化的CD3~+T细胞几乎没有促凋亡作用，但对抗CD3/CD28抗体联合刺激活化的CD3~+T细胞则有显著的促凋亡作用；黄芩素在5-20μM剂量范围内对未活化的和抗CD3/CD28抗体联合刺激活化的CD3~+T细胞均有显著的剂量依赖性的促凋亡作用，但对活化的CD3~+T细胞的促凋亡作用更强。
5、采用免疫磁珠纯化的CD19~+B细胞分析黄芩苷和黄芩素对B细胞的促凋亡作用，发现黄芩苷在5-20μM剂量范围内，作用24h，可以剂量依赖性地诱导CD19~+B细胞发生凋亡，而且LPS刺激活化的B细胞比Na ve B细胞对黄芩苷的促凋亡作用更加敏感；黄芩素促进CD19~+B细胞凋亡的作用更强，在5-20μM剂量范围内，黄芩素作用24h即能依赖性地诱导CD19~+B细胞发生凋亡（黄芩苷作用24h时对B细胞凋亡的影响极弱），而且对LPS刺激活化的B细胞的促凋亡作用明显强于Na ve B细胞。
6、脾细胞在不同浓度（0-20μM）的黄芩苷和黄芩素存在下培养24h，流式检测线粒体膜电位的变化以及脾细胞表达Fas蛋白及Fas配体的变化（Fas和FasL阳性细胞百分率）。结果发现：在5-20μM剂量范围内黄芩苷和黄芩素以剂量依赖的方式诱导脾细胞内线粒体膜电位下降，但在同样剂量范围内，脾细胞表面Fas及FasL表达的百分率未发生明显增加，表明黄芩苷和黄芩素主要通过激活线粒体介导的内源性的凋亡途径促进脾细胞的凋亡，而对死亡受体Fas介导的外源性凋亡途径无影响。
7、采用CIA小鼠为自身免疫性疾病模型，探讨黄芩苷的抗炎和免疫抑制效应及其体内诱导淋巴细胞凋亡的作用，发现黄芩苷治疗组小鼠的关节炎病理评分显著低于模型对照组，关节组织病理切片检查显示CIA模型小鼠关节组织有明显的炎性细胞浸润，滑膜增殖，关节软骨的破坏和骨质侵蚀等症状，给予黄芩苷治疗后，上述症状明显减轻或消失，说明黄芩苷有显著的抗关节炎效应；黄芩苷治疗组小鼠脾细胞在CⅡ体外再刺激下分泌IFN-γ、IL-17和IL-2的能力显著降低，血清抗CⅡ抗体水平（包括抗体总水平和IgG1、IgG2a亚型抗体水平）亦明显降低，表明黄芩苷治疗后，体内的细胞和体液免疫水平均受到抑制；脾脏冰冻切片免疫荧光分析表明黄芩苷治疗组小鼠脾细胞的凋亡明显增加，同时流式分析表明脾细胞中T和B细胞的百分率减少，T和B细胞的凋亡率增加，表明黄芩苷体内给药亦具有促进T和B细胞凋亡的效应。上述体内实验的结果表明：黄芩苷具有体内促进淋巴细胞凋亡和免疫抑制及抗关节炎效应。
8、采用ConA诱导的免疫性肝炎小鼠模型检测黄芩素的抗炎和体内促凋亡作用，发现黄芩素（100mg/kg）于ConA注射前2h腹腔注射给药可以降低ConA刺激所致的血清转氨酶升高，减轻肝组织的损伤和炎性细胞的浸润，同时增加脾脏内T、B淋巴细胞的凋亡，表明黄芩素具有体内促进淋巴细胞凋亡和防治免疫性肝炎的作用。
结论
黄芩苷和黄芩素体外对活化的T和B细胞具有选择性的促凋亡作用，其促凋亡机制与线粒体相关的内源性凋亡途径的激活有关。在CⅡ诱导的关节炎和ConA诱导的免疫性肝炎小鼠模型上，黄芩苷和黄芩素分别显示抗炎、免疫抑制和促进淋巴细胞凋亡的体内效应。这些发现拓展了对黄芩苷和黄芩素抗炎和免疫抑制作用机制的认识，进一步揭示了它们在自身免疫性疾病防治方面的应用潜力，为它们的临床应用提供了研究基础。
Background
Baicalein and its glycoside baicalin are major flavonoid constituents found in thetraditional Chinese medicinal herb Baikal skullcap (Scutellaria baicalensis Georgi), knownas “Huang qin” in China, which has been widely used for the treatment of various diseasessuch as pneumonia, hepatitis, and diarrhea. Previous studies have demonstrated thatbaicalin possess a wide range of pharmacological and biological activities such asanti-inflammatory, anti-tumor properties, anti-allergic, antimicrobial, antioxidant activities.Its anti-inflammatory activity has been estimated in various animal models of acute andchronic inflammation, including acute pancreatitis, asthma, ligature-induced periodontitis,embolism-induced acute lung injury, liver injury, hepatic fibrosis, and experimentalautoimmune encephalomyelitis. Baicalin administration generally could alleviateinflammatory tissue injury, reduce serum pro-inflammatory cytokine levels, and modulatecytokine production from immune cells, such as suppressing IL-4and IL-5but enhancingIL-10production in splenocytes in asthma model, suppressing IFN-γ but enhancing IL-4release from lymphocytes in experimental autoimmune encephalomyelitis model. In vitrostudy showed that baicalin inhibited lymphocyte proliferation stimulated by variousmotigens such as Con A, PMA/ionomycin, and LPS, further suggesting that lymphocytesmight be one of the important targets for baicalin. Evidence from studies using tumor celllines revealed that, in addition to anti-proliferating activity, baicalin also processpro-apoptotic activity. However, the influence of baicalin and baicalein on lymphocyteapoptosis has not been explored yet.
Apoptosis of immune cells represents one of the most important mechanisms ensuresthe development of protective immunity, maintenance of self-tolerance and prevention ofautoimmunity. Defects in immune cell apoptosis are closely associated with thedevelopment of various autoimmune diseases. Evidence from transgenic mice revealed thatselective knockdown of pro-apoptotic gene in immune cells resulted in their accumulationand, in turn, chronic lymphocyte activation and development of autoimmunity. Inautoimmune animal models, as well as patients with autoimmune diseases, various defectsof apoptosis in immune cells have been detected, which result in an overabundance oflymphocytes and repeat occurrence of inflammation. This might be a major cause ofinflammatory tissue injury and dysfunction of organs. Many classical immunosuppressive agents, such as cyclosporine A, rapamycin, dexamethasone, and1,25(OH)2D3, not onlyinhibit activation and proliferation of lymphocytes, but also promote their apoptosis, whichis attributed to their immunosuppressive effects. So far, the mechanisms ofactivation-induced cell death (AICD) that related to lymphocytes death have been widelyexplored, and it appears to serve as a negative immuno-regulatory mechanism that iscritical to ensure protective immunity and avoid autoimmunity. Therefore, interferencewith regulatory mechanisms of lymphocyte apoptosis represents a promising approach forthe development of new immune-modulatory therapies for autoimmune diseases.
Based on above background, the present study at first focused on the in vitro influenceof baicalin and baicalein on apoptosis of immune cells of different types and differentactivating status, and then further explored their in vivo anti-inflammatory andimmune-modulating activities, as well as their pro-apoptotic activities on lymphocytes, incollagen Ⅱ (CⅡ)-induced arthritic mice and Con A-induced immune-mediated hepatiticmice.
Aim
The present study is aimed to further understand the mechanisms underlying theanti-inflammatory and immune-modulating activities of baicalin and baicalein, and toexplore their therapeutic potential in treatment of autoimmune diseases, thus providingresearch basis for their clinical use.
Method
1. The in vitro effects of baicalin and baicalein on apoptosis of immune cells wereassayed using Con A-or LPS-stimulated mouse splenocytes, anti-CD3/CD28antibody-stimulated CD3~+T cell purified from mouse splenocytes with immunalmicrobeads, LPS-stimulated CD19~+B cells purified from mouse splenocytes with immunalmicrobeads, mouse thymocytes, bone-marrow cells, bone marrow-derived dendritic cells,PMA/ionomycin-stimulated Jurkat T cells, RAW264.7macrophages, bEnd.3endothelialcells, and L929mouse fibroblast cells. Cellular apoptosis was analyzed by flow cytometryafter stained with fluorescence-labeled antibodies against surface markers (such as CD3,CD19, and CD69) and with annexin V/propidium iodide (PI) or anti-active caspase3antibody. The effects of baicalin and baicalein on mitochondrial membrane potential insplenocyte and surface expression of Fas and FasL were assayed with flow cytometry to analyze the intracellular mechanisms of their pro-apoptotic activities.
2. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CⅡ-induced arthritic (CIA) mice. DBA/1mice were injectedintradermally at the base of the tail with0.1ml of the emulsion containing200μg of CⅡ,and then intravenously injected with0.1ml of PBS containing200ug of CⅡ at an intervalof21days to induced arthritis. Baicalin (50mg/kg) was administrated intraperitoneallydaily from days1to14after second CⅡ immunization. Arthritis symptoms were assesseddaily after drug administration by using a scoring system (0=no signs of arthritis;1=swelling and/or redness of the paw or one digit;2=two joints involved;3=more than twojoints involved; and4=severe arthritis of the entire paw and digits). The hind limbs of themice were removed postmortem, and histochemistry study with hematoxylin and eosinstaining, Micro-CT screening were performed to evaluate the anti-arthritic effects ofbaicalin. Levels of anti-CⅡ antibody in serum samples collected on day34weredetermined by ELISA. Splenocytes were prepared and cultured in medium containing CⅡ(50μg/ml) for48h, and Th1/Th2cytokines in the culture supernatants were measured byELISA. Apoptotic cells in spleen were identified by the TUNEL assay orimmunohistochemistry assay using fluorescence-labeled antibodies against CD3, CD19,and active caspase3. Lymphocyte apoptosis in splenocytes was also analyzed with flowcytometry after stained with FITC-annexin V and fluorescence-labeled antibodies againstCD3and CD19.
3. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. BALB/c mice wereintravenously injected with ConA（20mg/kg）to induced acute liver injury. Baicalein (100mg/kg) was administrated intraperitoneally2hours before ConA injection. Serum sampleswere collected8hours after ConA injection and measure for ALT levels. Liver and spleentissues were collected24hours after ConA injection, and histochemistry studies wereperformed to detect liver injury and apoptotic cells. Lymphocyte apoptosis in splenocyteswas also analyzed with flow cytometry after stained with FITC-annexin V andfluorescence-labeled antibodies against CD3and CD19.
Results
1. Baicalin and baicalein inhibited Con A or LPS-stimulated splenocytes proliferation dose-dependently in the dose range of5-40μM, indicating the inhibitory effects of baicalinand baicalein on lymphocytes function.
2. Baicalin and baicalein induced apoptosis of ConA-stimulated splenocytesdose-dependently in the dose range of5-20μM. Further analysis of cellular composition ofapoptotic cell showed that baicalin and baicalein mainly promote apoptosis in CD3~+T cellsand CD19~+B cells, but only mildly in CD11b+macrophages.
3. Comparison of pro-apoptotic activities of baicalin and baicalein (at10μM,exposure for24hours) on immune cells of different types and different activating statusrevealed that baicalin potently promote apoptosis in splenocytes, CD3~+T cells inConA-stimulated splenocytes, CD19~+B cells in LPS-stimulated splenocytes, bone-marrowcells, and PMA/ionomycin-stimulated Jurkat T cells, while only mildly in bonemarrow-derived dendritic cells, but showed almost no influence in non-stimulated Jurkat Tcells, RAW264.7macrophages, bEnd.3endothelial cells, and L929mouse fibroblast cells.These results demonstrated that baicalin does not unselectively promote apoptosis in allcells but selectively in T and B cells. It is noteworthy that Con A-activated CD3~+T cellswere more sensitive than na ve CD3~+T cells to the pro-apoptotic activity of baicalin.Similarly, PMA/ionomycin-stimulated Jurkat T cells were more sensitive thannon-stimulated Jurkat T cells, and LPS-activated CD19~+B cells were more sensitive thanna ve CD19~+B cells to the pro-apoptotic activity of baicalin. These results strongly suggestthat baicalin selectively promotes apoptosis in activated T and B cells. The action modes ofbaicalein are almost totally same as baicalin. These results suggest that baicalin andbaicalein mainly promote apoptosis in activated T and B cells.
4. The pro-apoptotic activities of baicalin and baicalein on T cell were further studiedusing CD3~+T cells purified from mouse splenocytes with immunal microbeads. The resultsshowed that, in dose range of5-20μM, baicalin had no influence on apoptosis of na veCD3~+T cells but dose-dependently promoted apoptosis in anti-CD3/CD28Ab-activatedones. Baicalein promote apoptosis in both na ve and anti-CD3/CD28Ab-activated T cellsdose-dependently in dose range of5-20μM, and anti-CD3/CD28Ab-activated T cells weremuch more sensitive to baicalein than na ve CD3~+T cells.
5. The pro-apoptotic activities of baicalin and baicalein on B cell were further studiedusing CD19~+B cells purified from mouse splenocytes with immunal microbeads. The results showed that, baicalin promote apoptosis in both na ve and LPS-activated CD19~+Bcells dose-dependently in dose range of5-20μM within24hours of exposure time, andLPS-activated CD19~+B cells were much more sensitive to baicalein than na ve ones.Baicalein had a more potent pro-apoptotic activity than baicalin. Within24hours ofexposure time, baicalein already significantly promote apoptosis in both na ve andLPS-activated B cells dose-dependently in dose range of5-20μM, and LPS-activated Bcells were much more sensitive to baicalein than na ve CD19~+B cells.
6. Exposure of baicalin or baicalein (5-20μM) for24hours resulted indose-dependent increase in the proportion of green fluorescence-positive cells (indicatingchange in mitochondrial membrane potential, that is low△φ) in JC-1stained splenocytes,indicating that baicalin and baicalein induced the loss of mitochondrial membrane potentialin splenocytes. However, within the same dose range, baicalin and baicalein did notenhance the expressions of Fas and FasL on cell surface of splenocytes. These resultssuggest that baicalin and baicalein promote lymphocyte apoptosis through activation ofmitochondrial-dependent endogenous pathway but not Fas-dependent exogenous pathway.
7. The in vivo anti-inflammatory, immune-modulating, and pro-apoptotic effects ofbaicalin were studied in CIA mice. The results showed that intraperitoneal administrationof baicalin (50mg/kg) decreased the severity of CⅡ-induced arthritis in mice. Thehistology of ankle joints from CIA mice showed severe proliferation of the synovium withsignificant infiltration of inflammatory cells, pannus formation, cartilages damage andbone erosion. Treatment with baicalin suppressed these pathologic changes, and the jointsshowed mild hyperplasia with reduced inflammatory cell infiltration, no pannus formation,and intact cartilage and bone. Baicalin treatment resulted in a significant decrease in serumlevel of anti-CⅡ IgG Abs. Production of IFN-γ, IL-2, and IL-17from collagen-stimulatedspleen cells was also reduced by baicalin treatment. These results indicated that bothhumoral and cellular immune responses against CⅡ in CIA mice were suppressed bybaicalin treatment. Immunohistochemistry and TUNEL staining of spleen sections showedthat baicalin treatment remarkably enhanced the apoptosis in lymphocytes. Flow cytometryanalysis of splenocytes revealed that baicalin treatment significantly reduced thepercentage of T and B cells in spleens, and increased their apoptosis at the same time.These results demonstrated that baicalin promotes the apoptosis of T and B lymphocytes invivo, thus exerts anti-inflammatory and immune-suppressing effects in CIA mice.
8. The in vivo anti-inflammatory and pro-apoptotic effects of baicalein were studied inCon A-induced immune-mediated hepatitis murine model. The results showed thatintraperitoneal administration of baicalein (100mg/kg) reduced serum ALT levels,alleviated Con A-induced liver injury and inflammatory infiltration, and enhancedapoptosis of T and B cells in spleens. These results demonstrated that baicalein promotesthe apoptosis of T and B lymphocytes in vivo, thus exerts anti-inflammatory inConA-induced hepatitis mice.
Conclusion
Baicalin and baicalein can selectively promote apoptosis in activated T and Blymphocytes possibly through modulation of mitochondrial-dependent endogenouspathway. Baicalin and baicalein exerts anti-inflammatory and immune-suppressing effectsthrough promote apoptosis of T and B lymphocytes in vivo, at least in part, in CⅡ-inducedarthritic mice and Con A-induced hepatitic mice, respectively. These findings extend ourunderstanding on the mechanism underlying the anti-inflammatory andimmune-suppressing effects of Baicalin and baicalein, and further reveal their therapeuticpotential in treatment of autoimmune diseases, thus providing research basis for theirclinical uses.
引文
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[3] Tian H, Zhang X, Wu C, et al. Effects of Baicalin and Octreotide on the serumTNF-alpha level and apoptosis in multiple organs of rats with severe acutepancreatitis. Inflammation,2009,32(3):191-201.
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