CN106038804B - 一种治疗强直性脊柱炎的中药制剂 - Google Patents
一种治疗强直性脊柱炎的中药制剂 Download PDFInfo
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Abstract
本发明公开了一种治疗强直性脊柱炎的中药组成物，有以下重量份的中药原料组成：熟地黄5‑25份，鹿角片1‑25份，麻黄3‑20份，桂枝6‑18份，川牛膝5‑20份，蜂房6‑23份；本发明的中药组成物诸药配伍共奏温肾养血，益精生髓，宣通血脉，散寒化湿的功效。
Description
技术领域
本发明属于治疗强直性脊柱炎领域，具体涉及一种治疗强直性脊柱炎的中药组合物领域。
背景技术
强直性脊柱炎(Ankylosing Spondylitis,AS)是一种侵犯骶髂关节、脊柱及外周关节的慢性进行性炎性疾病。炎性病变一般从骶髂关节开始，在中轴关节由下往上发展，最终可导致关节纤维性融合、骨性强直和变形，影响脊柱等关节的活动功能。临床主要见炎性腰背痛、僵硬与活动受限，随着病情进展可出现关节强直及畸形，并可伴发眼炎、炎性肠病、肌腱端病等关节外表现。炎症和新骨形成是本病的特征性病理表现。现代医学认为本病主要与遗传、感染、免疫等有关。
中医古籍中没有与本病对应的病名，认为本病属于“痹证”范畴，将其称为“骨痹”“肾痹”“大偻”“龟背风”“竹节风”。中医众多医家根据临床经验或者理论研究对本病分型分期，通过补益肝肾，调节免疫，配合祛风通络，活血散寒，消肿止痛，在减轻炎症，延缓骨质破坏方面具有显著的疗效，治疗前景广阔。
AS患者多见腰背疼痛、僵硬、恶寒，痛连颈项，活动不利，遇寒痛增，得温则舒，舌淡苔白，脉细弦。病机主要是肾督两虚，寒湿阻络。AS属中医“大偻”“肾痹”范畴，病在脊柱，《素问·骨空论》云：“督脉为病，脊强反折。”《中藏经·五痹》日:“骨痹者，乃嗜欲不节，伤于肾也，肾气内消……精气日衰,则邪气妄人。”《证治准绳》说“若因伤于寒湿，流注经络，结滞骨节。气血不和，而致腰胯脊疼痛。”《诸病源何论·背偻候》说“肝主筋而藏血，血为阴，气为阳，阳气精则养神，柔则养筋，阴阳合同则气血调适，共相荣养也，邪不能伤。若血虚则受风，风寒搏于脊膂之筋，冷则孪急，故令背偻”。纵观以上论述，可知本病的发病与肾督亏虚，阳气不足，风寒外侵，筋脉拘急有关。
目前对于AS尚无根治的方法，关键在于早期诊断，早期干预，包括药物和非药物治疗，晚期严重畸形需行外科手术。经典的治疗手段主要是使用非甾体抗炎药、慢作用药、生物制剂等。主要的治疗药物包括非甾体抗炎药(NSAIDS)抑制前列腺素合成而抗炎，慢作用药物(DMARDs)中的柳氮磺吡啶(SSZ)，甲氨蝶呤(MTX)，来氟米特(LEF)，羟氯喹(HCQ)，生物制剂中的依那西普，阿达木单抗，英夫利昔单抗及糖皮质激素等。治疗的目的主要是控制炎症，防止脊柱、髋关节僵直。
发明内容
本发明所要解决的技术问题是：提供一种治疗强直性脊柱炎的中药。
根据强直性脊柱炎“肾督两虚，寒湿阻络”的病机，治疗当温肾壮督、散寒祛湿，本发明的解决方案是，有以下重量计的原料药物制成：熟地黄10-25份，鹿角片1-19份，麻黄3-15份，桂枝6-18份，川牛膝5-20份，蜂房6-23份。
优选的，本发明的提供的中药由以下份重量的中药组成：熟地黄15份，鹿角片10份，麻黄10份，桂枝10份，川牛膝10份，蜂房10份。
有益效果：本发明组方中：
鹿角片味咸入肾，性温温阳，多用于治疗肾虚阳痿、腰脊疼痛及疮疡不消，补肝肾、益精血、强筋骨、活血消肿。现在根据其抗肿瘤、增强免疫、抗疲劳等作用，常用来治疗乳腺增生、骨质疏松症。王志兵通过检测鹿角盘多肽对小鼠单核巨噬细胞吞噬功能的影响，发现鹿角盘多肽可以明显地增加小鼠的吞噬功能。陈玉山实验观察鹿花盘多肽对巨噬细胞吞噬功能和T、B淋巴细胞的影响。用鹿花盘多肽腹膜给药，眼眶取血涂片后显微镜下观察淋巴细胞的个数，并且用小鼠腹腔注射鸡红细胞的方法观察吞噬细胞吞噬鸡红细胞的情况，计算吞噬系数。结果显示鹿花盘多肽可以显著地促进巨噬细胞的吞噬功能和T淋巴细胞的吞噬能力。
熟地黄是玄参科植物地黄经酒炖或蒸法炮制而成，具有填精益髓、补血滋阴的功效。其主要活性成分是多糖、5-羟甲基糖醛、氨基酸等。苗明三用熟地黄多糖检测对血虚模型免疫器官的影响，用环磷酰胺、放血致小鼠血虚模型。用熟地黄多糖灌胃后，取胸腺、脾脏做病理检查。发现模型小鼠的胸腺皮质增厚和皮质细胞数增多，脾小结变大和皮质细胞增多。结果说明熟地黄多糖可促进造血增强免疫功能。刘培建用小鼠胃部放血、腹腔注射环磷酰胺的方法制造小鼠气血两虚的模型，用熟地黄多糖灌胃，取尾部血检测全血细胞和血清粒-巨噬细胞击落刺激因子水平的变化，结果显示熟地黄多糖可提高粒-巨噬细胞击落刺激因子水平，可能与促进白介素1分泌刺激粒-巨噬细胞击落刺激因子的转录，或者促进白介素2分泌诱导T细胞产生粒-巨噬细胞击落刺激因子，或者直接刺激相应靶细胞刺激粒-巨噬细胞击落刺激因子有关。
牛膝味酸甘苦，酸甘化阴，酸苦涌泄，补益肝肾，活血化瘀，利尿通淋。根据产地有怀牛膝和川牛膝之分，怀牛膝补益肝肾之力为著，阳和汤中川牛膝补肝肾强腰膝基础上更偏于活血化瘀。主要药理组分为牛膝多糖和牛膝总皂苷。季敬彰通过实验表明牛膝多糖通过增强Th1类细胞因子的表达，抑制Th2类细胞因子的表达，促进Th1类免疫应答。王剑对川牛膝多糖体内免疫实验证实川牛膝多糖能够可以增强机体的非特异性免疫功能，并且可以促进免疫小鼠体内抗体的产生，多糖浓度越高抗体产生越强。另一项体外免疫实验发现牛膝多糖既有体液免疫功能，又能提高NK细胞活性和增强吞噬细胞的吞噬能力，其免疫活性广泛。高昌琨研究证实不同剂量下的牛膝总皂苷能够减轻大鼠或小鼠的炎症反应，降低大鼠琼脂肉芽肿数量，延长热板上小鼠舔足时间，改善血液流变性。
露蜂房性平，味甘，具有消肿散结、攻毒杀虫的作用。现代药理研究显示，露蜂房主要成分是蜂房油、蜂胶、蜂蜡，具有抗炎、抗菌的作用，其抗炎作用和氢化可的松类似。吴德全证实露蜂房可以抑制T细胞介导。程茂盛通过蜂房水提物对二甲苯所致的小鼠耳廓肿胀具有抑制作用，提示蜂房水提取物具有抑制炎性水肿，具有抗炎作用。孟海琴证实蜂房水提取物对醋酸致小鼠扭体反应有抑制，提示蜂房水提取物具有镇痛作用。
麻黄辛温散寒，具有祛风散寒，发汗解表，利水消肿等作用，主要成分为麻黄碱、多糖、黄酮、酚酸、挥发油等。陈荣明实验研究麻黄的不同成分对细胞免疫的抑制作用，结果显示麻黄-9905对细胞免疫有抑制，其作用一方面通过使胸腺萎缩使T淋巴细胞减少，另一方面与调整辅助T淋巴细胞与抑制T淋巴细胞比例有关。Kuang等发现，麻黄多糖具有免疫抑制作用，主要通过抑制脾细胞的增殖。王艳宏通过对麻黄水煎液及其对化学拆分组分对补体作用的实验证实，多糖组分对补体抑制作用最强，其次是水煎液，酚酸组分。
桂枝是樟科植物肉桂的干燥嫩枝，性味辛甘温，具有解表散寒、温通经脉的功效。主要活性物质是挥发油类，主要成分是桂皮醛。桂皮醛有明显的镇静、镇痛作用，作用与抑制花生四烯酸代谢、影响炎症介质生成和抗氧化有关。马锐颖等采用大鼠发热模型和小鼠IL-17β刺激模型，给药后测定大鼠下丘脑组织和细胞上清液中前列腺素(PGE2)的含量，结果显示桂皮醛能显著降低大鼠下丘脑PGE2的含量，抑制IL-1β刺激内皮细胞PGE2的释放。
附图说明
图1是高剂量组小鼠脊柱炎病理(HE染色*400)图
图2是中剂量组小鼠脊柱炎病理(HE染色*400图)
图3是低剂量组小鼠脊柱炎病理(HE染色*400)图
图4是空白对照组小鼠脊柱炎病理(HE染色*400)图
图5是阳性药组小鼠脊柱炎病理(HE染色*400)图
图6是模型组小鼠脊柱炎病理(HE染色*400)图
图7是高剂量组小鼠膝关节病理(HE染色*400)图
图8是中剂量组小鼠膝关节病理(HE染色*400)图
图9是低剂量组小鼠膝关节病理(HE染色*400)图
图10是空白对照组小鼠膝关节病理(HE染色*400)图
图11是阳性药组小鼠膝关节病理(HE染色*400)图
图12是模型组小鼠膝关节病理(HE染色*400)图
具体实施方式
实验验证
(一)实验材料
实验动物：BALB/c小鼠:6周龄，体重20±2g，雄性，48只，购自苏州，在南京中医药大学实验中心饲养，SPF条件，自由摄食饮水。
实验药品：柳氮磺吡啶，购自上海中西三维药业有限公司，批号：h31020450。鹿角片，熟地黄，麻黄，桂枝，蜂房，川牛膝等，均购自江苏省中医院中药房。
主要实验试剂：Mouse Interleukin 6(IL-6)ELISA Kit；Mouse Interleukin 17(IL-17)ELISA Kit；Mouse Interleukin 21(IL-21)ELISA Kit；Mouse anti-phosphoSTAT3 monoclonal antibodies；Aggrecan Peptide，北京博奥森生物技术有限公司，0.5mg，货号：bs-1223P；Freund's complete adjuvant，sigma公司,10ml，货号：F5881；Freund's incomplete adjuvant，sigma公司,10ml，货号：F5506；MaxVision TM试剂盒，福建迈新生物技术有限公司；PBS缓冲液：自行配制；其它试剂为进口分装或国产分析纯取。
实验仪器：樱花全自动真空组织脱水机；樱花包埋机；Thermo半自动切片机；樱花全自动染色机；Olympus显微镜BX41；超低温冰箱；超净工作台；台式高速冷冻离心机；旋转蒸发仪。
(二)实验方法
选用鹿角片10g，熟地黄15g，麻黄10g，桂枝10g，川牛膝10g，蜂房10g，加10倍水浸泡30min，煎煮2次，每次40min，合并滤液，用旋转蒸发仪浓缩至每毫升含生药分别0.2g，0.4g，0.8g，4℃冰箱保存备用。
分组:BALB/c雄性小鼠于SPF环境中饲养，动物自由进食、饮水一周后，8只小鼠不做任何处理设为正常对照组，其余小鼠进行蛋白聚糖诱导的佐剂关节炎造模。40只BALB/c小鼠随机分为5组，每组各8只：模型组，阳性药组，中药低剂量组，中药中剂量组，中药高剂量组。
模型建立:待小鼠9周龄时，无菌条件下用蛋白聚糖加弗氏完全佐剂(蛋白聚糖75ug+佐剂150ul)乳化振荡混匀后予小鼠腹腔注射，一周后加强免疫(初次免疫中的完全佐剂换成不完全佐剂，其余操作同初次免疫)，造模共需14天。
灌胃:各组小鼠在造模完成后一周，即11周龄时开始灌胃，剂量按小鼠成人系数9.019折算剂量
中药高剂量组：每天按体重给予新加阳和汤浓缩液(0.8g/ml)0.2ml/10g
中药中剂量组：每天按体重给予新加阳和汤浓缩液(0.4g/ml)0.2ml/10g
中药低剂量组：每天按体重给予新加阳和汤浓缩液(0.2g/ml)0.2ml/10g
阳性药组：每天按体重给予柳氮磺吡啶溶液(0.02g/ml)0.2ml/10g
正常对照组和模型组：每天按体重给予生理盐水0.2ml/10g
一般情况观察:小鼠的体重分别在分组前、造模前、造模后各测一次，正式用药后每周测一次；观察小鼠的毛色、食欲、精神状态、关节颜色、肿胀等变化。
小鼠血清中IL-6，IL-17，IL-21检测
灌胃91天后(小鼠23周龄)，眼眶后静脉丛取血，后置于离心管中，静置2小时，于离心机中以3000r/min离心10min，取血清。放置于于冷冻箱中保存。测定含量时，从冷冻箱中取出血清，常温环境中复温30min。用IL-6，IL-17，IL-21试剂盒测定，按照ELISA试剂说明书，具体步骤如下：
将浓缩洗涤液用蒸馏水稀释30倍后备用
取出酶标板，将其固定好，依次加上标准品50ul，此为标本孔。在待测样品孔中加入样品稀释液40ul，再加入待测样品10ul。另设空白孔，不加样品及酶标试剂
用封板膜将加样后的酶标板封板，后置于37℃恒温箱温育30分钟
揭膜，弃液，甩干，加洗涤液，静置，拍干，重复5次
除外空白孔其余孔加酶标试剂
用封板膜将加样后的酶标板封板，再次置于37℃恒温箱温育30分钟
揭膜，弃液，甩干，加洗涤液，静置，拍干，重复5次
加显色剂A，再加显色剂B，震荡，避光，显色
加终止液，终止反应
调零，测量各孔的吸光度(OD值)，测量须在15分钟以内进行
制定标准曲线，计算直线回归方程式，根据样品的OD值计算实际浓度。
病理形态学观察
小鼠眼眶取血后，处死小鼠，迅速解剖，取出脊柱、一侧膝关节，放于4％多聚甲醛固定后，以标准的病理组织包埋处理。切片，HE染色，在显微镜下观察炎性细胞的浸润度。请病理专业人员进行读片。
小鼠脊柱、膝关节磷酸化STAT3表达的检测
取小鼠脊柱关节、膝关节，剔除其附着的软组织，组织标本用4％多聚甲醛固定，常规脱水，石蜡包埋，免疫组化法测定磷酸化STAT3的含量，具体步骤如下：
石蜡切片，脱蜡至水
用PBS洗3遍，甩干
抗原修复
配置3％H2O2，室温下封闭，蒸馏水洗1遍
用PBS洗1遍，甩干
滴加一抗,室温下孵育或4℃恒温过夜
用PBS洗3次，甩干
滴加1滴或50ul MaxVision TM试剂，室温下孵育15分钟
PBS洗3次各5分钟
加2滴或100ul DAB溶液到切片，显微镜下观察1-3分钟，结果棕色即为阳性显色
用蒸馏水洗终止染色，用苏木素复染，0.5％盐酸分化，自来水冲洗，PBS冲洗返蓝
用梯度酒精脱水，透明，封片
每张切片取5个高倍视野，用Image Pro Plus 5.0.2图像分析软件测定各组阳性面积及光密度值。
统计学处理
用SPSS17.0统计分析，计量资料均用X±S表示，若数据符合正态分布，用单因素方差分析；若数据不符合正态分布，用秩和检验。所有结果均以p<0.05为差异有统计学意义。
(三)实验结果
新加阳和汤对Balb/c小鼠外周血IL-6、IL-17、IL-21含量的影响，如表一所示
表一IL-6、IL-17、IL-21的表达情况
注：IL-6统计分析，与空白组比较**P<0.01；与模型组比较▲P<0.05，▲▲P<0.01；组间比较(高剂量与低剂量比较)□□P<0.05；有统计学意义。组间比较(阳性药组与中药各剂量组比较)P>0.05，无统计学意义。IL-17统计分析，与空白组比较**P<0.05；与模型组比较▲P<0.05，▲▲P<0.01；组间比较(中药低剂量与高剂量组比较)□□P<0.01，有统计学意义。组间比较(阳性药组与中药各剂量组比较)P>0.05，无统计学意义。IL-21统计分析，与空白组比较**P<0.05；与模型组比较▲P<0.05，▲▲P<0.01；有统计学意义。组间比较(中药低剂量与高剂量比较)P>0.05；组间比较(阳性药组与中药各剂量组比较)P>0.05；无统计学意义。
病理观察
脊柱炎(见图一至图六)
外周关节炎(见图七至图十二)
(五)结论
新加阳和汤不仅可改善免疫亢进及抑制实验模型T细胞亚群，还可抑制抗体生成，降低抗体水平，来抑制亢进的体液免疫，说明新加阳和汤可以针对不同的免疫状态发挥调节作用。新加阳和汤能够多方面、多途径地抑制炎症、抑制骨质破坏及调节免疫，临床及实验疗效肯定，具有实际的临床意义。
Claims (4)
1.一种治疗强直性脊柱炎的中药组合物，其特征在于，由以下重量份的中药原料制成：熟地黄5-25份，鹿角片1-25份，麻黄3-20份，桂枝6-18份，川牛膝5-20份，蜂房6-23份。
2.根据权利要求1所述的治疗强直性脊柱炎的中药组合物，其特征在于，由以下重量份的中药原料制成：熟地黄10-25份，鹿角片5-19份，麻黄6-15份，桂枝6-15份，川牛膝5-15份，蜂房6-20份。
3.根据权利要求1所述的治疗强直性脊柱炎的中药组合物，其特征在于，由以下重量份的中药原料制成：熟地黄10-15份，鹿角片5-10份，麻黄6-10份，桂枝10-15份，川牛膝10-15份，蜂房6-12份。
4.根据权利要求1所述的治疗强直性脊柱炎的中药组合物，其特征在于，由以下重量份的中药原料制成：熟地黄15份，鹿角片10份，麻黄10份，桂枝10份，川牛膝10份，蜂房10份。
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| CN103394069A (zh) * | 2013-08-21 | 2013-11-20 | 宋立学 | 治疗强直性脊椎炎的中药组合物及其制备方法 |
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