【作者】 李凯；

【导师】 王育良；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 干眼症是以液性泪液产生减少、黏液分泌异常和睑板腺功能障碍等各种原因引起的泪膜液性成分的绝对和相对缺乏、眼表泪液分布异常、泪液蒸发增加为共同特征的综合征。此类患者常主诉眼部干燥感、异物感、畏光、视力模糊或波动等不适,严重者可导致视力明显下降而影响工作和生活。目前对干眼症的病因和发病机理尚未完全了解,检测方法有待于进一步提高,治疗尚缺乏行之有效的手段。免疫抑制剂的应用、激素类药物的推广和手术方法的更新以及一些新的检测仪器的发明都为干眼病的治疗带来新的希望。西医目前治疗以对症和局部治疗为主,多采用人工泪液替代物、永久或暂时封闭泪道系统、戴湿房眼镜等方法保存泪液来改善临床症状,但尚不能从根本上取得治愈效果。环抱霉素A又因明显的刺激限制了其在临床的运用,而颌下腺移植毕竟是一种手术,具有创伤性。人们不断在探索新的治疗干眼症的有效方法,能够既无创伤或创伤小,又能促进泪腺主动性分泌泪液。因此,找到一种安全可靠、疗效满意的干眼症治疗新方法是当前亟待解决的问题,中医药正是符合这种条件的传统治疗方法。上世纪九十年代,我们在中医药文献中发现鬼针草有“引起多泪的副作用”,从中受到启发,根据中医基础理论,筛选出鬼针草、枸杞子、菊花制成的中药口服颗粒制剂“润目灵”。方中主药鬼针草清热解毒,消肿散瘀;菊花归肺、肝经,可疏风散热、平肝明目；枸杞子归肝、肾经,能滋补肝肾,养阴明目,3药合用,共奏清热散瘀,养阴润目以治疗干眼症之功。我们曾将润目灵与阳性药泪然和必嗽平进行对照观察,结果显示其疗效优于阳性药对照组；又将鬼针草和润目灵进行对照研究,显示两者治疗干眼症有效,但润目灵疗效要优于鬼针草。上述的临床研究因为采用了阳性药物对照,没有应用盲法试验,因此不能排除主观因素影响导致的偏差,为了进一步客观判定润目灵治疗干眼症的绝对有效性,我们采用了安慰剂对照,将润目灵和安慰剂采用双盲双模拟的包装进行了临床研究。我们最早分离出小鼠泪腺肌上皮细胞,提出了拟胆碱能作用促进泪腺腺泡肌上皮细胞收缩,增加了泪液分泌。进一步我们应用鬼针草治疗阿托品致兔干眼症,结果显示鬼针草具有拮抗阿托品作用,能一定程度有效治疗局部滴用阿托品的兔干眼症。然而鬼针草治疗干眼症机制仍不明确,是否因其中含有的胆碱类成分作用于泪腺和睑板腺毒蕈碱样胆碱能受体(M受体)而引起上述腺体的分泌,本实验试图对这一机制进行探讨。一、临床研究部分中药润目灵治疗干眼症临床观察目的：将润目灵治疗组和安慰剂对照组进行对比,观察治疗前后干眼症的症状和体征的改变程度,从而了解润目灵治疗干眼症的确切疗效和安全性。方法：对75例干眼症患者进行了随机、平行对照临床研究。随机将其分入润目灵治疗组和安慰剂对照组,其中润目灵治疗组37例,安慰剂对照组38例。润目灵组使用鬼针草、枸杞子、菊花水煮喷雾干燥制成速溶颗粒剂分装,每袋剂量相当生药鬼针草15g,枸杞子10g,菊花6g。每次1袋,每天2次,疗程8周；安慰剂由生产上述颗粒剂厂家按双盲双模拟制作。每次1袋,每天2次,疗程8周。于入选时(第0天)和治疗开始后每2周对症状积分、泪液分泌量、泪膜破裂时间、角膜染色和不良反应情况进行观察评估。结果：1.治疗前两组性别、年龄、泪流量、泪膜破裂时间、角膜荧光染色和眼部症状总积分差异均无统计学意义(P＞0.05)；治疗前两组眼干、眼红和视疲劳差异均无统计学意义(P＞0.05)；异物感和畏光在治疗前差异有统计学意义(P＜0.05),但润目灵组严重程高于安慰剂组,可见润目灵组中高等级病人明显高于安慰剂组。提示影响两组疗效主要因素基本具有均衡性,具有可比性。2.治疗8周后,润目灵组显效率为62.2%,有效率为24.3%,无效率为13.5%；安慰剂组显效率28.9%,有效率50%,无效率21.3%。润目灵组显效率和总有效率高于安慰剂组,两组比较差异有统计学意义(P＜0.05)。3.润目灵治疗后泪流量增加、泪膜破裂时间延长、眼干症状改善,治疗前后差异有显著统计学意义(P＜0.01)；4.泪液分泌量：治疗8周后,两组的泪液分泌量均增加,且两组泪液分泌量增加值(润目灵治疗组为1.47±3.86mm／5min,安慰剂对照组为0.93士5.34mm／5min)之间的差异无统计学意义(P＞0.05)。泪膜破裂时间：治疗8周后,润目灵治疗组的平均泪膜破裂时间增加幅度(1.05±1.86秒)大于安慰剂对照组(0.14±1.90秒),且两组之间的差异有统计学意义(P＜0.01)。5.眼部症状：治疗8周后,润目灵治疗组眼干和视疲劳症状改善优于安慰剂对照组,差异有统计学上有意义(P＜0.05)。眼干症状在治疗后6周两组就出现统计学上差异(P＜0.05),润目灵治疗组优于安慰剂组对照组。6.在本研究期间未发生局部和全身性不良反应,而且无一例患者发生视力下降现象。结论：1.润目灵有促进泪液分泌、延长泪膜破裂时间和缓解眼干的作用,对干眼症有明显的治疗作用。2.润目灵治疗干眼症安全性好。二、实验研究部分鬼针草水提液对M受体的作用及机制研究目的：探讨中药润目灵主药鬼针草水提液对豚鼠回肠肌M受体动力学的作用。方法：采用豚鼠离体回肠纵肌累积剂量-效应曲线法,记录离体肠管加入药物前后的收缩情况,以肠管平均收缩张力(Mean Contractile Force, MCF)为统计指标,计算给药前后肠管收缩的变化百分率。1.加入鬼针草水提物,每次100μl,使其浓度分别为10,100,1000μg／ml终浓度,以加入生理盐水为空白对照组,记录各组回肠收缩情况。2.在两组浴槽中加入10-8mmol/L阿托品,10min后加入鬼针草水提物100uml,浓度分别为10,100,1000μg／ml终浓度,以加入等量生理盐水作为空白对照组,记录各组回肠收缩情况。3.以累计浓度法加入鬼针草水提物二氯甲烷部位(以下简称二氯甲烷部位),每次加入100μl,使浓度分别为0.0625g生药／L、0.125g生药／L、0.25g生药／L、0.5g生药／L、1g生药／L,每次间隔5min,制作量效关系曲线,然后用台氏液反复冲洗标本,待张力恢复正常后,再用相同方法加入乙酸乙酯部位、正丁醇部位、水部位、鬼针草水提物浓缩液、二甲基亚枫(DMSO)对照,制作等效关系曲线。结果：1.鬼针草提取液可显著促进豚鼠离体回肠的收缩,随着浓度的增加,收缩不断加强,1000ug／ml时收缩促进百分率达到32.7%。与空白对照组比较差异有显著统计学意义(P＜0.001)；2.阿托品空白对照组和阿托品加鬼针草水提液组收缩抑制百分率(%)的平均值分别为27.4%、10.1%,在使用鬼针草提取液处理后,阿托品对豚鼠离体肠管的抑制作用显著降低,与对照组比较差异有显著统计学意义(P＜0.001)；3.鬼针草水提液能够显著兴奋豚鼠离体回肠纵肌,使回肠收缩加强,平均肌张力增加,量效关系显著。鬼针草兴奋回肠主要活性成分集中在水部位及正丁醇部位。结论：鬼针草水提液可以明显减弱阿托品抑制豚鼠离体回肠纵肌收缩的幅度,这说明了鬼针草水提液和阿托品具有一定关系的拮抗作用,对于M受体具有兴奋作用,更多还原

【Abstract】 BackgroundXeroma is a syndrome characterized by absolute or relative deficiency of tear film aqueous constituents, abnormal tear distribution on the ocular surface and increased tear evaporation, which are caused by such factors as reduced aqueous tear production, inappropriate mucus secretion, meibomian gland dysfunction(MGD),etc. Xeroma patients often complain of discomforts such as sensation of dryness and foreign body in the eye, photophobia, blurred or fluctuating vision, and so on. Some serious ones may suffer from an obvious decline of eyesight that affect their work and life. For the time being the cause and mechanism of xeroma are not fully known to us. The methods of testing are to be improved, and effective treatments are still not present. The application of immunosuppressive agent and incretion, the innovation of surgery and the introduction of testing new instruments all bring hope to the treatment of xeroma. Western medicine mainly adopts a symptomatic and local treatment, which uses artificial tear substituent and eternal or temporary blockage of tear duct. to preserve tear so that clinical symptoms can be alleviated, but an ultimate curative effect cannot be achieved. The clinical application of Cyclosporine A is limited because of its apparently stimulant effect. The autologous submandibular gland transplantation is a surgery trauma anyway. We have been exploring new effective methods to treat xeroma, which not only are atraumatic or causing as little trauma as possible, but also stimulate the tear glands to secrete more tear. Therefore, it is an urgent task to find a new treatment of xeroma that is reliable and has a satisfactory effect. Traditional Chinese medicine is the answer that meets the above requirements. We have found in the literature of traditional Chinese medicine that Bidens Bipinnata L. has "a side effect of inducing excessive tear", which inspired us to develop Runmuling, an oral granule preparation composed of Bidens Bipinnata L., medlar and chrysanthemum. The remedium cardinale in the prescription, Bidens Bipinnata L., can clear away heat and toxic material, detumescence and eliminate stasis to activate blood circulation; chrysanthemum is for lung and liver channel tropism, which can remove heat, eliminate toxins and nourish the liver to improve eyesight and stop tears; medlar is for liver and kidney channel tropism, which can invigorate the liver and kidney and nourish the Yin to improve eyesight. The mixture of the three of them can have a combined effect of treating and curing xeroma. Clinical experiments have proved its quite good curative effect. To further our understanding of the prescription, the following clinical and animal experiments have been conducted to verify its curative effect, and to explore its mechanism.Part One Clinical research Clinical Observation of the Effect of Tradition Chinese Medicine Runmuling on XeromaResearch Purposes:To observe the extent to which the symptoms and signs of xeroma changes prior to and after Runmuling treatment, and compare the Runmuling group and the placebo group in order to investigate the exact curative effect and security of Runmuling on xeroma.Methods:75 patients with xeroma are investigated randomly and divided into parallel groups for comparison. They are randomly divided into Runmuling group (37 cases), and Placebo group (38 cases). The Runmuling group takes a packet of instant granules made of Bidens Bipinnata L. (15g), medlar (10g) and chrysanthemum (6g) each time and twice a day. The placebo is produced by the same manufacturer in a double-blind and double simulation method and taken by the placebo group with exactly the same dosage as Runmuling group. The treatment lasts for 8 weeks. Since the beginning of the treatment, every other week the patients are observed and evaluated in terms of symptom score, SIT, BUT, corneal staining and adverse reaction.Results:1.Prior to the treatment there is no statistically significant difference between the two groups in gender, age, tear flow, breakup time of tear film, corneal fluorescein staining and symptom score (P>0.05);nor in eye dryness, redness and fatigue (P>0.05); However, there is some statistically significant difference between the two groups in foreign body sensation and photophobia, with the Runmuling group more serious than the placebo group, signaling that the Runmuling group has more highly graded patients than the placebo group. The main factors which affect curative effects are balanced and comparable for the two groups.2. After 8 weeks of treatment, the Runmuling group has achieved 62.2% highly effective rate,24.3% effective rate and 13.5% ineffective rate; the placebo group has 28.9% highly effective rate,50% effective rate and 21.3% ineffective rate. Both the highly effective rate and the total effective rate of the Runmuling group are higher than those of the placebo group, and the difference between the two groups are statistically significant (P<0.05).3. After the Runmuling treatment, the SIT increases, the BUT prolongs, the eye dryness symptom is alleviated, and the difference prior to and after the treatment is statistically significant (p<0.01).4. SIT:After 8 weeks of treatment, the mean SIT value of both groups increases, and there is no statistically significant difference between the two groups’mean SIT increase (the Runmuling group being 1.47±3.86mm/5min, and the placebo group being 0.93±5.34mm/5min). BUT:after 8 weeks of treatment, the increase of the mean BUT value of the Runmuling group (1.05±1.86s) is larger than that of the placebo group, and the difference is statistically significant (P<0.01).5. eye symptoms:after 8 weeks of treatment, the Runmuling group does better in alleviating eye dryness and fatigue, and the difference is statistically significant (P<0.05). Specifically, there is statistically significant difference between the two groups in the symptom of eye dryness after 6 weeks of treatment, with the Runmuling group superior to the placebo group.6. During the course of the research neither local nor systemic adverse reaction has been found, and none of the patients suffers from eyesight decline.Conclusion:1.Runmuling can promote SIT, lengthen BUT, and alleviate eye dryness, and has an obvious therapeutical effect on xeroma.2. It is safe to use Runmuling for xeroma treatment.Part Two Experimental research Study on the Effect of the Water-extract Solution of Bidens Bipinnata L. on muscarinic-receptorsResearch Purposes:To investigate the effect of water-extract solution of Bidens Bipinnata L. on muscarinic-receptors in the guinea Pig ileum.Methods:The cumulative dose-response curve method is adopted to record the contraction of isolated longitudinal ileal muscles of guinea pigs, using the mean contractile force (MCF) of muscarinic-receptors as the statistical indicator to compute the percentage variation of ileum contraction prior to and after the treatment.1. Adding 100μl water-extract solution of Bidens Bipinnata L. each time to make solutions with different final concentrations, including 10,100,1000μg/ml respectively. For the control group, physiological saline is added. The ileum contractions of both groups are recorded.2. Adding atropine solution with a concentration 10-8mmol/L into the baths of both groups, and 10 minutes later adding 100uml water-extract solution of Bidens Bipinnata L.to make solutions with final concentrations of 10,100,1000μg/ml. For the control group, physiological saline is added. The ileum contractions of both groups are recorded.3. Using the cumulative dose method to add 100μl methylene dichloride fraction of the water-extract solution of Bidens Bipinnata L. each time, at an interval of 5 minutes, to make solutions with concentrations of 0.0625g crude drug/L、0.125g crude drug/L、0.25g crude drug/L、0.5g crude drug/L、1g crude drug/L. The cumulative dose-response curve is drawn. Tyrode’s solution is used to rinse the specimen until the contractile force returns to normal. The same process is applied to ethyl acetate fraction, normal butanol fraction, water fraction, concentrated water-extract solution of Bidens Bipinnata L. and dimethyl sulfoxide (DMSO) respectively, and equivalent curves are then drawn.Results:1. water-extract solution of Bidens Bipinnata L. can significantly promote the contraction of isolated ileal muscles of guinea pigs; the conctractile force is in proportion to the concentration, with MCF increased by 32.7% at a concentration of 1000ug/ml. the difference between the experiment group and the control group is statistically significant (P<0.001).2. The contraction suppressive percentage of atropine control group and atropine/water-extract solution of Bidens Bipinnata L. group is 27.4% and 10.1% respectively. After water-extract solution of Bidens Bipinnata L. treatment, the suppressive effect of atropine on the contraction of isolated ileal muscles is reduced, and the difference is statistically significant.3. Water-extract solution of Bidens Bipinnata L. can significantly excite the contraction of isolated longitudinal ileal muscles of guinea pigs. It strengthens the ileul contraction and increase the MCF. The dose-response relation is significant. The active ingredients of Bidens Bipinnata L.’s in exciting ileul muscles are concentrated in its water fraction and normal butanol fraction.Conclusions:Water-extract solution of Bidens Bipinnata L. can significantly reduce the suppressive effect of atropine on the contraction of isolated longitudinal ileal muscles of guinea pigs, which shows that water-extract solution of Bidens Bipinnata L. has an antagonistic effect on atropine and an exciting effect on muscarinic-receptors.更多还原