【作者】 陈瑜；

【导师】 黄子杰；

【作者基本信息】 福建医科大学 ， 药理学， 2009， 博士

【摘要】 肠易激综合征(Irritable Bowel Syndrome,IBS)作为临床常见病与多发病,严重影响患者的生存质量,对中国及全球的经济发展产生巨大影响。IBS的病因病理有待探讨,全球目前尚无有效的治疗方法。据报道,IBS女性患者约为男性患者的2~3倍,而且女性患者的症状随月经周期而波动。因此,性别与痛的关系一直受到科学研究者的关注。女性更易罹患各种疼痛疾病,以往认为,女性容易精神紧张、并易于就诊等是主要原因。但近年的研究提示,男女之间多种精神心理评分和就诊行为等并无差异,而女性的体表痛阈明显低于男性。关于不同性别之间内脏痛阈是否存在差异,罕见研究报道,结果也不一致。但多数学者认同性激素是这种差异的成因之一。多见以动物的动情周期或人的月经周期来研究体内的性激素水平与躯体疼痛关系的报道。但亦有文献报道月经周期不影响热刺激疼痛反应,而血浆高雌激素水平却与体表热刺激疼痛有关。可见生理周期并不能完全反映动物体内的性激素水平。因此,本研究在检测动物的动情周期的同时还直接测量其体内的雌激素水平。痛觉产生有赖于外周与中枢神经系统的相互作用,性激素可以在多水平上影响痛觉的产生和传递。众多研究表明,NMDA受体的激活在痛觉过敏以及异常痛的产生和维持中发挥着重要的作用。有研究报道,在急性内脏痛动物模型上,雌激素受体和N-甲基-D-门冬氨酸(N-methyl-D-aspartic acid,NMDA)受体的NMDAR1亚单位在脊髓背角上共表达,这支持雌激素对NMDA受体有直接作用。鞘内给予NMDA受体阻断剂(D(-)-2-amino-5-phosphonopentanoicacid,AP-5)能抑制结直肠扩张刺激引发的内脏痛反应,但在高雌激素组大鼠的抑制作用较低雌激素组大鼠的抑制作用弱。研究还发现,雌激素显著增加了腰骶段脊髓中NMDAR1蛋白的表达。以上数据提示,在急性内脏痛动物模型中,雌激素是通过上调NMDA受体的表达来增强脊髓的内脏伤害性传递的。众所周知,慢性内脏痛比急性内脏痛的产生机制要复杂得多,存在慢性痛觉的敏化现象。可以肯定,急性造模的动物不能很好地模拟IBS患者的慢性内脏高敏状态。2000年首次报道的Al-Chaer模型,采用新生期的结直肠扩张刺激,导致成年大鼠出现长期的内脏高敏状态,较好地模仿了IBS患者的特征,为研究IBS发病机制提供了可借鉴的动物模型。该动物模型的神经系统递质与受体功能状态,与急性造模的大鼠之间有很大的不同。在慢性内脏痛的动物模型体内的背根神经节和脊髓内脏相关神经元中,NMDA受体表达增强。在这种情况下,雌激素是否还能通过调节NMDA受体的表达与磷酸化来影响慢性内脏痛呢?针对以上问题,本研究中检测了雌性IBS模型大鼠的慢性内脏痛与血清雌激素水平,并做了相关性分析,同时比较了高雌激素与低雌激素模型雌鼠的慢性内脏痛反应的程度。为了排除孕激素等其它性激素对雌激素作用的干扰,本研究中只检测去卵巢和去卵巢加上雌激素替代的大鼠,以确定雌激素对慢性内脏痛的作用及其潜在的机制。本研究比较了AP-5(NMDA受体阻断剂)在高、低雌激素IBS模型大鼠上对结直肠扩张刺激的镇痛作用,同时对两组大鼠的脊髓、背根神经节上NMDA受体的表达情况进行检测。本研究将为女性IBS的发病机制的探讨,以及临床上对女性IBS患者的诊治提供理论依据。1慢性内脏痛大鼠模型的特点及评价1.1痛阈大鼠成年后模型雌鼠的痛阈显著低于对照雌鼠,模型雄鼠的痛阈亦显著低于对照雄鼠。雌雄模型大鼠痛阈差异无统计学意义。1.2腹壁撤退反射评分在20-60 mmHg压力的结直肠扩张(colorectal distension,CRD)刺激下,模型雄鼠腹壁撤退反射(abdominal withdrawal reflex,AWR)评分明显高于对照雄鼠,80 mmHg压力下的CRD刺激,两组雄鼠的AWR评分趋同,差异消失。在20 mmHg压力的CRD刺激下,模型雌鼠的AWR评分高于对照雌鼠,40-80 mmHg压力的CRD刺激,两组雌鼠的AWR评分趋同,差异消失。1.3甩尾时间以(50±1)℃恒温水浴为热致痛源,比较模型与对照大鼠的甩尾时间发现:模型大鼠的平均甩尾时间低于对照大鼠。1.4结肠组织病理改变比较对照和模型大鼠远端降结肠和直肠的局部组织学的病理改变。结果显示,对照与模型大鼠经过肉眼观察和显微镜检查,均未见明显病理改变。1.5模型雌鼠动情周期与血清雌激素水平的关系观察19只模型雌鼠的阴道涂片,分别在其处于动情前期(P)、动情期(E)、动情后期(M)、间情期(D)时,采集大鼠血液测定雌激素水平。雌鼠在P期的雌激素水平明显高于其它各期,此外,其它各期雌激素水平之间无显著差异。1.6动物分组模型组与对照组中又各自设立高雌激素组(E2组)与低雌激素组(OVx组),两组大鼠均去卵巢,E2组大鼠在测量内脏痛前两天腹腔注射雌激素100μg,OVx组大鼠则不补充雌激素。2慢性内脏痛与血清雌激素水平的关系2.1雌雄模型大鼠的放电比较大鼠放电反应随CRD压力增加而增加,各压力下放电的差异具有统计学意义。在15 mmHg,30 mmHg,45 mmHg的CRD刺激下,模型雌鼠腹外斜肌放电反应均高于模型雄鼠。2.2模型雌鼠的腹外斜肌放电与血清雌激素水平关系模型雌鼠的血清雌激素水平为115.70±26.34 pmol/L,最小值84.50pmol/L,最大值154.20 pmol/L。在20~80 mmHg压力的CRD刺激下,模型雌鼠的腹外斜肌放电平均峰值与血清雌激素水平正相关。2.3高雌激素大鼠与去卵巢大鼠内脏痛敏的比较2.3.1大鼠的阴道涂片结果高雌激素大鼠的阴道涂片见多边形的角化细胞,体积大,呈动情期表现,表明其体内雌激素水平高。去卵巢大鼠的阴道涂片见圆形上皮细胞,体积小,呈间情期表现,表明其体内雌激素水平低。2.3.2腹外斜肌放电水平比较在20mmHg的CRD压力下,模型E2组大鼠的的腹外斜肌放电水平高于模型OVx组大鼠,两组差别有显著性意义。另一方面,在20mmHg的CRD压力下,对照E2组大鼠的的腹外斜肌放电水平与对照OVx组大鼠之间没有明显差别。说明雌激素可加重模型大鼠内脏痛觉敏感性。3 AP-5鞘内给药对慢性内脏痛的抑制作用与血清雌激素水平的关系3.1 AP-5椎管给药的抑制作用3.1.1 AP-5抑制作用的时效关系AP-5的抑制作用在5min时最强,随时间渐渐减弱。在模型OVx组与E2组,给药5min、10min、15min后的内脏运动反应程度均低于给药前水平,差别有显著性意义。说明AP-5对模型大鼠的内脏痛有明显的抑制作用。3.1.2 AP-5对OVx组与E2组模型大鼠抑制作用的比较OVx组与E2组在给药5min、10min、15min、30min后,抑制百分比的差别无显著性意义。3.2 NMDAR1受体在OVx组与E2组模型大鼠的表达3.2.1 E2大鼠与OVx大鼠脊髓背根神经节NMDAR1受体表达采用免疫组织化学方法,比较E2大鼠与OVx大鼠背根神经节NMDAR1的表达。结果显示,大鼠腰骶段脊髓背根神经节阳性细胞,多为圆形或椭圆形的神经元,胞质呈棕黄色,呈强阳性表达。图像分析结果显示,与OVx大鼠相比,E2大鼠NMDAR1受体表达的平均光密度无明显差别。3.2.2 E2大鼠与OVx大鼠腰骶段脊髓NMDAR1受体表达采用免疫组织化学方法,比较E2大鼠与OVx大鼠脊髓背角神经元NMDAR1的表达。结果显示,E2大鼠与OVx大鼠腰骶段脊髓均可见NMDAR1阳性表达,胞质呈棕黄色,为强阳性表达。图像分析结果显示,E2大鼠腰骶段NMDAR1受体表达的平均光密度与OVx大鼠无明显差异。3.2.3肠道中NMDAR1受体表达在大鼠肠道内在神经丛中可见NMDAR1阳性表达,胞质呈棕黄色。综上所述,本工作提示:1.雌雄大鼠新生期接受结直肠扩张刺激,成年时(8 wk后)都可引发慢性内脏高敏,且其结直肠局部组织未见明显的病理改变,提示本模型可作为IBS样功能性慢性内脏痛的研究模型。2.雌性模型大鼠内脏疼痛的敏感性高于雄性模型大鼠,且与血清雌激素水平正相关,高雌激素模型大鼠的内脏痛敏感性高于去卵巢模型大鼠,提示雌激素增加了直肠感觉的敏感性。3.NMDA受体拈抗剂AP-5椎管给药可减轻内脏运动反应,且减轻的程度在去卵巢大鼠与高雌激素大鼠并无差别,两组大鼠的脊髓背角NMDAR1的表达差别无显著性。提示雌激素可能没有通过调节脊髓背角NMDAR1表达的改变在雌鼠慢性内脏痛中枢敏化机制中发挥作用。更多还原

【Abstract】 Irritable bowel syndrome(IBS) is a common and frequently encountered disease,lt has greatly affected the life quality of the patients,and also has great influence on Chinese and global social economy.The etiology and pathology of IBS are still not clear,so we still have no effective treatment for this disease.IBS is 2 - 3 times more prevalent in women than in men.The severity of pain symptoms in women with IBS fluctuates with the menstrual cycle.Therefore many researchers paid close attention to the relation between gender and pain.Women are easier to suffer from different kinds of pain disease.It was assumed that women are easier to be nervous and to go to see a doctor was the main reason.But the recent reports suggested,there were no difference in many psychiatry and psychology scores between men and women. The body surface pain threshold of women is significantly lower than that of men.But there were few researches on whether there was difference in visceral pain threshold between man and woman,and the results of these researches were contradictory.In the researches which considered there were difference in visceral pain threshold between man and woman,most of them thought gonadal hormone was the reason of the difference.Many researchers only used the estrous cycle of animal or the menstrual cycle of human being to deduce the gonadal hormone level,but didn’t measure the gonadal hormone level directly.Although there is some corresponding relation between these physiological cycle and the gonadal hormone level, they can’ t reflect the gonadal hormone level completely.As it was reported,though menstrual cycle didn’t influence thermo-pain refleetion,but high estrogen level inplasma didhaverelationwithbody surface thermo-pain.So it is more convinced to measure estrogen level than to deduce the gonadal hormone level by physiological cycle.In this study,the estrogen level as well as the estrus cycle of rats were measured.There was pheriphery and central nervous system interaction in pain transmit.Gonadal hormone can interact with this system on different levels.Many studies indicated that the activation of NMDA receptors played important roles in the producing and maintaining of hyperalgia and allodynia.Bin Tang reported in acute visceral pain animal model,estrogen receptor alpha and the NMDAR1 subunit of the NMDA receptor were co-expressed in dorsal horn neurons,supporting a direct action of estradiol on NMDA receptors.Intrathecal administration of the NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid(AP-5) dose-dependently attenuated the visceromotor response with greater potency in ovariectomized(OVx) rats compared to OVx with estradiol replacement(E2) rats.Estradiol significantly increased protein expression of NMDAR1 in the lumbosacral spinal cord compared to OVx rats. These datas suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NMDAR1 subunit expression in acute visceral pain animal model.As we all know,chronic visceral pain has chronic visceral hypersensitivity,the pathogenesy of which is by far more complex than those of acute visceral pain.Acute visceral pain model can hardly simulate the chronic visceral hypersensitivity state of IBS patients.In 2000 Al-Chaer first reported a new model.It was constructed by neonatal mechanical colorectal irritants,the adult rats had visceral hypersensitivity for a long time. Al-Chaer’ s model can simulate the characteristics of IBS patients.It provided a good animal model for the research on IBS pathogenesy.The nervous system transmitters and receptors functional status of this model is greatly different from those of the acute model.NMDA receptor expressions in the dorsal root gangalia(DRG) and spinal cord visceral correlated neurons of this chronic visceral pain animal model were upregulated.Under this condition,can estradiol influence chronic visceral pain by regulate the expression and phosphorylation of NMDA receptors?In this study,the chronic visceral pain and serum estrogen level of female IBS model rats were detected,the relation between them were analyzed.The chronic visceral pain degree of high estrogen and low estrogen female model rats were compared,ln order to avoid the interference of progestogen and other gonadal hormones,only ovariectomized rats and ovariectomized with estrogen substitute rats were used to determine the effect of estrogen on chronic visceral pain and the potential mechanism of it.The inhibit effects of AP-5(NMDA receptor antagonist) on chronic visceral pain and the NMDA receptor expression in the lumbosacral spinal cord and DRG between OVx rats and E2 rats were compared.This study will provide theory basis to the pathogenetic research and the clinical diagnosis and therapy of female IBS patients.1 The characteristics and evaluation of the chronic visceral pain model1.1 Pain thresholdMale and female model rats were made by improved Al-Chare’ s method. The pain thresholds of model female rats were significantly lower than those of control female rats.The pain thresholds of model male rats were significantly lower than those of control male rats.No significant difference was found between the male and female model rats’ pain thresholds. 1.2 Abdominal withdrawal reflex scoresThe AWR scores of model male rats were significantly higher than those of control male rats under 20-60mmHg CRD stimulation.The difference of AWR scores disappeared under 80 mmHg CRD stimulation.The AWR scores of model female rats were significantly higher than those of control female rats under 20 mmHg CRD stimulation.The difference of AWR scores disappeared under 40-80 mmHg CRD stimulation.1.3 Tail-flick latencyThe tail-flick latency of both model and control rats were compared.The tail-flick latency of model rats were significantly shorter than those of the control rats.1.4 The colorectal pathological changeThe distal colon descendens and rectal topographic histology of control rats and model rats were compared.Neither control nor model rats showed apparent pathological change under macroscopic observation and microscopic examination.1.5 The relation between serum estrogen level and estrus cycle of the female model ratsVaginal smear of 19 female model rats was observed.Serum estrogen level was detected in preoestrus,estrus,metoestrus and dioestrus.The serum estrogen level in preoestrus was significantly higher than those in estrus,metoestrus and dioestrus.No significant difference was found among the serum estrogen levels in estrus,metoestrus and dioestrus.1.6 Grouping of the animalsBoth model and control rats were divided into high estrogen(E2) group and low estrogen(OVx ) group.Both E2 and OVx rats were ovariectomized.Only E2 rats accepted estrogen(100μg,i.p.)two days before visceral pain measurement.2 The correlation between visceral pain and serum estrogen level 2.1 The electric discharge of male and female model ratsChange rate of electric discharge increased with CRD pressure.The difference of electric discharge change rates among each pressure was significant.Under 15 mmHg,30 mmHg,45 mmHg CRD pressure,electric discharge of EOMA of the model female rats was higher than those of the model male rats.2.2 The relation between electric discharge of EOMA of model female rats and their serum estrogen levelThe mean serum estrogen level of model female rat was 115.70±26.34 pmol/L,the minimun value was 84.50 pmol/L,the maximum was 154.20 pmol/L. A positive relation was detected between the electric discharge of EOMA of model female rats and their serum estrogen level under 20~80mmHg CRD pressure.2.3 The comparism of visceral pain sensitivity between high estrogen rats and OVx rats2.3.1 Vaginal smear results of ratsUnder microscopic examination,vaginal smear of E2 rats(high estrogen) showed many big polygonal keratinocytes,which is a typical estrous manifestation.This indicated high estrogen level in the rat,which can promote the maturation and cornification of the vagina epithelium cells. Vaginal smear of OVx rats(low estrogen) showed many small round epithelium cells,which is a typical dioestrus manifestation.This indicated low estrogen level in the rat.2.3.2 Comparism of electric discharge of EOMAUnder 20mmHg CRD pressure,the electric discharge of EOMAof E2 model rats was significantly higher than those of the OVx model rats.This demonstrated the visceral pain of E2 model rat was more sensitive than those of the OVx model rats.High estrogen level can lead to visceral pain sensitization.On the other hand,the electric discharge of EOMA of E2 control rats and OVx control rats didn’t show significant difference under 20mmHg CRD pressure.This indicated estrogen can add to the high visceral pain sensitivity of model rats.3 The inhibited effect of chronic visceral pain by intrathecal administration of AP-5 and its relation with estrogen level.3.1 Intrathecal administration of AP-5 attenuated the visceromotor response3.1.1 Time-effect relationship of AP-5 inhibitionThe peak effect occurred 5 min after AP-5 administration.The effect decreased as time went on.5min、10min、15min after AP-5 administration, the visceromotor response were all significantly lower than those before AP-5 administration.This demonstrated intrathecal administration of AP-5 significantly attenuated the visceral pain of rats.3.1.2 The inhibited effect of intrathecal AP-5 in OVx and E2 model rats.5min、10min、15min、30min after AP-5 administration,the inhibited percent of OVx and E2 model ratsdidn’ t show any significant difference.3.2 The expression of NMDARI in OVx and E2 model rats3.2.1 The expression of NMDARI in OVx and E2 model rats’ DRGThe NMDARI expression in OVx and E2 model rats’ DRG were compared by immunohistochemistry.The positive cells in the rat’ s lumbar and sacral DRG were round or elliptic,their kytoplasm stainedbrown.Image analysis showed,there were no significant difference between the average opticals which stands for the NMDAR1 expression in E2 rats and those of the OVx rats.3.2.2 The lumbar and sacral spinal cord expression of NMDAR1 in E2 and OVx ratsThe NMDAR1 expression in OVx and E2 model rats’ spinal cord dorsal horn neurons were compared by immunohistochemistry.NMDARI expression in both E2 and OVx rats’ lumbar and sacral spinal cord were found.The kytoplasm stained brown.Image analysis showed,there were no significant difference between the average opticals which standed for the NMDAR1 expression in E2 rats’ lumbar and sacral spinal cord and those of the OVx rats.3.2.3 Expression of NMDAR1 in intestinal tractNMDAR1 expression in intestinal tract internal nerve plexus was found.Its kytoplasm stained brown.In conclusion,this study indicates:1.Neonatal colonrectal distension can lead to chronic visceral sensitization in adult(after 8 wk) male and female rats.There was no conspicuous pathological change in colorectal topographic histology.This indicated that this model could be used to do research on IBS like chronic functional visceral pain.2.The visceral pain sensitivity of female model rats was higher than that of the male model rats.The discharging reactions of female rats’ EOMA to CRD were positively related to serum estrogen level.Visceral pain sensitivity of E2 model rats was higher than that of OVx model rats.These results all support the suppose that estrogen can increase colorectal sensitivity.3.Intrathecal administration of AP-5(NMDA receptor antagonist) attenuated the visceromotor response.There was no significant difference between the effect of AP-5 in E2 and OVx rats.No significant difference was found between the NMDARI expression in E2 and OVx rats’ spinal cord dorsal horn neurons.These datas suggest that estradiol doesn’t increase processing of visceral nociception by increasing NMDA receptor NMDAR1 subunit expression in spinal cord.更多还原