【作者】 彭圆；

【导师】 王平；

【作者基本信息】 湖北中医药大学 ， 中医基础理论， 2010， 博士

【摘要】 目的：通过皮下注射D-半乳糖、限水叠加复方地芬诺酯灌胃建立津亏便秘衰老模型,观察该模型动物相关的皮肤衰老指标,以评价便秘对皮肤衰老的影响,并通过增液承气汤作用于该复合模型,探讨其对皮肤作用的可能机制,从现代医学角度诠释中医学“肺外合皮毛,肺与大肠相表里”理论。方法：将144只2月龄昆明清洁级小鼠雌雄各半随机分为空白组、津亏便秘组(简称便秘组)、衰老组、津亏便秘衰老组(简称便秘衰老组)、增液承气汤(0.6g生药／ml)组、西沙必利组(西药组,2.5mg/kg)和增液汤组(0.51g生药／ml)。其中便秘组采用限水(1.5ml／d),叠加复方地芬诺酯(10mg/kg.d)灌胃建立津亏便秘模型,灌胃顺序依次为超纯水(20ml／kg.d)、复方地芬诺酯(10ml/kg.d)、超纯水(补充余量),间隔3小时；衰老组每天颈背部皮下注射等量D-半乳糖1000mg/kg造模；便秘衰老组及其治疗组两法同用建立便秘衰老复合证候模型；空白组以超纯水做对照；共6周。从第4周开始对增液承气汤组、增液组及西药组进行相应灌胃处理(将第一次的超纯水改为药液),持续3周。每周称量体重一次,实验结束后当天进行宏观体征观察并拍照,次日取材进行如下实验：皮肤的光电镜检测；小肠墨汁推进率观察；计算大肠和皮肤含水率；此外生化检测血清和皮、肺SOD、皮和血清HYP；用放射免疫方法检测皮肤组织匀浆HA含量；用免疫印迹检测皮肤AQP3和肺AQP1表达；用实时荧光定量PCR检测真皮TGFβRI/ⅡmRNA表达。结果：1、与空白组比较,便秘组、衰老组与便秘衰老组的各项指标显示有显著性差异(P<0.05)在宏观体征观察中各组表现出不同程度的衰老表现如形体消瘦,精神倦怠,活动后活动量减少,蜷缩聚堆,眯眼,弓背,且皮毛枯槁无光泽；相比,便秘组还表现出易激惹的症状；便秘衰老组的皮毛最为枯槁,形体最为消瘦,更急躁易怒。对组织标本进行检测,结果显示小肠推进率明显下降(P<0.01)；大肠和皮肤含水率下降(P<0.01)；皮肤明显变薄,胶原纤维数量减少；皮肤成纤维细胞存在明显线粒体水肿,异染色质发生边集,常染色质淡染或核染色致密成团的超微结构改变；血清、皮肤及肺组织中SOD活力明显下降(P<0.01),血清和皮肤组织中HYP含量减少(P<0.01)；皮肤组织匀浆中HA含量锐减；皮肤AQP3和肺AQP1表达明显减少(P<0.01)；真皮TGFβRI/ⅡmRNA表达强度明显下降(P<0.01)。且便秘组、衰老组差别不大(P>0.05),相比便秘衰老组变化程度较大。2、与便秘衰老模型相比,增液承气汤和增液汤对该模型小鼠的宏观体征如爪的形态、活动度等有一定改善,进食量和体重也有所增长,表现为爪甲淡红；西沙必利组小鼠仍爪甲偏红。对组织标本进行检测,结果显示增液承气汤、增液汤治疗、西沙必利对便秘衰老复合证候模型小鼠小肠推进率,血清、皮肤组织中SOD活力和HYP含量,皮肤AQP3和肺AQP1表达,真皮TGFβRI/ⅡmRNA表达,有显著改善(P<0.05,P<0.01)。在皮肤(大肠)含水实验中西沙必利的干预效果明显不及增液承气汤和增液汤(P<0.05)。表现为增液承气汤组小鼠大肠和皮肤含水率明显增加(P<0.01)；小鼠皮肤明显增厚,真皮胶原纤维数量增加,皮下脂肪填充减少；成纤维细胞损伤明显减轻,其核内染色质分布较均匀,核膜下异染色质聚集,胞浆内可见大量粗面内质网及线粒体；肺组织中SOD活力明显增强(P<0.01)；皮肤组织匀浆中HA含量明显增加(P<0.01)；皮肤AQP3和肺AQP1表达明显增强(P<0.01)；真皮TGFβRI/ⅡmRNA表达强度明显增加(P<0.01)。结论：1、便秘可导致皮肤衰老、加促皮肤衰老进程。2、D-半乳糖皮下注射法复合“灌水+复方地芬诺酯灌胃+灌水”的“三明治”法构建的小鼠模型是一种易于掌握、重复性高的津亏便秘衰老复合病证模型。3、增液承气汤具有明显的增水推舟、泄热通便功效,并能较好改善便秘衰老模型动物的宏观体征。4、机体自由基的清除能力下降、SOD活力下降,HYP含量降低、TGF-βRI/Ⅱ信号转导通路受阻；皮肤组织HA含量减少、AQP3表达下调,可能是津亏便秘衰老模型小鼠皮肤菲薄、干燥的病理机制。肺组织AQP1表达下降与SOD活力低下可能是津亏便秘衰老模型小鼠皮肤老化的中间环节。5、增液承气汤可有效改善证候模型小鼠的皮肤老化与皮肤组织改变,其作用机理可能与加强清除自由基,减少组织损伤；唤醒TGFβ信号转导通路,促进成纤维细胞的增殖与细胞外基质成分的合成,改善微循环血供；上调皮肤AQP3的表达及增加HA含量；增强肺组织SOD活力,促进肺组织AQP1合成,提高肺抵抗氧化应激的能力,恢复肺行水布津之能,达到濡养润泽皮肤,延缓皮肤衰老的作用。更多还原

【Abstract】 Objective:To establish the constipated and aged model mice of syndrome of fluid-deficiency by SC administration with D-galactose in the napes and backs, drink limiting and intragastric administration with diphenoxylate. It is to observe aged skin’correlative index and evaluate the effect of constipation on skin ageing. Moreover, we use prescription Zengyechengqitang to intervene the compound model of disease and syndrome, and discuss the effect and possible mechanism of Zengyechengqitang to explain the theory of "lung is associated with the large intestine" and "lung is governing the skin and hair" from the perspective of modern medicine.Methods:Kunming line mice (n=144)of equal numbers of male and female,weighing 20-22g,are randomly divided into seven groups:blank group, aged model group, constipated model group, constipated aged model group, Zengyechengqitang group, Cisapride group (western medicine control group ), Zengyetang group. The mice of blank group and constipated model group are injected with 0.1ml/10g ultrapure water in the napesand backs every day, while other five groups are injected subcutaneously with 0.1ml/10g 1000mg/kg D-galactose in the napes and backs, continuously for 42 days to establish the subacute aged models. At the same time, the mice of blank group and aged model group are given ultrapure water with every day, while other five groups are given 1.5 ml intragastric administration in sequence as follows: the ultra-pure water (20ml/kg.d), diphenoxylate (10ml/kg.d), ultra-pure water (to add the remaining amount),an interval of 3 hours every day, continuously for 42 days to establish the subacute aged constipation models. Since Zengyechengqitang (0.6g/ml), Zengyetang (0.51g/ml)and Cisapride group (2.5mg/kg) have been established model for three weeks, we give corresponding gavage therapy with 0.2ml/10g for three weeks(substituting the first ultra-pure water above in the morning for the counterparts),other groups are given isovolumic ultrapure water. During the experiment, we weigh the mice each weekend.Before the end of the experiment, the mice’s physical signs are observed in each group and are taken photoes. After that, do the following experiments with the materials I have got:Optical and transmission electron microscopic examination of the skin tissue was also performed to observe the ultrastructures;the effect on intestinal peristalsis is obsevred by the rate of small inetstine ink propelling test; water ratio of big inetstine and skin is calculated; both the changes of activity of SOD and the content of Hyp in mice’s skin & serum in all groups are tested with biochemical assay;in addition, the activity of SOD in lung tissue is carried out,and the content of HA in skin is investigated with radio-immunity method;the expressions of AQP3 in skin and AQP1 in the lung with Western blot analysis are detected, the expression of TGFβRⅠ/ⅡmRN in derma are examined with real-time fluorescence quantitative PCR technique.Results:1 Compared with the blank group, index on the aged model group,the constipated model group and the aged and constipated model group show remarkably different (P<0.05).The macroscopical signs in each group show aged performance in different degrees, for instance, emaciation and mental fatigue especially after activeities, the mice in the groups display the symtoms like fatigue,less mobility,crouching together, squinting and bowing back, their skin and fur are dry and dull.Compared with the aged model group & the constipated aged model group,the constipated group also display symptoms of irritability; compared with the aged model group and the constipated model group, the mice in the aged constipated model group are slimmer and easier to loss temper and their skin and fur are more haggard.The small inetstine ink propelling test shows that the effect on intestinal peristalsis remarkably decrease (P<0.01). Water ratio of big inetstine and skin decrease remarkably (P<0.01).Under the microscope, it is found that skin become thinner, and the amount of collagenous fibers in dermal significantly decrease. There is obvious mitochondrial swelling, chromatin margination occur, pale staining euchromatin or nuclear staining of the ultrastructural changes of dense clumps on skin fibroblasts by TEM. The SOD activity in serum,skin and lung tissue decrease remarkably (P<0.01); the HYP content of serum and skin tissue decrease remarkably (P<0.01); the HA content of skin tissue decline sharply. Western blot tests show that expression of AQP3 in skin and AQP1 in the lung remarkably reduce (P<0.01); real-time fluorescence quantitative PCR detect expression of TGFPRⅠ/ⅡmRNA in derma decrease remarkably(P<0.01). Three model groups show no significant difference (P> 0.05),but index of the aged constipation model group is the highest among of three model groups.2 Zengyechengqitang and Zengyetang can improve macroscopical signs of fluid-deficiency such as claw, food intake and body weight. The mice’s foot palm in Zengyetang group and Zengyetang is pink. The mice’s toes color in Cisapride group is still red.Compared with the constipated and aged model group, Zengyechengqitang , Zengyetang and Cisapride group can improve experiment index such as the small inetstine ink propelling test, the SOD activity and HYP content in mice’s skin & serum, the expression of AQP3 in skin and AQP1 in the lung ,and the expression of TGFβRⅠ/ⅡmRNA in derma (P<0.05, P<0.01), and the effect of Zengyechengqitang is better than that of Zengyetang. In other experiments, the effect of cisapride is not as good as the effect of Zengyechengqitang group and Zengyetang (P<0.05),and the effect of Zengyechengqitang is better than that of Zengyetang. Zengyechengqitang can remarkably increase water ratio of big inetstine and skin (P<0.01); Under the microscope, it is found that skin becomes thicker, the amount of collagenous fibers in dermal significantly increases, and fat cells in subcutaneous is less. The damage to the cell is lighter than the model group by TEM,which chromatin in nucleus is more well-distributed and heterochromatin is aggregated under the nuclear membrane and nucleoli is prominent.A large number of rough endoplasmic reticulum and mitochondria shows within the cytoplasm,it also can improve swelling of mitochondra. Zengyechengqitang can remarkably increase the activity of SOD in lung tissue (P<0.01),HA content of skin tissue (P<0.01), and expression of AQP3 in skin and AQP1 in the lung (P<0.01) and expression intensity of TGFβRⅠ/ⅡmRNA in derma (P<0.01).Conclusion:1 The constipation with syndrome of fluid-deficiency not only cause skin aging, but also promote an increasingly skin aging process.2 The compound model of disease and syndrome,which is made according to D-galactose with SC administration compound "drinking limited plus diphenoxylate plus drinking limited " with intragastric administration,is easy to operate.The compound model of disease and syndrome is a frequently repetitive animal model of constipated and aged model with syndrome of fluid-deficiency. 3 Zengyechengqitang has an remarkable effect on increasing Yin-fluid, dispersing heat and promoting inetstine movement,and improving macroscopical signs of the compound model mice.4 The decrease of the level of free radicals’scavenging and the activity of SOD, the decrease of the content of Hyp in mice’s skin & serum, the decrease of the HA content in skin and lowering of AQP3 expression, and the decrease of the expression intensity of TGFβRⅠ/ⅡmRNA in derma,are likely to be one of pathological mechanisms of steatosis cutis and watery impairment on skin aging’s performance with the compound model mice. The lowering of AQP1 in the mice and the low activity of SOD in lung tissue may be the intermediate link of constipation which causes skin aging.5 Zengyechengqitang can be more effective to improve the skin changes and the skin dryness of the compound model mice, and its pathological mechanism may be related to strengthen free radicals’scavenging, reduce tissue damage, enhance collagen metabolism, upregulate the expression of AQP3 and to the increase of the HA content of skin. Zengyechengqitang may play an important role in delaying skin aging by upregulating the expression levels of TGFβRⅠ/ⅡmRNA in derma, arousing TGFβsignal transduction pathway, promoting fibroblast proliferation and extracellular matrix synthesis, and improving blood supply on skin’s microcirculation.Zengyechengqitang may postpone lung aging through the enhancement of SOD activity in lung tissue and the improvement of the capacity against oxidative stress in the lung.Zengyechengqitang may promote AQP1 synthesis in lung tissue in order to compress Jin-fluid and moisturize skin.更多还原