The Research Of Zengyechengqitang's Effect On Skin Of The Constipated And Aged Model Mice With Syndrome Of Fluid-deficiency

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.