| Purpose:To explore the effect and possible therapeutic mechanism of Jianpi Yangxue Qufeng Formula in mice with chronic eczema on skin barrier function and immune mechanism.In order to provide experimental basis for Jianpi Yangxue Qufeng Formula in the treatment of chronic eczema with spleen deficiency.To further demonstrate Jianpi Yangxue Qufeng Formula as the important and feasible TCM treatment of chronic eczema. Material and method: 1 Materials: 1.1 Animals and Groups:Fifty healthy male mice, were randomly divided into 5 groups, namely normal group, model group, positive medicine group, Jianpi Yangxue Qufeng Formula high and low dose groups. 1.2 Drugs: Administered by intragastric administration. The Chinese medicine group were treated by Jianpi Yangxue Qufeng Formula, the positive medicine group were treated by levocetirizine hydrochloride. Normal group and model group were administered with normal saline.2 Method: 2.1 Modeling methods: Low-dose DNCB and Senna Fominm were used to establish mice models of chronic eczema with spleen deficiency. 2.2 Experimental method: Part 1: Study on the effects of the local inflammation of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Observing the effect of Jianpi Yangxue Qufeng Formula on mice with splenoasthenic chronic eczema in ear swelling and thickness.(2) Observing the effect of Jianpi Yangxue Qufeng Formula in spleen index and thymus index.(3) Using HE staining to observe the pathologica changes in tissue. Part 2: Study on the skin barrier function effects of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Using immunohistochemical method to observe the expression of AQP3 in skin tissue.(2)Using immunohistochemical method to observe the expression of BH in skin tissue. Part 3:Study on the regulation of the immune mechannism of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Using flow cytometry to detect the levels of CD4+ and CD8+ in spleen cells,and calculate the ratio of them.(2) Using immunohistochemical method to observe the expression of VCAM-1 and VCAM-1 in skin tissue.(3) Using immunohistochemical method WB to observe the expression of CysLTRl and CysLTR2 in skin tissue.(4) Using ELISA method to dectect the level of CCL17 and CCL18 in serum;and using RT-PCR method to dectect the expression of CCL17 and CCL18 in skin tissues. Results: Part 1: The inhibitory effect on the local inflammation of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Jianpi Yangxue Qufeng Formula can reduce the model mice ear swelling degree and the thickness of the lesion.There was significant difference between the model group and the normal group in ear swelling degree and ear thickness.(P<0.05); The treatment groups can significantly inhibit the mice ear swelling, compared with the model group, there was significant difference(P<0.01); There was no significant difference between positive group and every group of Chinese herbs(P>0.05).(2) Jianpi Yangxue Qufeng Formula can reduce the model mice spleen index and thymus index.Compared with normal group, spleen and thymus index in each model group were increased in varying degrees, there was significant difference(P<0.01); After drug intervention, compared with model group, the spleen index and thymus index were significantly decreased in the treatment groups with significant difference(P< 0.05); There was no significant difference between positive group and every group of Chinese herbs in the changes of spleen index and thymus index.(P>0.05).(3) Jianpi Yangxue Qufeng Formula has obvious inhibitory effect on the local inflammation, and can improve the pathological changes of skin lesions.Normal group: no hyperkeratosis, epidermal thickening and sponge formation, dermal inflammatory cell infiltration, no vascular dilatation. Model group: epidermal hyperkeratosis, acanthosis, intercellular edema was not obvious, no intraepidermal blisters, dermal perivascular inflammatory infiltration was obvious. The positive group and the high dose group of Chinese herbs: changes in the structure are similar. High dose group of Chinese herbs: hyperkeratosis of the epidermis is not obvious, acanthosis significantly reduced, dermal inflammatory infiltration was significantly reduced, the swelling of endothelial cells was reduced. Low dose group of Chinese herbs: epidermal hyperkeratosis and acanthosis, dermal inflammatory infiltration and endothelial cell swelling were reduced. Part 2: The repair effect on skin barrier function of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Jianpi Yangxue Qufeng Formula can significantly reduce the overexpression of AQP3 in the skin lesions.The average optical density of AQP3 in model group is significantly higher than normal group(P<0.01); Compared with model group, the treatment groups can reduce the expression of AQP3 in mice skin tissue, with significant difference(P<0.05). The positive group and high dose group of Chinese herbs had no statistical significance(P>0.05).(2) Jianpi Yangxue Qufeng Formula can significantly raise the weak epression of BH in the skin lesions.The average optical density of BH in model group was significantly lower than that of normal group(P<0.01); Compared with model group, the treatment groups can upregulate the expression of BH in mice skin tissue, with significant difference(P<0.05). The positive group and high dose group of Chinese herbs had no statistical significance(P>0.05). Part 3: The regulation on the immune mechannism of Jianpi Yangxue Qufeng Formula on mice with spleen-deficiency chronic eczema(1) Jianpi Yangxue Qufeng Formula can significantly raise the level of CD4+ and CD4+/CD8+ ratioCompared with normal group, the level of CD4+ and CD4+/CD8+ ratio were decreased significantly in model group, with significant difference(P<0.01). Compared with model group, the levels of CD4+ and CD4+/CD8+ were significantly increased in positive group and high dose group of Chinese herbs, with significant difference(P<0.01); the levels of CD4+ and CD4+/CD8+ were also increased in low dose group of Chinese herbs than those in model group with significant difference(P<0.05). Compared with positive group and high dose group of Chinese herbs, there was no significant difference(P>0.05). Compared with normal group, the level of CD8+ did not change significantly in other groups, there was no significant difference(P>0.05).(2) Jianpi Yangxue Qufeng Formula can obviously inhibited the overexpression of ICAM-1 and VCAM-1 in the skin lesions.Compared with normal group, the expression of ICAM-1 and VCAM-1 in skin tissue was significantly increased in model group, with significant difference(P<0.01). After administration,the expression of positive group and high dose group of Chinese herbs were significantly lower than model group(P<0.01). No statistically significant difference between the high dose group of Chinese herbs and positive group(P>0.05).(3) Jianpi Yangxue Qufeng Formula can obviously inhibited the overexpression protein of CysLTR1 and CysLTR2 in the skin lesions.Compared with normal group, the expression of CysLTR1 and CysLTR2 in skin tissue was significantly increased in model group, with significant difference(P<0.01). Compared with model group, the expression of positive group and high dose group of Chinese herbs were significantly decreased, with significant difference(P<0.01). Compared with positive group, the low dose group of Chinese herbs had stronger expression, with significant difference(P<0.01), and the high dose group of Chinese herbs had no significant difference with positive group(P>0.05). The expression intensity of CysLTR1 and CysLTR2 in each group had no statistical significance(P>0.05).(4) Jianpi Yangxue Qufeng Formula can obviously inhibit the level of CCL17 and CCL18 in serum,and weakened the expression of CCL17 and CCL18 in skin lesions.Compared with normal group, CCL17, CCL18 levels in serum increased significantly in model group, with significant difference(P<0.01). Compared with model group, the level of CCL17, CCL18 in serum of mice in each treatment group decreased significantly, with significant difference(P<0.01). Compared with positive group, the level of CCL17 and CCL18 significantly lower than low dose group of Chinese herbs, with statistically significant difference(PCCL17<0.05, PCCL18<0.01);The level of CCL17 and CCL18 has no significant difference between high dose group of Chinese herbs and positive group(P>0.05).Compared with normal group, the expression of CCL17 and CCL18 were significantly enhanced in lesional skin of mice in model group, with significant difference(P<0.01). Compared with model group, the expression of CCL17 and CCL18 in all treated groups were significantly decreased, with significant difference(P<0.01). Compared with positive group, the expression of CCL17 and CCL18 in low dose group of Chinese herbs was significantly stronger than positive group, with statistically significant difference(P<0.01); The expression of CCL17 and CCL18 has no significant difference between high dose group of Chinese herbs and positive group(P>0.05). Conclusion: 1.Using multi factor to establish animal model of chronic eczema with spleen deficiency, proving that the animal model of combination with disease and syndrome in the experimental study of Dermatology is feasible. 2. Jianpi Yangxue Qufeng Formula can significantly inhibit the mice ear swelling and decrease the ear thickness;can reduce the spleen index and thymus index; and has an obvious inhibitory effect on the local inflammation, and improvement on the pathological changes of skin lesions. Tip: Jianpi Yangxue Qufeng Formula has good inhibition effect on spleen-deficiency chronic eczema. 3. Jianpi Yangxue Qufeng Formula can significantly reduce the overexpression of AQP3 and strengthen the weak expression of BH. Tip: Jianpi Yangxue Qufeng Formula has good effect in repairing skin barrier function on spleen-deficiency chronic eczema. 4. Jianpi Yangxue Qufeng Formula can raise the level of CD4+ in spleen cells and the ratio of CD4+/CD8+; inhibit the expression of ICAM-1 and VCAM-1 in skin lesions; restrain the overexpression of protein of CysLTR1 and CysLTR2 in skin lesions. Regulate down the level of CCL17 and CCL18 in serum,and reduce the overexpression of CCL17 and CCL18 in skin lesions. Tip: Jianpi Yangxue Qufeng Formula has good effect in regulating the immune function on spleen-deficiency chronic eczema. |
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