桃红四物汤加味调控Th1/Th2平衡及卵巢雌二醇分泌干预银屑病的作用机制研究

Study on the Mechanism of Taohong Siwutang Jiawei in Regulating Th1/Th2 Balance and Ovarian Estradiol Secretion to Intervene in Psoriasis

关键词:  桃红四物汤加味  Th1/Th2平衡  卵巢  雌二醇  银屑病

Key words:  Taohong Siwutang Jiawei  Th1/Th2 balance  Ovary  Estradiol  Psoriasis

摘要:

目的 探讨桃红四物汤加味调控辅助性T细胞1（T helper cell 1，Th1）/Th2平衡及卵巢雌二醇分泌干预银屑病小鼠的作用机制。方法 将72只SPF级雌性BALB/c小鼠随机分为空白组、模型组、阳性对照组、桃红四物汤加味低、中、高剂量组，采用小鼠背部涂抹5%咪喹莫特乳膏的方法诱导小鼠银屑病样皮损模型，造模同时给予药物干预。给药后对小鼠背部皮损进行银屑病面积与严重性指数（Psoriasis area and severity index，PASI）评分；HE染色观察小鼠皮损组织病理和卵巢组织病理变化；TUNEL法检测小鼠卵巢组织颗粒细胞凋亡；IHC检测小鼠背部皮损组织中ki-67增殖抗原（ki-67 proliferation antigen，ki-67）表达；ELISA检测小鼠血清、皮损和卵巢组织中干扰素-γ（Interferon-γ，IFN-γ）、肿瘤坏死因子-α（Tumor necrosis factor-α，TNF-α）、白细胞介素（Interleukin，IL）-4、IL-10水平，血清和卵巢组织中雌二醇（Estradiol，E2）水平，以及卵巢组织中细胞色素P450胆固醇侧链裂解酶（Cytochrome?P450?cholesterol?side?chain?lyase，P450scc）、3β-羟基类固醇脱氢酶（3β-hydroxysteroid dehydrogenase，3β-HSD）、17β-HSD水平；Western blotting和Real-time PCR检测小鼠外周血中IFN-γ、IL-4、雌激素受体α（Estrogen receptor α，ERα）、ERβ蛋白及基因表达水平；流式检测小鼠外周血中Th1/Th2细胞比例。结果 与空白组相比，模型组小鼠PASI评分、Baker评分、ki-67阳性表达量和卵巢颗粒细胞凋亡百分率明显升高，血清、皮损和卵巢组织中IFN-γ、TNF-α浓度明显升高，IL-4、IL-10浓度明显降低，血清和卵巢组织中E2浓度明显降低，卵巢组织中P450scc、3β-HSD、17β-HSD浓度明显降低，外周血中IFN-γ蛋白及基因表达水平和Th1+细胞阳性率明显升高，IL-4、ERα、ERβ蛋白及基因表达水平和Th2+细胞阳性率明显降低，Th1/Th2细胞比例明显升高（P<0.05）；与模型组相比，各给药组小鼠PASI评分、Baker评分、ki-67阳性表达量和卵巢颗粒细胞凋亡百分率明显降低，血清、皮损和卵巢组织中IFN-γ、TNF-α浓度明显降低，IL-4、IL-10浓度明显升高，血清和卵巢组织中E2浓度明显升高，卵巢组织中P450scc、3β-HSD、17β-HSD浓度明显升高，外周血中IFN-γ蛋白及基因表达水平和Th1+细胞阳性率明显降低，IL-4、ERα、ERβ蛋白及基因表达水平和Th2+细胞阳性率明显升高，Th1/Th2细胞比例明显降低（P<0.05）。结论 桃红四物汤加味可以明显降低银屑病小鼠血清、皮损和卵巢组织中Th1细胞分化因子表达，升高Th2细胞分化因子表达，调节Th1/Th2免疫平衡，改善卵巢颗粒细胞凋亡，促进E2分泌及其受体ERα、ERβ表达，减轻炎性细胞浸润和表皮增厚程度，起到治疗银屑病的作用。

Abstract:

Objective To explore the mechanism of Taohong Siwutang Jiawei in regulating T helper cell 1 (Th1)/Th2 balance and ovarian estradiol secretion to intervene in psoriasis in mice. Methods Seventy-two SPF female BALB/c mice were randomly divided into blank group, model group, positive control group, and low, medium, and high-dose groups of Taohong Siwutang Jiawei. A mouse psoriasis-like skin lesion model was induced by applying 5% imiquimod cream to the back of the mice, and drug intervention was given simultaneously during modeling. After administration, the Psoriasis area and severity index (PASI) score was performed on the skin lesions on the back of the mice; HE staining was used to observe the histopathological changes of skin lesions and ovarian tissues in mice; TUNEL method was used to detect the apoptosis of granulosa cells in the mouse ovarian tissue; IHC was used to detect the expression of ki-67 proliferation antigen (ki-67) in the skin lesions on the back of the mice; ELISA was used to detect the levels of Interferon-γ (IFN-γ), Tumor necrosis factor-α (TNF-α), Interleukin (IL)-4, IL-10 in the serum, skin lesions, and ovarian tissue of the mice, the levels of Estradiol (E2) in the serum and ovarian tissue, and the levels of Cytochrome?P450?cholesterol?side?chain?lyase (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-HSD in the ovarian tissue; Western blotting and Real-time PCR were used to detect the protein and gene expression levels of IFN-γ, IL-4, Estrogen receptor α (ERα), and ERβ in the peripheral blood of the mice; flow cytometry was used to detect the proportion of Th1/Th2 cells in the peripheral blood of the mice. Results Compared with the blank group, the PASI score, Baker score, ki-67 positive expression level, and the percentage of ovarian granulosa cell apoptosis in the model group were significantly increased, the concentrations of IFN-γ and TNF-α in the serum, skin lesions, and ovarian tissue were significantly increased, the concentrations of IL-4 and IL-10 were significantly decreased, the concentrations of E2 in the serum and ovarian tissue were significantly decreased, the concentrations of P450scc, 3β-HSD, and 17β-HSD in the ovarian tissue were significantly decreased, the protein and gene expression levels of IFN-γ in the peripheral blood and the positive rate of Th1+ cells were significantly increased, the protein and gene expression levels of IL-4, ERα, and ERβ and the positive rate of Th2+ cells were significantly decreased, and the Th1/Th2 cell ratio was significantly increased (P<0.05). Compared with the model group, the PASI score, Baker score, ki-67 positive expression level, and the percentage of ovarian granulosa cell apoptosis in each drug administration group were significantly decreased, the concentrations of IFN-γ and TNF-α in the serum, skin lesions, and ovarian tissue were significantly decreased, the concentrations of IL-4 and IL-10 were significantly increased, the concentrations of E2 in the serum and ovarian tissue were significantly increased, the concentrations of P450scc, 3β-HSD, and 17β-HSD in the ovarian tissue were significantly increased, the protein and gene expression levels of IFN-γ in the peripheral blood and the positive rate of Th1+ cells were significantly decreased, the protein and gene expression levels of IL-4, ERα, and ERβ and the positive rate of Th2+ cells were significantly increased, and the Th1/Th2 cell ratio was significantly decreased (P<0.05). Conclusion Taohong Siwutang Jiawei can significantly reduce the expression of Th1 cell differentiation factors and increase the expression of Th2 cell differentiation factors in the serum, skin lesions, and ovarian tissue of psoriasis mice, regulate the Th1/Th2 immune balance, improve the apoptosis of ovarian granulosa cells, promote the secretion of E2 and the expression of its receptors ERα and ERβ, and reduce the degree of inflammatory cell infiltration and epidermal thickening, thus playing a role in the treatment of psoriasis.