归芍运脾汤对肝郁脾虚型乳腺增生大鼠的干预作用

Intervention of Guishao Yunpi decoction on mammary gland hyperplasia in rats with liver depression and spleen deficiency

目的基于雌激素受体α(ERα)/细胞外调节蛋白激酶(ERK)/环腺苷酸反应元件结合蛋白(CREB)通路探讨归芍运脾汤对肝郁脾虚型乳腺增生大鼠的干预作用及机制。方法将60只SPF级SD大鼠随机分为空白组10只和造模组50只。用苯甲酸雌二醇联合黄体酮复制乳腺增生大鼠模型,用夹尾刺激联合饥饱失常复制肝郁脾虚大鼠模型。将造模成功的大鼠随机分为模型组、对照组和高、中、低剂量实验组,每组10只。空白组和模型组均给予等量蒸馏水;高、中、低剂量实验组分别灌胃给予17.2、8.6、4.3 g·kg^(-1)·d^(-1)归芍运脾汤汤剂;对照组灌胃给予4 mg·kg^(-1)·d^(-1)他莫昔芬。6组大鼠每天给药1次,持续干预30 d。测定大鼠第2对乳头直径及高度,用酶联免疫吸附试验法检测大鼠乳腺组织中雌激素(E_(2))和孕激素(P)含量,用蛋白质印迹法测定乳腺组织中ERα、ERK1/2、CREB蛋白的表达水平。结果中、高剂量实验组和对照组、模型组、空白组的第2对乳头直径分别为(1.00±0.12)、(0.90±0.17)、(1.04±0.23)、(1.44±0.17)和(0.70±0.12)mm,乳头高度分别为(1.65±0.10)、(0.99±1.12)、(1.50±0.14)、(2.03±0.14)和(0.65±0.21)mm,乳腺组织中E2含量分别为(2020.26±382.09)、(1555.27±382.75)、(2072.20±289.64)、(3302.53±446.05)和(1376.44±319.81)pg·mL^(-1),ERα蛋白相对表达水平分别为0.75±0.08、0.57±0.07、0.65±0.08、0.96±0.08和0.49±0.05,ERK1/2蛋白相对表达水平分别为0.83±0.03、0.62±0.02、0.75±0.02、0.88±0.01和0.47±0.01,CREB蛋白相对表达水平分别为0.59±0.06、0.54±0.05、0.72±0.07、0.96±0.09和0.48±0.05;模型组的上述指标与中、高剂量实验组比较,差异均有统计学意义(均P<0.05)。结论归芍运脾汤能够有效地改善肝郁脾虚型乳腺增生大鼠的一般生存状况及乳腺增生程度,其具体机制可能是归芍运脾汤有效抑制乳腺组织中雌激素受体相关ERα/ERK/CREB信号通路过度激活而实现。

Objective To explore the intervention effect and mechanism of Guishao Yunpi decoction on mammary gland hyperplasia in rats with liver depression and spleen deficiency from estrogen receptorα(ERα)/extracellular regulated protein kinases(ERK)/cAMP-response element binding protein(CREB)pathway.Methods Sixty SPF SD rats were randomly divided into blank group(n=10 cases)and modeling group(n=50 cases).Estradiol benzoate combined with progesterone was used to rep licate the mammary hyperplasia rat model,and tail-clamping stimulation combined with hunger and satiety disorders was used to replicate the liver depression and spleen deficiency rat model.The successful rats were randomly divided into model group,control group and experimental-L,-M,-H groups,with 10 rats in each group.Blank group and model group were fed with distilled water.The rats in experimental-H,-M,-L groups of Guishao Yunpi decoction were given intragastric administration of 17.2,8.6,4.3 g·kg^(-1)·d^(-1) Guishao Yunpi decoction,respectively,and the positive control group was given tamoxifen(4 mg·kg^(-1)·d^(-1)).Six groups were treated for 30 days with once a day.The diameter and height of the second pair of nipples were measured,the contents of estrogen(E_(2))and progestational hormone(P)in breast tissue were detected by enzyme-linked immunosorbent assay,and the protein expression levels of estrogen receptorα(ERα),extracellular regulated protein kinases(ERK)and cAMP-response element binding protein(CREB)in breast tissue were determined by Western blotting.Results The diameters of the second pair of papillae in the experimental-M,-H groups,control group,model group and blank group were(1.00±0.12),(0.90±0.17),(1.04±0.23),(1.44±0.17)and(0.70±0.12)mm;nipple heights were(1.65±0.10),(0.99±1.12),(1.50±0.14),(2.03±0.14)and(0.65±0.21)mm;the breast tissue E_(2) levels were(2020.26±382.09),(1555.27±382.75),(2072.20±289.64),(3302.53±446.05)and(1376.44±319.81)pg·mL-1;ERαprotein expression levels were 0.75±0.08,0.57±0.07,0.65±0.08,0.96±0.08 and 0.49±0.05;ERK1/2 protein expression levels were 0.83±0.03,0.62±0.02,0.75±0.02,0.88±0.01 and 0.47±0.01;CREB protein expression levels were 0.59±0.06,0.54±0.05,0.72±0.07,0.96±0.09 and 0.48±0.05,respectively.Compared with model group,the differences of above indexes in the experimental-M and-H groups were statistically significant(all P<0.05).Conclusion Guishao Yunpi decoction can effectively improve the general living condition and the degree of mammary gland hyperplasia in rats with mammary gland hyperplasia of liver stagnation and spleen deficiency.The specific mechanism may be that Guishao Yunpi decoction can effectively inhibit the excessive activation of estrogen receptor-related ERα/ERK/CREB signal pathway in mammary gland tissue.