消核冲剂激活ASK1/MKK4/JNK通路促进乳腺上皮细胞凋亡改善大鼠乳腺增生分子机制

doi:10.12122/j.issn.1673-4254.2026.08.21

摘要/Abstract

摘要：

目的探究中药复方消核冲剂（XHGs）通过调控ASK1/MKK4/JNK信号通路促进细胞凋亡改善乳腺增生机制。方法苯甲酸雌二醇（0.5 mg·kg^-1·d^-1）和孕酮（5 mg·kg^-1·d^-1）肌肉注射未孕雌性SD大鼠构建乳腺增生模型，设置分组：对照组、模型组、低、中、高剂量消核冲剂灌胃组、高剂量消核冲剂加GS-444217灌胃组。分组给药处理后，记录乳头高度、直径，子宫指数；HE染色观察乳腺结构变化；免疫组化染色检测Ki-67和caspase-3蛋白表达；TUNEL染色检测组织凋亡；ELISA检测血清中E2、P、PRL、FSH、IL-1β和TNF-α的表达；RT-qPCR和Western blotting检测ASK1/MKK4/JNK及凋亡相关蛋白表达。结果结果显示模型组较对照组，乳房直径和乳头高度及子宫指数增加（P<0.05）；乳腺组织腺泡明显增生、导管显著扩张及炎性细胞浸润明显增多；Ki-67表达增加，caspase-3降低（P<0.05）；细胞凋亡降低（P<0.01）；E2、P、PRL、FSH、IL-1β和TNF-α水平也显著升高；Bcl-2表达升高，caspase-3、Bax降低；p-ASK1/ASK1、p-MKK4/MKK4、p-JNK/JNK、caspase-3和Bax蛋白降低，Bcl-2蛋白表达升高（P<0.01）。而给予消核冲剂治疗后，逆转了各指标的变化，且呈现出浓度依赖性改善（P<0.01）。同时GS-444217和XHGs-H灌胃处理组相较于XHGs-H又逆转了XHGs的改善效果。结论 XHGs通过激活ASK1/MKK4/JNK信号通路促进乳腺增生细胞凋亡改善大鼠乳腺增生。

关键词: 乳腺增生, 消核冲剂, 细胞凋亡, ASK1/MKK4/JNK通路, 雌激素, 孕激素

Abstract:

Objective To explore the mechanism of Xiaohe Granule (XHG) for alleviating mammary hyperplasia in rats. Methods A non-pregnant female SD rat model of mammary hyperplasia was established by intramuscular injections of estradiol benzoate (0.5 mg/kg) and progesterone (5 mg/kg). With untreated rats as the control group, the rat models were treated with saline (model group) or low-, medium- or high-dose XHG daily gavage, or with high-dose XHG plus oral administration of GS-444217. After the treatments, nipple height, diameter, and uterine index of the rats were measured, and histological changes in the mammary tissue were observed with HE staining. The expressions of Ki-67 and caspase-3 protein in the mammary tissue were detected using immunohistochemistry, and mammary epithelial cell apoptosis was examined using TUNEL staining. Serum levels of E2, P, PRL, FSH, IL-1β, and TNF-α were detected using ELISA, and the expressions of ASK1/MKK4/JNK and apoptosis-related proteins were analyzed using RT-qPCR and Western blotting. Results Compared with the normal control rats, the rats in the model group showed significantly increased mammary diameter, nipple height, and uterine index with obvious acinar hyperplasia, ductal dilation, and inflammatory cell infiltration in the mammary tissue. The rat models also showed significantly increased Ki-67 expression and serum levels of E2, P, PRL, FSH, IL-1β and TNF-α, markedly increased Bcl-2 expression, and lowered protein levels of p-ASK1/ASK1, p-MKK4/MKK4, p-JNK/JNK, caspase-3, and Bax in the mammary tissue. Treatment with XHG significantly reversed these changes in a dose-dependent manner. Treatment with GS-444217 significantly attenuated the ameliorative effect of high-dose XHG on mammary hyperplasia in the rat models. Conclusion XHG alleviates mammary hyperplasia in rats by promoting apoptosis of hyperplastic mammary epithelial cells via activating the ASK1/MKK4/JNK signaling pathway.

Key words: mammary gland hyperplasia, Xiaohe Granule, cell apoptosis, ASK1/MKK4/JNK pathway, estrogen, progesterone

张雨, 孙康, 刘举达, 王舒晗, 裴静. 消核冲剂激活ASK1/MKK4/JNK通路促进乳腺上皮细胞凋亡改善大鼠乳腺增生分子机制[J]. 南方医科大学学报, 2026, 46(8): 1936-1946.

Yu ZHANG, Kang SUN, Juda LIU, Shuhan WANG, Jing PEI. Xiaohe Granule improves breast hyperplasia in rats by promoting mammary epithelial cell apoptosis via activating the ASK1/MKK4/JNK signaling pathway[J]. Journal of Southern Medical University, 2026, 46(8): 1936-1946.

图/表 9

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene name Upstream primer (5'-3') Downstream primer (5'-3')

Product size (bp)

Rat-caspase3 AACTCTTCATCATTCAGGCCT CCATATCATCGTCAGTTCCAC

Rat-Bcl-2 CCGGGACGCGAAGTGCTATT GGGCGCGGAGTCTTCATCTC

Rat-Bax CCCAGGACGCATCCACCAAG TGTCAGCTGCCACACGGAAG

Rat-β-actin GTCCACCCGCGAGTACAACC CATACCCACCATCACACCCTGG

165

144

208

图1 各组大鼠子宫组织形态图及子宫指数的比较

Fig.1 Observation of the dissected uterine of the rats and comparison of body weight and uterine index among the 6 groups (Mean±SD, n=3). A: Control group; B: Model group; C: XHGs-L group; D: XHGs-M group; E: XHGs-H group; F: XHGs-H+GS-444217 group. *P<0.05, **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs model group; ▲P<0.01 vs XHGs-H group.

图2 各组大鼠左右乳腺组织大小形态图及左右乳房高度和直径的比较

Fig.2 Left and right mammary tissue size and morphology of the rats in each group and comparison of breast height and diameter among the 6 groups (Mean±SD, n=3). **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; ▲P<0.05, ▲▲P<0.01 vs XHGs-H group.

图3 大鼠乳腺组织病理HE染色

Fig.3 HE staining of rat mammary tissue in each group (Original magnification: ×400). A: Control group, B: Model group, C: XHGs-L group, D: XHGs-M group, E: XHGs-H group, F: XHGs-H+GS-444217 group.

图4 大鼠乳腺组织caspase-3和Ki67免疫组化染色及平均光密度值的比较

Fig.4 Immunohistochemical staining of caspase-3 and Ki67 in rat mammary gland tissue (×400) and comparison of average optical density values among the 6 groups (Mean±SD, n=3). **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; ▲▲P<0.01 vs XHGs-H group.

图5 各组大鼠乳腺组织样本TUNEL检测及细胞凋亡率的比较

Fig.5 TUNEL staining of mammary gland tissues of the rats from each group (×400) and comparison of cell apoptosis rate among the 6 groups (Mean±SD, n=3). **P<0.01 vs Control group; ##P<0.01 vs Model group; ▲▲P<0.01 vs XHGs-H group.

图6 Western blotting检测各组乳腺组织相关通路及凋亡基因蛋白表达条带图及灰度值表达的比较

Fig.6 Western blotting for detecting expressions of apoptosis-related proteins and the ASK1/MKK4/JNK pathway proteins in the breast tissues of the rats (Mean±SD, n=3). *P<0.05, **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; ▲▲P<0.01 vs XHGs-H group.

图7 XHGs对各组乳腺组织相关凋亡指标mRNA表达的影响

Fig.7 Effect of XHG on mRNA expression of apoptosis-related genes in the breast tissue of the rats (Mean±SD, n=3). **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; ▲▲P<0.01 vs XHGs-H group.

图8 XHGs对各组大鼠血清中相关指标表达的影响

Fig.8 Effects of XHG on serum biochemical indicators of the rats in each group (Mean±SD, n=3). **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; ▲P<0.05, ▲▲P<0.01 vs XHGs-H group.

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