芹菜素、针刺对LPS诱导的SD大鼠乳腺炎的抗炎效果及其机制研究

发布时间：2019-02-16 06:56

【摘要】：芹菜素是治疗乳腺炎要药的重要成分,属实际无毒、无遗传毒性的黄酮类化合物,且拥有较好的抗炎作用;针刺治疗疾病安全方便,该法已经广泛应用于炎症疾病的治疗,但是芹菜素或/和针刺治疗乳腺炎的效果还未见报道。本研究以SD大鼠为试验对象,在建立乳腺炎模型的基础上,对芹菜素或/和针刺对乳腺炎的治疗效果开展体内研究;并分离培养SD乳腺上皮细胞,进一步研究芹菜素对LPS诱导的乳腺上皮细胞炎性损伤的保护作用及机制,以期为临床应用芹菜素和针刺治疗乳腺炎提供科学的试验依据。首先建立SD大鼠乳腺炎模型。30只SD雌鼠于产后72 h随机分为对照组和5个试验组,每组5只,各试验组大鼠经第四对乳头灌注不同剂量LPS(10μg/侧、20μg/侧、30μg/侧、40μg/侧、50μg/侧),对照组灌注等量PBS。灌注后24 h观察各大鼠临床症状、乳腺组织病理学变化。结果发现,试验组大鼠第四对乳腺均可观察到不同程度炎症反应,且与LPS剂量呈依赖关系。结果表明,成功建立LPS诱导哺乳期SD大鼠乳腺炎模型。然后,利用建立的SD大鼠乳腺炎模型,探讨芹菜素、针刺对乳腺炎的治疗效果。将乳腺炎SD大鼠随机分为四组:LPS刺激组(LPS组)、芹菜素治疗组(Api组)、针刺治疗组(Acu组)、芹菜素联合针刺治疗组(Api+Acu组),正常SD大鼠为对照组(Con组),每组5只。在LPS灌注后的24 h开始实施治疗,每天一次,治疗5天,最后一次治疗后的24 h处死大鼠收集乳腺组织。观察乳腺组织的病理学变化;q RT-PCR和ELISA方法检测乳腺组织中TNF-α、IL-1β和IL-6的表达水平;western blot方法检测MPO表达水平和TLR4/NF-κB信号通路情况。结果显示,与LPS组相比,三个治疗组的乳腺组织病变明显减轻,MPO蛋白水平显著降低,TNF-α、IL-1β和IL-6的m RNA和蛋白水平均显著降低,且TLR4/NF-κB信号通路的激活被显著抑制。以上结果说明芹菜素、针刺能通过抑制TLR4/NF-κB信号通路来治疗LPS引起的SD大鼠乳腺炎。最后,分离培养SD大鼠乳腺上皮细胞,研究芹菜素对LPS诱导的乳腺上皮细胞炎性损伤的保护作用和机制。MTT法检测芹菜素对大鼠乳腺上皮细胞的毒性作用,q RT-PCR和ELISA方法检测芹菜素对大鼠乳腺上皮细胞中TNF-α、IL-1β和IL-6表达水平的影响。western blot和IIF法检芹菜素对SD大鼠乳腺上皮细胞中TLR4/NF-κB信号通路的影响。结果显示,芹菜素在1~10μg/m L的浓度下无细胞毒性作用;和LPS组相比,芹菜素预处理细胞能够显著降低TNF-α、IL-1β和IL-6的m RNA和蛋白水平;western blot结果显示芹菜素是通过抑制乳腺上皮细胞中TLR4/NF-κB信号通路的激活而发挥其抗炎作用的,IIF法检测结果证明,芹菜素可抑制p65的磷酸化及转移入核。本试验对芹菜素、针刺治疗乳腺炎的效果和作用机理进行了初步探索性研究,发现芹菜素、针刺能使TLR4的表达下调并对下游NF-κB信号通路的激活起到抑制作用,最终使乳腺中TNF-α、IL-1β和IL-6的mRNA和蛋白表达水平均显著下降。
[Abstract]:Apigenin is an important component in the treatment of mastitis. It belongs to non-toxic and non-genotoxic flavonoids and has good anti-inflammatory effect. Acupuncture has been widely used in the treatment of inflammatory diseases, but the effect of apigenin or / and acupuncture on mastitis has not been reported. In this study, the effects of apigenin or / and acupuncture on mastitis were studied in vivo on the basis of establishing mastitis model in SD rats. The protective effect and mechanism of apigenin on inflammatory injury induced by LPS were studied in order to provide scientific experimental basis for clinical application of apigenin and acupuncture in the treatment of mastitis. First, the mastitis model of SD rats was established. 30 SD female rats were randomly divided into control group and 5 experimental groups at 72 hours postpartum. Each group was perfused with different doses of LPS (10 渭 g / side, 20 渭 g / side, 30 渭 g / side) through the fourth pair of nipples, each group was injected with different doses of LPS (10 渭 g / side, 20 渭 g / side, 30 渭 g / side). 40 渭 g / side, 50 渭 g / side), the control group was perfused with the same amount of PBS.. The clinical symptoms and histopathological changes of mammary gland were observed 24 hours after perfusion. The results showed that different degree of inflammation was observed in the fourth pair of mammary glands in the experimental group, which was dependent on the dose of LPS. The results showed that the model of mastitis induced by LPS was successfully established in SD rats. Then, the effects of apigenin and acupuncture on mastitis in SD rats were studied. Mastitis SD rats were randomly divided into four groups: LPS stimulation group (LPS group), apigenin treatment group (Api group), acupuncture treatment group (Acu group), apigenin combined acupuncture treatment group (Api Acu group) and normal SD rats as control group (Con group). There were 5 rats in each group. After 24 hours of LPS perfusion, the rats were sacrificed to collect mammary gland tissue once a day for 5 days and 24 hours after the last treatment. The expression levels of TNF- 伪, IL-1 尾 and IL-6 in breast tissues were detected by Q RT-PCR and ELISA methods. MPO expression and TLR4/NF- 魏 B signal pathway were detected by; western blot method. The results showed that compared with the LPS group, the breast lesions in the three treatment groups were significantly reduced, the MPO protein level was significantly decreased, and the m RNA and protein levels of TNF- 伪, IL-1 尾 and IL-6 were significantly decreased. Moreover, the activation of TLR4/NF- 魏 B signaling pathway was significantly inhibited. These results suggest that apigenin and acupuncture can inhibit TLR4/NF- 魏 B signaling pathway to treat SD mastitis induced by LPS. Finally, mammary epithelial cells of SD rats were isolated and cultured to study the protective effect and mechanism of apigenin on the inflammatory injury induced by LPS. The toxic effect of apigenin on mammary epithelial cells was detected by MTT assay. The effects of apigenin on the expression of TNF- 伪, IL-1 尾 and IL-6 in rat mammary epithelial cells were detected by Q RT-PCR and ELISA. The effects of. Western blot and IIF assay on TLR4/NF- 魏 B signaling pathway in SD rat mammary epithelial cells were detected. The results showed that apigenin had no cytotoxicity at the concentration of 1 渭 g / mL, and that apigenin pretreatment could significantly reduce the m RNA and protein levels of TNF- 伪, IL-1 尾 and IL-6 compared with LPS group. Western blot results showed that apigenin inhibited the activation of TLR4/NF- 魏 B signaling pathway in mammary epithelial cells. IIF assay demonstrated that apigenin could inhibit p65 phosphorylation and metastasis into the nucleus. The effect and mechanism of apigenin and acupuncture on mastitis were studied. It was found that apigenin and acupuncture could down-regulate the expression of TLR4 and inhibit the activation of downstream NF- 魏 B signaling pathway. Finally, the expression levels of mRNA and protein of TNF- 伪, IL-1 尾 and IL-6 in breast were significantly decreased.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332;R655.8

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