【摘要】：芹菜素是治療乳腺炎要藥的重要成分,屬實(shí)際無(wú)毒、無(wú)遺傳毒性的黃酮類化合物,且擁有較好的抗炎作用;針刺治療疾病安全方便,該法已經(jīng)廣泛應(yīng)用于炎癥疾病的治療,但是芹菜素或/和針刺治療乳腺炎的效果還未見(jiàn)報(bào)道。本研究以SD大鼠為試驗(yàn)對(duì)象,在建立乳腺炎模型的基礎(chǔ)上,對(duì)芹菜素或/和針刺對(duì)乳腺炎的治療效果開展體內(nèi)研究;并分離培養(yǎng)SD乳腺上皮細(xì)胞,進(jìn)一步研究芹菜素對(duì)LPS誘導(dǎo)的乳腺上皮細(xì)胞炎性損傷的保護(hù)作用及機(jī)制,以期為臨床應(yīng)用芹菜素和針刺治療乳腺炎提供科學(xué)的試驗(yàn)依據(jù)。首先建立SD大鼠乳腺炎模型。30只SD雌鼠于產(chǎn)后72 h隨機(jī)分為對(duì)照組和5個(gè)試驗(yàn)組,每組5只,各試驗(yàn)組大鼠經(jīng)第四對(duì)乳頭灌注不同劑量LPS(10μg/側(cè)、20μg/側(cè)、30μg/側(cè)、40μg/側(cè)、50μg/側(cè)),對(duì)照組灌注等量PBS。灌注后24 h觀察各大鼠臨床癥狀、乳腺組織病理學(xué)變化。結(jié)果發(fā)現(xiàn),試驗(yàn)組大鼠第四對(duì)乳腺均可觀察到不同程度炎癥反應(yīng),且與LPS劑量呈依賴關(guān)系。結(jié)果表明,成功建立LPS誘導(dǎo)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,探討芹菜素、針刺對(duì)乳腺炎的治療效果。將乳腺炎SD大鼠隨機(jī)分為四組:LPS刺激組(LPS組)、芹菜素治療組(Api組)、針刺治療組(Acu組)、芹菜素聯(lián)合針刺治療組(Api+Acu組),正常SD大鼠為對(duì)照組(Con組),每組5只。在LPS灌注后的24 h開始實(shí)施治療,每天一次,治療5天,最后一次治療后的24 h處死大鼠收集乳腺組織。觀察乳腺組織的病理學(xué)變化;q RT-PCR和ELISA方法檢測(cè)乳腺組織中TNF-α、IL-1β和IL-6的表達(dá)水平;western blot方法檢測(cè)MPO表達(dá)水平和TLR4/NF-κB信號(hào)通路情況。結(jié)果顯示,與LPS組相比,三個(gè)治療組的乳腺組織病變明顯減輕,MPO蛋白水平顯著降低,TNF-α、IL-1β和IL-6的m RNA和蛋白水平均顯著降低,且TLR4/NF-κB信號(hào)通路的激活被顯著抑制。以上結(jié)果說(shuō)明芹菜素、針刺能通過(guò)抑制TLR4/NF-κB信號(hào)通路來(lái)治療LPS引起的SD大鼠乳腺炎。最后,分離培養(yǎng)SD大鼠乳腺上皮細(xì)胞,研究芹菜素對(duì)LPS誘導(dǎo)的乳腺上皮細(xì)胞炎性損傷的保護(hù)作用和機(jī)制。MTT法檢測(cè)芹菜素對(duì)大鼠乳腺上皮細(xì)胞的毒性作用,q RT-PCR和ELISA方法檢測(cè)芹菜素對(duì)大鼠乳腺上皮細(xì)胞中TNF-α、IL-1β和IL-6表達(dá)水平的影響。western blot和IIF法檢芹菜素對(duì)SD大鼠乳腺上皮細(xì)胞中TLR4/NF-κB信號(hào)通路的影響。結(jié)果顯示,芹菜素在1~10μg/m L的濃度下無(wú)細(xì)胞毒性作用;和LPS組相比,芹菜素預(yù)處理細(xì)胞能夠顯著降低TNF-α、IL-1β和IL-6的m RNA和蛋白水平;western blot結(jié)果顯示芹菜素是通過(guò)抑制乳腺上皮細(xì)胞中TLR4/NF-κB信號(hào)通路的激活而發(fā)揮其抗炎作用的,IIF法檢測(cè)結(jié)果證明,芹菜素可抑制p65的磷酸化及轉(zhuǎn)移入核。本試驗(yàn)對(duì)芹菜素、針刺治療乳腺炎的效果和作用機(jī)理進(jìn)行了初步探索性研究,發(fā)現(xiàn)芹菜素、針刺能使TLR4的表達(dá)下調(diào)并對(duì)下游NF-κB信號(hào)通路的激活起到抑制作用,最終使乳腺中TNF-α、IL-1β和IL-6的mRNA和蛋白表達(dá)水平均顯著下降。
[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- 偽, 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- 偽, 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- 偽, 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- 偽, 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- 偽, IL-1 尾 and IL-6 in breast were significantly decreased.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R-332;R655.8

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