丹參水提物對LPS誘導(dǎo)子宮內(nèi)膜上皮細(xì)胞炎癥的抗炎機制研究
發(fā)布時間：2019-03-22 08:50
【摘要】：子宮內(nèi)膜上皮細(xì)胞的炎性病變是奶牛子宮內(nèi)膜炎發(fā)病的根本所在,探究炎癥過程中上皮細(xì)胞結(jié)構(gòu)與功能的變化,將有助于闡明疾病發(fā)病機制和治療藥物的研發(fā)。丹參是廣泛用于治療奶牛子宮內(nèi)膜炎的中藥之一,但從子宮內(nèi)膜上皮細(xì)胞研究丹參的作用機制的報道較少。為了探討丹參水提物對LPS誘導(dǎo)的炎癥模型的保護(hù)機制,開展了以下研究。1.篩選LPS誘導(dǎo)炎癥模型的最佳劑量和作用時間。設(shè)置5個不同濃度LPS分別作用于山羊EEC 6h、12h、24h后,MTT法檢測EEC的細(xì)胞活性。結(jié)果表明5μg/m L LPS作用12h時促進(jìn)細(xì)胞增殖作用最為明顯,在此基礎(chǔ)上,建立EEC炎癥模型,ELISA檢測模型組中TNF-α和IL-1β含量顯著高于對照組(P0.01),故確定LPS最佳致炎劑量為5μg/m L,最佳作用時間是12h。2.篩選SME對EEC最佳作用劑量。以6個不同濃度的SME作用EEC 24h,結(jié)果表明SME對EEC未產(chǎn)生明顯毒性,且促進(jìn)EEC增殖。由此確定SME作用濃度分別為250μg/m L(高劑量組)、100μg/m L(中劑量組)、10μg/m L(低劑量組)。3.SME對EEC炎癥模型中TNF-α、IL-1β、NO、PGE2、TLR4、CD14、MMP-2、MMP-9等蛋白表達(dá)的影響。分別用高、中、低劑量的SME處理EEC細(xì)胞炎癥模型,觀察SME作用0h、4h、6h、12h、18h、24h、36h后各項指標(biāo)的變化。結(jié)果表明,模型組中TLR4、CD14、TNF-α、IL-1β、NO、PGE2、MMP-2和MMP-9等蛋白在各自到達(dá)峰值時含量均明顯高于對照組(P0.01);不同濃度SME作用炎癥模型后,均能使這些指標(biāo)降低,且高劑量SME作用效果最為明顯(P0.01)。4.SME對EEC炎癥模型中相關(guān)基因表達(dá)的影響。實時熒光定量PCR檢測SME對EEC炎癥模型中相關(guān)基因表達(dá),結(jié)果表明,模型組中TNF-α、IL-1β、TLR4、CD14、MMP-2、MMP-9 m RNA基因表達(dá)水平到達(dá)峰值時顯著高于對照組(P0.01)。用高、中、低劑量的SME處理后均能降低細(xì)胞炎性因子TNF-α、IL-1β、TLR4、CD14、MMP-2、MMP-9的表達(dá),且高劑量SME作用最為明顯(P0.01)。另外,隨著作用時間的延長,MMP-2、MMP-9表達(dá)逐漸降低,24h后明顯低于對照,SME作用后又逐漸恢復(fù)到正常水平。綜上所述,EEC炎癥反應(yīng)的發(fā)生可能與LPS誘導(dǎo)激活TLR信號通路(TLR4、CD14),進(jìn)而上調(diào)細(xì)胞因子(TNF-α、IL-1β)、炎癥介導(dǎo)因子(NO、PGE2)和基質(zhì)金屬蛋白酶(MMP-2、MMP-9)的表達(dá)有關(guān)。而SME作用于炎癥細(xì)胞后,通過降低炎癥介導(dǎo)因子、TLR信號通路的表達(dá),達(dá)到減緩炎性反應(yīng)的程度;在炎癥反應(yīng)初期,SME通過下調(diào)MMPs的表達(dá),抑制了子宮內(nèi)膜細(xì)胞結(jié)構(gòu)被降解的炎性損傷,并在炎癥反應(yīng)后期通過上調(diào)MMP-2、MMP-9的表達(dá),從而提高子宮自我修復(fù)重塑的能力。以上可能是LPS誘導(dǎo)炎性反應(yīng)的發(fā)生機制和丹參提取物的抗炎作用機制發(fā)揮的途徑,但其具體機制尚需進(jìn)一步研究。
[Abstract]:The inflammatory lesion of endometrial epithelial cells is fundamental to the pathogenesis of endometritis in dairy cows. Exploring the changes of the structure and function of epithelial cells in the course of inflammation will help to clarify the pathogenesis of the disease and the research and development of therapeutic drugs. Salvia miltiorrhiza is one of the widely used Chinese herbs in treating cow endometritis, but there are few reports about the mechanism of salvia miltiorrhiza from endometrial epithelial cells. In order to investigate the protective mechanism of Salvia miltiorrhiza water extract on LPS-induced inflammatory model, the following studies were carried out. The optimal dose and time of LPS-induced inflammation model were screened. Five different concentrations of LPS were added to goat EEC for 6 h, 12 h, and 24 h later, the cell activity of EEC was detected by MTT assay. The results showed that the effect of 5 渭 g / m L LPS on cell proliferation was the most obvious at 12h. On this basis, the inflammatory model of EEC was established. The contents of TNF- 偽 and IL-1 尾 in the model group were significantly higher than those in the control group (P0.01). Therefore, the optimal dose of LPS was 5 渭 g / mL and the optimal time was 12 h 路2.2.The optimal inflammatory dose was 5 渭 g / mL. The best dose of SME on EEC was screened. After EEC was treated with 6 different concentrations of SME for 24 h, the results showed that SME had no obvious toxicity to EEC and promoted the proliferation of EEC. The concentrations of SME were 250 渭 g / m L (, 100 渭 g / m L (, and 10 渭 g / m L (, respectively. The effects of 3.SME on TNF- 偽, IL-1 尾, NO,PGE2,TLR4,CD14,MMP-2, in the inflammatory model of EEC were determined to be 250 渭 g / m L (, 100 渭 g / m L (and 10 渭 g / m L (, respectively. The effect of MMP-9 and other proteins expression. The inflammatory models of EEC cells were treated with high, middle and low doses of SME respectively. The effects of SME at 0 h, 4 h, 6 h, 12 h, 18 h, 24 h and 36 h were observed. The results showed that the contents of TLR4,CD14,TNF- 偽, IL-1 尾, NO,PGE2,MMP-2 and MMP-9 in the model group were significantly higher than those in the control group (P0.01). Different concentrations of SME could decrease these indexes, and the effect of high dose SME was the most obvious (P0.01). The effect of 4.SME on the expression of related genes in the inflammatory model of EEC was affected. Real-time fluorescence quantitative PCR was used to detect the expression of TNF- 偽, IL-1 尾, TLR4,CD14,MMP-2, in the inflammatory model of EEC. The results showed that TNF- 偽, IL-1 尾, TLR4,CD14,MMP-2, were detected in the model group. The expression level of MMP-9 m RNA gene was significantly higher than that of the control group (P0.01). The expression of TNF- 偽, IL-1 尾 and TLR4,CD14,MMP-2,MMP-9 was decreased after treatment with high, middle and low dose of SME, and the effect of high dose SME was the most obvious (P0.01). In addition, with the prolongation of the treatment time, the expression of MMP-2,MMP-9 decreased gradually, was significantly lower than that of the control 24 hours later, and returned to the normal level gradually after SME treatment. In conclusion, the pathogenesis of EEC inflammation may be related to the activation of TLR signaling pathway (TLR4,CD14) induced by LPS, and the upregulation of cytokines (TNF- 偽, IL-1 尾), inflammatory mediators (NO,PGE2) and matrix metalloproteinases (MMP-2,). The expression of MMP-9 is related. After SME acted on inflammatory cells, it decreased the expression of inflammatory mediators and TLR signaling pathway to the extent of attenuating inflammatory response. In the early stage of inflammatory reaction, SME inhibited the degradation of endometrial cell structure by down-regulating the expression of MMPs, and up-regulated the expression of MMP-2,MMP-9 at the later stage of inflammatory reaction, thus improving the ability of uterine self-repair and remodeling. These may be the mechanisms of LPS-induced inflammatory reaction and the anti-inflammatory mechanism of Salvia miltiorrhiza extract, but the specific mechanisms need to be further studied.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7
本文編號：2445448
[Abstract]:The inflammatory lesion of endometrial epithelial cells is fundamental to the pathogenesis of endometritis in dairy cows. Exploring the changes of the structure and function of epithelial cells in the course of inflammation will help to clarify the pathogenesis of the disease and the research and development of therapeutic drugs. Salvia miltiorrhiza is one of the widely used Chinese herbs in treating cow endometritis, but there are few reports about the mechanism of salvia miltiorrhiza from endometrial epithelial cells. In order to investigate the protective mechanism of Salvia miltiorrhiza water extract on LPS-induced inflammatory model, the following studies were carried out. The optimal dose and time of LPS-induced inflammation model were screened. Five different concentrations of LPS were added to goat EEC for 6 h, 12 h, and 24 h later, the cell activity of EEC was detected by MTT assay. The results showed that the effect of 5 渭 g / m L LPS on cell proliferation was the most obvious at 12h. On this basis, the inflammatory model of EEC was established. The contents of TNF- 偽 and IL-1 尾 in the model group were significantly higher than those in the control group (P0.01). Therefore, the optimal dose of LPS was 5 渭 g / mL and the optimal time was 12 h 路2.2.The optimal inflammatory dose was 5 渭 g / mL. The best dose of SME on EEC was screened. After EEC was treated with 6 different concentrations of SME for 24 h, the results showed that SME had no obvious toxicity to EEC and promoted the proliferation of EEC. The concentrations of SME were 250 渭 g / m L (, 100 渭 g / m L (, and 10 渭 g / m L (, respectively. The effects of 3.SME on TNF- 偽, IL-1 尾, NO,PGE2,TLR4,CD14,MMP-2, in the inflammatory model of EEC were determined to be 250 渭 g / m L (, 100 渭 g / m L (and 10 渭 g / m L (, respectively. The effect of MMP-9 and other proteins expression. The inflammatory models of EEC cells were treated with high, middle and low doses of SME respectively. The effects of SME at 0 h, 4 h, 6 h, 12 h, 18 h, 24 h and 36 h were observed. The results showed that the contents of TLR4,CD14,TNF- 偽, IL-1 尾, NO,PGE2,MMP-2 and MMP-9 in the model group were significantly higher than those in the control group (P0.01). Different concentrations of SME could decrease these indexes, and the effect of high dose SME was the most obvious (P0.01). The effect of 4.SME on the expression of related genes in the inflammatory model of EEC was affected. Real-time fluorescence quantitative PCR was used to detect the expression of TNF- 偽, IL-1 尾, TLR4,CD14,MMP-2, in the inflammatory model of EEC. The results showed that TNF- 偽, IL-1 尾, TLR4,CD14,MMP-2, were detected in the model group. The expression level of MMP-9 m RNA gene was significantly higher than that of the control group (P0.01). The expression of TNF- 偽, IL-1 尾 and TLR4,CD14,MMP-2,MMP-9 was decreased after treatment with high, middle and low dose of SME, and the effect of high dose SME was the most obvious (P0.01). In addition, with the prolongation of the treatment time, the expression of MMP-2,MMP-9 decreased gradually, was significantly lower than that of the control 24 hours later, and returned to the normal level gradually after SME treatment. In conclusion, the pathogenesis of EEC inflammation may be related to the activation of TLR signaling pathway (TLR4,CD14) induced by LPS, and the upregulation of cytokines (TNF- 偽, IL-1 尾), inflammatory mediators (NO,PGE2) and matrix metalloproteinases (MMP-2,). The expression of MMP-9 is related. After SME acted on inflammatory cells, it decreased the expression of inflammatory mediators and TLR signaling pathway to the extent of attenuating inflammatory response. In the early stage of inflammatory reaction, SME inhibited the degradation of endometrial cell structure by down-regulating the expression of MMPs, and up-regulated the expression of MMP-2,MMP-9 at the later stage of inflammatory reaction, thus improving the ability of uterine self-repair and remodeling. These may be the mechanisms of LPS-induced inflammatory reaction and the anti-inflammatory mechanism of Salvia miltiorrhiza extract, but the specific mechanisms need to be further studied.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S853.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 吳明樓,蘇輝,方必春,朱滿琪,劉士喜,姚智深;奶牛子宮內(nèi)膜炎發(fā)生的相關(guān)因素及防治措施[J];安徽農(nóng)業(yè)科學(xué);1996年03期
2 李朝霞,王地;丹參水溶性成分的研究進(jìn)展[J];北京中醫(yī);2004年03期
3 張彥明;牛子宮內(nèi)膜炎的研究與臨床進(jìn)展[J];黃牛雜志;1998年01期
4 許臘梅;馮萬宇;王巖;王志明;段瑩瑩;侯振中;;奶牛子宮內(nèi)膜炎研究進(jìn)展[J];動物醫(yī)學(xué)進(jìn)展;2008年08期
5 梁書文;;奶牛子宮內(nèi)膜炎的病因與綜合防制[J];中國畜牧獸醫(yī);2011年03期
6 杜冠華,張均田;丹參水溶性有效成分——丹酚酸研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;2000年05期
7 王春梅,喬延江;細(xì)胞模型發(fā)展現(xiàn)狀及應(yīng)用于中藥研究的探討[J];世界科學(xué)技術(shù);2004年03期
8 張倩媚;馬穎;;子宮內(nèi)膜干細(xì)胞的研究進(jìn)展[J];山西醫(yī)藥雜志(下半月刊);2013年06期
9 姜靜宜;單悅梅;高明奇;;丹酚酸A對缺血再灌注大鼠缺血期胃粘膜血流量及再灌注期胃粘膜脂質(zhì)過氧化的影響[J];微循環(huán)學(xué)雜志;2008年02期
10 崔燎,鄒麗宜,劉鈺瑜,艾春媚,吳鐵,吳怡;丹參水提物和丹參素促進(jìn)成骨細(xì)胞活性和防治潑尼松所致大鼠骨質(zhì)疏松[J];中國藥理學(xué)通報;2004年03期
相關(guān)博士學(xué)位論文 前1條
1 劉珊;雌二醇和孕酮對牛子宮內(nèi)膜上皮細(xì)胞TLRs表達(dá)及功能的影響[D];吉林大學(xué);2012年
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