丹参水提物对LPS诱导子宫内膜上皮细胞炎症的抗炎机制研究

发布时间：2019-03-22 08:50

【摘要】：子宫内膜上皮细胞的炎性病变是奶牛子宫内膜炎发病的根本所在,探究炎症过程中上皮细胞结构与功能的变化,将有助于阐明疾病发病机制和治疗药物的研发。丹参是广泛用于治疗奶牛子宫内膜炎的中药之一,但从子宫内膜上皮细胞研究丹参的作用机制的报道较少。为了探讨丹参水提物对LPS诱导的炎症模型的保护机制,开展了以下研究。1.筛选LPS诱导炎症模型的最佳剂量和作用时间。设置5个不同浓度LPS分别作用于山羊EEC 6h、12h、24h后,MTT法检测EEC的细胞活性。结果表明5μg/m L LPS作用12h时促进细胞增殖作用最为明显,在此基础上,建立EEC炎症模型,ELISA检测模型组中TNF-α和IL-1β含量显著高于对照组(P0.01),故确定LPS最佳致炎剂量为5μg/m L,最佳作用时间是12h。2.筛选SME对EEC最佳作用剂量。以6个不同浓度的SME作用EEC 24h,结果表明SME对EEC未产生明显毒性,且促进EEC增殖。由此确定SME作用浓度分别为250μg/m L(高剂量组)、100μg/m L(中剂量组)、10μg/m L(低剂量组)。3.SME对EEC炎症模型中TNF-α、IL-1β、NO、PGE2、TLR4、CD14、MMP-2、MMP-9等蛋白表达的影响。分别用高、中、低剂量的SME处理EEC细胞炎症模型,观察SME作用0h、4h、6h、12h、18h、24h、36h后各项指标的变化。结果表明,模型组中TLR4、CD14、TNF-α、IL-1β、NO、PGE2、MMP-2和MMP-9等蛋白在各自到达峰值时含量均明显高于对照组(P0.01);不同浓度SME作用炎症模型后,均能使这些指标降低,且高剂量SME作用效果最为明显(P0.01)。4.SME对EEC炎症模型中相关基因表达的影响。实时荧光定量PCR检测SME对EEC炎症模型中相关基因表达,结果表明,模型组中TNF-α、IL-1β、TLR4、CD14、MMP-2、MMP-9 m RNA基因表达水平到达峰值时显著高于对照组(P0.01)。用高、中、低剂量的SME处理后均能降低细胞炎性因子TNF-α、IL-1β、TLR4、CD14、MMP-2、MMP-9的表达,且高剂量SME作用最为明显(P0.01)。另外,随着作用时间的延长,MMP-2、MMP-9表达逐渐降低,24h后明显低于对照,SME作用后又逐渐恢复到正常水平。综上所述,EEC炎症反应的发生可能与LPS诱导激活TLR信号通路(TLR4、CD14),进而上调细胞因子(TNF-α、IL-1β)、炎症介导因子(NO、PGE2)和基质金属蛋白酶(MMP-2、MMP-9)的表达有关。而SME作用于炎症细胞后,通过降低炎症介导因子、TLR信号通路的表达,达到减缓炎性反应的程度;在炎症反应初期,SME通过下调MMPs的表达,抑制了子宫内膜细胞结构被降解的炎性损伤,并在炎症反应后期通过上调MMP-2、MMP-9的表达,从而提高子宫自我修复重塑的能力。以上可能是LPS诱导炎性反应的发生机制和丹参提取物的抗炎作用机制发挥的途径,但其具体机制尚需进一步研究。
[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- 伪 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- 伪, 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- 伪, 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- 伪, IL-1 尾, TLR4,CD14,MMP-2, in the inflammatory model of EEC. The results showed that TNF- 伪, 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- 伪, 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- 伪, 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.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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