【作者】 金凤；

【导师】 曹金山； 刘博；

【作者基本信息】 内蒙古农业大学 ， 临床兽医学， 2021， 硕士

【摘要】 前列腺素（prostaglandins,PGs）广泛存在于人和动物机体的各个组织器官中,具有多种生物活性,如在炎症反应中,可大量诱导其合成分泌,进而对炎症反应的进程发挥一定的调控作用。宿主天然免疫系统中模式识别受体（pattern recognition receptor,PRR）的激活在细胞因子的分泌过程中发挥了关键性的作用。有研究认为,PGs的合成分泌与模式识别受体,如TLRs（Toll-Like Receptors）和NLRP3（NLR pyrin domain-containing 3）介导的宿主天然免疫应答具有紧密的联系。但在LPS（lipopolysaccharide）、BLP（Braun lipoprotein）及大肠杆菌诱导的小鼠炎症反应过程中,PGD₂（prostaglandin D₂）的合成分泌与模式识别受体是否具有紧密的关联及其是否对信号通路激活和细胞因子的分泌具有影响,目前尚不清楚。本研究中,使用LPS、BLP及大肠杆菌分别刺激C57BL/6J WT、TLR2基因敲除小鼠(TLR2^-/-)、TLR4基因敲除小鼠(TLR4^-/-)及NLRP3基因敲除小鼠(NLRP3^-/-)小鼠腹腔巨噬细胞后,通过实时荧光定量PCR和Western blot方法分别检测COX-2、H-PGDS基因和蛋白的表达水平。实验结果表明COX-2基因和蛋白的表达水平均发生下调（P<0.01）;并使用ELISA方法检测PGD₂的分泌情况,实验结果表明PGD₂的分泌也发生显著下调（P<0.001）。此外,本研究分析了内源性PGD₂合成酶抑制剂（H-PGDS和HQL-79）预处理对LPS、BLP及大肠杆菌分别诱导的C57BL/6J小鼠腹腔巨噬细胞促炎性细胞因子（IL-1β、TNF-α）、趋化因子（RANTES）和抗炎因子（IL-10）分泌水平的影响。实验结果表明,当内源性PGD₂被抑制后,LPS或大肠杆菌诱导腹腔巨噬细胞分泌IL-1β、TNF-α、RANTES和IL-10的水平均发生显著下调（P<0.05）;BLP诱导腹腔巨噬细胞分泌IL-1β和TNF-α的水平均发生显著下调（P<0.001）;而RANTES和IL-10的分泌水平均发生显著上调（P<0.001）。此外,本研究分析了外源性PGD₂预处理后,LPS、BLP及大肠杆菌分别诱导的C57BL/6J小鼠腹腔巨噬细胞NF-κB p65、MAPKs（ERK、p38）的磷酸化水平、Caspaes-1激活情况以及炎性介质的分泌情况。实验结果表明,LPS或大肠杆菌刺激15 min和30 min后,与PGD₂未预处理实验组相比,外源性PGD₂预处理可显著上调小鼠巨噬细胞ERK、p38和p65的磷酸化水平（P<0.05）,这与TNF-α和IL-1β的分泌水平上调的趋势是相对应的。在刺激30 min和60 min后,外源性PGD₂可显著下调LPS或大肠杆菌诱导的巨噬细胞ERK、p38和p65磷酸化水平（P<0.05）,与IL-10和RANTES分泌下调的趋势可相互对应。外源性PGD₂可显著下调BLP刺激15 min、30 min和60 min后巨噬细胞ERK和p38的磷酸化水平（P<0.05）,同时显著上调刺激15 min和30 min后巨噬细胞p65的磷酸化水平（P<0.001）,这与RANTES的分泌水平是相一致的。当使用外源性PGD₂预处理后,LPS、BLP和大肠杆菌均不能诱导小鼠巨噬细胞Caspase-1的激活,在ATP存在的情况下,大肠杆菌感染诱导的小鼠巨噬细胞Caspase-1的激活,但并没有发生显著变化。综上所述,TLR2、TLR4和NLRP3参与了LPS、BLP及大肠杆菌诱导的巨噬细胞PGD₂合成分泌过程,且内外源性PGD₂对LPS、BLP及大肠杆菌诱导的小鼠巨噬细胞激活均具有调控作用。更多还原

【Abstract】 Prostaglandins（PGs）is widely present in various tissues and organs of humans and animals,and have a variety of biological activities.PGs can be synthesized and secreted in large quantities,and then they can play a certain regulatory role in the process of inflammation.The activation of pattern recognition receptors in the host’s innate immune system plays a key role in the secretion of cytokines.The synthesis and secretion of PGs is closely connection to the host’s innate immune response mediated by pattern recognition receptors,such as Toll-Like receptors（TLRs）and NLR pyrin domain-containing3（NLRP3）.However,in the process of inflammation in mice induced by lipopolysaccharide（LPS）,Braun lipoprotein（BLP）and（Escherichia coli）E.coli,the synthesis and secretion of prostaglandin D₂（PGD₂）and pattern recognition receptor.Whether there is a close connection and whether it has an effect on the activation of signaling pathways and the secretion of cytokines is still unclear.In this study,LPS,BLP and E.coli was used to respectively stimulate peritoneal macrophages of C57BL/6J WT,TLR2^-/-,TLR4^-/-,and NLRP3^-/-in mice.The expression of COX-2 and H-PGDS were detected by q PCR and Western blot.The results showed that the expression of COX-2 gene and protein were down-regulated（P<0.01）,and the secretion of PGD₂ was also down-regulated by ELISA（P<0.001）.In addition,analyzed the effects of pretreatment with endogenous PGD₂ synthase inhibitors（H-PGDS and HQL-79）on the secretion of pro-inflammatory cytokines（IL-1βand TNF-α）,chemokines（RANTES）and anti-inflammatory cytokine（IL-10）in peritoneal macrophages of C57BL/6J mice induced by LPS,BLP and E.coli.The results showed that PGD₂ was inhibited,IL-1β,TNF-α,RANTES and IL-10 secreted by peritoneal macrophages were significantly down-regulated by LPS or E.coli（P<0.05）;BLP induced peritoneal macrophages IL-1βand TNF-αsecreted by cells were significantly down-regulated（P<0.001）;while after PGD₂ pretreatment the secretion of RANTES and IL-10 were significantly up-regulated（P<0.001）.In addition,analyzed the phosphorylation of NF-κB p65 and MAPKs（ERK,p38）,caspaes-1activation and secretion of inflammatory mediators in peritoneal macrophages of C57BL/6J mice induced by LPS,BLP and E.coli.The results shown that 15 min and30 min stimulation with LPS or E.coli,compared with the PGD₂ unpretreated experimental group,exogenous PGD₂ pretreatment can significantly up-regulate the phosphorylation of ERK,p38 and p65 in mouse macrophages（P<0.05）,which corresponds to the upward regulation of the secretion levels of TNF-αand IL-1β.After 30 min and 60 min stimulation,exogenous PGD₂ could significantly reduce the phosphorylation levels of ERK,p38 and p65（P<0.05）induced by LPS or E.coli,which could be correlated with the trend of down-regulation of IL-10 and RANTES.Exogenous PGD₂ can significantly down-regulate the phosphorylation of ERK and p38 of macrophages after 15 min,30 min and 60 min of BLP stimulation（P<0.05）,and significantly up-regulate of phosphorylation of p65 of macrophages after 15 min and 30 min stimulation（P<0.001）,which is consistent with the secretion of RANTES.After pretreatment with exogenous PGD₂,LPS,BLP and E.coli could not induce the activation of caspase-1 in mouse macrophages.In the presence of ATP,the activation of caspase-1 in mouse macrophages induced by E.coli infection did not change significantly.In conclusion,TLR2,TLR4 and NLRP3 are involved in the synthesis and secretion of PGD₂ induced by LPS,BLP and E.coli,and exogenous PGD₂ can regulate the activation of mouse macrophages induced by LPS,BLP and E.coli.更多还原