趋化因子MCP-1在EAN中的作用及雷公藤多甙的影响

目的 探讨单核细胞趋化蛋白-1(MCP-1)在实验性变态反应性神经炎(experimental autoimmune neuritis,EAN)中的作用及雷公藤多甙(TWP)对其影响.方法 用兔坐骨神经匀浆免疫Wistar大鼠建立EAN模型,TWP灌胃治疗,观察大鼠发病情况和组织病理改变,用免疫组化技术检测MCP-1在坐骨神经中的表达.结果 EAN组大鼠在第15天发病达到高峰,且病理改变可见炎性细胞浸润及脱髓鞘,其MCP-1表达高峰在疾病早期(9 d),随后逐渐下降,与对照组相比有显著差异性(P＜0.001).EAN TWP组大鼠发病程度较EAN NS组轻,MCP-1表达的整体趋势较EAN NS组降低.结论 MCP-1可能对EAN 发病起始动作用.TWP可能通过抑制趋化因子MCP-1的表达来减轻EAN.     

雷公藤多甙对实验性自身免疫性脑脊髓炎的治疗及炎症浸润细胞凋亡的影响

目的 观察雷公藤多甙（TWP）能否抑制实验性自身免疫性脑脊髓炎（EAE）模型的发病、病变程度以及对中枢神经系统（CNS）炎症浸润细胞凋亡的影响，为临床应用提供实验依据。方法将雌性豚鼠随机分为对照组、EAE组和喂服TWP组，EAE组和TWP组豚鼠采用颈背部皮下多点注射完全弗氏佐剂-豚鼠全脊髓匀浆（CFA-GPSCH），并辅以注射百日咳疫苗（BPV），诱导豚鼠建立EAE模型。观察各组EAE的发病情况，在光学显微镜下计数中枢神经系统炎症细胞浸润程度，并用3’末端脱氧核糖核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）检测其凋亡情况。结果 TWP治疗组豚鼠发病数较EAE对照组减少，起病时间相对延迟，临床表现明显减轻，炎性细胞浸润明显减少。与EAE对照组相比，TWP治疗组的中枢神经系统炎症浸润细胞的凋亡率明显增高。结论 TWP可有效地抑制EAE的发病，其机制可能与其抑制中枢神经系统炎症细胞的浸润及增加炎症细胞的凋亡有关，提示该药有临床应用前景。     

雷公藤多甙对IL-1β诱导大鼠滑膜细胞株RSC-364表达JNK1蛋白的影响

目的探讨雷公藤多甙（Tripterygium Glycosides，TG）对IL-1β诱导大鼠滑膜细胞株RSC-364 JNK1的影响，进一步阐明雷公藤多甙治疗类风湿性关节炎的作用机制。方法运用Western—blot检测不同浓度的雷公藤多甙（0、5、10、20mg／L）对IL-1β诱导大鼠滑膜细胞株RSC．364JNK1蛋白的表达。结果雷公藤多甙明显地抑制IL-1β诱导大鼠滑膜细胞株RSC-364对JNK1蛋白的表达。结论雷公藤多甙抑制IL—1β诱导大鼠滑膜细胞株RSC．364对JNK1蛋白的表达，可能是其治疗类风湿性关节炎的重要作用机制之一。     

NK-κB在EAN中的作用及鼻粘膜耐受的影响

目的 :探讨核转录因子NF κB在EAN中的作用以及鼻粘膜耐受的影响。方法 :采用RT PCR和Western blot法检测Wistar大鼠脾脏淋巴细胞IFN γ、TGF β1、IL 10mRNA表达和P6 5蛋白表达。 结果 :IFN γmRNA在发病早期表达明显增加 ,恢复期下降 ;IL 10、TGF β1mRNA在恢复期表达最明显 (P <0 0 5 )。鼻粘膜耐受后大鼠发病率和发病程度减轻 ,IL 10、TGF β1mRNA表达增加 (P <0 0 5 )。P6 5蛋白表达与IL 10、TGF β1mRNA表达呈负相关 ,与IFN γmRNA表达呈正相关。 结论 :P6 5蛋白可能参与EAN的发病 ,鼻服抗原诱导T细胞产生IL 10、TGF β1,通过抑制P6 5蛋白表达这一途径 ,有效预防EAN发生。     

雷公藤多甙对大鼠海马内注射β-淀粉样蛋白后星形胶质细胞增生的影响

目的：探讨大鼠海马内注射v淀粉样蛋白1-40(Aβ1-40)后海马星形胶质细胞表达GFAP的变化及雷公藤多甙(TWP)对其的影响。方法：在大鼠左侧海马定向注射Aβ1-40作为Aβ组,注射生理盐水作为对照组,雷公藤多甙腹腔注射治疗海马内注入了Aβ1-40的大鼠为TWP组。用免疫组织化学方法观察各组大鼠海马星形胶质细胞GFAP的表达。结果：Aβ组海马星形胶质细胞增生、肥大,GFAP阳性细胞数增多,细胞截面积明显增大。TWP组星形胶质细胞数较Aβ组减少,并且细胞截面积明显缩小。结论：雷公藤多甙能抑制Aβ诱导的海马星形胶质细胞的反应性增生。     

雷公藤多甙对佐剂性关节炎模型大鼠ICAM-1的影响

目的:探讨口服雷公藤多甙对大鼠佐剂性关节炎(adjuvant arthritis AA)的治疗作用,以及对外周淋巴器官中细胞黏附分子ICAM-1水平的影响.方法:制备大鼠AA模型,应用免疫组化法检测口服和未口服雷公藤多甙的大鼠淋巴器官中ICAM-1水平的变化;观察口服雷公藤多甙前后关节的肿胀度.结果:口服雷公藤多甙的AA大鼠的外周淋巴器官中ICAM-1的水平明显低于未口服雷公藤多甙的AA大鼠,关节炎症状明显改善.结论:口服雷公藤多甙可以有效降低ICAM-1水平,从而治疗佐剂性关节炎.     

外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠TLR4表达的影响

目的:观察外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠TLR4和MD-2表达的影响,探讨其可能的作用机制。方法:以MOG35-55免疫C57BL/6J小鼠建立EAE模型,多时间点皮下注射TGF-β1作为干预,RT-PCR检测实验小鼠脑中TLR4的mRNA表达;ELISA法检测外周血清中MD-2的水平。结果:与EAE组相比,TGF-β1组在发病初期TLR4和MD-2表达差异无统计学意义(P>0.05,P>0.05);高峰期TLR4表达增高(P<0.05),MD-2表达无差异(P>0.05);慢性期TLR4和MD-2表达均降低(P<0.05,P<0.05)。结论:外源性TGF-β1可能通过上调TLR4的表达而影响EAE小鼠的病情。     

EphA4在海人酸诱导大鼠大脑皮层神经元凋亡中的表达及天麻素的影响

目的： 探讨EphA4在海人酸诱导大鼠大脑皮层神经元凋亡过程中的表达变化,及天麻素对EphA4表达的影响。方法：提取新生大鼠大脑皮层神经元体外培养,7 d后随机分为对照组、海人酸模型组、海人酸+天麻素干预组;采用倒置相差显微镜观察神经元形态学变化,Hoechst 33258荧光染色、AO/EB荧光染色、LDH活性测定检测神经元凋亡和坏死情况,CY3荧光染色检测EphA4表达变化。结果：海人酸模型组较对照组神经元凋亡率显著升高（P<0.01）,EphA4表达显著增高（P<0.01）;海人酸+天麻素干预组较海人酸模型组神经元凋亡率显著下降,EphA4表达上调显著受抑。结论：海人酸诱导大鼠大脑皮层神经元凋亡过程中EphA4表达增高,天麻素在这一过程中抑制EphA4表达,对神经元具有一定保护作用。     

低氧对大鼠肺动脉平滑肌细胞血红素加氧酶-1 mRNA 表达及细胞增殖的影响

探讨血红素加氧酶－1（HO－1）mRNA在低氧大鼠肺动脉平滑肌细胞（PASMC0的表达及HO－1／一氧化碳（HO－1／CO）体系对PASMC增殖的影响。应用荧光定量RT－PCR法测定HO－1mRNA表达。用双波长法检测碳氧血红蛋白（HbCO)吸光值。应用免疫细胞化学方法检测细胞增殖核抗原（PCNA）及核转录因子－κB（NF-κB）的表达。发现HO－1 mRNA在常氧大鼠PASMC有低水平的表达，低氧12h HO-1mRNA水平是常氧时的1．5倍，且HbCO产量随之显著增高（P＜0．01）；低氧24h HO-1 mRNA表达呈回落趋势，HbCO产量亦有所减少，但两者仍高于常氧水平。低氧12h及24h PASMC PCNA核阳性反应颗粒表达较常氧时增强（P＜0．01，P＜0．001），使用HO抑制剂ZnPP-9，其PCNA该阳性反应颗粒表达较单纯低氧时增加更多（P＜0．001，P＜0．01）。低氧组核NF－κB阳性染色较常氧组增强（P＜0．001），使用ZnPP-9，其表达则比低氧时更多（P＜0．01）。低氧通过诱导大鼠PASMC的HO－1 mRNA基因表达，上调HO／CO体系活性，使内源性CO含量增高，抑制PASMC增殖；NF－κB参与了PASMC增殖的调控机制。     

雷公藤内酯醇对大鼠胶原性关节炎及足爪组织基质金属蛋白-2表达的影响

目的:研究雷公藤内酯醇(TP)对大鼠胶原性关节炎(CIA)及足爪组织基质金属蛋白酶-2(MMP-2)表达的影响。方法:采用鸡Ⅱ型胶原建立CIA大鼠模型,观察药物对CIA大鼠体重及足肿胀度的影响,免疫组化法观察大鼠足爪组织中MMP-2蛋白的表达。结果:TP可有效缓解CIA大鼠体重减轻及明显抑制CIA大鼠足爪肿胀;治疗后TP组足爪组织MMP-2阳性表达比模型对照组明显降低(P<0.01)。结论:TP对CIA大鼠具有治疗作用,其机制可能与其下调MMP-2表达、降低血清炎性细胞因子水平有关。     

Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune disease and an animal model of multiple sclerosis. To investigate how upstream TLR signals influence autoimmune T cell responses, we studied the role of individual TLR and MyD88, the common TLR adaptor molecule, in the initiation of innate and adaptive immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in CFA. MyD88(-/-) mice were completely EAE resistant. Purified splenic myeloid DC (mDC) from MyD88(-/-) mice expressed much less IL-6 and IL-23, and serum and T cell IL-17 were absent. TLR4(-/-) and TLR9(-/-) mice surprisingly exhibited more severe EAE symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were higher in TLR4(-/-) mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6 expression by splenocytes was higher in TLR9(-/-) mice. Our data suggest that MyD88 mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization, which in turn drives IL-17-producing encephalitogenic Th17 cell activation. Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOG-induced EAE.     

硝普钠对内毒素诱导的U937细胞TLR4表达的影响

目的:研究一氧化氮(NO)供体硝普钠(SNP)对内毒素(LPS)诱导的人单核巨噬细胞株U937表达TLR4的影响,明确其抗炎机制.方法:佛波脂(PMA)诱导U937成熟后分为四组,即对照组(空白对照)、LPS组(10 ng/ml LPS+100 ng/ml rhLBP)、低剂量SNP组(LPS+rhLBP+50 μmol/L SNP)和高剂量SNP组(LPS+rhLBP+500 μmol/L SNP).用RT-PCR和Western blot测定TLR4 mRNA和蛋白表达.ELISA法测定细胞上清中的肿瘤坏死因子α(TNF-α)的含量.结果:低、高剂量SNP组TLR4 mRNA的OD值较LPS组(0.308±0.050、0.138±0.004 4 vs 0.342±0.098,P<0.05、P<0.01)低,较对照组高(0.030±0.012,均P<0.01).低、高剂量SNP组TLR4 蛋白的OD值较LPS组(4.42±1.01、3.06±0.07 vs 6.02±1.19,均P<0.01)低,较对照组(2.01±0.09,均P<0.01)高.低、高剂量SNP组TNF-α较LPS组显著降低(105.5±2.56、71.5±7.75 vs 128.67±39.67,均P<0.05),较正常组(60±17.2,P<0.05、P>0.05)高.结论:SNP可能通过抑制TLR4介导的LPS信号跨膜转导,降低其引起的炎症反应,提示SNP对急性肺损伤或脓毒血症可能具有预防和早期治疗的作用.     

人参二醇组皂苷对LPS致休克大鼠肝脏TLR2、TLR9 mRNA表达的影响

目的：观察人参二醇组皂苷(PDS)对TLR2和TLR9 mRNA表达的影响,探讨PDS抗休克的分子生物学机制。方法:大鼠随机分为对照(control)组、LPS休克(LPS)组、人参二醇组皂苷小剂量(LPS+PDSL)组和人参二醇组皂苷中剂量(LPS+PDSM)组。大鼠舌下静脉注射LPS(4 mg/kg)4 h后测定血清中NOS活性、NO含量，肝组织中LPO含量、SOD活性以及TLR2、TLR4 mRNA的表达。结果:LPS+PDSL组和LPS+PDSM组NOS活性、NO含量和LPO含量明显低于LPS组，SOD活性明显高于LPS组(P<0.05)；LPS+PDSL组和LPS+PDSM组TLR2 mRNA表达明显低于LPS组，TLR9 mRNA表达无变化。结论:PDS通过下调肝组织中TLR2 mRNA表达，降低NOS活性、NO含量，对肝脏有保护作用。     

Toll-like receptor-2, CD14 and heat-shock protein 70 in inflammatory lesions of rat experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a well-known animal model of Guillain-Barré syndrome (GBS) characterized by inflammation and demyelination in the peripheral nervous system. Toll-like receptors (TLRs) together with their co-receptors form the first line of the self-defense, and play important roles in innate immune responses and inflammation. TLRs can be activated by endogenous ligands, like heat shock protein 70 (HSP70). In this study, we examined the spatiotemporal expressions of TLR2, CD14 and Hsp70 in EAN rats using immunohistochemistry and RT-PCR. A significant up-regulation of TLR2, CD14 and Hsp70 was seen in sciatic nerves of EAN rats and correlated with disease severity. Furthermore, activated macrophages were the main cellular resource of TLR2, CD14 and Hsp70 in EAN. Our results suggest that TLR2-, CD14- or Hsp70-based immunomodulation might have potential in the control of unwanted innate immune system activation in inflammatory neuropathies.     

Brucella abortus induces Irgm3 and Irga6 expression via type-I IFN by a MyD88-dependent pathway,without the requirement of TLR2, TLR4, TLR5 and TLR9

The innate immune system senses bacterial pathogens by pattern recognition receptors, such as the well-characterised Toll-like Receptors (TLR). The activation of TLR signalling cascades depends on several adaptor proteins, among which MyD88 plays a key role in triggering innate immune responses. Here, we show in murine macrophages that Brucella abortus triggers expression of the interferon-inducible resistance proteins (IRGs, p47 GTPases) via type-I IFN secretion at late time points, when Brucella has reached its replication niche. This induction requires the adaptor molecule MyD88 but does not involve the TLRs normally implicated in sensing Gram-negative bacteria, namely TLR2, TLR4, TLR5 and TLR9. Brucella mutants lacking the functional VirB type-IV secretion system were not capable of inducing Irgm3 and Irga6 expression, suggesting that the type-IV secretion system is part of the triggering of the activation process. Our data suggest that Brucella is recognized intracellularly by an unknown receptor, different from the conventional ones used for Gram-negative sensing, but one that nevertheless signals through MyD88.     

自身免疫调节因子(AIRE)对TLRs表达的影响

目的：探讨AIRE对外周抗原提呈细胞（APC）TLRs表达的影响。方法：①脂质体法将pEGFPC3-AIRE质粒转染RAW264．7细胞。②共聚焦荧光显微镜观察转染效果。③RT—PCR法检测转染后不同时间点（36、48、72、96小时）RAW264．7细胞上AIRE及TLR1-9的表达水平。结果：①成功将质粒转入RAW264．7细胞，转染效率约为60％～70％。②建立了AIRE转染的RAW264．7细胞，确定转染后72小时为转染最佳时间点。③AIRE转染后72小时，RAW264．7细胞上TLR1、4、5、9表达升高，TLR3、7、8表达降低，转染后96小时，TLR2、6表达升高。结论：AIRE可能通过调控APC上TLRs的表达，调节机体的免疫应答。通过对不同的TIJRs的影响，来维持对病原微生物有效应答的同时，维持对自身组织的耐受状态。     

Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection

Toll-like receptors (TLRs) expressed by mucosal epithelium play an essential role in the defense against microbes by recognizing conserved bacterial molecules. For the first time TLR4, TLR5 and TLR9 have been microanatomically localized in patients with noninflamed gastric mucosa and Helicobacter pylori gastritis by immunohistochemistry. Because polarized expression of TLRs in apical and basolateral epithelial compartments is thought to modulate mucosal immunity, subcellular TLR distribution by gastric epithelium was investigated using confocal microscopy. TLR4, TLR5 and TLR9 were expressed by gastric epithelium in antrum and corpus of all patients with H. pylori gastritis (n = 14) and with noninflamed gastric mucosa (n = 5). TLR4 was expressed at the apical and the basolateral pole of the gastric epithelium as well in noninflamed gastric mucosa as in H. pylori gastritis. TLR5 and TLR9 expression in the noninflamed gastric mucosa was identical to that of TLR4 with localization at the apical and the basolateral epithelial pole. However, in H. pylori gastritis TLR5 and TLR9 expression on the gastric epithelium changed to an exclusive basolateral localization without detectable expression at the apical pole. In the human stomach, the gastric epithelium expressed TLR4, TLR5 and TLR9, which gives it the possibility to interact with H. pylori. Furthermore, gastric epithelial TLR4 expression is highly polarized in an apical and a basolateral compartment, whereas TLR5 and TLR9 polarization seems to be a process dynamically influenced by H. pylori infection. This polarized and dynamically regulated gastric epithelial expression of TLRs supports a sentinel role for these receptors in the mucosal immunity to H. pylori.     

Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is the animal model of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) that is the most common subtype of Guillain-Barre syndrome (GBS). While neuropathic pain is a common symptom of GBS, its underlying mechanisms remain elusive. Central sensitization, particularly spinal glia (microglia and astrocytes) activation, is important for the initiation and maintenance of neuropathic pain. P2X(4) receptor (P2X(4)R) is an ATP-gated ion channel and its spinal up-regulation has been found to be crucial for the development of neuropathic pain following peripheral nerve injury. The initiation of mechanical allodynia in rat EAN was observed at day 9 before the onset of neurological signs. Maximal level of mechanical allodynia was observed from days 17-19 and then a slow recovery, long after the cessation of typical neurological signs of EAN, until day 37 was observed. Expression of P2X(4)R in lumbar spinal cords was studied by immunohistochemistry. P2X(4)R immunoreactivity (IR) was mainly seen in gray matter, particularly in the dorsal horn. Accumulation of P2X(4)R(+) cells in the lumbar dorsal horn was observed at day 9, reached the maximal level at day 17 and remained elevated until day 37 after immunization. Furthermore, a negative correlation between the density of P2X(4)R(+) cells in the lumbar dorsal horn with mean hind-paw withdrawal threshold in EAN rats was seen, indicating that P2X(4)R might contribute to EAN mechanical allodynia. Double staining revealed that almost all P2X(4)R(+) cells co-expressed CD68, a marker for reactive microglia, but not the astrocyte marker, glial fibrillary acidic protein (GFAP). Our data demonstrate that EAN induces mechanical allodynia and P2X(4)R expression in spinal microglia, suggesting that EAN is a good animal model for neuropathic pain in polyneuropathy and spinal microglia activation might participate in EAN-induced neuropathic pain.     

α-MSH对脂多糖诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA 表达的影响

目的：观察α-黑色素细胞刺激素(α-MSH)对脂多糖（LPS）诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达的影响，探讨α-MSH拮抗LPS的作用机制。方法：用半定量逆转录多聚酶链反应（RT-PCR）的方法检测LPS诱导体外培养的小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达水平和给予α-MSH后对CD14和TLR4 mRNA表达的影响。结果：正常静息小鼠腹腔巨噬细胞只表达少量的CD14和TLR4 mRNA,给予LPS刺激后6 h，两者表达明显强于正常对照（P<0.01），并且其表达量随着LPS刺激时间的增加维持在高水平，24 h达到峰值，在48 h CD14 mRNA的表达降到正常水平，而TLR4 mRNA的表达仍然维持在高水平。在LPS刺激的同时给予α-MSH，CD14和TLR4 mRNA的表达则明显低于LPS组（P<0.05），而且α-MSH这种效应与其使用浓度有关，0.1 nmol/L α-MSH不影响LPS诱导的CD14和TLR4 mRNA的表达，而当α-MSH的浓度达到1、10、100 nmol/L则能显著影响CD14和TLR4 mRNA的表达（P<0.05），但各个浓度组之间的作用没有明显差别（P>0.05）。结论：α-MSH抗LPS的效应可能与其下调LPS信号转导通路关键受体CD14和TLR4 mRNA的表达有关，从而干扰LPS跨膜信号转导，阻碍巨噬细胞活化。