脊髓NF-κB/p65介导的神经免疫调节在大鼠佐剂性关节炎发病中的作用及机制研究
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摘要
研究背景
类风湿性关节炎(Rheumatoid arthritis, RA),是一种由自身免疫障碍引致免疫系统攻击关节的长期慢性炎症。这种炎症会造成关节变形直至残废,并会因关节疼痛及磨损而失去部分的活动能力,会极大影响患者的生活质量。尽管目前存在很多抗风湿类药物,如非甾类抗炎药(Nonsteroidal Antiinflammatory Drugs, NSAIDs)、慢作用抗风湿药、肾上腺糖皮质激素等,其中新近开发研究的也为数不少,包括肿瘤坏死因子-a(Tumor necrosis factor, TNF-a)阻断剂、抗CD20单抗、炎性因子的中和抗体[白介素-1β(interleukin-1β, IL-1β)、白介素-1(interleukin-1,IL-1)]。但是探索类风湿致病机理,并且依此研发相关新药仍是医学界的热点。
众所周知,外周组织损伤或炎症会引起脊髓一系列的变化,在关节炎的发生发展过程中,受损的关节组织以及滑膜炎可激活外周伤害性感受器,导致连续和强烈的伤害性信号向上传入脊髓进行调制、整合,这样可能会诱发脊髓的神经免疫反应,从而引起细胞因子、兴奋性氨基酸、环氧化酶-2(cyclooxygenase, COX-2)和前列腺素的产生和分泌,进而导致脊髓伤害性神经元的持续兴奋性增强(即中枢敏化)。有意思的是,脊髓也可以通过信号传递调节外周炎症。有确切证据表明,鞘内给予的相关的一些化合物可减轻外周炎症,这些化合物包括腺苷、色胺、氯胺酮和吗啡、沙利度胺、星形胶质细胞和小胶质细胞抑制剂、促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK) p38拮抗剂等。而这些化合物具有一个共同的效应：减少脊髓神经元的兴奋性。另外,一些涉及调节外周炎症的通路已经被阐明,其中包括由交感神经和副交感神经分支组成的自主系统,其支配免疫器官,并可以影响外周的免疫反应。另一种机制即背根反射(dorsal root reflexes, DRR),可沿躯体传入神经纤维逆向传递信号,促使感觉神经末梢释放神经肽,如血管活性肠肽、P物质和降钙素基因相关肽,从而影响周围炎症。此外,下丘脑-垂体-肾上腺(Hypothalamic-Pituitary-Adrenal, HPA)轴也可以对炎症形成反馈机制,其往往涉及自身免疫和炎性相关疾病,如类风湿性关节炎。
核因子-κB (Nuclear factor-kappa B, NF-κB)是一种关键性的转录因子,参与了中枢神经系统(central nervous system, CNS)许多生理活动或病理过程,如炎症、神经元可塑性、突触传递、学习、记忆和疼痛。NF-κB通过调控众多基因来实现这些过程,其中包括细胞因子(IL-1β、IL-6、TNF-α),促炎症酶COX-2和诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS),趋化因子和粘附因子。NF-κB的5个亚基已经确定,包括gp105/p50(NF-κB1), p100/p52(NF-κB2)、p65(RelA)、RelB和c-REL, NF-κB的活性形式是这些亚基组成的二聚体。NF-κB最常见和最具特点的二聚体是p50/p65,其广泛表达于中枢神经系统,在基因表达的调节中起着重要的作用。先前的研究表明,外围组织损伤或炎症可活化脊髓NF-κB/p65,我们的早期工作也发现NF-κB/p65与TNF-α共表达于慢性压迫性损伤模型大鼠的脊髓背角,下调脊髓NF-κB/p65的表达可显著降低坐骨神经结扎引起的机械痛觉过敏和热痛觉过敏。这些研究结果表明NF-κB/p65参与了中枢敏化。然而,尚不确定脊髓NF-κ/p65是否也参与了外周炎症的发生发展。
本研究首先构建NF-κB/p65基因RNA干扰(RNA interference, RNAi)'慢病毒载体并转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察其沉默效应以及对IL-1β、TNF-α和COX-2表达的影响。在体建立大鼠佐剂性关节炎(adjuvant-induced arthritis, AIA)模型,鞘内注射慢病毒重组体,探讨脊髓NF-κB/p65在AIA大鼠外周炎症和痛觉过敏中作用。另外,我们还评估了NF-κB/P65对脊髓IL-1β、 TNF-α和COX-2表达的调控作用,以阐明脊髓NF-KB/p65参与外周炎症和痛觉过敏的相关机制。
第一部分慢病毒介导RNA干扰对体外培养大鼠脊髓神经元和星形胶质细胞NF-κB/p65基因的沉默效应
目的
构建NF-κB/p65基因RNA干扰慢病毒载体并转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察其沉默效应以及对IL-1β、TNF-α和COX-2表达的影响
方法
1.针对大鼠NF-κB/p65基因特异性序列,设计3条NF-κB/p65-siRNA序列及1条阴性对照序列,合成包含各正反义靶序列的互补DNA链,退火形成双链DNA,插入到经BamH Ⅰ和EcoR Ⅰ酶切后的pFU-GW慢病毒载体中,PCR筛选阳性克隆,DNA测序鉴定。与慢病毒包装质粒pHelper1.0、pHelper2.0通过lipofectamine2000共转染至包装细胞293T,滴度测定后-80°冰箱保存备用。
2.体外培养神经元和星形胶质细胞,用免疫荧光技术进行细胞纯度鉴定。
3.慢病毒重组体转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察转染效率。然后用含1μg/ml脂多糖(Lipopolyssacride, LPS)的培养液刺激细胞24h。根据干预条件不同,实验分为6组：阴性对照组、LPS组、LPS+LV-NC组、LPS+LV-shNF-κB/p65-1组、LPS+LV-shNF-κB/p65-2组和LPS+LV-shNF-κB/p65-3组。
4.倒置显微镜下观察神经元和星形胶质细胞的形态改变,Real-Time PCR和Western blotting技术分别检测慢病毒重组体对神经元和星形胶质细胞NF-κB/p65mRNA和蛋白的干扰效率,ELISA法检测培养基中星形胶质细胞分泌的炎性因子(IL-1β、 TNF-α)的变化,Western blot技术检测神经元和星形胶质细胞COX-2的表达情况。
结果
1.测序结果证实合成的寡核苷酸链插入正确,重组载体构建成功,测定慢病毒滴度为108～9TU/mL。
2.培养神经元7d后使用神经元特异性烯醇化酶(neuron-specific enolase, NSE)特异性抗体对其进行纯度鉴定,结果显示85%以上为神经元。培养星形胶质细胞12d后进行传代,传代2次后使用神经胶质酸性蛋白(glial fibrillary acidic protein, GFAP)特异性抗体对其进行纯度鉴定,结果显示达90%以上为星形胶质细胞。
3.慢病毒重组体能成功转染大鼠脊髓神经元和星形胶质细胞,神经元转染效率大于90%,与LV-NC组比较,LPS+LV-shNF-κB/p65-1组和LPS+LV-shNF-κB/p65-2组NF-κB/P65mRNA和蛋白的表达都明显下调,mRNA水平干扰效率达95%(P<0.01),蛋白水平干扰效率达85%以上(P<0.01),而LPS+LV-shNF-κB/p65-3组无明显下调(P>0.05)。相比阴性对照组,神经元的LPS组COX-2表达无明显变化,LV-shNF-κB/p65-1和LV-shNF-κB/p65-2能下调COX-2的表达,而LV-shNF-κB/p65-3则无明显作用。另外,星形胶质细胞转染效率大于85%,与LV-NC组比较,LPS+LV-shNF-κB/p65-1组和LPS+LV-shNF-κB/p65-2组NF-κB/p65mRNA和蛋白的表达明显下调,mRNA水平干扰效率达92%(P<0.01),蛋白水平干扰效率达83%以上(P<0.01),而LPS+LV-shNF-κB/p65-3组无明显变化(P>0.05)。相比阴性对照组,星形胶质细胞LPS组的TNF-α、IL-1β和COX-2蛋白表达明显上调(TNF-α, P<0.01; IL-1β,,P<0.01; COX-2, P<0.05), LV-shNF-κB/p65-1和LV-shNF-κB/p65-2能抑制这种上调效应(P<0.01),而LV-shNF-κB/p65-3组无这种作用。
结论
成功构建了大鼠NF-κB/P65基因的RNA干扰慢病毒载体,其可使大鼠脊髓神经元和星形胶质细胞的NF-κB/p65基因表达下调,并能抑制LPS导致的TNF-α、IL-1β和COX-2的上调效应。这样为我们下一步动物体内实验奠定了坚实的基础,也为神经系统NF-κB/p65基因功能研究提供一种有效的工具。
第二部分脊髓NF-κB/P65对大鼠佐剂性关节炎外周炎症以及痛觉过敏的调控作用
目的
炎症组织的伤害性刺激可能增加脊髓背角神经元的兴奋性(即中枢敏化),兴奋的神经元可返回信号并调控外周炎症,但是其中的机制尚不明确。最近的一些研究表明,脊髓NF-κB/p65参与了中枢敏化以及相关疼痛。我们的目的是研究脊髓NF-KB/p65是否也参与了大鼠佐剂性关节炎的外周炎症反应及相关痛觉过敏。
方法
1.构建大鼠佐剂性关节炎模型,在完全性弗氏佐剂(complete Freund's adjuvant,CFA)注射后第3d或10d,鞘内给予10u1慢病毒重组体(LV-NF-KB/p65或LV-NC)。根据干预条件不同,实验分为8组：对照组、CFA组、CFA+LV-NC (3d)组、CFA+LV-shNF-KB/p65-1(3d)组、CFA+LV-shNF-KB/p65-2(3d)组、CFA+LV-NC (10d)组、CFA+LV-shNF-KB/p65-1(10d)组、CFA+LV-shNF-KB/p65-2(10d)组。
2.CFA注射后第14d,使用Western blot技术、免疫荧光技术以及EMSA技术评估脊髓NF-KB/p65蛋白的表达和转录活性。在CFA注射前1d和注射后第6d、第8d、第10d、第12d、第14d、第16d、第18d以及第20d,对大鼠的疼痛相关行为、足跖肿胀程度进行了评估,在第20d,观察评估大鼠的关节组织病理学改变。此外,在CFA注射后第14d,检测脊髓炎性介质TNF-α, IL-1β和COX-2的表达。
结果
1.外周炎症上调了脊髓NF-κB/p65蛋白的表达,其主要分布于脊髓背角的神经元和星形胶质细胞,CFA注射后第3d或10d,鞘内注射LV-shNF-KB/p65-1和LV-shNF-κB/p65-2都能下调NF-κB/p65蛋白的表达和转录活性。
2.相比阴性对照组,CFA组大鼠的机械痛阈、热痛阈明显下降(P<0.01),而足跖肿胀明显增加(P<0.01),CFA注射后第3d或10d,鞘内给予LV-shNF-KB/p65-1和LV-shNF-κB/p65-2都能显著抑制这些效应(P<0.01)。另外,相比CFA+LV-NC (3d)组,大鼠关节炎症和组织破坏程度在CFA+LV-shNF-KB/p65-2(3d)组显著降低(P<0.01),而LV-shNF-κB/p65-1(10d)组轻微降低,但未达统计学意义(P>0.05)；此外,相比CFA+LV-NC (10d)组,CFA+LV-shNF-KB/p65-1(10d)组和LV-shNF-κB/p65-2(10d)组的关节炎症和组织破坏程度无明显变化(P>0.05)。CFA注射后第3d,鞘内给予LV-shNF-κB/p65-1能下调脊髓TNF-α和IL-1p的表达(P<0.01),但对COX-2的表达无明显作用(P>0.05),而LV-shNF-KB/p65-2能抑制脊髓TNF-α, IL-1β和COX-2表达(TNF-α, P<0.01; IL-1β, P<0.01; COX-2, P<0.05)。CFA注射后第10d,鞘内注射LV-shNF-KB/p65-1能下调脊髓TNF-α的表达(P<0.05),对IL-1β无显著作用(P>0.05),而LV-shNF-KB/p65-2能同时抑制TNF-α和IL-1β的表达(P<0.05),但两者都对COX-2无明显作用(P>0.05)。
结论
外周炎症上调了脊髓NF-κB/p65蛋白的表达,其主要分布于脊髓背角的神经元和星形胶质细胞。鞘内注射LV-shNF-KB/p65能下调NF-κB/P65蛋白的表达,并且能降低大鼠的痛觉过敏、足跖肿胀以及关节破坏。此外,其也能减少脊髓TNF-α, IL-1β和COX-2的过表达。这些表明脊髓NF-κB/p65在AIA大鼠外围炎症和痛觉过敏的发生发展过程中起着重要作用。因此,抑制脊髓NF-κB/p65的表达对外周炎症性疾病可能是一种潜在的治疗方法。
Background
Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks flexible (synovial) joints. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility. The disease greatly affects the quality of life of patients. There are many drugs in the RA treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), slow-acting anti-rheumatic drugs, adrenal corticosteroids, and specific agents come into sight just these years, which cover a large part of recent research. These agents include:tumor necrosis factor (TNF)-a blocking agents, inflammatory factors specific neutralizing antibodies (interleukin (IL)-1, IL-6). A lot of effort is made to explore the pathogenesis of rheumatoid and develop new agents.
It is known that peripheral tissue damage or inflammation may induce a series of activation events in the spinal cord. During the development of experimental arthritis, the peripheral nociceptors may be sensitized by inflamed synovium and damaged articular tissue, and continuous and intense nociceptive input from inflamed joints may induce neural-immune interactions. This leads to the production and secretion of cytokines, excitatory amino acids, cyclooxygenase (COX-2), and prostaglandins, which can increase the excitability of nociceptive neurons at the spinal level, including the central terminals of the primary sensory afferents (i.e., central sensitization).
In addition, the spinal cord can also signal to the periphery to regulate inflammation. There is convincing evidence that a variety of spinally administered compounds attenuate peripheral inflammation. These compounds include adenosine, serotonin, ketamine and morphine, thalidomide, antagonists of microglia and inhibitors of astrocytes, and blockers of p38mitogen-activated protein kinase (MAPK). Interestingly, all these compounds have another effect in common:a reduction of neuronal excitability in the spinal cord. This common mechanism for both phenomena, i.e. for pain and maintenance of inflammation, might in fact be a clue as to the pathways involved. A variety of neuronal pathways that modulate peripheral inflammation have been implicated, including the sympathetic and the parasympathetic branches of the autonomic system. The branches of the vagal nerve and sympathetic fibers innervate immune organs, wherein they can influence peripheral immune responses. Another mechanism, the dorsal root reflex, involves antidromic signaling along somatic afferent fibers that influences peripheral inflammation by releasing neuropeptides, such as vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide, from the sensory nerve endings. Moreover, the Hypothalamic-Pituitary-Adrenal (HPA) axis, which can also provide a feedback mechanism on inflammation, is often blunted in a wide range of autoimmune and inflammatory diseases such as rheumatoid arthritis.
Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a pivotal role in the central nervous system (CNS), in processes such as inflammation, neuronal plasticity, synaptic transmission, learning, memory, and pain(1). NF-κB contributes to the regulation of these CNS processes by positively regulating the transcription of numerous genes including cytokines (IL-1β, IL-6, and TNF-a), pro-inflammatory enzymes COX-2and inducible nitric oxide synthase (iNOS)), chemokines, and adhesion factors. Five subunits of NF-κB have been identified, namely, gp105/p50(NF-κB1), p100/p52(NF-κB2), p65(RelA), RelB, and c-Rel. The active form of NF-κB is a dimer formed from2of these subunits. The most common and best characterized form of NF-κB is the p50/p65heterodimer, which is widely expressed in the CNS and plays an important role in the regulation of gene expression. Previous studies have indicated that activation of spinal NF-KB/p65occurs in peripheral tissue damage or inflammation. In the rat chronic constriction injury (CCI) model, we previously observed extensive co-localization of NF-KB/p65with TNF-a in the spinal dorsal horn, and down-regulation of spinal NF-KB/p65expression significantly attenuated sciatic nerve ligation-induced mechanical and thermal hyperalgesia. These findings suggest NF-KB/p65has a role in central sensitization. However, it is still unknown if spinal NF-KB/p65can also facilitate a peripheral inflammatory response.
In the present study, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-κB/p65) and transfect it into rat spinal neurons and astrocytes to silence NF-KB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-α, and COX-2. Moreover, arthritis was induced by CFA inoculation of the rats. Rats with adjuvant-induced arthritis (AIA) were administered spinally with LV-shNF-κB/p65. Thereafter, we explored the roles of spinal NF-KB/p65in the peripheral inflammation and hyperalgesia in AIA rats. In addition, we also evaluated the expression of IL-1β,TNF-α, and COX-2in the spinal cord of these rats to gain insight into the mechanism of how NF-κB/p65contributes to peripheral inflammation and hyperalgesia in this model system.
Part I Inhibitory effects of Lentivirus-mediated RNA Interference on NF-KB/p65gene in cultured Rat Spinal Neurons and Astrocytes
Objective
To construct lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-KB/p65)and transfect it into rat spinal neurons and astrocytes to silence NF-κB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-a, and COX-2.
Methods
1. Three siRNA sequences and a negative control sequence were designed according to NF-KB/p65gene sequence of rats. The complementary DNA containing both sense and antisense DNA oligos of the targeting sequence was synthesized and cloned into the pFU-GW vector, which were digested by BamW I and EcoR I. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with pHelper1.0and pHelper2.0packaging plasmids into293T cells by use of Lipofectamine2000, then the virus titer was measured.
2. Rat spinal neurons and astrocytes were cultured in vitro. Immunofluorescence staining was used to identify spinal neurons and astrocytes purity
3. The recombinant lentivirus was transfected into spinal neurons and astrocytes. The transfection efficiencies were observed in each group. Thereafter, the cells were treated with1μg/ml of LPS for24h. These cells were divided into five groups:negative control group, LPS group, LPS+LV-NC group, LPS+LV-shNF-κB/p65-1group, LPS+LV-shNF-KB/p65-2group and LPS+LV-shNF-κB/p65-3group.
4. Morphologic change of neurons and astrocytes were observed under invert microscop.5days after transfected, the expression level of NF-κB/p65mRNA and protein in cultured neurons and astrocytes was detected by Real-time PCR and Western Blot. ELISA was used to detect the changes of inflammatory cytokines (IL-1β,TNF-α) in the cultured astrocytes. Western blotting was used to determine the expression of COX-2in cultured neurons and astrocytes.
Results
1. DNA sequencing results demonstrated that the inserted sequences were correct. The titer of virus was108-9TU/mL.
2. Neurons were identitied with NSE (neuron-specific enolase) antibody. The NSE-positive cells of these cells were above85%. Astrocytes were identitied with GFAP (glial fibrillary acidic protein) antibody. The GFAP-positive cells of these cells were over90%.
3. Recombinant lentivirus could be successfully transfected into spinal neurons and astrocytes with the transduction rate higher than90%. LV-shNF-KB/p65-1or LV-shNF-KB/p65-2groups showed a lower expression level of NF-κB/p65mRNA and protein in the cultured neurons (p<0.01), while LV-shNF-κB/p65-3group didn't reach statistical significance compared with LV-NC groups (p>0.05). The interference efficiency of mRNA and protein in neurons were above95%and85%respectively. Compared to the negative control group, neuronal COX-2expression in LPS group has no significant changes (p>0.05). LV-shNF-KB/p65-1and LV-shNF-κB/p65-2can inhibit the expression of COX-2(p<0.01), but not LV-shNF-κB/p65-3(p>0.05). Compared with the LV-NC group, LPS+LV-shNF-KB/p65-1group and LPS+LV-shNF-κB/p65-2group showed a lower expression level of NF-κB/p65mRNA and protein in the cultured astrocytes (p<0.01), whereas LPS+LV-shNF-κB/p65-3group has no significant changes (p>0.05). The interference efficiency of mRNA and protein in astrocytes were above92%and83%respectively (p<0.01). Compared to the negative control group, astrocytic TNF-a, IL-1β and COX-2in LPS protein group expression was significantly increased (TNF-α,p<0.01; IL-1β,p<0.01; COX-2, p<0.05), LV-shNF-κB/p65-1and LV-shNF-κB/p65-2can inhibit this effect (p<0.01), but not LV-shNF-κB/p65-3(p>0.05).
Conclusion
The lentivirus RNAi vector targeting rat spinal NF-κB/p65gene has been constructed successfully. It may down-regulate NF-KB/p65expression in spinal neurons and astrocytes and subsequent inhibiting LPS-induced spinal TNF-a, IL-1β and COX-2expression. Taken together, our reults suggest that the lentiviral vector derived shRNA approach shows a great promise for the study of functional NF-κB/p65gene expression.
Part II Activation of Spinal NF-KB/p65Contributes to Peripheral Inflammation and Hyperalgesia in Rat Adjuvant-Induced Arthritis
Objective
It is known that noxious stimuli from inflamed tissues may increase the excitability of spinal dorsal horn neurons (central sensitization), which can signal back and contribute to peripheral inflammation. However, the underlying mechanisms have yet to be fully defined. A number of recent studies indicate that spinal nuclear factor-kappaB (NF-κB) p65is involved in central sensitization as well as pain-related behavior. Thus, our aim was to determine whether NF-κB/p65also can facilitate a peripheral inflammatory response in rat adjuvant-induced arthritis (AIA).
Methods
1. Arthritis was induced by CFA inoculation of the rats on day0. The animals which received intrathecal10ul of the lentiviral construct encoding either the scrambled shRNA sequence (LV-NC) or encoding the NF-κB/p65shRNA sequence (LV-shNF-κB/p65) were separated into2treatment groups. One group was treated on day3, and one group was treated on day10. Thus, Rats were randomly assigned to8groups, consisting of control group, CFA group, CFA+LV-NC (3d) group, CFA+LV-shNF-κB/p65-1(3d) group, and CFA+LV-shNF-κB/p65-2(3d) group, CFA+LV-NC (10d) group, CFA+LV-shNF-KB/p65-1(10d) group, and CFA+LV-shNF-KB/p65-2(10d) group.
2. In order to confirm the knockdown of NF-κB/p65expression by spinally delivered LV-shNF-κB/p65, on day14, the expression and activation of spinal NF-κB/p65was assayed by Western blot analysis, immunofluorescence, and electrophoretic mobility shift assay (EMSA). Pain-related behavior and paw swelling were assessed at baseline (1day before CFA injection) and day0was the time point of CFA injection. Measurements were also obtained at6,8,10,12,14,16,18and20days following CFA injection. On the day20, joint histopathological changes were evaluated. Moreover, the expression of spinal TNF-a, IL-1β and COX-2were assessed at14days after CFA treatment.
Results
1. Peripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65-1and LV-shNF-κB/p65-2both knocked down NF-κB/p65expression (p<0.01).
2. CFA group showed an obvious increase in mechanical hyperalgesia, thermal hyperalgesia and paw edema compare to control group. The CFA+LV-shNF-κB/p65-1and CFA+LV-shNF-KB/p65-2groups treated on day3or10can inhibite this effect(all p <0.01). The severity of Joint inflammation and destruction was significantly lower in the group treated with LV-shNF-κB/p65-2on day3, whereas these changes were modestly, but not significantly, less severe in the group treated with LV-shNF-KB/p65-1on day3(p>0.05). Furthermore, the histologic features of arthritis were not statistically significantly different between the CFA+LV-NC (10d) group and the CFA+LV-shNF-κB/p65-1(10d) or CFA+LV-shNF-KB/p65-2(10d) groups (p>0.05). The IL-1β and TNF-a protein levels in the CFA+LV-shNF-KB/p65-1(3d) and CFA+LV-shNF-κB/p65-2(3d) groups were significantly lower than those in the CFA+LV-NC (3d) group (p<0.01). Moreover, TNF-a expression levels in the CFA+LV-shNF-KB/p65-1(10d) and CFA+LV-shNF-κB/p65-2(10d) groups were also obviously lower than those in the CFA+LV-NC (10d) group (p <0.05). Furthermore, when compared with the CFA+LV-NC group (10d), the expression of IL-1β was observed to be significantly lower in the CFA+LV-shNF-κB/p65-2group (10d)(p<0.05), whereas the expression of IL-1β was modestly, but not significantly, lower in the LV-shNF-κB/p65-1(10d) group (p>0.05). Moreover, the LV-shNF-κB/p65-2group showed a decreased expression of COX-2protein (p<0.05), while the LV-shNF-KB/p65-1group (3d) showed a modestly reduced expression of COX-2protein, but the difference did not reach statistical significance (p>0.05). In addition, in the CFA+LV-shNF-KB/p65-1and CFA+LV-shNF-κB/p65-2groups treated on day10, the COX-2protein levels were also not statistically significantly different when compared with those in the CFA+LV-NC group (10d)(p>0.05).
Conclusion
Peripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65knocked down NF-κB/p65expression and significantly attenuated hyperalgesia, paw edema, and joint destruction. In addition, spinal delivery of LV-shNF-κB/p65reduced the over-expression of spinal TNF-α, IL-1β and COX-2. These indicate that spinal NF- KB/p65plays an important role in the initiation and development of both peripheral inflammation and hyperalgesia. Thus, inhibition of spinal NF-κB/p65expression may provide a potential treatment to manage painful inflammatory disorders.
类风湿性关节炎(Rheumatoid arthritis, RA),是一种由自身免疫障碍引致免疫系统攻击关节的长期慢性炎症。这种炎症会造成关节变形直至残废,并会因关节疼痛及磨损而失去部分的活动能力,会极大影响患者的生活质量。尽管目前存在很多抗风湿类药物,如非甾类抗炎药(Nonsteroidal Antiinflammatory Drugs, NSAIDs)、慢作用抗风湿药、肾上腺糖皮质激素等,其中新近开发研究的也为数不少,包括肿瘤坏死因子-a(Tumor necrosis factor, TNF-a)阻断剂、抗CD20单抗、炎性因子的中和抗体[白介素-1β(interleukin-1β, IL-1β)、白介素-1(interleukin-1,IL-1)]。但是探索类风湿致病机理,并且依此研发相关新药仍是医学界的热点。
众所周知,外周组织损伤或炎症会引起脊髓一系列的变化,在关节炎的发生发展过程中,受损的关节组织以及滑膜炎可激活外周伤害性感受器,导致连续和强烈的伤害性信号向上传入脊髓进行调制、整合,这样可能会诱发脊髓的神经免疫反应,从而引起细胞因子、兴奋性氨基酸、环氧化酶-2(cyclooxygenase, COX-2)和前列腺素的产生和分泌,进而导致脊髓伤害性神经元的持续兴奋性增强(即中枢敏化)。有意思的是,脊髓也可以通过信号传递调节外周炎症。有确切证据表明,鞘内给予的相关的一些化合物可减轻外周炎症,这些化合物包括腺苷、色胺、氯胺酮和吗啡、沙利度胺、星形胶质细胞和小胶质细胞抑制剂、促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK) p38拮抗剂等。而这些化合物具有一个共同的效应：减少脊髓神经元的兴奋性。另外,一些涉及调节外周炎症的通路已经被阐明,其中包括由交感神经和副交感神经分支组成的自主系统,其支配免疫器官,并可以影响外周的免疫反应。另一种机制即背根反射(dorsal root reflexes, DRR),可沿躯体传入神经纤维逆向传递信号,促使感觉神经末梢释放神经肽,如血管活性肠肽、P物质和降钙素基因相关肽,从而影响周围炎症。此外,下丘脑-垂体-肾上腺(Hypothalamic-Pituitary-Adrenal, HPA)轴也可以对炎症形成反馈机制,其往往涉及自身免疫和炎性相关疾病,如类风湿性关节炎。
核因子-κB (Nuclear factor-kappa B, NF-κB)是一种关键性的转录因子,参与了中枢神经系统(central nervous system, CNS)许多生理活动或病理过程,如炎症、神经元可塑性、突触传递、学习、记忆和疼痛。NF-κB通过调控众多基因来实现这些过程,其中包括细胞因子(IL-1β、IL-6、TNF-α),促炎症酶COX-2和诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS),趋化因子和粘附因子。NF-κB的5个亚基已经确定,包括gp105/p50(NF-κB1), p100/p52(NF-κB2)、p65(RelA)、RelB和c-REL, NF-κB的活性形式是这些亚基组成的二聚体。NF-κB最常见和最具特点的二聚体是p50/p65,其广泛表达于中枢神经系统,在基因表达的调节中起着重要的作用。先前的研究表明,外围组织损伤或炎症可活化脊髓NF-κB/p65,我们的早期工作也发现NF-κB/p65与TNF-α共表达于慢性压迫性损伤模型大鼠的脊髓背角,下调脊髓NF-κB/p65的表达可显著降低坐骨神经结扎引起的机械痛觉过敏和热痛觉过敏。这些研究结果表明NF-κB/p65参与了中枢敏化。然而,尚不确定脊髓NF-κ/p65是否也参与了外周炎症的发生发展。
本研究首先构建NF-κB/p65基因RNA干扰(RNA interference, RNAi)'慢病毒载体并转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察其沉默效应以及对IL-1β、TNF-α和COX-2表达的影响。在体建立大鼠佐剂性关节炎(adjuvant-induced arthritis, AIA)模型,鞘内注射慢病毒重组体,探讨脊髓NF-κB/p65在AIA大鼠外周炎症和痛觉过敏中作用。另外,我们还评估了NF-κB/P65对脊髓IL-1β、 TNF-α和COX-2表达的调控作用,以阐明脊髓NF-KB/p65参与外周炎症和痛觉过敏的相关机制。
第一部分慢病毒介导RNA干扰对体外培养大鼠脊髓神经元和星形胶质细胞NF-κB/p65基因的沉默效应
目的
构建NF-κB/p65基因RNA干扰慢病毒载体并转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察其沉默效应以及对IL-1β、TNF-α和COX-2表达的影响
方法
1.针对大鼠NF-κB/p65基因特异性序列,设计3条NF-κB/p65-siRNA序列及1条阴性对照序列,合成包含各正反义靶序列的互补DNA链,退火形成双链DNA,插入到经BamH Ⅰ和EcoR Ⅰ酶切后的pFU-GW慢病毒载体中,PCR筛选阳性克隆,DNA测序鉴定。与慢病毒包装质粒pHelper1.0、pHelper2.0通过lipofectamine2000共转染至包装细胞293T,滴度测定后-80°冰箱保存备用。
2.体外培养神经元和星形胶质细胞,用免疫荧光技术进行细胞纯度鉴定。
3.慢病毒重组体转染体外培养的大鼠脊髓神经元和星形胶质细胞,观察转染效率。然后用含1μg/ml脂多糖(Lipopolyssacride, LPS)的培养液刺激细胞24h。根据干预条件不同,实验分为6组：阴性对照组、LPS组、LPS+LV-NC组、LPS+LV-shNF-κB/p65-1组、LPS+LV-shNF-κB/p65-2组和LPS+LV-shNF-κB/p65-3组。
4.倒置显微镜下观察神经元和星形胶质细胞的形态改变,Real-Time PCR和Western blotting技术分别检测慢病毒重组体对神经元和星形胶质细胞NF-κB/p65mRNA和蛋白的干扰效率,ELISA法检测培养基中星形胶质细胞分泌的炎性因子(IL-1β、 TNF-α)的变化,Western blot技术检测神经元和星形胶质细胞COX-2的表达情况。
结果
1.测序结果证实合成的寡核苷酸链插入正确,重组载体构建成功,测定慢病毒滴度为108～9TU/mL。
2.培养神经元7d后使用神经元特异性烯醇化酶(neuron-specific enolase, NSE)特异性抗体对其进行纯度鉴定,结果显示85%以上为神经元。培养星形胶质细胞12d后进行传代,传代2次后使用神经胶质酸性蛋白(glial fibrillary acidic protein, GFAP)特异性抗体对其进行纯度鉴定,结果显示达90%以上为星形胶质细胞。
3.慢病毒重组体能成功转染大鼠脊髓神经元和星形胶质细胞,神经元转染效率大于90%,与LV-NC组比较,LPS+LV-shNF-κB/p65-1组和LPS+LV-shNF-κB/p65-2组NF-κB/P65mRNA和蛋白的表达都明显下调,mRNA水平干扰效率达95%(P<0.01),蛋白水平干扰效率达85%以上(P<0.01),而LPS+LV-shNF-κB/p65-3组无明显下调(P>0.05)。相比阴性对照组,神经元的LPS组COX-2表达无明显变化,LV-shNF-κB/p65-1和LV-shNF-κB/p65-2能下调COX-2的表达,而LV-shNF-κB/p65-3则无明显作用。另外,星形胶质细胞转染效率大于85%,与LV-NC组比较,LPS+LV-shNF-κB/p65-1组和LPS+LV-shNF-κB/p65-2组NF-κB/p65mRNA和蛋白的表达明显下调,mRNA水平干扰效率达92%(P<0.01),蛋白水平干扰效率达83%以上(P<0.01),而LPS+LV-shNF-κB/p65-3组无明显变化(P>0.05)。相比阴性对照组,星形胶质细胞LPS组的TNF-α、IL-1β和COX-2蛋白表达明显上调(TNF-α, P<0.01; IL-1β,,P<0.01; COX-2, P<0.05), LV-shNF-κB/p65-1和LV-shNF-κB/p65-2能抑制这种上调效应(P<0.01),而LV-shNF-κB/p65-3组无这种作用。
结论
成功构建了大鼠NF-κB/P65基因的RNA干扰慢病毒载体,其可使大鼠脊髓神经元和星形胶质细胞的NF-κB/p65基因表达下调,并能抑制LPS导致的TNF-α、IL-1β和COX-2的上调效应。这样为我们下一步动物体内实验奠定了坚实的基础,也为神经系统NF-κB/p65基因功能研究提供一种有效的工具。
第二部分脊髓NF-κB/P65对大鼠佐剂性关节炎外周炎症以及痛觉过敏的调控作用
目的
炎症组织的伤害性刺激可能增加脊髓背角神经元的兴奋性(即中枢敏化),兴奋的神经元可返回信号并调控外周炎症,但是其中的机制尚不明确。最近的一些研究表明,脊髓NF-κB/p65参与了中枢敏化以及相关疼痛。我们的目的是研究脊髓NF-KB/p65是否也参与了大鼠佐剂性关节炎的外周炎症反应及相关痛觉过敏。
方法
1.构建大鼠佐剂性关节炎模型,在完全性弗氏佐剂(complete Freund's adjuvant,CFA)注射后第3d或10d,鞘内给予10u1慢病毒重组体(LV-NF-KB/p65或LV-NC)。根据干预条件不同,实验分为8组：对照组、CFA组、CFA+LV-NC (3d)组、CFA+LV-shNF-KB/p65-1(3d)组、CFA+LV-shNF-KB/p65-2(3d)组、CFA+LV-NC (10d)组、CFA+LV-shNF-KB/p65-1(10d)组、CFA+LV-shNF-KB/p65-2(10d)组。
2.CFA注射后第14d,使用Western blot技术、免疫荧光技术以及EMSA技术评估脊髓NF-KB/p65蛋白的表达和转录活性。在CFA注射前1d和注射后第6d、第8d、第10d、第12d、第14d、第16d、第18d以及第20d,对大鼠的疼痛相关行为、足跖肿胀程度进行了评估,在第20d,观察评估大鼠的关节组织病理学改变。此外,在CFA注射后第14d,检测脊髓炎性介质TNF-α, IL-1β和COX-2的表达。
结果
1.外周炎症上调了脊髓NF-κB/p65蛋白的表达,其主要分布于脊髓背角的神经元和星形胶质细胞,CFA注射后第3d或10d,鞘内注射LV-shNF-KB/p65-1和LV-shNF-κB/p65-2都能下调NF-κB/p65蛋白的表达和转录活性。
2.相比阴性对照组,CFA组大鼠的机械痛阈、热痛阈明显下降(P<0.01),而足跖肿胀明显增加(P<0.01),CFA注射后第3d或10d,鞘内给予LV-shNF-KB/p65-1和LV-shNF-κB/p65-2都能显著抑制这些效应(P<0.01)。另外,相比CFA+LV-NC (3d)组,大鼠关节炎症和组织破坏程度在CFA+LV-shNF-KB/p65-2(3d)组显著降低(P<0.01),而LV-shNF-κB/p65-1(10d)组轻微降低,但未达统计学意义(P>0.05)；此外,相比CFA+LV-NC (10d)组,CFA+LV-shNF-KB/p65-1(10d)组和LV-shNF-κB/p65-2(10d)组的关节炎症和组织破坏程度无明显变化(P>0.05)。CFA注射后第3d,鞘内给予LV-shNF-κB/p65-1能下调脊髓TNF-α和IL-1p的表达(P<0.01),但对COX-2的表达无明显作用(P>0.05),而LV-shNF-KB/p65-2能抑制脊髓TNF-α, IL-1β和COX-2表达(TNF-α, P<0.01; IL-1β, P<0.01; COX-2, P<0.05)。CFA注射后第10d,鞘内注射LV-shNF-KB/p65-1能下调脊髓TNF-α的表达(P<0.05),对IL-1β无显著作用(P>0.05),而LV-shNF-KB/p65-2能同时抑制TNF-α和IL-1β的表达(P<0.05),但两者都对COX-2无明显作用(P>0.05)。
结论
外周炎症上调了脊髓NF-κB/p65蛋白的表达,其主要分布于脊髓背角的神经元和星形胶质细胞。鞘内注射LV-shNF-KB/p65能下调NF-κB/P65蛋白的表达,并且能降低大鼠的痛觉过敏、足跖肿胀以及关节破坏。此外,其也能减少脊髓TNF-α, IL-1β和COX-2的过表达。这些表明脊髓NF-κB/p65在AIA大鼠外围炎症和痛觉过敏的发生发展过程中起着重要作用。因此,抑制脊髓NF-κB/p65的表达对外周炎症性疾病可能是一种潜在的治疗方法。
Background
Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks flexible (synovial) joints. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility. The disease greatly affects the quality of life of patients. There are many drugs in the RA treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), slow-acting anti-rheumatic drugs, adrenal corticosteroids, and specific agents come into sight just these years, which cover a large part of recent research. These agents include:tumor necrosis factor (TNF)-a blocking agents, inflammatory factors specific neutralizing antibodies (interleukin (IL)-1, IL-6). A lot of effort is made to explore the pathogenesis of rheumatoid and develop new agents.
It is known that peripheral tissue damage or inflammation may induce a series of activation events in the spinal cord. During the development of experimental arthritis, the peripheral nociceptors may be sensitized by inflamed synovium and damaged articular tissue, and continuous and intense nociceptive input from inflamed joints may induce neural-immune interactions. This leads to the production and secretion of cytokines, excitatory amino acids, cyclooxygenase (COX-2), and prostaglandins, which can increase the excitability of nociceptive neurons at the spinal level, including the central terminals of the primary sensory afferents (i.e., central sensitization).
In addition, the spinal cord can also signal to the periphery to regulate inflammation. There is convincing evidence that a variety of spinally administered compounds attenuate peripheral inflammation. These compounds include adenosine, serotonin, ketamine and morphine, thalidomide, antagonists of microglia and inhibitors of astrocytes, and blockers of p38mitogen-activated protein kinase (MAPK). Interestingly, all these compounds have another effect in common:a reduction of neuronal excitability in the spinal cord. This common mechanism for both phenomena, i.e. for pain and maintenance of inflammation, might in fact be a clue as to the pathways involved. A variety of neuronal pathways that modulate peripheral inflammation have been implicated, including the sympathetic and the parasympathetic branches of the autonomic system. The branches of the vagal nerve and sympathetic fibers innervate immune organs, wherein they can influence peripheral immune responses. Another mechanism, the dorsal root reflex, involves antidromic signaling along somatic afferent fibers that influences peripheral inflammation by releasing neuropeptides, such as vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide, from the sensory nerve endings. Moreover, the Hypothalamic-Pituitary-Adrenal (HPA) axis, which can also provide a feedback mechanism on inflammation, is often blunted in a wide range of autoimmune and inflammatory diseases such as rheumatoid arthritis.
Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a pivotal role in the central nervous system (CNS), in processes such as inflammation, neuronal plasticity, synaptic transmission, learning, memory, and pain(1). NF-κB contributes to the regulation of these CNS processes by positively regulating the transcription of numerous genes including cytokines (IL-1β, IL-6, and TNF-a), pro-inflammatory enzymes COX-2and inducible nitric oxide synthase (iNOS)), chemokines, and adhesion factors. Five subunits of NF-κB have been identified, namely, gp105/p50(NF-κB1), p100/p52(NF-κB2), p65(RelA), RelB, and c-Rel. The active form of NF-κB is a dimer formed from2of these subunits. The most common and best characterized form of NF-κB is the p50/p65heterodimer, which is widely expressed in the CNS and plays an important role in the regulation of gene expression. Previous studies have indicated that activation of spinal NF-KB/p65occurs in peripheral tissue damage or inflammation. In the rat chronic constriction injury (CCI) model, we previously observed extensive co-localization of NF-KB/p65with TNF-a in the spinal dorsal horn, and down-regulation of spinal NF-KB/p65expression significantly attenuated sciatic nerve ligation-induced mechanical and thermal hyperalgesia. These findings suggest NF-KB/p65has a role in central sensitization. However, it is still unknown if spinal NF-KB/p65can also facilitate a peripheral inflammatory response.
In the present study, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-κB/p65) and transfect it into rat spinal neurons and astrocytes to silence NF-KB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-α, and COX-2. Moreover, arthritis was induced by CFA inoculation of the rats. Rats with adjuvant-induced arthritis (AIA) were administered spinally with LV-shNF-κB/p65. Thereafter, we explored the roles of spinal NF-KB/p65in the peripheral inflammation and hyperalgesia in AIA rats. In addition, we also evaluated the expression of IL-1β,TNF-α, and COX-2in the spinal cord of these rats to gain insight into the mechanism of how NF-κB/p65contributes to peripheral inflammation and hyperalgesia in this model system.
Part I Inhibitory effects of Lentivirus-mediated RNA Interference on NF-KB/p65gene in cultured Rat Spinal Neurons and Astrocytes
Objective
To construct lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-KB/p65)and transfect it into rat spinal neurons and astrocytes to silence NF-κB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-a, and COX-2.
Methods
1. Three siRNA sequences and a negative control sequence were designed according to NF-KB/p65gene sequence of rats. The complementary DNA containing both sense and antisense DNA oligos of the targeting sequence was synthesized and cloned into the pFU-GW vector, which were digested by BamW I and EcoR I. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with pHelper1.0and pHelper2.0packaging plasmids into293T cells by use of Lipofectamine2000, then the virus titer was measured.
2. Rat spinal neurons and astrocytes were cultured in vitro. Immunofluorescence staining was used to identify spinal neurons and astrocytes purity
3. The recombinant lentivirus was transfected into spinal neurons and astrocytes. The transfection efficiencies were observed in each group. Thereafter, the cells were treated with1μg/ml of LPS for24h. These cells were divided into five groups:negative control group, LPS group, LPS+LV-NC group, LPS+LV-shNF-κB/p65-1group, LPS+LV-shNF-KB/p65-2group and LPS+LV-shNF-κB/p65-3group.
4. Morphologic change of neurons and astrocytes were observed under invert microscop.5days after transfected, the expression level of NF-κB/p65mRNA and protein in cultured neurons and astrocytes was detected by Real-time PCR and Western Blot. ELISA was used to detect the changes of inflammatory cytokines (IL-1β,TNF-α) in the cultured astrocytes. Western blotting was used to determine the expression of COX-2in cultured neurons and astrocytes.
Results
1. DNA sequencing results demonstrated that the inserted sequences were correct. The titer of virus was108-9TU/mL.
2. Neurons were identitied with NSE (neuron-specific enolase) antibody. The NSE-positive cells of these cells were above85%. Astrocytes were identitied with GFAP (glial fibrillary acidic protein) antibody. The GFAP-positive cells of these cells were over90%.
3. Recombinant lentivirus could be successfully transfected into spinal neurons and astrocytes with the transduction rate higher than90%. LV-shNF-KB/p65-1or LV-shNF-KB/p65-2groups showed a lower expression level of NF-κB/p65mRNA and protein in the cultured neurons (p<0.01), while LV-shNF-κB/p65-3group didn't reach statistical significance compared with LV-NC groups (p>0.05). The interference efficiency of mRNA and protein in neurons were above95%and85%respectively. Compared to the negative control group, neuronal COX-2expression in LPS group has no significant changes (p>0.05). LV-shNF-KB/p65-1and LV-shNF-κB/p65-2can inhibit the expression of COX-2(p<0.01), but not LV-shNF-κB/p65-3(p>0.05). Compared with the LV-NC group, LPS+LV-shNF-KB/p65-1group and LPS+LV-shNF-κB/p65-2group showed a lower expression level of NF-κB/p65mRNA and protein in the cultured astrocytes (p<0.01), whereas LPS+LV-shNF-κB/p65-3group has no significant changes (p>0.05). The interference efficiency of mRNA and protein in astrocytes were above92%and83%respectively (p<0.01). Compared to the negative control group, astrocytic TNF-a, IL-1β and COX-2in LPS protein group expression was significantly increased (TNF-α,p<0.01; IL-1β,p<0.01; COX-2, p<0.05), LV-shNF-κB/p65-1and LV-shNF-κB/p65-2can inhibit this effect (p<0.01), but not LV-shNF-κB/p65-3(p>0.05).
Conclusion
The lentivirus RNAi vector targeting rat spinal NF-κB/p65gene has been constructed successfully. It may down-regulate NF-KB/p65expression in spinal neurons and astrocytes and subsequent inhibiting LPS-induced spinal TNF-a, IL-1β and COX-2expression. Taken together, our reults suggest that the lentiviral vector derived shRNA approach shows a great promise for the study of functional NF-κB/p65gene expression.
Part II Activation of Spinal NF-KB/p65Contributes to Peripheral Inflammation and Hyperalgesia in Rat Adjuvant-Induced Arthritis
Objective
It is known that noxious stimuli from inflamed tissues may increase the excitability of spinal dorsal horn neurons (central sensitization), which can signal back and contribute to peripheral inflammation. However, the underlying mechanisms have yet to be fully defined. A number of recent studies indicate that spinal nuclear factor-kappaB (NF-κB) p65is involved in central sensitization as well as pain-related behavior. Thus, our aim was to determine whether NF-κB/p65also can facilitate a peripheral inflammatory response in rat adjuvant-induced arthritis (AIA).
Methods
1. Arthritis was induced by CFA inoculation of the rats on day0. The animals which received intrathecal10ul of the lentiviral construct encoding either the scrambled shRNA sequence (LV-NC) or encoding the NF-κB/p65shRNA sequence (LV-shNF-κB/p65) were separated into2treatment groups. One group was treated on day3, and one group was treated on day10. Thus, Rats were randomly assigned to8groups, consisting of control group, CFA group, CFA+LV-NC (3d) group, CFA+LV-shNF-κB/p65-1(3d) group, and CFA+LV-shNF-κB/p65-2(3d) group, CFA+LV-NC (10d) group, CFA+LV-shNF-KB/p65-1(10d) group, and CFA+LV-shNF-KB/p65-2(10d) group.
2. In order to confirm the knockdown of NF-κB/p65expression by spinally delivered LV-shNF-κB/p65, on day14, the expression and activation of spinal NF-κB/p65was assayed by Western blot analysis, immunofluorescence, and electrophoretic mobility shift assay (EMSA). Pain-related behavior and paw swelling were assessed at baseline (1day before CFA injection) and day0was the time point of CFA injection. Measurements were also obtained at6,8,10,12,14,16,18and20days following CFA injection. On the day20, joint histopathological changes were evaluated. Moreover, the expression of spinal TNF-a, IL-1β and COX-2were assessed at14days after CFA treatment.
Results
1. Peripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65-1and LV-shNF-κB/p65-2both knocked down NF-κB/p65expression (p<0.01).
2. CFA group showed an obvious increase in mechanical hyperalgesia, thermal hyperalgesia and paw edema compare to control group. The CFA+LV-shNF-κB/p65-1and CFA+LV-shNF-KB/p65-2groups treated on day3or10can inhibite this effect(all p <0.01). The severity of Joint inflammation and destruction was significantly lower in the group treated with LV-shNF-κB/p65-2on day3, whereas these changes were modestly, but not significantly, less severe in the group treated with LV-shNF-KB/p65-1on day3(p>0.05). Furthermore, the histologic features of arthritis were not statistically significantly different between the CFA+LV-NC (10d) group and the CFA+LV-shNF-κB/p65-1(10d) or CFA+LV-shNF-KB/p65-2(10d) groups (p>0.05). The IL-1β and TNF-a protein levels in the CFA+LV-shNF-KB/p65-1(3d) and CFA+LV-shNF-κB/p65-2(3d) groups were significantly lower than those in the CFA+LV-NC (3d) group (p<0.01). Moreover, TNF-a expression levels in the CFA+LV-shNF-KB/p65-1(10d) and CFA+LV-shNF-κB/p65-2(10d) groups were also obviously lower than those in the CFA+LV-NC (10d) group (p <0.05). Furthermore, when compared with the CFA+LV-NC group (10d), the expression of IL-1β was observed to be significantly lower in the CFA+LV-shNF-κB/p65-2group (10d)(p<0.05), whereas the expression of IL-1β was modestly, but not significantly, lower in the LV-shNF-κB/p65-1(10d) group (p>0.05). Moreover, the LV-shNF-κB/p65-2group showed a decreased expression of COX-2protein (p<0.05), while the LV-shNF-KB/p65-1group (3d) showed a modestly reduced expression of COX-2protein, but the difference did not reach statistical significance (p>0.05). In addition, in the CFA+LV-shNF-KB/p65-1and CFA+LV-shNF-κB/p65-2groups treated on day10, the COX-2protein levels were also not statistically significantly different when compared with those in the CFA+LV-NC group (10d)(p>0.05).
Conclusion
Peripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65knocked down NF-κB/p65expression and significantly attenuated hyperalgesia, paw edema, and joint destruction. In addition, spinal delivery of LV-shNF-κB/p65reduced the over-expression of spinal TNF-α, IL-1β and COX-2. These indicate that spinal NF- KB/p65plays an important role in the initiation and development of both peripheral inflammation and hyperalgesia. Thus, inhibition of spinal NF-κB/p65expression may provide a potential treatment to manage painful inflammatory disorders.
引文
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