TLR4,TLR9 mRNA在P0 180-199诱导Lewis大鼠EAN模型中的动态表达及TWP对其的影响

目的:观察TLR4,TLR9 mRNA在P0 180-199诱导Lewis大鼠FAN模型中动态表达,以及雷公藤多甙对其的影响.方法:以周围神经髓鞘蛋白P0 180-199免疫Lewis大鼠,给予雷公藤多甙灌胃,TWP 40 mg/(kg·d)干预治疗.观察免疫后大鼠症状和组织病理学改变及TWP对其的影响,利用RT-PCR检测TLR4,TLR9的mRNA表达水平.结果IEAN组出现EAN行为学改变、炎性细胞浸润和髓鞘脱失,TLR4、TLR9的表达高于ETA组(P＜0.05).EAN+TWP组大鼠症状较FAN+NS组稍改善,炎性浸润减少,FAN+TWP组,TLR4和TLR9的表达低于EAN+NS组(P＜0.05).CFA组大鼠无症状,TLR4和TLR9的表达高于NS组(P＜0.05).结论:TLR4和TLR9可能参与EAN发病;雷公藤多甙可能通过抑制TLR4和TLR9的功能来减轻EAN的发病.     

L-161982对实验性自身免疫性神经炎大鼠趋化因子的影响

探讨PGE2-EP4受体拮抗剂L-161982对实验性自身免疫性神经炎(EAN)临床及趋化因子表达的影响。用周围神经鞘磷脂(BPM)免疫Lewis大鼠,随机将21只大鼠分为3组,治疗A组、治疗B组和对照组。两个治疗组均以剂量为5mg/kg的PGE2-EP4受体拮抗剂L-161982腹腔注射治疗。治疗A组于免疫阶段(即免疫前1天至免疫后第8天)每日行L-161982处理,治疗B组于发病阶段(免疫后第5天至第14天)每日行L-161982处理,对照组在整个实验阶段每日给予相同体积的L-161982溶媒DMSO腹腔注射。观察各组EAN模型大鼠的临床评分变化,于疾病高峰期即免疫后第15天取坐骨神经,对其趋化因子CXCL-12、MCP-1的表达水平进行免疫组化检测。与对照组比较,不同阶段处理的两个治疗组均能延迟EAN的发病时间(P0.05),降低高峰期临床评分(P0.05),减少坐骨神经中CXCL-12和MCP-1的表达(P0.05)。PGE2-EP4拮抗剂L-161982对EAN有治疗作用。     

雷公藤多苷降低糖尿病肾病大鼠炎性细胞因子的表达

目的观察雷公藤多苷(TWP)对糖尿病肾病(DN)大鼠核因子κB(NF-κB)、C-C型趋化因子配体5(CCL5)、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)等炎性细胞因子表达的影响。方法链脲佐菌素(STZ)腹腔注射建立DN大鼠模型。动物随机分为正常组、模型组、1.8 g/kg/d TWP治疗组。用药8周末,计算肾脏指数(KI)、全自动生化分析仪检测大鼠血糖(BG)、糖化血红蛋白(HbA1c)、血肌酐(Scr)、血尿素氮(BUN)等生化指标。ELISA检测大鼠血清超敏C反应蛋白(hs-CRP)和肾匀浆IL-6、TNF-α和CCL5的含量;HE染色观察大鼠肾脏病理学改变;免疫组织化学染色检测大鼠肾组织NF-κB的表达。结果与模型组相比,TWP可改善DN大鼠一般状况;明显降低DN大鼠空腹BG、HbA1c、KI、BUN及Scr水平,增加DN大鼠体质量(P0.01);与模型组比较,TWP明显降低DN大鼠血清hs-CRP水平及肾匀浆中IL-6、TNF-α和CCL5的水平,明显下调DN大鼠肾组织NF-κB的表达(P0.01),经TWP治疗后DN大鼠肾脏病理变化减轻。结论 TWP可降低糖尿病大鼠血清及肾脏炎性细胞因子表达,减轻肾脏病变。     

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。     

Ⅱ型胶原蛋白和雷公藤多甙对佐剂性关节炎治疗作用的实验研究

目的探讨口服Ⅱ型胶原(typeⅡcollagen,CⅡ)、雷公藤多甙(Tripterygium Wilfordii Polyglycosidium,TWP)对大鼠佐剂性关节炎(adjuvant arthritis,AA)的治疗作用及在治疗过程中对大鼠外周淋巴器官中白介素10(IL-10)的影响。方法建立动物模型,采用关节炎评分法对关节肿胀程度进行评估,采用免疫组化法检测大鼠外周淋巴器官中IL-10水平的变化。结果口服Ⅱ型胶原及雷公藤多甙可以明显减轻病变关节的炎症反应,降低发病率,推迟AA的发病并且使病程明显缩短,而且大鼠外周淋巴器官中IL-10水平也较模型组显著升高。结论口服CⅡ及TWP可有效减轻佐剂型关节炎大鼠的关节病变,并能使AA大鼠外周淋巴结中IL-10水平升高。     

雷公腾多甙对实验性变态反应性神经炎周围神经的影响

目的:了解雷公腾多甙(Tripterygium polyglucoside,TPG)对实验性变态反应性神经炎(experimental allergic neuritis,EAN)周围神经的影响。方法:在模型大鼠出现EAN临床症状后连续给予TPG14d,观察EAN临床症状、周围神经电生理与组织病理的变化。结果:免疫后23d EAN坐骨神经的运动神经传导速度和复合肌肉动作电位(compound muscle action potential,CMAP)的波幅明显降低,潜伏期和时限显著延长(P0.05,与对照组比较)。大量炎性细胞侵入坐骨神经,多数神经纤维呈现严重的髓鞘脱失和相当程度的轴索变性;位于郎飞结间隙和近结旁段的轴膜钠、钾离子通道基本检测不到(P0.05,与对照组比较)。TPG干预显著减轻了CMAP波幅的降低和潜伏期与时限的延长,坐骨神经炎性反应和脱髓鞘减轻,部分轴膜钠、钾离子通道得以保留(P0.05与EAN组比较)。结论:TPG作为一种可能减少格林-巴利综合征持久性神经功能损害的药物值得进一步研究。     

雷公藤甲素对糖尿病肾病大鼠肾组织TNF-α与MCP-1表达的影响

目的观察雷公藤甲素对糖尿病肾病大鼠肾组织肿瘤坏死因子-α(TNF-α)与单核细胞趋化因子蛋白-1(MCP-1)表达的影响。方法选取50只雄性SD大鼠,随机分为正常组(n=10)、模型组(n=20)及雷公藤甲素组(n=20)。模型组与雷公藤甲素组大鼠给予高糖高脂饮食,并以链脲佐菌素腹腔注射建立糖尿病肾病大鼠模型。8周后处死大鼠,检测相应生化指标并计算肾脏指数,免疫组织化学方法和Western blot方法检测TNF-α与MCP-1的蛋白表达。结果模型组大鼠肾脏指数、血糖、血肌酐和24 h尿蛋白较对照组显著升高,P0.05;与模型组相比,雷公藤甲素组大鼠的各项指数显著降低,P0.05。与对照组比较,模型组大鼠肾脏组织TNF-α与MCP-1的蛋白表达显著升高,P0.05;与模型组相比,雷公藤甲素组大鼠肾脏组织TNF-α与MCP-1的蛋白表达显著降低,P0.05。结论雷公藤甲素能降低糖尿病肾病大鼠肾组织TNF-α与MCP-1的水平,具有一定的肾保护作用。     

雷公藤多甙对佐剂性关节炎模型大鼠的作用

目的探讨雷公藤多甙(TWH)对佐剂性关节炎(AA)模型大鼠的预防、治疗作用。方法用氟氏完全佐剂(FCA)诱导大鼠形成关节炎,经TWH进行预防和治疗处理后,观察大鼠足跖关节肿胀度,关节炎症指数及踝关节滑膜的病理改变。结果TWH预防组能阻止大鼠继发性病变的发生,而治疗组能显著减轻大鼠足跖关节肿胀度,关节炎症指数,减轻踝关节滑膜病理改变。结论TWH不仅能改善大鼠佐剂性关节炎模型关节炎症状、抑制炎症反应,而且能预防继发性病变的出现。     

刺五加注射液对大鼠坐骨神经损伤的作用及可能机制

目的研究刺五加注射液对大鼠坐骨神经损伤的机制及可能机制。方法 SD大鼠随机分为正常组、模型组、治疗组1(20mg/kg·d)和治疗组2(40mg/kg·d)。大鼠左侧坐骨神经夹持法建立坐骨神经损伤模型,手术当天开始腹腔注射给药,连续6周。观察大鼠左足皮肤温度、坐骨神经功能指数(SFI)和坐骨神经传导速度(SNCV)及神经组织形态学的改变,免疫组织化学染色法及Western Blot观测大鼠坐骨神经生长因子的表达。结果治疗组大鼠皮肤温度、坐骨神经功能指数、坐骨神经传导速度及神经组织形态学改变均优于模型组,神经生长因子的表达水平明显高于模型组。结论刺五加注射液对损伤后大鼠坐骨神经有促进修复的作用,其机制可能与促进神经生长因子的表达有关。     

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。     

Expression of interleukin-16 in sciatic nerves, spinal roots and spinal cords of experimental autoimmune neuritis rats

Experimental autoimmune neuritis (EAN) is a well-known animal model of Guillain-Barré Syndrome. In this study, we studied the spatiotemporal expression of interleukin-16 (IL-16) in the nervous system of EAN rats and pharmacological effects of minocycline on IL-16 expressions in EAN rats. In sciatic nerves and dorsal/ventral roots of EAN rats, IL-16⁺ cells, identified as macrophages and T cells, were mainly found to concentrate around blood vessels. However, in spinal cords, IL-16⁺ microglial cells were mainly found in lumbar dorsal horns. Massive IL-16⁺ cell accumulation in sciatic nerves and spinal roots was temporally correlated with severity of neurological signs of EAN. Furthermore, a strong correlation of IL-16⁺ cell accumulation with local demyelination in perivascular areas of sciatic nerves, and significant reduction of IL-16⁺ cell numbers in sciatic nerves and spinal cords by minocycline suggested a pathological contribution of IL-16⁺ cells in EAN. Taken together, robust IL-16⁺ cell accumulation in the nervous system and its temporal correlation with severity of neurological signs in EAN might suggest a pathological role of IL-16 in EAN, which makes IL-16 a potential pharmacological target.     

Augmented expression of daintain/allograft inflammatory factor-1 is associated with clinical disease: dynamics of daintain/allograft inflammatory factor-1 expression in spleen, peripheral nerves and sera during experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is an animal model of Guillain-Barré syndrome, characterized by inflammation and demyelination of the peripheral nervous system (PNS). Daintain/allograft inflammatory factor-1 (daintain/AIF-1) is a novel interferon-gamma-inducible protein expressed by macrophages during organ specific autoimmune diseases. To study the involvement of daintain/AIF-1 in EAN we induced EAN in Lewis rats by immunizing with bovine PNS myelin (BPM) and complete Freund's adjuvant (CFA). The expression of daintain/AIF-1 was examined in the spleen, peripheral nerves and sera during the course of EAN by immunohistochemistry and radioimunoassay (RIA). The expression of daintain/AIF-1 in the spleen and in the sciatic nerves peaked at the preclinical stage (day 7 post immunization (p.i.)) and at the height (day 15 p.i.) of clinical EAN, consistent with a disease promoting role for daintain/AIF-1. Daintain/AIF-1 expressing cells represented a subset of ED1+ or CD11b/c+ mononuclear cells. A significant increase of daintain/AIF-1-like immunoreactivity in sera occurred at the preclinical stage of EAN. Taken together, these data indicate that daintain/AIF-1 may play a proinflammatory role in the pathogenesis of EAN.     

Upregulation of osteopontin in Schwann cells of the sciatic nerves of Lewis rats with experimental autoimmune neuritis

We examined the expression of osteopontin (OPN) in the sciatic nerves of rats with experimental autoimmune neuritis (EAN), using immunohistochemistry and immunoblotting, to study its involvement in the pathogenesis of autoimmune peripheral nervous system diseases. Constitutive OPN expression was detected in some Schwann cells; expression was increased after immunization with adjuvant alone. At day 14 after induction of EAN, many Schwann cells had a granular pattern of immunoreactivity, whereas very few inflammatory cells were OPN-positive. Even after recovery from hindlimb paralysis, at 24 days post-immunization, OPN expression remained elevated in the Schwann cells. The results suggest that OPN expression in Schwann cells is easily induced by immunostimulation, and further enhanced by the inflammatory reaction in EAN. Continued elevation of OPN after recovery may represent a functional recovery after a transient inflammatory insult.     

Treatment for experimental autoimmune neuritis with clodronate (Bonefos)

Experimental autoimmune neuritis (EAN) serves as an animal model for human Gullain–Barre syndrome (GBS), an autoimmune disease causing demyelination and inflammation of peripheral nerves. Macrophages, which play a major role in this autoimmune inflammatory process, can be selectively targeted by high doses of bisphophonates. The goal of this study was to examine the effect of the bisphosphonate, clodronate, on the severity of the EAN model. EAN was induced in female adult rats by immunization with bovine peripheral myelin. A number of treatment protocols with clodronate were used based on the common dosage regimen of 20 mg/kg in humans starting with the appearance of clinical signs on day 10 post-immunization. The clinical parameters measured included a clinical score, a motor performance test performed on a Rotarod and body weight. The expression of the matrix metaloprotease (MMP-9) in the sciatic nerves was measured as a marker of inflammatory macrophages. Treatment with clodronate, 20 mg/kg daily and 40 mg/kg every 2 days, significantly reduced the disease severity (a 75 % decrease in severity, p < 0.01 by ANOVA) as measured by the clinical score compared to controls. Performance on the Rotarod test and body weight confirmed the clinical score findings. MMP-9 expression levels were significantly lower in the sciatic nerves of clodronate-treated rats. The present findings support the efficiency of clodronate in inflammatory diseases of the peripheral nervous system. The mechanism of action includes inhibition of inflammatory macrophages. The results suggest the use of bisphosphonates be considered in humans with GBS.     

Protective effect of Rolipram in experimental autoimmune neuritis: protection is associated with down-regulation of IFN-gamma and inflammatory chemokines as well as up-regulation of IL-4 in peripheral nervous system

Rolipram, a phosphodiesterase type 4 inhibitor, is reported to have anti-inflammatory effects. It can markedly downregulate antigen-driven T cell proliferation and suppress TNF-(alpha and TNF-beta production in vitro and in vivo, which have led to its use in the treatment of a number of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). EAN is a CD4+ T cell-mediated demyelinating autoimmune disease of peripheral nervous system (PNS) that represents an animal model for the study of the immunopathogenesis and immunotherapy of Guillain-Barré syndrome (GBS) in human. In the previous study, we reported that suppression of EAN by Rolipram was associated with down-regulated myelin antigen-induced T cell responses as well as downregulated IFN-gamma and TNF-alpha production. Here we report that EAN induced in Lewis rats by inoculation with the PNS P2 protein peptide 57-81 and Freund's complete adjuvant (FCA), was strongly suppressed by Rolipram administered twice daily intraperitoneally from day 9 post immunization (p.i.), i.e. after onset of clinical EAN to day 18 p.i. This clinical effect was associated with dose-dependent down-regulated production of IFN-gamma and the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha, MIP-2 and monocyte chemotactic protein-1(MCP-1) as well as up-regulated IL-4 production in sciatic nerve sections from Rolipram-treated EAN rats at maximum of clinical EAN, i.e. on day 14 p.i.. These findings suggest that Rolipram may be useful in certain T cell-dependent autoimmune diseases and inflammatory neuropathies. These observations call for further studies on the potential role of Rolipram in the treatment of autoimmune diseases.     

Intranasal administration of recombinant mouse interleukin-12 increases inflammation and demyelination in chronic experimental autoimmune neuritis in Lewis rats

To examine whether interleukin (IL)-12 modulates ongoing chronic experimental autoimmune neuritis (EAN), we evaluated the effects of recombinant mouse IL-12 (rmIL-12) in Lewis rats with chronic EAN, induced by immunization with P0 peptide (180-199) plus complete Freund's adjuvant. Rats were treated intranasally with either 0.1 or 1 microg/rat/day rmIL-12 for 6 days from the onset of clinical chronic EAN, on days 5-10 postimmunization (p.i.). Only high-dose rmIL-12 exacerbated chronic EAN. This clinical effect was associated with higher numbers of inflammatory cells and more severe demyelination in sciatic nerve sections on days 15 and 80 p.i. compared with low-dose rmIL-12-treated rats and phosphate-buffered saline (PBS)-treated control rats. High-dose rmIL-12 increased significantly the lymph node mononuclear cell proliferation in response to P0 peptide 180-199 and IFN-gamma production in the sciatic nerves. These data indicate that intranasally administered IL-12 acts as a proinflammatory cytokine in chronic EAN. Effective inhibition of IL-12 in vivo could be considered for therapeutic use in chronic inflammatory demyelinating polyradiculoneuropathy.     

INCREASED EXPRESSION OF PHOSPHOLIPASE D1 IN THE SCIATIC NERVE OF RATS WITH EXPERIMENTAL AUTOIMMUNE NEURITIS

Phospholipase D1 (PLD1) expression in the sciatic nerve was studied in induced experimental autoimmune neuritis (EAN) in Lewis rats. PLD1 immunoreactivity was seen in some Schwann cells in the sciatic nerves of normal rats. In parallel with the progression of EAN, PLD1-positive Schwann cells significantly increased in number and showed intense immunoreactivity. PLD1 was also detected in some ED1+ macrophages in EAN lesions. These results suggest that PLD1 in macrophages and Schwann cells plays an important role in the activation of these cells in the pathogenesis of EAN, an animal model of human peripheral demyelinating disease.     

Increased expression of phospholipase D1 in the sciatic nerve of rats with experimental autoimmune neuritis

Phospholipase D1 (PLD1) expression in the sciatic nerve was studied in induced experimental autoimmune neuritis (EAN) in Lewis rats. PLD1 immunoreactivity was seen in some Schwann cells in the sciatic nerves of normal rats. In parallel with the progression of EAN, PLD1-positive Schwann cells significantly increased in number and showed intense immunoreactivity. PLD1 was also detected in some ED1+ macrophages in EAN lesions. These results suggest that PLD1 in macrophages and Schwann cells plays an important role in the activation of these cells in the pathogenesis of EAN, an animal model of human peripheral demyelinating disease.     

Activation of extracellular signal-regulated kinases in the sciatic nerves of rats with experimental autoimmune neuritis

To investigate whether the phosphorylation of extracellular signal-regulated kinase (ERK) is involved in autoimmune injury of the peripheral nervous system (PNS), the expression of phosphorylated ERK (p-ERK) was analyzed in experimental autoimmune neuritis (EAN) in rats. Western blot analysis showed that the level of p-ERK was increased significantly in the sciatic nerves of rats on days 14 (p<0.05) and 24 (p<0.01) post-immunization, compared with controls, and its reaction declined at day 30 post-immunization. Immunohistochemistry showed that p-ERK protein was weakly expressed in Schwann cells and vascular endothelial cells in the sciatic nerves of CFA-immunized control rats. In EAN-affected sciatic nerves, p-ERK immunoreactivity was found mainly in ED1-positive macrophages on days 14 and 24 post-immunization. Moreover, on days 24 and 30 post-immunization, p-ERK immunoreactivity increased gradually in the Schwann cells of rat sciatic nerves with EAN. Based on these results, we postulated that the phosphorylation of ERK has an important role in the differentiation and survival of cells, including inflammatory cells and Schwann cells, in the rat sciatic nerve in EAN. Specifically, the activation of ERK in the recovery phase of EAN paralysis seems to be related in the survival of Schwann cells.     

Nasal administration of recombinant rat IL-4 ameliorates ongoing experimental autoimmune neuritis and inhibits demyelination

Experimental autoimmune neuritis (EAN) is a CD4(+)T cell-mediated demyelinating disease of the peripheral nervous system (PNS) and serves as an experimental model for human immune-demyelinating neuropathies. In this study, we examined the effect of recombinant rat interleukin-4 (rrIL-4) on chronic EAN in Lewis rats induced by immunization with P0 peptide 180-199 and complete Freund's adjuvant (CFA). We estimated that nasal administration of rrIL-4, in dose ranges of 0.1-1 microg/rat/day in the initial phase of EAN, decreased the severity and the duration of clinical EAN. Hyporesponsiveness of T cells, downregulation of Th1 cell responses (INF-gamma), but increased levels of specific IgG1 isotypes document that nasal administration of rrIL-4 was systemically immune effective. Low grade inflammation and complete lack of regional demyelination within the sciatic nerves were seen in rrIL-4 treated rats. Based on these observations we suggest that nasal administration of IL-4 could be further evaluated, considering its possible use in human immune-demyelinating neuropathies.