大黄多糖对内毒素诱导急性前葡萄膜炎TLR4/NF-κB传导通路干预作用的实验研究

Experimental study of the effects of Rhubarb polysaccharides on TLR4/NF-κB signal transaction in LPS-induced acute anterior uveitis

目的通过观察大黄多糖对内毒素(LPS)诱导的急性前葡萄膜炎TLR4通路的影响,揭示大黄多糖对急性前葡萄膜炎TLR4通路的分子干预机制,为TLR4通路介导的炎症治疗提供新的思路和途径。设计实验研究。研究对象野生型Wistar大鼠、RAW264.7巨噬细胞系。方法野生型Wistar大鼠15只,随机分为3组,正常对照组、模型组(LPS组)、大黄多糖处理组,每组5只,大黄多糖处理组腹腔注射400 mg/kg大黄多糖,对照组和模型组注射等量PBS。各组腹腔注射2小时后模型组和大黄多糖处理组每只大鼠足底注射0.1 ml霍乱弧菌内毒素LPS,对照组注射等体积的PBS,24小时后裂隙灯下观察大鼠眼前节炎症反应并按炎症分级评分,RT-PCR观察大鼠虹膜TLR4表达的变化。RAW264.7巨噬细胞系进行体外培养,用大黄多糖、LPS分别刺激,通过Western blot、RT-PCR、ELISA、免疫荧光技术观察两者对巨噬细胞TLR4及相关分子的影响。主要指标大鼠眼前节炎症反应程度、巨噬细胞TLR4表达变化。结果裂隙灯下观察,LPS组相比于正常对照组虹膜充血严重,前房闪辉及瞳孔区纤维素渗出均较多;大黄多糖干预组大鼠仅轻度虹膜充血,瞳孔缘无明显渗出膜,瞳孔无缩小。RT-PCR示大黄多糖干预组TLR4 mRNA表达较LPS组明显降低。Western blot和RT-PCR结果显示大黄多糖在体外分别能引起巨噬细胞TLR4、髓样分化蛋白-88(MyD88)、核因子-κB(NF-κB)p65蛋白和mRNA表达量的增加;ELISA显示大黄多糖100μg/ml作用于Raw264.7细胞24小时后能引起上清液炎症因子IL-17,IL-10,TNF-α,INF-γ,IL-1β的表达增高;免疫荧光显示大黄多糖能引起培养细胞的激活、TLR4复合物及NF-κB p65表达。结论大黄多糖对内毒素诱导的大鼠急性前葡萄膜炎模型具有明显的抑制作用。体外实验显示大黄多糖同LPS一样,在体外具有激活巨噬细胞TLR4及其下游MyD88和NF-κB、调节细胞因子表达的作用。

Objective TLRg-mediated acute anterior uveitis is a serious ocular inflammatory disease. New targeted therapies are its most advanced treatments. Our objective was to study experimentally the molecular intervention mechanism of Rhubarb polysaccha- rides （RP） on endotoxin Lipopolysaccharides （LPS）-induced acute anterior uveitis TLR4 pathway and evaluate their interest as a new treatment. Design Experimental Study. Participants Wild-type Wistar rats and RAW264.7 macrophage cell line. Method （1） 15 wild-type Wistar rats were randomly divided into three groups：one normal control group, and two model groups： LPS group and RP treatment group （n=5）. RP group was treated with 400 mg/kg rhubarb polysaccharides by intraperitoneal injection, the control group and the model group were injected with equivalent PBS. Two hours later both LPS group and RP group was injected with 0.1 ml LPS, the control group was injected with a isovolumetric of PBS. After 24 hr we observed the rat anterior segment inflammation using a slit-lamp and giving an inflammation grading score. The TLR4 expression in the rats iris was analyzed by Real time （RT）-PCR. （2） RAW264.7 macrophage ceils were cultured in vitro. RP and LPS were used to stimulate cells. Then macrophages and their related molecules were evaluated by Western blot analysis, RT-PCR, ELISA and immunofluorescence techniques. Main Outcome Measures The degree of inflammation of the rat anterior segment, and TLR4 expression changes on macrophages. Results （1） Under slit-lamp observation of rats we found in the LPS group, severe iris hyperemia, anterior chamber flare, pupil fibrin exudate, while in the RP intervention group showed only mild iris hyperemia, no obvious pupil exudative membrane and no pupil to shrink. Using RT-PCR, the TLR4 mRNA expression in RP intervention group was significantly lower as compared with the LPS group. （2） Western blot and RT-PCR results showed that RP in vitro could induce an increase expression of TLR4, myeloid differentiation protein-88 （MyD88）, nuclear factor-KB （NF-KB） p65 protein and mRNA by macrophages. ELISA showed that RP can increase supernatant inflammatory cytokines IL-17, IL-10, TNF-α, IFN-γ IL-1β expression by Raw264.7 cells. Immunofluorescence showed that RP could cause activation of cultured cells of TLR4 complex and increased NF-κB p65 expression. Conclusion RP significantly inhibited the activation of TLR4 pathway in endotoxin-induced acute anterior uveitis rats model. In vitro study, we found that RP have the same role as LPS on the activation of TLR4 and its downstream MyD88 and NF-κB and can also regulate the expression of cytokine.