Experimental study of the effects of Rhubarb polysaccharides on TLR4/ NF-κB signal transaction in LPS-induced acute anterior uveitis
ZHANG Xiao-Long, Wang-Jing, Xu-Zhuo-Zai, Li-Zhong-Qiu, Lu-Hong-
2013, 22(2):
134-140.
Asbtract
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1792 )
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Objective TLR4-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 polysaccharides (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 cells 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 immuno-fluorescence 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-κB(NF-κB) 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.(Ophthalmol CHN, 2013, 22: 134-140)