红芪多糖干预内毒素诱导的葡萄膜炎模型中糖原合成酶3-β的表达及其作用机制

Expression of glycogen synthase kinase 3-β in radix hedysari polysaccharide pre-treated lipopolysaccharide-induced uveitis in rats

目的通过观察红芪多糖(radix hedysari polysaccharide,HPS)和氯化锂(Li Cl)对霍乱弧菌内毒素(lipopolysaccharide,LPS)诱导的葡萄膜炎的抗炎作用,探讨糖原合成酶3-β(glycogen synthase kinase 3-β,GSK3-β)在葡萄膜炎中的作用机制。方法200只Wistar大鼠随机分为4组(n=50):空白对照组(negative control,NC)组、LPS诱导的葡萄膜炎组(LPS组)、HPS治疗组(LPS+HPS组)和Li Cl治疗组(LPS+Li Cl组)。LPS+HPS组腹腔注射400 mg·kg^(-1)HPS,LPS+Li Cl组腹腔注射0. 5 mol·L^(-1)的Li Cl 100μL,对照组和LPS组注射等量PBS。2 h后,LPS组、LPS+HPS组和LPS+Li Cl组每只大鼠足底注射0. 1 mL LPS注射液,NC组注射等体积的PBS。应用临床评分、裂隙灯照相、HE染色等检查评价炎性反应程度; Western blot和RT-PCR检测虹膜睫状体GSK3-β和核因子-κB(neuclear factor-κB,NF-κB) p65表达水平;酶联免疫吸附试验(enzyme-linked immunosorbent as-say,ELISA)检测大鼠前房房水中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)、白细胞介素10(interleukin-10,IL-10)、白细胞介素1β(interleukin-1β,IL-1β)等细胞因子的水平。结果 LPS注射后6 h、12 h、24 h、48 hLPS组的炎症评分分别为(2. 3±0. 2)分、(3. 6±0. 7)分、(3. 9±0. 3)分、(3. 2±0. 4)分,显著高于其他三组(均为P <0. 05),而LPS+HPS组、HPS+Li Cl组和NC组之间差异均无统计学意义(均为P> 0. 05),HPS或Li Cl预处理对LPS诱导的大鼠葡萄膜炎产生了抗炎效果。经过HPS或Li Cl处理,LPS诱导的葡萄膜炎大鼠虹膜睫状体磷酸化GSK3-β水平上调,NF-κB p65表达明显被抑制,前房房水中抗炎因子IL-10水平上调,而TNF-α、IL-6、IL-1β等炎症细胞因子受到抑制。结论 HPS或Li Cl预处理可以抑制LPS诱导的大鼠葡萄膜炎炎性反应,这一抗炎作用与GSK3-β的抑制性磷酸化密切相关。

Objective To evaluate the effect of hedysari polysaccharide(HPS)and lithium chloride(LiCl,a selective glycogen synthase kinase 3-βinhibitor)in endotoxin-induced uveitis(EIU)and to explore this anti-inflammatory mechanism.Methods A total number of 200 Wistar rats were randomly divided into four groups:normal control(NC)group,lipopolysaccharide(LPS)-induced uveitis group(LPS group),HPS-treated group(LPS+HPS group)and LiCl-treated LPS(LPS+LiCl)group.Two hours before LPS injection,LPS+HPS group received intraperitoneal injection of 400 mg·kg-1 HPS and LPS+LiCl group received intraperitoneal injection of 100μL LiCl of 0.5 mol·L^-1.LPS group and NC group both was intraperitoneally injected with 100μL saline solution only.After 2 h,the three groups received intraperitoneally injection of 200μg of LPS in 100μL of sterile saline simultaneously.NC group was intraperitoneally injected with 100μL saline solution only.Clinical score,slit-lamp photography,hematoxylin and eosin staining were used to determine the degree of inflammatory reaction.The protein levels of GSK3-βand nuclear factor-kappa B(NF-κB)in iris-cilliary body were examined by Western blot.The mRNA expression of GSK3-βand NF-κB was examined by real-time PCR(RT-PCR).Tumor necrosis factor-α(TNF-α),interleukin-10(IL-10),interleukin-6(IL-6)and interleukin-1β(IL-1β)in aqueous humor were detected by enzyme-linked immunosorbent assay(ELISA).Results The inflammatory scores of LPS group at 6 h,12 h,24 h and 48 h after LPS injection were 2.3±0.2,3.6±0.7,3.9±0.3 and 3.2±0.4,respectively,which were significantly higher than those in the other three groups(all P<0.05).However,there was no significant difference in LPS+LiCl,LPS+HPS and NC groups(all P >0.05).Pretreatment with HPS or LiCl both produced an anti-inflammatory effect during endotoxin-induced uveitis.With HPS or LiCl treatment,the level of P-GSK3-βin irisciliary body was upregulated and the expression of NF-κB p65 was significantly suppressed.Also,HPS or LiCl treatment suppressed the production of pro-inflammatory cytokine TNF-α,IL-1βand IL-6,while enhanced the production of anti-inflammatory cytokine IL-10 in the aqueous humor endotoxin-induced uveitis.Conclusion HPS or LiCl pretreatment can suppress intraocular inflammatory responses in rats with endotoxin-induced uveitis.Mechanistically,this anti-inflammatory effect may be related to the inhibitory phosphorylation of GSK3-β.