鸡矢藤提取物抗尿酸钠晶体诱导急性痛风性关节炎的作用及机制研究
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摘要
痛风性关节炎(Gouty arthritis, GA)是嘌呤代谢障碍引起的血尿酸浓度过高导致尿酸盐结晶沉积于关节所致的一种代谢性关节疾病。本课题采用的鸡矢藤提取物(Extracts from Paederia Scandens, EPS)是从茜草科鸡矢藤属植物鸡矢藤中提取的一类有效成分,其主要活性成份为鸡矢藤苷、鸡矢藤次苷、车叶草苷、r-谷甾醇及挥发油等。本室前期研究表明EPS具有抗炎作用。本课题主要研究了EPS对实验性GA模型的影响。主要实验结果如下:
1 EPS对MSU晶体诱导的大鼠急性痛风性关节炎模型的影响
将大鼠行麻醉(10%水合氯醛0.03ml·kg-1, ip)后仰卧固定,向左后踝关节腔注入尿酸钠溶液50uL (20mg·mL-1),制备大鼠急性GA模型。观察大鼠全身状况;用足爪容积测量仪测定造模后不同时间踝关节肿胀度;造模24小时后进行行为学评分;光学显微镜观察关节病理变化;放射免疫分析法检测TNF-α和IL-1β的含量;逆转录聚合酶链反应法检测TNF-αmRNA和IL-1βmRNA的表达;免疫组化法检测NF-κBp65的表达。
结果显示,EPS (4500, 2250 mg·kg-1, ig, qd×9d)能明显降低GA大鼠的关节肿胀度,改善大鼠造模后的步态异常。EPS (4500, 2250 mg·kg-1, ig, qd×9d)可减轻GA大鼠踝关节的组织水肿、减少关节组织炎性细胞浸润,以及改善滑膜增生等病理改变。表明EPS在一定程度上可以改善大鼠GA的炎症状态,表现较好的抗GA效应。EPS (4500, 2250 mg·kg-1, ig, qd×9d)可显著降低GA大鼠滑膜组织TNF-α和IL-1β的水平、降低GA大鼠滑膜组织TNF-αmRNA和IL-1βmRNA的表达及抑制NF-κB向核内转移,表明EPS可有效保护炎性细胞因子介导的滑膜组织损伤,这可能是它发挥抗GA效应的重要机制。
2 EPS对MSU晶体诱导的家兔急性痛风性关节炎模型的影响
将家兔行麻醉(20%乌拉坦5ml·kg-1,iv)后仰卧固定,向左后膝关节腔注入尿酸钠溶液50uL(25mg·mL-1),制备家兔急性GA模型。测量造模后不同时间膝关节肿胀度;造模12小时后对关节灌洗液中白细胞进行计数;光学显微镜观察关节病理变化;紫外分光光度法检测PGE2的含量;免疫组化法检测COX-2的含量;逆转录聚合酶链反应法检测COX-2mRNA的表达。
结果显示,EPS (2300, 1150 mg·kg-1, ig, qd×7d)能明显降低GA家兔的关节肿胀度,并可显著减轻GA家兔滑膜组织的水肿、炎性细胞浸润,改善滑膜增生等病理改变。表明EPS在一定程度上可以改善家兔GA的炎症状态,表现较好的抗GA效应。EPS (2300, 1150 mg·kg-1, ig, qd×7d)可显著降低GA家兔滑膜组织PGE2的含量,降低GA家兔滑膜组织COX-2含量及抑制COX-2mRNA的表达。表明EPS可有效保护COX-2介导的滑膜组织的炎性损伤,这可能是它发挥抗GA效应的另一重要机制。
3结论
总之,本文研究结果显示:EPS对尿酸钠晶体诱导大鼠和家兔急性痛风性关节炎模型均具有明显的抗GA作用。EPS抗GA作用机制可能是:①降低滑膜组织炎性细胞因子TNF-α和IL-1β的含量,抑制滑膜组织TNF-αmRNA和IL-1βmRNA的表达,抑制核转录因子NF-κB向核内转移,终止炎性细胞因子的恶性循环。②抑制关节腔液内中性粒细胞的募集,降低关节滑膜组织PGE2含量,抑制滑膜细胞COX-2酶的活性以及抑制滑膜细胞COX-2mRNA的表达,从而抑制关节炎的发生、发展。
Acute gouty arthritis (GA) is thought to be an inflammatory response to microcrystals of monosodium urate monohydrate (MSU) that precipitate in joint tissues from supersaturated body fluids or are shed from preexisting articular deposits. Extracts from Paederia scandens (LOUR.) MERRILL (Rubiaceae) (EPS) are paederoside, asperuloside, paederosidic acid, deacety lasperuloside, scandoside, asperulosidic acid and so on. Our former studies indicated that EPS have protective effects against inflammation. Effects of EPS on GA model were investigated. The main results are as following:
1 Effect of EPS on GA rat model
We studied the effects of EPS on acute gouty arthritis induced by monosodium urate crystal in rats. Monosodium urate monohydrate (MSU) crystals were administered into rat ankle joints. Ankle joint volume of rats was measured by volume meter; the level of TNF-αand IL-1βwas determined by radioimmunoassay; mRNA expression of TNF-αand IL-1βof synovial tissue in GA rats was analyzed by RT-PCR, and expression of NF-κB by immunohistochemistry was detected.
The results showed that EPS (4500, 2250 mg·kg-1, ig, qd×9d) could significantly inhibit joint swelling of rats, improve the gaits and the histopathologic changes, could inhibited the increase of TNF-αand IL-1βlevel; EPS possessed antigout arthritis effects; its mechanism may be related to inhibit the product of IL-1βand TNF-α; At the same time, 4500, 2250 mg·kg-1 of EPS inhibited mRNA expression of TNF-αand IL-1βof synovial tissue and decreased the biologically activity of NF-κB. The results suggested that EPS possesses antiinflammatory effects by modulating pro-inflammatory mediators’production from synovial tissue and inactivate NF-κB pathway transmembrane signal transduction which plays a crucial role in pathogenesis of this disease.
2 Effect of EPS on GA rabbit model
We studied the effects of EPS on acute gouty arthritis induced by monosodium urate in rabbits. Monosodium urate monohydrate (MSU) crystals were administered into rabbits’joints. Increases in joint size, and leukocyte infiltration to synovial fluids were analyzed. PGE2 activity was measured after increasing periods of time; expression of COX-2 by immunohistochemistry; COX-2 mRNA levels were monitored by RT-PCR.
The results showed that EPS (2300, 1150 mg·kg-1, ig, qd×7d) could significantly inhibit joint swelling of rabbits, improve the histopathologic changes, could inhibited the expression of COX-2 and COX-2mRNA in synovium of GA rabbits; These results suggested that EPS could inhibit PGE2 activity, COX-2 expression and PGE2 activity and down-regulate COX-2mRNA expression in synovial tissue from GA rabbits, which might be one of molecular mechanisms of its anti-inflammatory effects in GA rabbits.
3 Conclusions
In summary, our results demonstrated that EPS had anti-inflammatory action on animal model. The mechanisms of its effect on GA might be associated with its action on down-regulatation of MSU crystals-induced TNF-αand IL-1βproduction by inhibiting their expressions at the gene level. Furthermore, these inhibitions might be correlated with the blockage of NF-κB activation in synovial tissue, In addition, EPS was also down-regulate COX-2 and COX-2mRNA expressions.
1 EPS对MSU晶体诱导的大鼠急性痛风性关节炎模型的影响
将大鼠行麻醉(10%水合氯醛0.03ml·kg-1, ip)后仰卧固定,向左后踝关节腔注入尿酸钠溶液50uL (20mg·mL-1),制备大鼠急性GA模型。观察大鼠全身状况;用足爪容积测量仪测定造模后不同时间踝关节肿胀度;造模24小时后进行行为学评分;光学显微镜观察关节病理变化;放射免疫分析法检测TNF-α和IL-1β的含量;逆转录聚合酶链反应法检测TNF-αmRNA和IL-1βmRNA的表达;免疫组化法检测NF-κBp65的表达。
结果显示,EPS (4500, 2250 mg·kg-1, ig, qd×9d)能明显降低GA大鼠的关节肿胀度,改善大鼠造模后的步态异常。EPS (4500, 2250 mg·kg-1, ig, qd×9d)可减轻GA大鼠踝关节的组织水肿、减少关节组织炎性细胞浸润,以及改善滑膜增生等病理改变。表明EPS在一定程度上可以改善大鼠GA的炎症状态,表现较好的抗GA效应。EPS (4500, 2250 mg·kg-1, ig, qd×9d)可显著降低GA大鼠滑膜组织TNF-α和IL-1β的水平、降低GA大鼠滑膜组织TNF-αmRNA和IL-1βmRNA的表达及抑制NF-κB向核内转移,表明EPS可有效保护炎性细胞因子介导的滑膜组织损伤,这可能是它发挥抗GA效应的重要机制。
2 EPS对MSU晶体诱导的家兔急性痛风性关节炎模型的影响
将家兔行麻醉(20%乌拉坦5ml·kg-1,iv)后仰卧固定,向左后膝关节腔注入尿酸钠溶液50uL(25mg·mL-1),制备家兔急性GA模型。测量造模后不同时间膝关节肿胀度;造模12小时后对关节灌洗液中白细胞进行计数;光学显微镜观察关节病理变化;紫外分光光度法检测PGE2的含量;免疫组化法检测COX-2的含量;逆转录聚合酶链反应法检测COX-2mRNA的表达。
结果显示,EPS (2300, 1150 mg·kg-1, ig, qd×7d)能明显降低GA家兔的关节肿胀度,并可显著减轻GA家兔滑膜组织的水肿、炎性细胞浸润,改善滑膜增生等病理改变。表明EPS在一定程度上可以改善家兔GA的炎症状态,表现较好的抗GA效应。EPS (2300, 1150 mg·kg-1, ig, qd×7d)可显著降低GA家兔滑膜组织PGE2的含量,降低GA家兔滑膜组织COX-2含量及抑制COX-2mRNA的表达。表明EPS可有效保护COX-2介导的滑膜组织的炎性损伤,这可能是它发挥抗GA效应的另一重要机制。
3结论
总之,本文研究结果显示:EPS对尿酸钠晶体诱导大鼠和家兔急性痛风性关节炎模型均具有明显的抗GA作用。EPS抗GA作用机制可能是:①降低滑膜组织炎性细胞因子TNF-α和IL-1β的含量,抑制滑膜组织TNF-αmRNA和IL-1βmRNA的表达,抑制核转录因子NF-κB向核内转移,终止炎性细胞因子的恶性循环。②抑制关节腔液内中性粒细胞的募集,降低关节滑膜组织PGE2含量,抑制滑膜细胞COX-2酶的活性以及抑制滑膜细胞COX-2mRNA的表达,从而抑制关节炎的发生、发展。
Acute gouty arthritis (GA) is thought to be an inflammatory response to microcrystals of monosodium urate monohydrate (MSU) that precipitate in joint tissues from supersaturated body fluids or are shed from preexisting articular deposits. Extracts from Paederia scandens (LOUR.) MERRILL (Rubiaceae) (EPS) are paederoside, asperuloside, paederosidic acid, deacety lasperuloside, scandoside, asperulosidic acid and so on. Our former studies indicated that EPS have protective effects against inflammation. Effects of EPS on GA model were investigated. The main results are as following:
1 Effect of EPS on GA rat model
We studied the effects of EPS on acute gouty arthritis induced by monosodium urate crystal in rats. Monosodium urate monohydrate (MSU) crystals were administered into rat ankle joints. Ankle joint volume of rats was measured by volume meter; the level of TNF-αand IL-1βwas determined by radioimmunoassay; mRNA expression of TNF-αand IL-1βof synovial tissue in GA rats was analyzed by RT-PCR, and expression of NF-κB by immunohistochemistry was detected.
The results showed that EPS (4500, 2250 mg·kg-1, ig, qd×9d) could significantly inhibit joint swelling of rats, improve the gaits and the histopathologic changes, could inhibited the increase of TNF-αand IL-1βlevel; EPS possessed antigout arthritis effects; its mechanism may be related to inhibit the product of IL-1βand TNF-α; At the same time, 4500, 2250 mg·kg-1 of EPS inhibited mRNA expression of TNF-αand IL-1βof synovial tissue and decreased the biologically activity of NF-κB. The results suggested that EPS possesses antiinflammatory effects by modulating pro-inflammatory mediators’production from synovial tissue and inactivate NF-κB pathway transmembrane signal transduction which plays a crucial role in pathogenesis of this disease.
2 Effect of EPS on GA rabbit model
We studied the effects of EPS on acute gouty arthritis induced by monosodium urate in rabbits. Monosodium urate monohydrate (MSU) crystals were administered into rabbits’joints. Increases in joint size, and leukocyte infiltration to synovial fluids were analyzed. PGE2 activity was measured after increasing periods of time; expression of COX-2 by immunohistochemistry; COX-2 mRNA levels were monitored by RT-PCR.
The results showed that EPS (2300, 1150 mg·kg-1, ig, qd×7d) could significantly inhibit joint swelling of rabbits, improve the histopathologic changes, could inhibited the expression of COX-2 and COX-2mRNA in synovium of GA rabbits; These results suggested that EPS could inhibit PGE2 activity, COX-2 expression and PGE2 activity and down-regulate COX-2mRNA expression in synovial tissue from GA rabbits, which might be one of molecular mechanisms of its anti-inflammatory effects in GA rabbits.
3 Conclusions
In summary, our results demonstrated that EPS had anti-inflammatory action on animal model. The mechanisms of its effect on GA might be associated with its action on down-regulatation of MSU crystals-induced TNF-αand IL-1βproduction by inhibiting their expressions at the gene level. Furthermore, these inhibitions might be correlated with the blockage of NF-κB activation in synovial tissue, In addition, EPS was also down-regulate COX-2 and COX-2mRNA expressions.
引文
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