乌梢蛇Ⅱ型胶原蛋白对大鼠佐剂型关节炎滑膜细胞因子的作用

The Effect of the Type Ⅱ Collagen Protein from Zaocys on Cytokines Production by Synoviocytes in Rats with Adjuvant Arthritis

目的:观察乌梢蛇Ⅱ型胶原蛋白(tyPeⅡcollagen,CⅡ)对大鼠佐剂型关节炎(adjuvant arthritis,AA)滑膜细胞因子的作用。方法:限制性胃蛋白酶降解法提取乌梢蛇CⅡ,SDS-PAGE凝胶电泳法和紫外分光光度计法鉴定;采用完全弗氏佐剂(complete Freud′s adjuvant,CFA)足垫皮下注射诱导AA大鼠模型;收集滑膜细胞上清液,L929细胞毒性法检测滑膜细胞上清液中TNF-α生物活性,MTT法检测滑膜细胞上清液中IL-1β生物活性;ELISA法检测滑膜细胞上清液细胞因子水平。结果:提取的CⅡ分子量约为118kD^120 kD,紫外光谱吸收仪测定提取的CⅡ吸收峰为230nm,均与标准的牛鼻中隔CⅡ相符;乌梢蛇CⅡ各剂量组体外对滑膜细胞上清液TNF-α和IL-1β活性没有直接作用;乌梢蛇CⅡ中剂量组可抑制AA大鼠的滑膜细胞和派氏淋巴结共培体系上清液TNF-α和IL-1β活性(P<0.01),乌梢蛇CⅡ低、高剂量可抑制AA大鼠的滑膜细胞和派氏淋巴结(PP)共培体系上清液IL-1β活性(P<0.05);灌服乌梢蛇CⅡ低、中剂量组与模型组比较,TNF-α活性下降(P<0.05);中剂量组与模型组比较,IL-1β活性下降(P<0.05);模型组与空白对照组比较,滑膜上清液TNF-α和IL-1β水平显著升高(P<0.01);乌梢蛇CⅡ中、高剂量组与模型组比较,均能抑制滑膜上清液TNF-α和IL-1β水平(P<0.01);乌梢蛇CⅡ低剂量组能显著抑制滑膜上清液IL-1β水平(P<0.01);乌梢蛇CⅡ中剂量能显著升高滑膜上清液TGF-β(P<0.01);结论:乌梢蛇CⅡ体外对滑膜细胞炎症因子无直接作用,灌服乌梢蛇CⅡ可以抑制滑膜细胞炎症因子的水平和活性。

Objective ： To investigate the effect of the type Ⅱ collagen （ CⅡ ） protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis（AA）. Methods ：Type Ⅱ collagen protein was abstracted and purificated from Zaocys. Adjuvant arthritis （AA） was induced by a single intradermal injection of 0. 1 mL of complete Freund＇s adjuvant into the left hind paw. Synovioeytes＇ supernatants were harvested and synoviocytes-Peyer＇s Patches （PP） cells coculture system were developed. Tumor necrosis factor-α（TNF-α） activity was measured by L929 cytotoxicity bioassay and Interleukin （IL）-1β activity was measured by MTT dye reduction. The synovioeytes＇ supernatants cytokines＇ levels were detected by ELISA. Results：Each concentration of C Ⅱ from Zaocys had no effect on IL-1β and TNF-α production by synoviocytes in vitro. Middle concentration of C Ⅱ suppressed the activity of IL-1β and TNF- α production by synoviocytes-PP cells coculture system （ P 〈 0. 05 ）. Treating with low and high dose of CⅡ suppressed the activity of TNF-α and IL-1β producing by synoviocyte （ P 〈 0. 05 ）, significantly suppressed in the group of AA rats treated with middle dose of CⅡ （P 〈 0. 01 ）. Treating with middle and high dose of CⅡ decreased the level of synovioeytes＇ supernatants TNF-α （ P 〈 0. 05 ） , the level of synoviocytes＇ supernatants IL-1β decreased in all treating groups （P 〈 0. 05 ）. Treating with middle dose of CⅡ increased the level of serum TGF-β（ P 〈 0. 05 ）. Middle concentration of CⅡ suppressed the activity of IL-1 and TNF production by synoviocytes-PP cells eocuhure system （ P 〈 0. 05 ）. Conclusions ： CⅡ from Zaocys has no direct effect on the activity of IL-1β and TNF production by synoviocytes in vitro. Oral administration of type Ⅱ collagen protein from Zaocys can effectively suppressed the activity and level of the cytokines production by synoviocytes in rats with adjuvant arthritis （AA）.