消化酶在口服Ⅱ型胶原蛋白治疗类风湿性关节炎中的作用

Effects of the digestive enzymes on treating rheumatoid arthritis by oral type Ⅱ collagen

目的:观察消化酶在体内和体外对天然Ⅱ型胶原蛋白(native typeⅡcollagen,N-CⅡ)和变性Ⅱ型胶原蛋白(denatured typeⅡcollagen,D-CⅡ)的酶解作用,探讨口服Ⅱ型胶原蛋白(typeⅡcollagen,CⅡ)治疗类风湿性关节炎的机制。方法:在体外将胃蛋白酶、胰蛋白酶和α-糜蛋白酶分别作用于N-CⅡ和D-CⅡ,采用SDS-PAGE电泳和免疫印迹法分析酶解产物。将N-CⅡ和D-CⅡ注入鼠胃内和十二指肠,把不同时间收集的胃和肠内容物进行SDS-PAGE电泳分析。结果:N-CⅡ分子和亚基都能与兔抗鸡CⅡ抗体反应;D-CⅡ可被分解成十多条免疫印迹反应呈阳性的蛋白片段。CⅡ在胃、肠道能处于溶解状态。在体外,N-CⅡ在胃蛋白酶和α-糜蛋白酶作用下电泳和免疫印迹图谱无变化,而被胰蛋白酶作用后出现10条免疫印迹为阳性的条带;D-CⅡ易被上述消化酶分解。N-CⅡ不被胃内环境水解,可被肠道消化酶缓慢降解;D-CⅡ可被胃、肠道消化酶迅速分解。结论:N-CⅡ可被胰蛋白酶水解产生多条活性片段,但对胃内环境、胃蛋白酶和α-糜蛋白酶具有抵抗力;D-CⅡ易被胃、肠道消化酶水解。结果提示口服CⅡ诱导免疫耐受、治疗类风湿性关节炎可能与CⅡ特性和胰蛋白酶对它的作用有关。

Objective： To observe the enzymolysis of digestive enzymes on the native type Ⅱ collagen（N-C Ⅱ） and the denatured type Ⅱcollagen（D-C Ⅱ） in vitro and in vivo in order to study the mechanism of treating rheumatoid arthritis（RA） by oral administration of type Ⅱ collagen（CⅡ）.Methods： N-C Ⅱ and D-C Ⅱ were respectively treated with pepsin,trypsin and α-chymotrypsin in vitro and the products were analyzed by SDS-PAGE and Western blotting.N-C Ⅱ and D-C Ⅱ were infused to the murine stomach and duodenum.The gastric contents and intestinal contents were collected at different time and analyzed by SDS-PAGE.Results： N-C Ⅱ and its subunits could all react with the rabbit anti-C Ⅱ antibody.D-C Ⅱwas pyrolyzed to more than 10 fragments showed positive in immunoblotting.CⅡcould be with the lyso-state in the gastrointestinal tract.After the pepsin and α-chymotrypsin treatments,the N-C Ⅱ SDS-PAGE and immunoblotting graphs had no obvious change,but for D-C Ⅱ the positive bands in immunoblotting vanished.After the trypsin treatment,10 positive bands in immunoblotting could be seen for N-C Ⅱ and no positive bands for D-C Ⅱ.N-C Ⅱcould not be hydrolyzed in gastric environment and could be slowly degraded in intestinal tract.D-C Ⅱcould be easily hydrolyzed by the digestive enzymes in the gastrointestinal tract.Conclusion： N-C Ⅱcan be produced active fragments by the trypsin treatment and had resistance to gastric environment,pepsin and α-chymotrypsin.D-C Ⅱcan be easily hydrolyzed by the digestive enzymes in the gastrointestinal tract.The results suggest that the inducing immunotolerance to treat RA by oral administration of CⅡ is possibly concerned with the CⅡ characteristics and trypsin enzymolysis.