狭鳕鱼皮胶原蛋白肽经由水通道蛋白质3信号分子对过氧化氢诱导的HaCaT细胞氧化损伤的保护作用

摘要

目的：探讨狭鳕鱼皮胶原蛋白肽对H2O2诱导HaCaT细胞氧化损伤保护作用及分子作用机制。方法狭鳕鱼皮胶原蛋白肽50，100，200 mg·L-1组预处理12 h后，加入300μmol·L-1的H2O2培养12 h。采用酶生化法测定细胞上清液中过氧化氢酶（CAT），谷胱甘肽过氧化物酶（GSH-Px），总超氧化物歧化酶（T-SOD）和总抗氧化能力（T-AOC）的水平。Western蛋白印迹法检测细胞内水通道蛋白质3（AQP3）表达水平。结果与正常组相比，狭鳕鱼皮胶原蛋白肽浓度小于300 mg·L-1时无细胞毒性。H2O2300μmol·L-1组的细胞存活率降低到54.0%（P<0.01）。预先给狭鳕鱼皮胶原蛋白肽50，100，200 mg·L-1组处理12 h使得细胞存活率相应增加到69.4%，79.4%和89.1%（P<0.05，P<0.01）。与模型组相比，狭鳕鱼皮胶原蛋白肽50，100，200 mg·L-1组的CAT，GSH-Px，T-SOD和T-AOC含量提高，其中200 mg·L-1组的CAT，GSH-Px， T-SOD和T-AOC含量相应升高了56.90%，48.68%，48.24%和71.43%（P<0.01）。与模型组相比，狭鳕鱼皮胶原蛋白肽200 mg·L-1组AQP3表达量减少得最多，降低到67.1%（P<0.01）。结论狭鳕鱼皮胶原蛋白肽可保护H2O2诱导的氧化损伤，其机制可能与增强抗氧化能力和减少AQP3表达水平有关。%OBJECTIVE To investigate the protective effect of collagen peptides from walleye pollock skin(CPWPS)in the hydrogen peroxide(H2O2)-induced oxidative stress damage model of the human keratinocyte cell line (HaCaT). METHODS HaCaT cells were pretreated with CPWPS 50,100 and 200 mg · L-1 for 12 h. Then H2O2 was added to the final concentration of 300μmol · L-1. After 12 h,the activity of catalase(CAT),glutathion peroxidase(GSH-Px),total superoxide dismutase(T-SOD)and total antioxidant capacity (T- AOC) were detected by enzymes and biochemical method. The expression of aquaporin3 (AQP3) in HaCaT cells was measured by Western blotting. RESULTS Compared with control group,CPWPS group did not exhibit significant cytotoxicity at a concentration up to 300 mg·L-1. The viability of cells treated with H2O2 300μmol·L-1 for 12 h decreased to 54.0%(P<0.01). The viability of cells pretreated with CPWPS 50,100 and 200 mg · L-1 for 12 h increased to 69.4%,79.4%and 89.1%(P<0.05,P<0.01),respectively. Compared with the model group,the activity of CAT,GSH-Px,T-SOD and T-AOC in HaCaT cells pretreated with CPWPS 50,100 and 200 mg·L-1 for 12 h increased. Compared with model group,the activity of CAT,GSH-Px,T-SOD and T-AOC of CPWPS 200 mg · L-1 pretreatment group increased by 56.90%,48.68%,48.24% and 71.43%(P<0.01),respectively. Western blotting revealed that the expression of AQP3 in H2O2 model group was in⁃creased relative to that in control group,while the expression of AQP3 in CPWPS pretreatment groups was decreased. The expression of AQP3 in CPWPS 200 mg · L-1 pretreatment group decreased to 67.1%(P<0.01) compared to the H2O2 model group. CONCLUSION CPWPS can protect HaCaT cells against H2O2- induced oxidative stress damage. The cytoprotective effects are associated with enhanced antioxidant capacity and decreased expression of AQP3.