The Study On The Mechanism Of Oxidative Damage Induced By Soybean Trypsin Inhibitor In Pancreatic Mitochondria Of Mice

Soybean contains a variety of anti-nutritional ingredients which have aversive effects on human and animal digestion, absorption and nutrient utilization. Among them, soybean trypsin inhibitors(STI) is most significant and it can greatly increase free radical level of pancreas, keeping body in oxidative stress state. And it is at Mitochondria that energy conversion takes place, which must be affected by the overdose of oxygen free radicals, however, there has no available reference related to this area so far. Therefore, with pancreatic mitochondria as target, this assay studied the effects of STI on its oxidation and antioxidant level as well as the resulting change of mitochondrial structure and function. The experimental results will provide theoretical basis for further discussion on oxidative damage mechanism of STI to pancreas. 1. The effect of soybean trypsin inhibitor on the balance between oxidant and antioxidant of pancreatic mitochondria in miceFor this purpose, 60 mice were randomly divided into three groups as follows: control group was fed on control diet, STI group was fed on control diet containing 4.8mg/g STI, VC group was fed on STI diet supplemented with 1500mg/kg vitamin C. After feeding three weeks to slaughter and extract pancreatic mitochondria were used to detect the changes of Malondialdehyde(MDA), etc. levels in pancreas mitochondrial. Using fluorescence quantitative PCR technique, the changes of SOD and GSH-Px gene mRNA expression level in pancreatic tissue were detected. The results showed that compared with control group, soybean trypsin inhibitor could significantly increase the MDA content and nitric oxide synthase(NOS) activity in pancreas mitochondria of mice, increased by 38.65% and 94.00%. At the same time, the levels of SOD, GSH-Px, CAT, T-AOC and GSH were significantly reduced(P<0.05) by 31.30%, 58.5%, 28.57%, 82.96%, 45.00%. And after adding antioxidants it was presented the opposite trend. Fluorescence quantitative PCR detection results showed that compared with control group, the levels of SOD and GSH-Px mRNA expression in STI group were significantly decreased(P < 0.05), after adding antioxidants the mRNA expression level was significantly increased(P<0.05). 2. The effect of STI on structure and function in pancreatic mitochondriaFor this purpose, selected 120 male mice, experimental design above. And then the structure and function indexes(Na+, K+ ATPase, etc.) of mitochondria were detected; The apoptosis index(cytochrome c, caspase 3, ect.) mRNA expression levels and protein level were analyzed. The results showed that compared with control group, the Ca2+ concent in STI group was significantly increased in pancreas mitochondria of mice, Na+, K+ ATPase and other functional enzyme activities were significantly decreased(P<0.05) by 75.51%, 81.48%, 38.36%. After adding antioxidants, Ca2+ concent was significantly decreased(P<0.05), and the functional enzyme activities were significant increased(P<0.05), but still lower than the control group. Light microscope and electron microscope results showed that compared with control group, the irreversible damage in STI group was observed, such as mitochondria swelling cavity, the nucleus dissolved and so on, after adding antioxidants, the structure damage of pancreas were slightly changed. Western blot results showed that the cyt c and Caspase 3 protein expression levels in STI group were significantly higher than those of control group and STI+VC group, and the difference had a statistical significant(P<0.05). Mitochondrial depolarization of pancreatic cells in STI group was obviously higher than that of control group(P<0.05), after adding antioxidants, it was significantly decreased(P<0.05), but still higher than the control group, which suggested STI could result in decrease of mitochondria membrane potential. RT-PCR detection results showed that compared with control group, Na+, K+ ATPase, Ca2+ ATPase and ATPase mRNA expression levels in STI group were significantly decreased(P<0.05), after adding antioxidants, the mRNA expression level was significantly increased(P<0.05).All above results showed that STI induced oxidative stress in the pancreatic mitochondria, and further caused the oxidative damage of mitochondria. In addition, vitamin C may interfere with the effect of STI on organisms, and effectively improve the oxidative stress state of pancreas mitochondrial.