| Diabetic liver disease refers to liver abnormity of histology and function.It also belongs to one kind of diabetic chronic complications induced by diabetes mellitus(DM).The hepatic pathology in diabetes includes:(1)liver glycogen accumulation(2)appearance of lipid droplets(3)hepatic cirrhosis: collagen fiber emerges in early stage of liver adipose degeneration(4)non-specific cellular denaturation(5)changes like microangiopathy, adipose granuloma and so on.Professor Brownlee claims that oxidative stress is the main mechanism of DM.Reactive oxygen species(ROS) caused by high blood glucose breaks out and contributes to microangiopathy.Nrf2-ARE pathway,as one of the most elements against oxidative stress, regulates specific antioxidative proteins and therefore prevents lipid deposition and strengths liver function.Nrf2 binds to ARE and regulates antioxidant enzymes and phase II enzymes(such as heme oxygenase-1, catalase, thioredoxin reductase and so on) to resist oxidative stress.However no reports of this pathway are found in diabetic liver injury.Dl-3-n-butylphthalide(NBP) is broadly used on cerebrovascular disorders,which improves oxidative stress induced by Ischemia reperfusion in brain tissues.It helps to decrease the release of glutamicacid, the contentration of calcium and ROS and increase the activity of anti-oxidative proteins.There are no studies of NBP in diabetic liver injury have been reported.Objective:The study intervened rats with diabetic liver injury by oral administration of NBP.It emphasized on:① the potential relation between diabetic liver injury and Nrf2-ARE pathway②the theraputic mechanism of NBP on oxidative stress,which provided the basis on prevention and treatment of diabetic liver injury.Methods: 60 male Sprague-Dawley rats were randomly separated into 3 groups:control group,diabetes mellitus group(model group) and NBP group.All rats except control group were intraperitoneally injected with 1%STZ after feeding 1 week for adjustion,while control group was administrated with rather dosage of citrate buffer. Blood samples were collected from the tail vein after 72 hours. Fasting blood glucose was calculated by using one touch glucometer and blood glucose(BG) ≥16.7mmol/L was taken as the standard model.The control rats were treated with drink water for 12 weeks.At the same time,the model rats were feed with corn oil and the NBP rats were treated with NBP(80mg/kg).Then the animals were sacrificed and serum samples were tested for GLU、TG、TC、HDL、LDL、MDA、T-SOD. Also, the liver of each animal was excised and fixed in 10% formalin solution, and then subjected to PAS staining to observe the histopathological changes under a light microscopy. The expressions of FN in liver tissues were analyzed by immunohistochemical staining. Part of liver tissues were washed in PBS buffer and reserved under-800 C for detection of MDA, SOD. The expressions of Nrf2, HO-1,Catalase in rat liver tissues were examined by Western Blot.Results:1 Effects of high blood glucose on rats.There were both a few deaths in model group and NBP group.10 rats were found dead in model group and 8 in NBP group. The main reason for death was multiple organ dysfunction and ketoacidosis caused by high glucose. Body weight in control group increased progressively. Normal drinking, urine volume, and excrements were observed in control group. After being injected with STZ, rats in other groups showed decreased body weight, fatigue, poor reaction and diarrhea.2 Histopathological examination.The gastric mucosa in animals pretreated with HPTP or omeprazole(group 3–4) displayed increased PAS staining intensity compared to the rats in group 2, indicating an increase in the glycoprotein content of gastric mucosa in pretreated rats3 Effects of NBP on the serum ALT, AST, GLU, TC, TG, LDL and HDL activities of diabetic rats.The serum level of ALT ALT, AST, GLU, TC, TG, LDL: model group and NBP group both had a significantly high level compared with control group(P<0.05);NBP group had a decreased level compared with model group(P<0.05).The serum level of HDL: HDL decreased in model and NBP group in comparison with control group(P<0.05).NBP had a higher level in comparison with model group(P<0.05).Results above indicated that NBP decreased serum level of ALT, AST,GLU, TC, TG, LDL and increased intensity of HDL which might be potential mechanism of treatment.4 Effects of NBP on MDA and T-SOD in serum and liver tissues.Values of MDA in serum and liver tissues of the rats: The MDA content increased after treating with STZ(P<0.05).NBP group increased in serum and liver compared with model group(P < 0.05).Injection of STZ had a significant effect on T-SOD activity in serum and tissue of rats(P< 0.05). Specifically, model group and NBP group differed significantly and increased in serum when compared with control group, while T-SOD activity increased in comparison with model group(P < 0.05).Results above showed that NBP could reduce the MDA content and increased T-SOD activity and thus relieved oxidative stress of diabetic liver injury rats.5 Effects of NBP on Nrf2, HO-1, Catalase, Fn in liver tissues.① Immunohistochemistry:Nrf2 expressed mainly in hepatocyte nuclear.The content of nuclear nrf2 increased in model and NBP group in comparison with control group, especially in NBP group(P<0.05).The expression of HO-1 and Catalase were detected in three groups, more especially in NBP group(P<0.05). Fn was expressed in portal area and central vein area. NBP group showed a decreasing level of Fn in comparison with model group(P<0.05).②Western Blot: The expression of nuclear Nrf2 increased in model and NBP group compared with control group(P<0.05).The expression of Nrf2 increased in NBP group in comparison with model group(P<0.05).The levels of anti-oxidant proteins such as HO-1 and Catalase increased in both model and NBP group, especially in NBP group(P<0.05).Results above implied that NBP regulated Nrf2 and its related anti-oxidant proteins and delayed the development of diabetic liver injury.Conclusions:1 Oxidative stress and disorder of lipid metabolism was severely asso- ciated to type 1 diabetes.2 Nrf2 and its downstream anti-oxidant proteins such as HO-1 and Catalase involved in anti-oxidant mechanism of diabetic liver injury.3 NBP specifically activated the expression of Nrf2, HO-1 and Catalase, improved disorder of glucose and lipid metabolism and alleviated diabetic liver injury. |
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