| Objective:A rat model of Diabetes Mellitus with liver necrosis was constructed by high fat and high sugar diet plus STZ induction(NAFLD、NASH、Liver Fibrosis),to investigate the mechanism of DMOC on diabetic liver necrosis and liver fibrosis.Methods:Healthy SD male rats aged 5 weeks(weighted about 180 ± 10g)were purchased by Taiwan Lusco Biotechnology Co.,Ltd.After one week of adaptive feeding,the rats were randomly divided into 4 groups:①Group Con(basic diet,n=10),②Group DMOC(basic diet +DMOC,n=10),③Group DMF(high fat and high glucose+STZ,n=10)and ④Group DMF+DMOC(high fat and high glucose+STZ+DMOC,n=10),①② as control group;③④as induction group.Rats in each group were fed with the corresponding feed and provided with reverse osmosis.After 4 weeks of feeding,the fats were injected intraperitoneally with STZ twice(25 mg/kg+m2,72h).On the fourth day.Instrument to measure fetal blood glucose after injection,screening fasting blood glucose(FBG>11.1mmol/L、HbAlc>7%)for the success of diabetic model rats.The results of the Group ③④ induction rats showed hyperglycemia,hyperlipidemia and abnormal liver function.After 36 weeks of feeding,the liver biopsy showed tendency of liver fibrosis significantly and the Group② ④ intervention was treated with DMOC at 36-44 weeks.Weighed once a week adjusted for weight according to body weight and fed for up to 44 weeks.The health status,body weight,organ weight,serological test about the levels of FBG、TC、TG、AST、ALT、ROS、TNF-α、IL-6 and TGF-β1 were measured in each group.HE and Masson’s Trichrome stain were used to observe the liver necrosis and fibrosis.The changes of α-SMA、Fibrinogen、Ⅳ=Collagen in liver tissue were detected by IHC method.The protein expression of Smad 2、3、pSmad 2、3、α-SMA、MMP1、2、9 were detected by WB assay.Results:1 DMOC intervention in Diabetes Mellitus with liver necrosis1.1 General observation of rats:Group Con rats generally good,normal eating,bright color shiny,increase body weight steadyly,stool was granular.Group induction rats were varying degrees of eating reduction,apathetic,lethargy,dull skin,weight gain,loose stools.1.2 Changes of body weight in rats:Before DMOC was induced,the body weight of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,the body weight of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.05).The results show that DM will increase weight,while DMOC has improved weight gain changes.1.3 Changes of liver tissue weight in rats:Before DMOC was induced,the liver tissue weight of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.05);After induction,the liver tissue weight of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results show that DM will make liver tissue weight enlargement and DMOC has improved liver tissue weight changes.1.4 Changes of abdominal fat weight in rats:Before DMOC was induced,the abdom,inal fat weight of the Group DMF was significantly higher than that of the Group Con,thedifference was statistically significant(P<0.01);After induction,abdominal fat weight of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.05).The results show that DM will make liver tissue weight enlargement and DMOC has improved abdominal fat weight changes.1.5 Changes of fetal blood glucose in rats:Before DMOC was induced,the fetal blood glucose of Group DMF were significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,the fetal blood glucose of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results showed that fetal blood glucose(FBG>11.1mmol/L)for diabetic rats and DMOC can effectively regulate the fetal blood glucose function of diabetic rats.1.6 Changes of blood lipid in rats:Before DMOC was induced,total cholesterol and triglyceride of Group DMF were significantly higher than that of the Group Con,the difference was statistically significant(all P<0.01);After induction,total cholesterol and triglyceride of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.05、P<0.01).The results show that DM duraction increases and blood lipids have increased trend and DMOC increases the delay of total cholesterol and lowering the triglyceride of Diabetic rats.1.7 Changes of liver function in rats:Before DMOC was induced,AST and ALT of Group DMF were significantly higher than that of the Group Con,the difference was statistically significant(all P<0.01);After induction,AST of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.05)and ALT decreased by 20%,the difference was not statistically significant(P>0.05).The results show that DM can cause liver necrosis and DMOC can slow damage.1.8 HE stain of liver tissue in rats:DMOC can significantly reduce the foam-like changes,cell swelling,unclear boundaries,hepatic sinus stenosis,central vein around the portal area with a small amount of lymphocyte infiltration.2 DMOC intervention in Diabetes Mellitus with liver fibrosis2.1 Changes of serum free radical ROS in Rats:Before DMOC was induced,the free radical ROS of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,free radical ROS of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results show that DM will infiltrate inflammatory cells in liver tissue and DMOC can improve free radical ROS changes after intervention.2.2 Changes of serum cytokines TNF-α in rats:Before DMOC was induced,the cytokines TNF-α of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,cytokines TNF-α of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results show that DM will infiltrate inflammatory cells in liver tissue and DMOC can improve cytokines TNF-α changes after intervention.2.3 Changes of serum inflammatory cytokines IL-6 in rats:Before DMOC was induced,the inflammatory cytokines IL-6 of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,cytokines IL-6 of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results show that DM will infiltrate cells and DMOC can improve cytokines IL-6 changes after intervention.2.4 Changes of serum fibrosis factor TGF-β1 in rats:Before DMOC was induced,the fibrosis factor TGF-β1 of the Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(P<0.01);After induction,fibrosis factor TGF-β1 of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(P<0.01).The results show that DM will infiltrate inflammatory cells and DMOC can improve fibrosis factor TGF-β1 changes after intervention.2.5 Changes of Masson’s trichrome stain in liver tissue:DM can cause liver fibrosis then DMOC can improve inflammatory cell infiltration and collagen hyperplasia.2.6 Changes of α-SMA、Fibrinogen、Ⅳ-Collagen was observed by IHC in liver tissue:DM can cause liver fibrosis and DMOC inhibit α-SMA、Fibrinogen、Ⅳ-Collagen activation has a significant inhibitory effect for liver fibrosis.2.7 Changes of relative density of pSmad 2/Smad 2、pSmad 3/Smad 3、α-SMA/β-actin in rates liver tissue:Before DMOC was induced,the relative density of pSmad 2/Smad2、pSmad3/Smad3、a-SMA/β-actin in Group DMF was significantly higher than that of the Group Con,the difference was statistically significant(All P<0.01);After induction,the relative density of pSmad2/Smad2、pSmad3/Smad3、α-SMA/β-actin of Group DMF+DMOC was significantly lower than that of Group DMF,the difference was statistically significant(All P<0.01).The results showed that DM can cause liver fibrosis,although Smad 2、Smad 3 were expressed but no significant difference in each group and DMOC significantly inhibited the protein expression of pSmad 2,pSmad 3 and a-SMA.2.8 Changes of relative density of MMP 1、MMP 2、MMP 9 in rates liver tissue:Before the drug was induced,the relative density of MMP1/β-actin、MMP2/β-actin、MMP9/β-actin in Group DMF was significantly lower than that of the Group Con,the difference was statistically significant(All P<0.01);After induction,the relative density of MMP1/β-actin、MMP2/β-actin、MMP9/β-actin of Group DMF+DMOC was significantly higher than that of Group DMF,the difference was statistically significant(All P<0.01).The results show that DM can cause liver fibrosis and DMOC can increase the protein expression of MMP 1、MMP2、MMP 9 and promote the decomposition of collagen.Conclusion:1 High fat and high glucose diet + STZ model of diabetic rates with liver necrosis can damage the pancreas leading to the secretion of insulin damage and finally induced hyperglycemia and observed significant liver fibrosis continuely.This model is associated with "Western dietary" habits caused by T2DM with liver necrosis and fibrosis.2 DMOC improves the Body weight、Liver weight、Abdominal adipose weight、FBG、TC、TG、AST、ALT、ROS、TNF-α、IL-6、TGFβ1dysfuction in diabetic rats with liver necrosis and fibrosis.3 DMOC can improve diabetic rats with liver necrosis and fibrosis,cell swelling,unclear boundaries,hepatic sinus stenosis,a small amount of lymphocyte infiltration in the central venous infusion area,reduction of α-SMA、Fibrinogen、Ⅳ-Collagen and collagen deposition.The mechanism may be related to inhibit cytokines and fibrosis signaling pathways of ROS→TNF-a、IL-6 and TGF-β1→Smads →α-SMA in diabetic rats and promote the protein expression of MMP 1、MMP 2、MMP 9 to increase degradation of collagen. |
PDF Full Text Request