| BackgroundDiabetes mellitus(DM)is a chronic,metabolic disease characterized by elevated blood glucose,which is mainly caused by insufficient insulin secretion or insulin resistance(IR)in the body,and its prevalence showed a trend of increasing year by year,is threatening human health seriously.Type 2 Diabetes Mellitus(T2DM)is the most common form and accounts for around 90% of all DM cases worldwide.Prolonged hyperglycemia and IR of target organs during T2 DM causes pathological damage to multiple tissues and organs,including the liver.It has been established that the pathogenesis of Diabetic Liver Injury(DLI)is related to many factors including IR,oxidative stress,inflammation,and lipid metabolism disorder.Aldo-keto Reductase Family 1 Member B1(AKR1B1),also known as Aldose Reductase(AR),is the first key enzyme in the polyol pathway,one of the glucose metabolic pathways,which can regulate target genes to participate in vital life activities,including cell metabolism,oxidative stress,inflammation,and immune response,and is also associated with the development of multiple diabetic complications.Recently,AKR1B1 has been identified as a potential therapeutic target for non-alcoholic fatty liver disease,but little is known about its roles in the progression of DLI.There is currently no effective drug treatment for DLI in clinical practice,and oral hypoglycemic agents for T2 DM,as well as hepatoprotective medications were still the main treatment modality.Therefore,it is of great importance to elucidate the pathogenesis of DLI and identify novel therapeutic targets.Linarin(LN)is an active ingredient of Chrysanthemum,which has a wide range of pharmacological activities such as anti-inflammatory,antioxidant,antihypertensive,anticancer,anti-osteoporosis,and neuroprotective effects,among others.However,its role in DLI has previously not been investigated.Therefore,the effects of LN on DLI were evaluated by measuring inflammation and oxidative stress indicators both at the cellular level and with animals.Further transfection of AKR1B1 overexpression plasmid will be performed in vitro to explore this potential mechanism of LN efficacy.Objects and methodsThe first part of the experiment was designed to construct a rat model for T2 DM and to investigate the protective effects of LN on liver injury in T2 DM rats.After one week of adaptive feeding,male SD rats were randomly divided into normal control group(NC group)and T2 DM modeling group according to body weight and fed with and high-fat diet for five weeks except for the NC group.Then,a rat model of T2 DM was established via intraperitoneal injection of low-dose of streptozotocin(STZ;30 mg/kg).Venous blood was collected at 24,72 h,and 7 d after STZ injection in order to assess whether the T2 DM model has been successfully established(blood glucose level ≥ 16.7 mmol/L).The diabetic rats were randomly divided into T2 DM group,LN 15 mg/kg group,LN 30 mg/kg group and LN 60mg/kg group.The rats in the respective LN treatment groups were given by gavage with the corresponding dose of LN once a day,lasting four weeks,the NC group and T2 DM group were given the same dose of solvent via intragastric administration.The 24 h food intake,water intake,and urine output of rats were collected by metabolism cages once a week.Both the body weight and fasting blood glucose(FBG)of rats were measured weekly during the experimental period.Liver tissues were collected after rats were sacrificed at the end of the experiment and whole blood was collected from the apex of the heart;Serum levels of transaminase(ALT and AST),lipid parameters(TC,TG,HDL-C,and LDL-C)were measured by using chemistry reagent kits;Serum levels of insulin,inflammatory factors(TNF-α,IL-1β,and IL-6)and oxidative stress indicators(SOD,GSH-Px,and MDA)in the liver homogenates were measured with the corresponding test kits;Liver tissues were sectioned at 5 μm and then were examined by H&E staining and Oil red O staining respectively.The second part of the experiment was designed to elucidate that the effects of LN on inflammation and oxidative stress in hepatic stellate cells LX-2 induced by high glucose and palmitic acid.LX-2 cells were treated with 33.3 mmol/L glucose and 0.1 mmol/L palmitic acid for 24 h to establish the in vitro DLI model.The cell experiments were divided into three groups,including control group,model group and LN(20 μmol/L)intervention group;The levels of oxidative stress indicators(SOD,GSH-Px,and MDA)in the cells and inflammatory factors(TNF-α,IL-1β,and IL-6)in the cell supernatants were measured using ELISA kits;The levels of reactive oxygen species(ROS)and apoptosis of LX-2 cells in each group were measured by flow cytometry.The third part of the experiment was designed to elucidate that LN ameliorates DLI by alleviating inflammation and oxidative stress through inhibition of AKR1B1.The AKR1B1 protein expression of rat liver tissues in the different experimental groups were detected by Western blot.The m RNA expression level of AKR1B1 in LX-2 cells in each group was measured by real-time fluorescent quantitative PCR.Then,AKR1B1 overexpression plasmids were constructed and then transfected into LX-2 cells.This experiment was divided into five groups,including control group,model group,LN(20 μmol/L)intervention group,transfected with empty plasmid group(LN 20 μmol/L+oe-NC)and transfected with AKR1B1 overexpression plasmid group(LN 20 μmol/L+oe-AKR1B1).The levels of oxidative stress indicators(SOD,GSH-Px,and MDA)in the cells and inflammatory factors(TNF-α,IL-1β,and IL-6)in the cell supernatants were remeasured using ELISA kits after plasmid transfection.The levels of ROS and apoptosis of LX-2 cells in each group were retested by flow cytometry after transfection was completed.Results1.After 4 weeks LN intervention,compared with the NC group,the 24 h food intake,water intake,urine output and FBG of rats in the T2 DM group were significantly increased,whereas the body weight was markedly decreased;In addition,the serum levels of insulin in the T2 DM group was significantly decreased(P<0.01),HOMA-IR was significantly increased(P<0.01),the serum levels of TC,TG and LDL-C were significantly increased(P<0.01),and the serum levels of HDL-C was decreased significantly(P<0.01).Obvious pathological changes can be found in the liver tissue of T2 DM rats as shown in H&E and oil red O staining results,such as structural disorders of hepatic lobules,hepatocyte necrosis and lipid accumulation,and others.These results above indicated that the T2 DM rat model was established successfully through low-dose STZ injection combined with high-fat diet and showed significant injury in the liver;Compared with the T2 DM group,the 24 h water intake and urine output of rats in the LN group were decreased gradually.The FBG of rats in the LN60mg/kg group were markedly decreased(P<0.01)and the body weight of rats in the LN60mg/kg group were markedly increased(P<0.01),the serum levels of insulin of rats in the LN 60mg/kg group was significantly increased(P<0.05),HOMA-IR of rats in the LN60mg/kg group was significantly decreased(P<0.01),the serum levels of TC,TG,and LDL-C of rats in the LN 60mg/kg group were significantly decreased(P<0.01).There was no significant difference in HDL-C between the LN intervention groups and the T2 DM group(P>0.05).Moreover,LN treatment significantly improved liver pathological damage in T2 DM rats.After 4 weeks LN intervention,compared with the NC group,the liver index and the levels of hepatic TNF-α,IL-1β,and IL-6 were significantly increased in the rats of T2 DM group(P<0.01).The activities of hepatic SOD and GSH-Px were significantly decreased(P<0.01),but the levels of hepatic MDA were significantly increased(P<0.01)in the rats of T2 DM group.The serum levels of ALT and AST were significantly raised(P<0.01).These results suggest that the liver of T2 DM rats developed severe inflammation and oxidative stress;Compared with the T2 DM group,the liver index and the levels of hepatic TNF-α,IL-1β and IL-6 were significantly decreased in the rats of LN 30 mg/kg group and LN 60mg/kg group(P<0.01).The activities of hepatic SOD and GSH-Px were significantly increased(P<0.01),whereas the levels of hepatic MDA were significantly decreased(P<0.01)in the rats of LN 30 mg/kg group and LN 60 mg/kg group.The serum levels of ALT and AST of rats in the LN 60mg/kg group were significantly decreased(P<0.01).These results suggest that LN treatment ameliorates T2DM-associated liver injury by alleviating inflammation and oxidative stress.2.Compared with the control group,the levels of TNF-α,IL-1β,and IL-6 in the LX-2cell supernatants of model group were significantly increased by 33.3 mmol/L glucose and 0.1mmol/L palmitic acid treatment(P<0.01);Compared with the model group,the levels of TNF-α,IL-1β,and IL-6 in the cell supernatants of LN intervention group were significantly decreased(P<0.01),indicating that treatment with LN can inhibit the inflammatory response induced by high glucose and high palmitic acid.Compared with the control group,the levels of SOD and GSH-Px in LX-2 cells of model group were significantly decreased(P<0.01),but the content of MDA and the levels of ROS were significantly increased(P<0.01);Compared with the model group,the levels of SOD and GSH-Px in cells were significantly increased(P<0.01),and the content of MDA and the levels of ROS were decreased significantly after the LN intervention(P<0.01),indicating that treatment with LN can inhibit the oxidative stress induced by high glucose and high palmitic acid.Compared with the control group,the apoptotic rate of LX-2 cells in the model group was increased significantly(P<0.01);Compared with the model group,the apoptotic rate of LX-2 cells in the LN intervention group was significantly decreased(P<0.01),indicating that LN can inhibit the apoptosis induced by high glucose and high palmitic acid.3.The results of Western blot indicated that the protein expression levels of AKR1B1 were significantly increased in the liver of T2 DM rats compared with the control group(P<0.01),and significantly downregulated AKR1B1 protein expression levels were found in the LN intervention groups compared with the model group(P<0.01).In addition,real-time fluorescence quantitative PCR assay showed that the levels of AKR1B1 expression in LX-2cells was significantly increased in the model group compared with that in the control group(P<0.01),whereas it decreased significantly in the LN(10 μmol/L,20 μmol/L,and 50 μmol/L)intervention groups(P<0.01).Compared with the LN intervention group,the levels of TNF-α,IL-1β,and IL-6 in the LX-2 cell supernatants were significantly increased in the LN+oe-AKR1B1 group,the levels of SOD and GSH-Px in cells were significantly decreased in the LN+oe-AKR1B1 group(P<0.01),the content of MDA and the levels of ROS were significantly increased(P<0.01)and the apoptotic rate of LX-2 cells was significantly increased with AKR1B1 overexpression(P<0.05).These results suggest that LN can inhibit the expression of AKR1B1 in DLI as well as the overexpression of AKR1B1 can reverse the anti-inflammatory and anti-oxidant effects of LN.Conclusion1.LN treatment can improve the general condition of T2 DM rats,alleviate the trend of weight loss,reduce FBG and improve dyslipidemia.2.LN can ameliorate liver inflammation and oxidative stress induced by T2 DM,resulting in improved transaminase activity and hepatic tissue injury found in rats with DLI.3.LN can reduce inflammatory response,oxidative stress and apoptosis of LX-2 cells induced by high glucose and high palmitic acid,and that such protective effects of LN against DLI are related to its inhibitory effects on AKR1B1. |
PDF Full Text Request