| Objective:Type 2 diabetes,as a chronic disease with a worldwide threat to human health,is difficult to be cured due to its complicated pathogenesis.Although at present many hypoglycemic drugs with a variety of therapeutic targets have been applied in clinical practice such as metformin,sulfonylureas,?-glycosidase inhibitor and insulin,long-term use of these drugs usually lead to some adverse effects.With a long history in the treatment of diabetes,many Traditional Chinese Medicines(TCMs)as well as their extracts have been found to carry an apparent antidiabetic effect.Therefore,to find the active ingredients of these antidiabetic TCMs and to clarify their mechanisms are of great significance.The present study aims to clarify the anti-diabetic effect and the mechanisms of honokiol by exploring the influence of honokiol on blood glucose,blood lipid,oxidative stress in the liver,the activity of CYP450 enzymes,and the level of mRNA expression of liver and kidney transporters in type 2 diabetic rats,as well as by investgating the pharmacokinetic differences of honokiol in diabetic model rats and normal rats.Method:(1)The hypoglycemic&hypolipidemic effect and anti-oxidative stress test:Type 2 diabetic rat model was established using male Wistar rats after 6-week of high-fat diet followed by single intraperitoneal injection of low dose(30 mg/kg)strepotozotocin.Well-established rat models were randomly assigned into the model group,honokiol groups(low dose,25 mg/kg/d;medium dose,50 mg/kg/d;high dose,100 mg/kg/d),metformin(250 mg/kg/d)positive control group,and normal control group,with eight rats in each of the groups.After ig administration as above for consecutive 8 weeks,OGTT test was conducted.Blood was collected from orbital venous plexus for blood glucose and lipid test,and liver was removed after the rats were sacrificed by cervical vertebra dislocation to prepare liver homogenate and hepatic microsome for the detection of changes in anti-oxidative related indicators including activity of SOD,CAT,GSH-Px and CYP2E1 as well as content of MDA.(2)Test of the effect on activity of CYP 450 in liver and mRNA expression level of transporters in liver and kidney:Same method was used to build rat models.Type 2 diabetic rats were randomly divided into the model control group,honokiol groups(low dose,25 mg/kg/d;medium dose,50 mg/kg/d;and high dose,100 mg/kg/d)and normal control group,with 6 rats in each of the groups.At the end of the treatment with honokiol for 8 weeks,the rats were sacrificed,and part of their livers were used to prepare liver microsome for activity detection of CYP1A2,CYP2E1,CYP2C,CYP2B,CYP3A and CYP4A.Total RNA was extracted from part of the liver and kidney tissues to determine the mRNA expression level of the uptake and efflux transporters using fluorescence quantitative PCR method.(3)Pharmacokinetic test:Same method was used to build rat models.The model rats and normal rats were randomly divided into 4 groups,respectively,with 6 rats in each group.Two groups with each from the model rats and normal rats were aintragastric administration of 50 mg/kg honokiol,and blood was collected from the orbital venous plexus respectively at 5,10,15,30,45,60,120,240,480,720 min after administration to determine the plasma concentration of honokiol and its metabolites and to calculate the pharmacokinetic parameters.The rest groups of model rats and normal rats were intragastrically given 50 mg/kg honokiol,and sacrificed by dislocation of the cervical vertebra respectively at 5,15 and 240 min after administration.Liver,kidney and brain tissues were collected to prepare homogenate to determine the content of honokiol and its metabolites using HPLC.Results:(1)Hypoglycemic and hypolipidemic effect and anti-oxidative stress test:Compared to the normal groups,a higher blood glucose level and greater blood glucose area under the concentration-time curve were observed in the model groups with significant difference(p<0.01)respectively at 30,60,90 and 120 min after intragastrical administration of 2 g/kg glucose.The increased blood glucose was lowered in the diabetic rats after administration of honokiol at different doses.Compared to the model control group,the low,medium and high dose honokiol groups respectively showed a decrease of 32.7%,35.4%and 32.7%in the area under the curve.The model control group had a significantly increased blood glucose and blood lipid levels in comparison with the normal group(p<0.01),along with a decreased CAT,SOD and GSH-Px activity and elevated MDA level(p<0.01)as well as a significant increase in CYP2E1 enzyme activity(p<0.01).Compared to the model control group,the honokiol groups had a 29%-41%decrease in blood glucose,a 38%-49%decrease in TC level,a 74%-86%decrease in TG level,as well as a 34%-43%decrease in LDL-C level after the 8-week intragastric administration,all with statistical difference(p<0.05 or p<0.01)but absent of dose dependent effect.No significant difference was found in level of HDL-C.In addition,there was no significant difference observed between honokiol and metformin in their hypoglycemic and hypolipidemic effect.Meanwhile,compared with the model group,the dosage group showed a significant decrease of 45%-53%in liver MDA level,as well as a non dosage-dependent increase by 15-29%,12-22%and 30-22%respectively in the activity of CAT,SOD and GHS-Px to a level which still failed to match the normal range.No significant difference was observed in CAT,SOD and GSH-Px activity between the dosage group and the metformin group.The activity of CYP2E1 also decreased by 11.4%,51.3%and 11.2%respectively in low,medium and high dose of honokiol groups compared to the model group.(2)The effect on activity of CYP 450 in liver and mRNA expression level of transporters in liver and kidney:Type 2 diabetes model group showed a similar CYP2B and CYP3A enzyme activity to the normal group but a significantly enhanced activity in CYP1A2,CYP2E1,CYP4A and CYP2C(p<0.01 or p<0.05)2.36,2.10,2.55 and 1.86 times to the normal group,respectively.The mRNA expression of Oat2,Oatp2b1 and Oatp1a5 was significantly down-regulated(p<0.05 or p<0.01),while the expression of mRNA of Octn2,Oatp3al,Oatplal and Mdr2 increased significantly(p<0.05)in liver;an up-regulation mRNA expression of Oat2,Mdr2 and Ntcp(p<0.05)as well as a significant down-regulation of Octl,Octn2,Oatp2b1 and Oatp1a5(p<0.05 orp<0.01)in kidney was also observed.Compared to the model group,honokiol showed no significant effect on CYP2B activity in the liver of diabetic rats,in contrast,it was found to dose-dependently inhibit the activity of CYP1A2,CYP2E1,CYP4A and CYP2C,particularly in the medium and high dose groups where a significant difference was observed(p<0.05 or p<0.01);Honokiol also up-regulated the mRNA expression of liver Oat2 and Oatp2b1,with a significant difference in high dose group(p<0.05);the down-regulation of the mRNA of renal Bcrp and Mrp4 were found in honokiol groups(p<0.05).(3)Pharmacokinetic test:Honokiol as well as its glucuronidation metabolite M2 was detected in plasma and kidneys in the rats of normal group and model group;while honokiol,its glucuronidation metabolite M2 as well as the hydroxylation/glucuronidation/sulfation metabolite Ml was found in liver,and only honokiol detected in brain.Compared with the normal group,honokiol showed a significantly reduced AUC0-12h and Cmax in diabetic groups(p<0.05),and a decreased mean retention time(MRT)and elimination half-life(t1/2z)but with no statistical differences.AUC0-12h and Cmax of the metabolite M2 were significantly increased(p<0.05 or p<0.01).The concentration of honokiol was higher in the liver of the rats in diabetic model groups than in the normal group at all the three time points,but with no statistical significance;a higher honokiol concentration was measured in the kidney of the rats in diabetic groups compared to the normal group,with an extremely significant difference at 5 min(p<0.01)and significant difference at 15 min(p<0.05);The diabetic model groups also showed a higher honokiol concentration in the brain of rats than normal group,with extremely significant difference at 15 min(p<0.01).The concentration of M1 was higher in the liver in diabetic model groups than in the normal group,with a significant difference at 5 min and 240 min(p<0.05),and the concentration of M2 was significantly higher than in normal group at 5 min(p<0.05),but with no significant difference at 15 min and 240 min;In the kidney,the concentration of M2 was significantly higher in diabetic model groups than in the normal group at 15 min(p<0.05),but with no obvious difference at 5 min and 240 min.Conclusion:Honokiol could reduce the level of blood glucose and lipid in high-fat diet and streptozotocin induced type 2 diabetic model rats,improve their oral glucose tolerance,increase the activity of hepatic antioxidant enzymes such as SOD,CAT and GSH-Px,as well as reduce the activity of CYP2E1 which is related to oxidative stress,and decrease the level of lipid peroxidation products MDA,indicating that honokiol has anti-diabetic effect.In addition,honokiol can inhibit the activity of CYP1A2,CYP2E1,CYP2C and CYP4A which are associated with diabetes and its complications,up-regulate the mRNA expression of Oat2 and Oatp2b1 in liver,down-regulate the mRNA expression of Bcrp and Mrp4 in kidney,suggesting that honokiol might play its role in treating diabetes through its influence on activity of CYP450 and the mRNA expression of the hepatic and renal transporters which are associated with the occurrence and development of diabetes.Study on the pharmacokinetics and tissue distribution of honokiol and its metabolites in diabetes condition and in normal state revealed a certain extent of changes in pharmacokinetic parameters and tissue distribution under the pathological condition,which could be explained by the changes in CYP450 enzyme activity and transporter mRNA expression under the diabetic condition. |
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