| BackgroundWith the arrival of the 21 st century,aging problem has become a huge challenge around the world.China is a populous country which has the highest proportion of aged people in the world,so anti-aging is obviously important.On the other hand,organ degenerative changes following aging pathology were critical risk factors responsible for many chronic diseases,such as neurodegenerative disease,hypertension,and diabetes.Among these,brain injury caused by aging was the most principal and easily to occur.In recent years,the fields of aging and age-related disease researches have made great progress.Although aging is an irresistible process of human life,the gosl to improve the quality of life and slow down the process of aging can be arrived.More and more researchers from basic to clinical have focused their sight on the physiopathologic mechanisms of aging and developing potential anti-aging drug though modern technological methods.Er-zhi-wan(EZW),which is composed by Eclipta alba L.,fresh leaves of(Linn)Hassk.(Asteraceae),and Fructus Ligustri Lucidi,fruit of Ligustrum lucidum Ait.(Oleaceae),has been used as a famous prescription in China for thousands of years,which is identified to tonify the liver and the kidney and strengthening the bones and muscles.Some active compounds have been found in it,such as salidroside,ecliptasaponin,apigenin,specnuezhenide.They were proved to have antioxidate,anticancer,and anti-fibrosis effects,but the pharmacokinetic behavior in body of it has not clear.The objective of this paper was to investigate the effects of EZW on brain injury caused by aging which was produced by D-galactose though modern molecular biological,pharmacological and pathological methods.MethodA.42 male rats were randomly divided into 6 groups: the blank control group(normal saline,1 ml/100g),the model group(normal saline,1 ml/100g),the positive group(12.26 mg/kg/day,1 ml/100g),three groups which administered of EZW(7.412 g/kg/day,3.706 g/kg/ day,1.853 g/kg/ day,respectively),and each group was administered for successive seven days.The aging model subcutaneously produced with D-galactose continuously for 40 days(0.125 g/kg/day,).Meanwhile,each group was given as the dosage above for 40 days.At the 41 th day,morris maze test was carried out for continuous five days.At the 46 th day,animals were sacrificed by anesthesia to obtain the whole brains.Half of each brain was fixed in 10% formaldehyde and embed in paraffin,observed histological change with optical microscope.Electron microscopy observed changes in the morphology and mitochondrial structure of neurons in the brain.Another part of each brain was homogenated,then the concentration of malondialdehyde(MDA),the total activities of superoxide dismutase(SOD),the level of reactive oxygen species(ROS),adenosine triphosphate(ATP)and mitochondrial membrane potential(MMP)in the mitochondria of brain tissues were all detected;the expression of PGC-1?,Foxo3?,Sirt1,P53,Caspase3,Bcl2 and Bax in hippocampus were determined by western blot;the m RNA level of PGC-1?,Foxo3?,Sirt1,p53,Caspase3,Bcl2 and Bax were detected by real-time fluorescence quantitative PCR(RT-PCR).B.Thirty SD male rats were randomly assigned into three dose groups according to their weight.A LC-MS/MS method was established for simultaneous determination of salidroside,ecliptasaponin,apigenin,and specnuezhenide,the four major ingredients in EZW in rat plasma,and the pharmacokinetic behavior of these components were investigated.SD rats were oral administrated(7.412 g/kg/d?3.706 g/kg/d?1.853 g/kg/d,1ml/100g)of the formula.At different time points(0min,5min,10 min,20min,30 min,60min and 2h,4h,8h,12 h,24h),the concentrations of these five components in rat plasma were determined and main pharmacokinetic parameters including Cmax,Tmax,t1/2,MRT,CL,Vd,AUC were estimated using the DAS 2.1.1 software.Result(1)EZW can significantly descrease the content of MDA and increase the the activity of SOD;(2)EZW can significantly enhanced the activity of ATP in the hippocampus and decreased the level of ROS;(3)EZW can obviously relieve the brain hippocampus neurons and microglia degeneration necrosis,and can obviously relieve hippocampus nerve damage and mitochondrial swelling necrosis;(4)Marris maze experiment shows that EZW can obviously reduce the escape latent period(P<0.01),and significantly increased the time to across platform(P<0.05);(5)Western blot and q PCR experiments showed that compared to the blank group,the expression of p53 and caspase3 were obviously reduced by administration of EZW,and the expression of sirt1,PGC-1?,Foxo3?,Bcl2,Bax were increased by EZW intervention.ConclusionEZW can significantly reduce brain injury caused by aging,such as clearing excrescent oxygen free radicals,improving mitochondrial functions and activities.And these effects were finished by regulating the sirt1 pathway to reduce the cell apoptosis which caused by D-galactose,adjusting gene and protein expression of sirt1,PGC-1?,Foxo3?,p53,caspase3,Bcl2,Bax.A precise,accurate and selective method was built by LC-MS/MS,and the pharmacokinetic behaviors in plasma of salidroside,apigenin,and specnuezhenide which were main components in EZW were accorded with a two-compartment model,and the behavior of ecliptasaponin was accorded with one-compartment model by using DAS 2.1.1 software. |
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