The Effects And The Mechanisms Of Heshouwuyin And Its Effective Components On Delaying The Natural Aging Of Cerebral Cortex Of Rats

Brain aging is a precursor of aging in various organs of the body.It plays a leading role in the aging process,and affects the aging process of various systems.Slowing down the senescence of the central nervous system to a certain extent will reduce the incidence of degenerative diseases of the central nervous system in the elderly(such as Alzheimer 's disease,Parkinson' s Disease,Huntington's disease).Because of the aging of the central nervous system and the irreversibility of the neurodegenerative diseases,there is still a lack of drugs to effectively alleviate the progress of the disease.Therefore,the mechanism of brain aging and the development of drugs to effectively delay brain senescence and protect the brain tissue are explored,and the antiaging effects of drugs are analyzed,and the incidence of degenerative diseases of the nervous system is reduced.Improving the quality of life of the elderly is of great scientific,economic and social significance in the coming aging society to achieve healthy aging.The theory of traditional Chinese medicine to postpone brain senescence and prevent senile disease is the method of invigorating Kidney and invigorating Brain.Based on the theory of syndrome differentiation and treatment,TCM compound has the characteristics of multiple targets and multi-links and has good prospects for delaying senility,preventing and treating degenerative diseases of the central nervous system.In recent years,the study of senescence and pharmacodynamics has penetrated into the effective components of single herb medicine,in order to avoid the side effects caused by the complex components of plant medicine,develop effective anti-aging target for mono-drug,effectively delay human natural aging.Styrene glycosides(TSG),phenylethyl alcohol glycosides(PhGs)and Epimedium Glycoside(Icariin)are the main components ofHeshouwuyin(Polygonum Multiflorum 15 g,Cistanche Deserticum 10 g,Herniated Epimedium 10 g,Achyranthes Bidentata 15 g,Radix Salviae Miltiorrhizae 25 g,and Poria Cocos 15 g).Heshouwuyin has been proved to be effective on antiaging of reproductive system,but its anti-aging of the central nervous system needs further study.The correlation Heshouwuyin and gonad axis was reported and the hypothalamic-pituitary-testicular(ovary)axis is closely related to the aging and functional abnormalities of the cerebral cortex.Therefore,this topic studies the effect and mechanism of Heshouwuyin and its effective components in delaying the cerebral cortex of natural aging rats.It provides a theoretical reference for Chinese medicine to postpone the senescence of the central nervous system and develop effective anti-aging target for mono-drugs and reduce the senile degenerative diseases.Part one The Effects and the Mechanisms of Heshouwuyin and Its Effective Components on Delaying the Natural Aging of Cerebral Cortex of RatsObjective: To investigate the effects of Heshouwuyin and its effective components on the learning and memory ability,the cortex senescence,the apoptosis of nerve cells,the anti-oxidation capability,and the expressions of apoptosis related factors in the cerebral cortex of natural aging rats.Methods: SD rats of 16-month-old were randomly divided into 5 groups,including old group,TSG group,PhGs group,Icariin group,and Heshouwuyin group,respectively.30 rats were in each group.The rats in the four drug groups were administered with TSG,PhGs,Icraiin by a dosage of100 mg/kg,and Heshouwuyin by a dosage of 48g/kg for 60 days,respectively,when the rats are 16 month old of age.The rats in the the old groups were intragastrically administered with equal amount of saline for 60 days.SD rats enter the experiment at 18-month-old of age.30 rats of 3-month-old with the same origin and the same genetic background were selected as the young group,the same amount of physiological saline was given for 30 days,and enter the experiment at 4-month-old of age.The Morris water maze tests wereperformed for looking at the effects of TSG,Ph Gs,Icraiin and Heshouwuyin on the behavioral changes of the natural aging rats.These rats were then sacrificed and their brain tissues were removed,individually.The morphological changes of their cerebral cortexes,the positive rates of SA-?-gal expressions and the rates of the apoptosis of the cerebral cortex cells were detected by using HE,SA-?-gal and TUNEL staining,respectively.The amounts of ROS produced,the activities of SOD and GSH-Px,and the MDA in the cerebral cortexes in each group were measured by using kits.The mRNA transcribed with the genes of Bax,Bcl-2,Caspase-3,and also the protein translated in the cerebral cortexes of the natural aging rats were measured by using Real-time PCR and Western-blotting,respectively.Results:1.The learning and memory abilities of the rats in each group were evaluated by using Morris water maze tests.On the first stage of the experiments(scored in the 1st to the 5th day),the escape latencies demonstrated by the rats in the old group were found to be longer significantly than those of the rats in the young group(P<0.01).In the 5th day,the escape latencies of the rats in the TSG group,the PhGs group and the Heshouwuyin group were significantly shorter than those of the rats in the old group(P<0.05);the escape latencies of the rats in the Icraiin group were found to be extended slightly,but there were no significant difference(P>0.05).Compared with the rats in the young group,the numbers of platform crossing of the rats in the old group were significantly reduced(P<0.01).Compared with the rats in the old group,the numbers of platform crossing of the rats were significantly increased by the rats in the TSG,the PhGs and the Heshouwuyin groups,but the numbers of platform crossing of the rats in the Icraiin group had no obvious change(P>0.05).2.HE staining: In the young group,cell structures of cerebral cortex were shown clearly with the obvious neurites,the well-distributed chromatin,and the normal nuclear structure.The numbers of cells were also higher.In the old group,the cerebral cortex cells were smaller,the neurites were shorter,and the cytoplasm were stained deeply.The shapes of the cerebral cortex cells were irregular with condensed chromatin.Compared the four drug groups with the old group,it was observed that the volumes of some cells were larger,the numbers of protuberances were increased,the cytoplasm were stained lighter,the nucleus were more regular,the chromatin condensation was not obvious,and the distribution was even.The changes of Heshouwuyin group were the most obvious.3.SA-?-gal staining in cerebral cortex:The positive rate of SA-?-gal in the old group were significantly higher than that of the young group(P<0.01).The positive rates of SA-?-gal in the PhGs group and in the Heshouwuyin group were significantly lower than that in the old group(P<0.05 or 0.01).4.TUNEL staining in cerebral cortex:The rates of apoptotic cells in the old group were increased significantly when compared with those in the young group(P<0.01).The apoptosis rates of PhGs,Icraiin and Heshouwuyin groups were significantly lower than that of old group(P<0.05)..5.ROS content was detected by DCFH-DA method: The content of ROS in cerebral cortex of the old group was significantly higher than that of the young group(P<0.01).The relative contents of ROS in the PhGs and the Heshouwuyin groups were significantly lower than that in the old group(P<0.05).6.The activity of SOD was detected by XOD method:The activity of SOD in cerebral cortex of the old group was significantly lower than that of the young group(P<0.01).The activities of SOD in the Icraiin and the Heshouwuyin groups were significantly higher than that of the old group(P<0.05).7.The activity of GSH-Px was detected by DNTB method:The activity of GSH-Px in cerebral cortex of the old group was significantly lower than that of the young group(P<0.01).The activities of GSH-Px in the PhGs,the Icraiin and the Heshouwuyin groups were significantly higher than that of the old group(P<0.05).8.MDA content was detected by TBA method:The content of MDA incerebral cortex of the old group was significantly higher than that of the young group(P<0.01).The contents of MDA in the TSG,the PhGs and the Heshouwuyin groups were significantly lower than that of the old group(P<0.05).9.Real-time PCR and Western-blot showed: compared with the young group,mRNA and protein expressions of Bax,Caspase-3 in cerebral cortex of the old group were increased,mRNA and protein expression of Bcl-2 was decreased,the difference was statistically significant(P<0.01).Compared with the old group,mRNA and protein expressions of Bax,Caspase-3 in the Icraiin group and the Heshouwuyin group were decreased,mRNA and protein expression of Bcl-2 was increased,the difference was statistically significant(P<0.05).Summary: TSG,Ph Gs and Heshouwuyin will shorten the escape latency of rats and increase the number of platform crossing obviously.So it is considered that they contributed to improve the learning and memory ability of natural aging rats,and the effect of Heshouwuyin is the best.The four drugs can improve the morphology of brain aging,especially Heshouwuyin,the brain tended to remain young.PhGs and Heshouwuyin reduce the positive expression of SA-?-gal in rat cerebral cortex and delay the process of natural aging.PhGs,Icraiin and Heshouwuyin can reduce the apoptosis of cerebral cortex cells in natural aging rats.PhGs increased the activity of GSH-Px and reduced the content of ROS and MDA in the cerebral cortex of natural aging rats.Icraiin improved SOD and GSH-Px activity.Heshouwuyin affected the above four indexes and improved the antioxidant capacity of the cerebral cortex in natural aging rats.Icraiin and Heshouwuyin may inhibit apoptosis and delay the natural aging of the cerebral cortex by upregulating the mRNA and protein expression of Bcl-2 and downregulating the mRNA and protein expression of Bax and Caspase-3.Part two Establishment and identification of Neuron Aging Model in vitroObjective: To establish the natural aging neurons model by exploring the time rule of primary neurons of natural aging rats and to determine the time point of neuronal aging by extending the culture time.Methods: Cortical neurons of newborn SD rats were cultured primarily by using Neurolbasal+B27 medium,and the purity of the neurons cultivated was identified by immunocytochemistry staining.The numbers of the neurons were counted and their morphological changes were observed in different culture time points(7,9,11,13,15,17,19,21 d).SA-?-gal cell chemical staining method was used to detect the positive rate of neurons.The early apoptosis rates of neurons during cultivations were detected by TUNEL terminal marker method.The expressions of Caspase-3 protein was detected by Western-blotting.The activities of Caspase-3 were detected by spectrophotometry.Results: morphological observation using Optical microscope: the neurons cultured in the 7th day were found to be conical,triangular,fusiform,elliptical and full typical axons and dendrites.The cell contour was clear,the structure was solid,the cytoplasm was uniform and bright,and the nucleus was big and apparent.In the10 th day of cultivation,the neuronal protrusion was clearest and the network was dense.In the15 th day of cultivation,no significant changes with the numbers of neurons were seen.The morphology of neurons was regular with crosslinking of nerve fibers,and the neurons seemed to aggregate.In the 17 th day of cultivation,,the numbers of living neuron cells decreased,the numbers of pyknosis neurons increased,the protrusion of the neuron cells were thin,short,broken and blur,and some of them were shown beaded.A large number of suspended cell fragments can be seen in the medium.In the 15 th day of cultivation,the positive rate of SA-?-gal was 60.56±9.20% and the early apoptosis rate was 31.48±6.05%,both of them were significantly higher than those measured in the 7th and13 th day of cultivations.The level of Caspase-3 protein was 1.27±0.015 which was more than 2 folds increasing compared to the expressions by the neuron cells in the 7th day of cultivations,significantly different from those inthe 7th and in the 13 th day of cultivation.The Caspase-3 activity of the neuron was 2.21±0.24 which was 1.5 to more than 2 times of the 7th to 13 th days,and the difference was significant.When the neurons were cultured to15 th days in vitro,60% of the neurons were old and more than 30% of the neurons were apoptotic.The whole cell culture system enters the natural aging stage.Summary: In the absence of drugs added and damages induced,15 th day of neurons cultivation is the time point of entering the natural aging stage in Neurolbasal+B27 culture system.The time point in cultivation was reliable and was used in the subsequent anti-aging researches by using drugs in vitro.Part three Effect and Mechanism of Effective Components of Heshou-wuyin and their Compatibility Applications on the delay of cultured cortical neurons aging in vitroObjective: To investigate the effects of Compatibility active components(TSG,PhGs,Icariin)and their compatibility applications(TSG+Ph Gs+Icraiin)on the morphology,SA-?-gal and TUNEL positive cell rates,apoptosis and oxidative stress related factors,?-catenin,REST of natural aging neurons.Through preliminarily comparison of natural aging neural network construction situation,oxidative stress resistance,the role of ageing and apoptosis,analyze its function and mechanism of delaying neuron natural aging.Methods: Cortical neurons of newborn SD rats were cultured primarily in Neurolbasal+B27 medium.The samples were divided into 6 groups:(1)ST;(2)LT;(3)TSG(50 ?mol/l);(4)PhGs(50 ?mol/l);(5)Icariin(50 ?mol/l);(6)TSG+PhGs+Icariin(50 ?mol/l).The neurons cultivated for 7 days were assumed as the ST group,and the neurons cultivated for 15 th days were assumed as the LT group.Distyrene,benzyl glycoside,icariin and their compatibility of 50 ?mol/l was given to different drug group neurons for 72 hours on cultured 12 th to14th days.Before the experiment,the neurons in the ST and LT group were treated with the same amount of media with DMSOand PBS for 72 hours.The neurons of each group on 7th and 15 th day were fixed or collected.The morphological changes and growth conditions of the neurons were observed and photographed under the inverted microscope.SA-?-gal cell staining and TUNEL immunocytochemistry staining were performed too.ROS content was detected by DCFH-DA method,SOD activity was detected by XOD method,GSH-Px activity was detected by DNTB method,and MDA level was detected by TBA method.Using immunocytochemistry,spectrophotometry,western blot and PCR methods,the expression of Bcl-2,Bax,Caspase-3,?-catenin?REST protein and the vitality of Caspase-3 enzyme were detected.Results:1.Morphological changes under the inverted microscope: On the 15 th days of the neuron culture,the polymerization of TSG and PhGs group was significantly less than that of LT group.No polymerization of neurons was found in the Icariin and Compatibility group.On the 17-19 th days,pyknosis neurons in the four drug groups reduced,and there was a complete neural network structure,especially the change of Icariin group was obvious.On the21 st days,there was almost no neuron in the Icariin group,and the cell debris was full of vision.There were clear neurons structure in the Compatibility group occasionally.2.SA-?-gal cell staining: Compared with the ST group,the positive rate of SA-?-gal was significantly increased in the LT group(P<0.01).Compared with the LT group,the SA-?-gal positive rate of the four drug groups was significantly reduced(P<0.01).The level of blue dyeing of neurons in PhGs and Compatibility group was significantly light,indicating that the expression of SA-?-gal decreased.3.TUNEL immunocytochemistry staining: Compared with the ST group,the apoptosis rate of LT group was significantly higher(P<0.01).Compared with the LT group,the apoptosis rate of TSG group was not significantly reduced(P>0.05).The apoptosis rate of Ph Gs,Icraiin and Compatibility group was significantly reduced(P<0.01),and the neurons was light stained,indicating that the TUNEL expression was decreased.4.ROS content was detected by DCFH-DA method: The relative content of ROS in the LT group was significantly higher than that in the ST group,and the difference was statistically significant(P<0.05).The relative content of ROS in PhGs and Compatibility group was significantly lower than that in the LT group(P<0.01)..5.The activity of SOD was detected by XOD method: The activity of SOD in the LT group was significantly lower than that in the ST group(P<0.01).The activity of SOD in PhGs,Icraiin and Compatibility group was significantly higher than that in the LT group(P <0.05 or 0.01).6.The activity of GSH-Px was detected by DNTB method: The activity of GSH-Px in the LT group was significantly lower than that in the ST group(P<0.05 or 0.01).The activity of GSH-Px in Ph Gs,Icraiin and Compatibility group was significantly higher than that in the LT group(P<0.01).The activity of GSH-Px in TSG group was higher than that in the LT group(P<0.05),and the difference were significant.7.MDA content was detected by TBA method: The MDA content of the LT group was significantly higher than that of the ST group(P<0.01).The MDA content of neurons in TSG,Ph Gs and Compatibility group was significantly lower than that in the LT group(P<0.01).8.Bax and Bcl-2 were detected by Western-blot: The ratio of Bax/Bcl-2in the LT group was more than 2 times that of the ST group(P<0.01).The expression of Bax and Bcl-2 proteins in PhGs,Icraiin,and Compatibility group were significantly different,and the ratio of Bax/Bcl-2 was significantly lower than that in the LT group(P<0.05 or 0.01).9.Bax and Bcl-2 mRNA were detected by RT-PCR: The relative level of Bax mRNA in the LT group was more than twice as that of the ST group,while the expression of Bcl-2 mRNA was significantly less(P<0.01).Compared with LT group,the expression of Bax mRNA decreased,while Bcl-2 mRNA increased in four drug groups(P<0.05).The changes of Bax and Bcl-2 mRNA in Ph Gs,Icraiin and Compatibility groups were significant(P<0.01).10.Caspase-3 was detected by immunocytochemical staining: The positive rate of Caspase-3 in the LT group was significantly higher than that in the ST group(P<0.01).Compared with the LT group,the positive rates of Caspase-3 in four drug groups was significantly lower(P<0.05).11.Caspase-3 activity was detected by spectrophotometry:The activity of Caspase-3 in the LT group was about 2 times that of the ST group,and the difference was significant(P<0.01).Compared with LT group,the relative activity of Caspase-3 in PhGs,Icraiin and Compatibility groups was significantly reduced(P<0.01).12.?-catenin was detected by Western-blot : Compared with the ST group,the expression of ?-catenin in the LT group was significantly increased(P<0.01).The expression of intranuclear ?-catenin was higher than that of cytoplasm.Compared with the LT group,the expression of intranuclear ?-catenin in PhGs,Icraiin and Compatibility group increased,and the expression of ?-catenin in cytoplasm decreased significantly(P<0.05).13.REST was detected by Western-blot:Compared with the ST group,the expression of REST in the LT group was significantly increased(P<0.01),The expression of intranuclear REST was higher than that of cytoplasm.?-catenin is metastasized with REST protein.Compared with the LT group,the expression of intranuclear REST in Icraiin and Compatibility group increased,and the expression of REST in cytoplasm decreased significantly(P<0.05).Summary: All four drugs can maintain the normal morphology of the neurons and show different anti-aging effect on neurons.The effect of Compatibility was longer,and Icraiin had a significant anti-aging effect in the early stages of aging.All four drugs can delay the natural aging process of cultured neurons in vitro by reducing the expression of SA-?-gal.PhGs,Icraiin and Compatibility can reduce the TUNEL expression and reduce the apoptosis of natural aging neurons.With the decreasing of SOD and GSH-Px antioxidant enzyme activity in natural aging neurons,intracellular MDAcontent increased obviously.It's a sign of the aging of neurons.In natural aging neurons,TSG can improve the activity of GSH-Px and reduce MDA content.Icraiin can improve the activity of SOD and GSH-Px.PhGs and Compatibility can reduce ROS,increase the activity of SOD and GSH-Px and reduce MDA content,and increase anti-oxidative stress ability.Four drugs are able to reduce the apoptosis of natural aging neurons by affecting the expression of Bax,bcl-2 protein or mRNA expression,reducing Bax/ bcl-2ratio,reducing the expression or activity of caspase-3.PhGs,Icraiin and Compatibility may increase self-repair and anti-oxidative stress,anti-apoptotic ability and delay aging by promoting the nuclear transfer of?-catenin and(or)REST in the natural aging neurons.Conclusion: In the non-serum Neurolbasal+B27 system,the 15 th day of primary culture of cortical neurons can be regarded as the time node of natural senescence of neuron culture system.Heshouwuyin and its effective components delay the natural aging of the cerebral cortex through improving the antioxidant ability of neurons,regulating apoptosis related factor expression,and the ability of learning and memory of the natural aging rat,especially Heshouwuyin.Among the effective components,Icraiin has a prominent anti-apoptosis effect.PhGs has good anti-oxidation and anti-apoptosis effects.PhGs,Icariin and Compatibility promote intracellular transfer of neuronal ?-catenin and/or REST,and delay the natural aging process of neurons through the Wnt/?-catenin pathway.