| Aging is a natural phenomenon, that comprehensive complex recessionphysiological of body molecules, cells, organs and the whole function, thepathological changes in the process of change is the abbreviation of old age.Organism have three vital process phase, growth and developement phase,reproductive period and senescence phase. Different species have similarmechanism of duration of life decsision. Aging is a natural process in all livingorganisms, can not resist, but can slow down. Anti-aging has already become amajor public issue with the increasing elderly population in the world.Aging is caused by multiple factors and it is always associated withdiverse chronic diseases in the process of life. A number of theories have beenproposed to explain the nature of the aging process. Currently300kinds such asthe free radical theory, genetics, error catastrophe theory, theory of cross-linking,metabolic theory, trauma theory, neuroendocrine theory, etc., are discussed fromdifferent researches, but the free radical theory is the most accepted. Effects offree radicals on proteins and nucleic acids involved in a wide range, theconsequences are serious and complex, but also the most abundant experimentalevidence to support this theory, free radicals is one of the important reasons forthe formation of aging. A large of number of experimental studies have shown that reducing the free radicals produced, suppressing inflammation can preventdiseases of aging. Nearly50years of theory and experiment confirmed that thefree radical theory of aging is one of the most self-convincing, while in recentyears studies have shown that as a normal aging and complex life processes,involving different tissues of the body, a series of specific program changes thecell. Numerous studies have shown that cell senescence as a basic process ofaging that was achieved by means of signal transduction pathways, these factorsalso involved in the signaling pathway of which the most classic cellularsenescence pathway is p16INK4a-Rb and p53-p21WAF1/CIP1-Rb pathway. It hadbeen found that these two aging pathways p16or p21overexpression can makesenescent cells reactive oxygen species (ROS) levels rise and accumulationinduced senescence.Yulangsan [Millettia pulchra Kurz var laxior (Dunn) Z. Wei, YLS],Guangxi Zhuang folk conventional medicine, mainly in Guangxi, Guangdongand other places, it has the ability of tonify the qi, blood, lungs etc. People oftenused to treat dyspepsia, skinny, weak after the illness, mental retardation,malnutrition treatment, postpartum weakness, back pain, rheumatism andchronic hepatitis, but also for mental deterioration, physical health, fatigue,forgetfulness, mainly for the elderly, infirm and sick are also used afterpostpartum weakness other nourishing, pediatric treatment of diseases such asmental retardation, enhance immunity and improve memory and other brainfunction. Yulangsan polysaccharide [Millettia pulchra Kurz var laxior (Dunn) Z.Wrei polysaccharides YLSPS] is one of the main effective components extractedfrom Yulangsan. Previous studies showed that YLSPS has strong anti hydroxylfree radical and oxygen free radicals, and obvious anti-tumor effect on sarcomabearing mice, which an regulate the immune function of mice and affect the behavior of chronic stress depression rats and hippocampal structure. The anti-aging effect of YLSPS has not been reported in literature.Objective: In the present study, we study the effect and its mechanism ofYLSPS on aging mice induced by D-galactose(1) Detecte activity level of SOD, GSHPx, CAT, T-AOC in serum, liver andbrain tissue, and observe the histopathological changes in liver, brain;(2)investigate the effect of YLSPS on D-galactose model mice immune functionand detect the content of advanced glycation end products (AGEs) in serum;(3)Evaluated effect of YLSPS on the improvement memory behavior of aging miceinduced by D-galactose;(4) To detect the level of mRNA p16and p21of liverand brain, and examined the expression of p53and p21protein of liver and brain,to explore the cell senescence signal transduction pathways p53-p21and p16regulatory factors involved, begin to find out the mechanism of YLSPS in anti-aging body from the perspective of molecular biology.Method1. Experimental animal:60Kunming mice (18-22gam) were randomly divided into six groups:Normal group (Control), D-galactose group (200mg/kg), YLSPS high-dosegroup (600mg/kg), YLSPS middle-dose group (300mg/kg), YLSPS low-dosegroup (150mg/kg) and vitamin E group200mg/kg),10mice in each group.Aging model mice were induced by D-galactose (20mg/ml), neck backinjection for eight weeks of continuous treatment. Positive group and thetreatment group injected D-gal solution while vitamin E, YLSPS wereintragastric gavage. 2. Specimen collection and research methods:(1) After eight weeks of continuous treatment, activities SOD, GSHPx, CAT andT-AOC as well as level of MDA in serum, liver and brain were determined byspectrophotometry method. While take each group of mice liver and brain werefixed with formaldehyde, paraffin tissue sections, and observed thehistopathological of liver and brain tissues by hematoxylin and eosin staining.(2) Application the formular: thymus weight/body weight, spleen weight/body weight to detected thymus index and spleen index. While detected IL2, IL6,AGEs content in serum by ELISA method.(3) Learning and memory function was evaluated by the Morris water maze.Immunohistochemical method to detect the expression of BDNF and ChATprotein of brain tissue.(4) Application qRT-PCR to detect expression of p16, p21gene in liver andbrain tissue.(5) Western blotting was used to detect the expression of p53, p21protein inliver and brain tissue.Result1. Effect of YLSPS on protective oxidative damage and histopathology inaging mice induced by D-galactose.1.1Result of anti-oxidant index：The activity of SOD，GSHPx, CAT, and T-AOC in serum, liver and brain tissue of D-gal group were significantlydecreased than Control group (p<0.01)．All YLSPS groups could significantlyincrease these indexes of serum, liver and brain tissue (p<0.01orp<0.05)，YLSPS-H group was the best in all YLSPS groups．The activity of MDA in serum, liver and brain tissue of D-gal group weresignificantly increased than Control group (p<0.01)．All YLSPS groups could significantly decrease these indexes of serum, liver and brain tissue (p<0.01orp<0.05)，YLSPS-H group was the best in all YLSPS groups1.2Result of histopathology:Changes in macroscopic anatomy: In normal group liver, the surface issmooth, reddish brown color, soft and brittle, look no exception. Surface ofliver in D-gal group had necrosis, the color of liver was partial white. YLSPSeach dose group was nomal and it color was reder than the D-gal group.By microscope: the structure of liver cells in normal group was normal.hepatic cord arrangement rules, sinusoidal normal central venous no exception.Liver cell of D-gal group was shrinkage, nuclear also reduced, enhancedeosinophilic cytoplasm, some liver cells loosely connected, sinusoidal dilatedhepatic cord stenosis. Liver cells of YLSPS group showed structure, size,sinusoid, hepatic cords was normal.Hippocampus neurons of normal group arranged in neat rows, levelsclosely, form a complete, clear cell edge. D-gal group hippocampus cell showedthat pyralmidal neuron either presented a densely stained shrunken apperaranewith minimal cytoplasm or had disappeared. The model group were smallnumber of cells, intercellular growth, loosely arranged heartbeat, the volume ofnerve cells decreases, shrinkage. In contrast, majority of the neurons wererescued in mice treat with YLSPS, the number of neurons increased, mostcomplete cell morphology, cell edge clear, uniform chromatin, nucleoli stained.2. Effect of YLSPS in thymus index, spleen index and content of IL2, IL6,AGEs in serum of aging mice induced by D-galactose.2.1Thymus index, spleen index of D-gal group was significantly lower than thecontrol group, the difference was statistically significant (P <0.01). Comparedwith the D-gal group, the thymus index, spleen index of vitamin E group, YLSPS group was significantly increased, the difference was statisticallysignificant (P<0.05or p<0.01).2.2The result of ELISA showed that concentrations of IL-2in serum of D-galwas decrease than control group, the difference was significant (p <0.01).Concentrations of IL-2in serum of all YLSPS group was increased comparedwith the D-gal group, there were significant differences (P<0.05orP<0.01).(3) The result of ELISA had shown that concentrations of IL-6in serum of D-gal was increase than control group, the difference was significant (p <0.01).Concentrations of IL-6in serum of all YLSPS group was decreased comparedwith the D-gal group, there were significant differences (P<0.05or P<0.01).(4) The result of ELISA showed that concentrations of AGEs in serum of D-galwas increase than control group, the difference was significant (p <0.01).Concentrations of AGEs in serum of all YLSPS group was decreased comparedwith the D-gal group, there were significant differences (p<0.05or p<0.01).3. Effect of YLSPS on memory improvement of aging mice induced by D-galactose3.1Effect of YLSPS on the learning and memoryUsing the Morris water maze test to exam the influence of YLSPS oncognitive ability of aged mice induced by D-galactose and we found the declinein locomotor activity and exploring ability, passive learning and memory andspatial cognitive ability on D-galactose induced aging mice. Treated withYLSPS after8weeks, the learning and memory ability of aging mice wassignificantly improved. By the latency comparison between each group and themodel group in the fifth days: normal control group compared with the modelgroup, the latency significantly difference (p<0.01); compared with the modelgroup and the treatment group of YLSPS and vitamin E, the escape latency also significantly difference (p<0.05), suggesting that the ability of learning andmemory improved in D-galactose induced aging mice after treated with YLSPSand vitamin E.3.2Effect of YLSPS on the expression of CHAT and BDNF in the brain of agingmice induced by D-galactose.The immunohistochemical result showed that the expression of the bothChAT and BDNF in brain hippocampus of D-galactose group mice wassignificant lower than Control group (p <0.01). The expression of the both ChATand BDNF in brain hippocampus of YLSPS was significantly increasedcompared with D-gal group (p<0.01or0.05).The results showed that the learning, and memory in the aging miceinduced by D-galactose may be improved by YLSPS, suggested that theimprovement of learning and memory may be related to the increase in theexpression of hippocampal BDNF and ChAT in the aging mice induced by D-galactose.4. Effect of YLSPS-H on the expression of p16, p21mRNA in the brain andliver of aging mice induced by D-galactose.The result of qRT-PCR showed that expression of p16and p21genes in bothbrain and liver of D-gal group was increase than control group and thedifference was significant (p <0.01). Expression of p16and p21genes in bothbrain and liver of YLSPS-H group was decreased compared with D-gal groupand the difference was significant (p <0.05).5. Effect of YLSPS-H on the expression of p53, p21protein in the brain andliver of aging mice induced by D-galactose.The result of Westernblotting showed that expression of p53and p21protein inboth brain and liver of D-gal group was increase than control group and the difference was significant (p <0.01). Expression of p53and p21protein in bothbrain and liver of YLSPS-H group was decrease compared with D-gal group andthe difference was significant (p <0.01or0.05).Conclusion1. YLSPS can increase antioxidant enzyme activity, remove excessive oxygenfree radicals, and reduce or stop the destruction of cells and tissues, thusprotecting the normal function of cells and tissue and delaying the aging processinduced by D-galactose.2. YLSPS can improve immune function by increasing the thymus index, spleenindex and serum interleukin2(IL-2) levels, while decreasing serum interleukin6（IL-6）levels.3. YLSPS can improve learning and memory deficit in the D-Gal-induced mouseaging model, which might be related by increasing the expression of ChAT andBDNF protein in brain.4. YLSPS at the cellular level has slow down the aging process effects bydecrease expression of p16, p21gene in liver and brain tissue, while reducingexpression of p53, p21protein in aging mice induced by D-galactose. |
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