| Aim of the study: Anoectochilus roxburghii(A.roxburghii)is a folk medicine and is rich in flavonoid compound that have a better ability to scavenge free radicals both in vitro and vivo,and improve the memory on natural aging mice in vivo.This study is to have a further investigation of the anti-aging effect of Anoectochilus roxburghii flavonoid extract(ARF)in elder mice(18-month-old)and its neuron-protective effect and the underlying molecular mechanism in senescent mouse brain.In addition,we investigate SIRT1 signaling pathway in D-gal SH-SY5 Y cells,to determine whether the neuron-protective effect of ARF is SIRT1-dependent.Materials and methods:1.Fifty-two 18-month-old Kun-Ming(KM)mice were treated with ARF(94.18 and 276.72 mg/kg,BW),vitamin E(100 mg/kg,BW)and normal saline(as an aging model group),and thirteen 2-month-old Kun-Ming(KM)mice were treated with normal saline(as an sham group)for 8 weeks.The body weight,appearance and behavior of mice were observed and recorded every 7 days.After treatment and behavioral study of Morris water maze(MWM),all mice were sacrificed by cervical dislocation under anesthesia.Then the index of Vital organ,the levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),monoamine oxidase(MAO)and acetylcholinesterase(Ach E)in cortex were determined.Hippocampus histopathological morphology were observed by HE,Nissl,SA-β-gal and TUNEL staining.Moreover,SIRT1,p53,p21 and apoptosis-related molecular expression levels in the hippocampus were performed by western blotting.2.D-gal actose(D-gal)was used to accelerate aging and establish oxidative stress in SH-SY5 Y cells.Cytotoxic effects of ARF on SH-SY5 Y cells were measured by using Cell Counting Kit-8(CCK-8)method.After determining the dosage of flavonoids extract from A.roxburghii,the ROS levels both in D-gal induced SH-SY5 Y cell aging model and the aging model treated with EX527(a SIRT1 inhibitor)was measured.Western blotting was performed to determined the expression of SIRT1 and p53 both in D-gal induced SH-SY5 Y cell aging model and the aging model treated with EX527.Results:1.The effect on general signs of natural aging mice: The young mice had a normal diet,lustrous hair,tight and elastic skin,and more autonomous activity.Compared with natural aging mice,both of low dose(94.18 mg/kg,BW)and high dose(376.72 mg/kg,BW)of ARF could significantly improve the changes in aging mice due to aging process,including inappetence,hair color change,skin relaxation and less autonomous activity.2.The effects on improving memory loss in brain of natural aging mice:(1)In the behavioral study performed by MWM,compared with the young mice,the escape latency of aging mice were significantly longer,and need more time to find the target platform,while the number of platform crossings and the time taking in the target quadrant were significantly decreased.Compared with natural aging mice,ARF not only could significantly improved the learning ability,but also significantly shortened the escape latency and the length of distance to reach the target platform in aging mice.Moreover,ARF-treated mice showed more platform crossings and spent more time in the target quadrant.(2)In Nissl staining,compared with young mice,the number of Nissl bodies in the hippocampal CA1 region of aging mice was markedly reduced.But after treated with ARF,the number of Nissl bodies was obviously increased.(3)The effect on ACh-E level: compared with young mice,natural aging mice had high level of ACh-E in the cortex.Compared with aging mice,ARF effectively reduced ACh-E level in aging mice.3.The effect on viscera index of natural aging mice: The results showed that compared with young mice,natural aging mice,the organ indexes of the vital organs(heart,liver)and immune organs(thymus)of the naturally aging mice were significantly decreased,while the organ indexes of the spleen were significantly increased.However,ARF could significantly reduce the index of heart,liver and thymus,but failed to reduce the index of spleen,compared with aging mice.4.The effects on oxidative damage in the brain of natural aging mice:(1)The effect on histological morphological changes in hippocampus(HE and SA-β-gal staining): HE staining results showed that the arrangement of nerve cells was disordered and the nuclei are wrinkled and stained deeply in hippocampus CA1 regionin of aging mice.And SA-β-gal staining results showed that the numbers of senescence cells in hippocampal CA1 region of aging mice were significantly increased.However,compared with aging mice,ARF significantly improved hippocampus pathological alterations and reduced the numbers of senescent cells.(2)The effect on oxidative stress in the brain: Compared with young mice,the levels of ROS,MDA and MAO were higher,while SOD was low level in the cortex of aging mice.However,ARF could effectively reduced ROS,MDA and MAO levels,and increased SOD activities.5.The effect on apoptosis of neuron in the brain of natural aging mice:(1)The effect on histological morphological changes in hippocampus(TUNEL staining): In TUNEL staining,compared with young mice,the numbers of apoptosis positive cells of hippocampal CA1 in natural aging mice were significantly increased.Compared with aging mice,ARF could significantly reduced the numbers of positive cells of hippocampal CA1.(2)The effect on protein expression in hippocampus:Western blot results showed that the expression of SIRT1 in the hippocampus of aging mice was significantly decreased,compared with young mice.Meanwhile,the expression of p53,p21 and apoptosis marker Caspaes-3 were increased and the apoptosis-related Bcl-2/Bax ratio was decreased in the hippocampus of aging mice.However,ARF could effectively reversed these changes above.6.The effect on oxidative damage of D-gal-induced SH-SY5 Y aging cell model: In D-gal-induced SH-SY5 Y aging cell model,compared with D-gal-induced aging model group,ARF could significantly reduce the intracellular ROS level and increase the expression of SIRT1 and inhibit the expression of p53.However,after the addition of SIRT1 inhibitor EX527,compared with each administration group,ARF had no effects on the up-regulation of SIRT1,the reduction both of p53 expression and the ROS level.Conclusions:1.ARF had a certain anti-aging effect on 18-month-old natural aging mice,including improving the general signs changes caused by aging,decreased behavioral activity,and ameliorating atrophy of important organs(heart,liver)and immune organs(thymus),but failed to improve splenomegaly lesions caused by aging.2.ARF had the effect on improving learning and memory in natural aging mice,and its mechanism may be related to the ability of reducing the loss of Nissl bodies and ACh-E activity inhibition.3.ARF had protective effects on oxidative damage in the brain of natural aging mice,its mechanism may be associated with ameliorating the pathological morphology of hippocampal nerve cells,reducing the numbers of aging cells,oxidative stress and the level of MAO,as well as regulating the antioxidant enzyme system.4.The protective effects of ARF on neurons of natural aging mice were related to regulating SIRT1 and inhibiting apoptosis.Its mechanism is to inhibit neuronal apoptosis by activating SIRT1 expression,inhibiting p53、p21,and then increasing the expression Bcl-2 apoptotic protein,and inhibiting the expression of apoptotic protein Bax and apoptosis marker Caspaes-3.5.ARF could reduced oxidative stress damage in D-gal induced SH-SY5 Y cells.The protective effect on neurons was related to up-regulation of SIRT1 expression,inhibition of p53 protein expression.And its protective effect depended on the regulation of SIRT1. |
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