Effects Of Cistanche Tubulosa Glycosides And Echinacoside On Oxidative Stress-induced Alzheimer's Disease Models And Mechanisms In Vivo And In Vitro

Alzheimer's Disease (AD) is one of the dementia diseases commonly happened insenior people,and pathologically characterized by the generation of senile plaquesand loss of neuron where the effects of oxidative stress are thought to play criticalroles.Researchs have shown that H₂O₂ and oxygen free radicals can be generated asbyproducts of normal and aberrant metabolic processes that utilize molecular oxygen,including amyloid aggregation,dopamine oxidation,and brain ischemia/reperfusion.Formed H₂O₂ can be converted into highly toxic hydroxyl radical to attack proteins,deoxynucleic acid,and lipid membrane,thereby leading to mitochondrialdysfunction,calcium imbalance,and apoptosis of neuronal cells.In recent years,researchers have focused more on effective substances for anti-oxidation andanti-free radicals from natural products for the treatment of AD.In China,Cistanche tubulosa glycosides(CTG) isolated from the stems ofCistanche tubulosa (schrenk) Wight have been approved as a treatment for vasculardementia and produced by Sinphar TianLi Pharmaceutical Company (Hangzhou).This is the first government-approved drug containing phenylethanoid glycosides.Recent studies have shown that phenylethanoid glycosides are effective at scavengingfree radicals and protecting against glutamate-induced neurotoxicity.CTG alsoshowed antioxidant properties and neuroprotective function with echinacoside (ECH)as a quality control compound (content is more than 25%).ECH is the first compoundthat was defined as a phenylethanoid glycoside.It was first extracted from Echinaceaangustifolia in 1950 and exists in several other plants.ECH has recently beendemonstrated to protect hepatotoxicity and is a potent promoter of neuronal survivalinduced by TNF-αand MPTP.However,these studies provide limit information aboutthe protective effects of ECH on oxidative stress-induced injury in vitro.This research is to study the protective effects of CTG and ECH on oxidativestress-induced injuries in vivo and in vitro,and disclose the related mechanisms forfurther developing CTG and ECH in the treatment of dementia diseases such as AD. The results are shown as following:1.CTG significantly ameliorated the learning and memory disorders induced byD-gal and sodium nitrite in mice with the increases of Na⁺-K⁺ATPase,GSH-Px andSOD activity and the decreases of the level of NO in mouse or rat brain.2.Protective effects of ECH on H₂O₂-induced cytotoxicity in PC 12 cells weredemonstrated by the experiments as follows:(1) The cell viability was markedlyelevated and the leakage amount of LDH descended by ECH in H₂O₂-injured PC12cells;(2) Apoptosis and necrosis status was improved by ECH,which was confirmedby flow cytometry (FCM) and Hoechst 33342 staining.3.The GSH-P_X and Na⁺-K⁺ ATPase activity were enhanced by ECH in H₂O₂-injuredPC12 cells,however,Caspase-3 activity was inhibited.4.When PC 12 cells were exposed to H₂O₂,the intracellular ROS level wassignificantly increased with MMP decreasing.However,treatment with ECHeffectively reduced ROS generation and a significant increase of Rhodamine123fluorescence intensity was detected compared to H₂O₂ incubation alone.5.Data obtained from LCSM suggested that the exposure to H₂O₂ irritation increased[Ca²⁺]i and NO in PC12 cell rapidly.Treatment of ECH for 30 min prior to H₂O₂addition supressed the time course curve and caused a significant decrease of[Ca²⁺]iand NO peak values.The study suggested that[Ca²⁺]i and NO tightly involved in theprocess of apoptosis in H₂O₂-injured PC12 cells.6.JC-1 and LCSM were used to confirm the effect of ECH on MMP in H₂O₂-injuredPC 12 cells as described above.R₀/R_t was used as the indicator of the change of MMP,in which R₀ is the ratio of red fluorescence intensity to green fluorescence intensity atthe beginning,and Rt is the ratio at one time after H₂O₂ irritation.The results showedthat ECH inhibited the increase of R₀/R_t in 20 min after H₂O₂ irritation.7.We also measured the effect of ECH on H₂O₂-induced up-regulation of theBax/Bcl-2 ratio by Western blot.Bax protein expression in PC 12 cells wassignificantly increased,whereas the Bel-2 level was decreased.Pre-and co-treatmentwith ECH dramatically reduced the up-regulated Bax/Bcl ratio induced by H₂O₂.8.To investigate whether there are any apoptotic gene expression changes involved in the apoptosis and the effects of ECH,the expression levels of Bcl-2,p53,p65 andiNOS mRNA were examined using RT-PCR.Decrease of Bcl-2 mRNA and increaseof p53,p65 and iNOS mRNA under the treatment of H₂O₂ were reversed by thetreatment of ECH to PC 12 cells.Based on the above results,it is concluded that:(1) CTG significantlyameliorated the learning and memory disorders induced by oxidative stress,themechanisms may be related to the increase of Na⁺-K⁺ ATPase,GSH-Px and SODactivity as well as the decrease of the level of NO in brain.(2) the results suggestedthat ECH had markedly protective effects on H₂O₂-induced injury and apoptosis inPC12 cells by enhancing the GSH-P_X and Na⁺-K⁺ ATPase activity,inhibitingCaspase-3 activity and ROS,NO production,attenuating the MMP loss,preventingthe influx of Ca²⁺,up-regulating the expressions of the Bcl-2 protein and Bcl-2mRNA,and down-regulating the expressions of p53,p65 and iNOS mRNA.(3) CTGcan be used to prevent and treat senile dementia such as AD based on the experimentsand frether clinical tests.The data obtained from this study provided a sound basis toconfirm that ECH is a potential candidate for intervention in neurodegenerativediseases such as Alzheimer's and Parkinson's disease.