Molecular Mechanism Of Anti-brain Aging Of Coptis Chinensis And Its Traditional Chinese Medicine Monomers

Objective:Brain aging is a series of degenerative changes in the function and structure of the human brain with the increase of the age.In the process of brain aging,brain cells show signs of bioenergy damage,neuronal network abnormalities,oxidatively modified organelles,and inflammation in analysis,which will further lead to neurodegeneration including dementia,Parkinson’s and cerebrovascular diseases.There is no denying that China has already entered into an aging society,and the number of deaths caused by aging neurological diseases is growing rapidly.Therefore,it is of great importance to find effective methods to alleviate the process of senile encephalopathy and brain aging.The proportion of neuronal cells in the human brain is only about 10%,while glial cells account for the majority of brain cells.Glial cells physiologically perform various functions,mainly assisting neurons in their protection,support,nutrition and regulation.General aging of brain glial cells might induce the acceleration of brain aging,changes in neuro homeostasis and regulation-related gene expression,which could even cause severe neuronal damage,the occurrence of neurodegenerative diseases and the decline of brain cognitive ability.Therefore,it is urgent to fight against aging of brain glial cells for aging process of the brain.Coptis and Cistanche are commonly used in the practice of traditional Chinese medicine,and they are used simultaneously in various prescriptions.Rhizoma Coptidis,also known as"Chicken Claw Lian" and "Wei Lian",is a perennial herb of the Ranunculaceae family.Its taste is bitter and cold in nature.There are abundant components including alkaloids,flavonoids and lignans in it.Berberine is one of the most representative and highest active substances in Coptis alkaloids.Modern pharmacological studies have found that berberine has beneficial effects on cardiovascular system and could play a crucial role in anti-tumor,anti-inflammatory and anti-bacterial process.Cistanche cistanche,also known as "cunyun","cistanche",belongs to the Lamiaceae plant.Its sweet and salty taste endow it with warm trait and functions of nourishing kidney,nourishing essence and bloo.Cistanche cistanche contains plenty of chemical components such as phenethyl alcohol glycosides,iridoids and glycosides,among which Echinacoside is one of the main active ingredients.It has been reported with anti-aging,anti-oxidan properties and neuroprotective effects of the drug.Accroding to the above,this study draws on these two components based on clinical experience of traditional Chinese medicine.In vixo,Coptis combined with Cistanche are used on normal aging mice to observe its effect on the morphology of hippocampal neurons.In vitro,Ber and Ech are applied to protect glial cells HEB from senility induced by topoisomerase Ⅱ Etoposide(Eto).These exploration including molecular mechanism of anti-brain aging provides experimental and theoretical evidence for traditional Chinese medicine against brain aging.Method:1.HE staining to observe the effect of berberine combined with Cistanche on hippocampal neurons of aging mice;2.β-galactosidase staining method to detect the optimal concentration of Eto for HEB cell senescence;3.CCK8 cell proliferation test the effect of Ber combined with Ech on the viability of senescent HEB cells induced by Eto;4.β-galactosidase staining method to detect the effect of Ber combined with Ech on the senescence state of senescent HEB cells induced by Eto;5.Flow cytometry to detect the effect of Ber combined with Ech on Eto-induced senescent HEB cell cycle;6.Laser confocal to observe the effect of Bell combined with Ech on ROS oxidative stress level and nuclear morphology of senescent HEB cells induced by Eto.7.Western Blot to detect the effect of Ber combined with Ech on the expression of FoxO1 protein in Eto-induced senescent HEB cells and the effect of the downstream pathway of the transformation of antioxidant protein catalase,SOD2 expression and antioxidant protein Bcl-2 expression.Result:1.The effect of berberine combined with Cistanche on the neuronal cells of the hippocampus of aging miceHE staining results showed that most of the cells in the hippocampus CA1 and CA3 regions of normal aging mice had nuclear membrane dissolution,nuclear pyknosis,cell morphology changes,loose cell distribution and a small number.Mice in the administration group had normal cell morphology in the hippocampus CA1 and CA3 regions.The nuclear membrane is obvious and the nucleus is located in the center of the cell,with a large number of cells.There is no obvious difference between two groups in the hippocampus CA2 area.2.The optimal concentration of Eto for HEB cell senescence modeling and the appropriate concentration of Ber and Ech for HEB cellsThe results of β-galactosidase staining method showed that with the concentration of Eto increased,the staining of HEB cells gradually deepened,and the proportion of senescence-positive HEB cells increased.Finally,10-4 μM Eto was selected as the concentration of HEB cell senescence modeling;CCK8 cell proliferation was tested in Both Ber and Ech within the concentration range of 10-8-10-4 μM have no obvious toxic effects on HEB cells(P>0.05).10-4 μM was choosed as dosing concentration for the single use and 5×10-5 μM was used as dosing concentration for combined use.3.The effect of Ber combined with Ech on the viability of senescent HEB cells induced by EtoThe results of the CCK8 cell proliferation test showed that compared with the normal group,HEB cell viability was significantly reduced after Eto modelling for 24 h(P<0.05);Eto modelling was added for 24 h after treatment,compared with Eto modelling,Ber combined with Ech and Ech alone.The drug group can effectively restore the decreasing trend of HEB cell viability induced by Eto(P<0.05).The difference of HEB cell viability between the Ber alone drug group and the Eto model group was not statistically significant(P>0.05).4.The effect of Ber combined with Ech on the senescence state of senescent HEB cells induced by EtoThe staining results of β-galactosidase staining method showed that the proportion of senescence-positive cells increased after 24 h of Eto modeling(P<0.01).The Ber single drug group,the Ech single drug group and the Ber combined Ech group were all treated after 24 h of drug treatment,which can effectively reduce the proportion of senescence-positive HEB cells caused by Eto(P<0.01).5.The effect of Ber combined with Ech on the cell cycle of senescent HEB induced by EtoThe results of flow cytometry after PI staining showed that most of the HEB cell cycle was blocked in G2/M phase after 24 h of Eto modeling.Ech alone and Ber combined with Ech group can effectively reduce Eto block after 24 h.The proportion of HEB cells in G2/M phase(P<0.05),and the Ber alone treatment group had no significant effect on the G2/M phase cycle block of HEB cells caused by Eto(P>0.05).6.The effect of Ber combined with Ech on the oxidative stress level of ROS and the nucleus morphology of senescent HEB cells induced by EtoThe results of laser confocal fluorescence showed that compared with the ROS levels in HEB cells of the normal group,the fluorescence intensity of ROS in HEB cells increased significantly after 24 hours of Eto modeling,and the oxidative stress level increased significantly.Ber alone dosing group,Ech alone dosing group and combination group could significantly inhibit the increase of ROS levels in HEB cells caused by Eto after 24 hours of treatment.The results of Hoechst33342 fluorescence staining showed that compared with the normal group,there was no significant difference in the nucleus morphology of each group.7.The effect of Ber combined with Ech on the anti-apoptosis and anti-oxidant protein expression regulated by FoxO1 pathway in Eto-induced senescent HEB cellsWestern Blot showed that compared with the Eto model group,the Ech single-dosed group and the Ber combined Ech dosed group significantly promoted the expression of FoxO1,Bcl-2,Catalase and SOD2(P<0.05).The Ber group could significantly promote the expression of Foxol,Catalase and Bcl-2 in HEB cells(P<0.05),however,there was no significant effect on the expression of SOD2(P>0.05).Conclusion:1.The combination of berberine and Cistanche could effectively inhibit the depletion of neurons in the hippocampus of aging mice.2.Ber combined with Ech significantly increased the viability of senescent HEB cells induced by Eto and the proportion of senescence-positive HEB cells.3.Ber combined with Ech significantly reduced the proportion of HEB cells that were blocked in G2/M phase induced by Eto.4.Ber combined with Ech significantly reduced the oxidative stress level of ROS in HEB cells induced by Eto.5.Ber combined with Ech significantly up-regulated the anti-apoptosis and anti-oxidant protein expression regulated by FoxO1 pathway in HEB cells induced by Eto.