The Role Of Aciatic Acid And Andrographolide In Targeting Mitochondria Against Neurodegenerative Diseases And Related Mechanisms

Background:It has been shown that oxidative stress,mitochondrial defects,and inflammatory responses are involved in the development of neurodegenerative diseases such as Alzheimer’s disease(AD)and Parkinson’s disease(PD).The typical pathological features of AD are senile plaques(SP),neurofibrillary tangles(NFT),and selective neuronal cell death in the brain.The main deposition of senile plaques in the brain is Aβprotein.The α-synuclein protein(α-syn)associated with PD is the major component of Lewy bodies.Overexpression of α-syn in cells leads to overproduction of mitochondrial oxygen free radicals and abnormal mitochondrial structure and function.In the pathogenesis of AD and PD,mitochondrial damage is an important link,and the neuroprotective mechanism targeting mitochondria has rarely been reported.Asiatic acid(AA)is an extract of Centella asiatica,a pentacyclic triterpene compound that has the effect of scavenging free radicals and protecting mitochondria,and can prevent depression.Our previous research found that AA also has neuroprotective effects,but its mechanism remains to be further studied.Andrographolide,the main active ingredient of Andrographolide,has antitumor activity by inhibiting tumor cell cycle and apoptosis,and also fights oxidative stress and improves diabetic dementia in mice.In addition,in APP/PS1 Alzheimer’s disease mice,andrographolide protects synaptic plasticity,promotes LTP inhibition of LTD,improves AD symptoms,and promotes neuronal regeneration in APP/PS1 mice by activating Wnt,inhibiting GSK-3β,and alleviating neurodegenerative diseases,however,the mechanism is unknown.Objective: In view of oxidative stress,mitochondrial dysfunction and inflammation are important factors in the development of AD and PD,protection of mitochondria,mitigation of oxidative stress and inhibition of inflammation are important for the prevention and treatment of such diseases.Our previous studies have shown that andrographolide protects mitochondrial homeostasis and inhibits the activation of inflammatory bodies in uncontrolled inflammation by inducing mitochondrial autophagy;andrographolide also scavenges ROS and protects rat brain mitochondria,or protect cerebral ischemia-reperfusion rat neuronal cells by inhibiting microglial inflammatory response.On this basis,this paper intends to analyze the role of asiatic acid and andrographolide in improving the symptoms of AD and PD by establishing AD and PD in vivo and in vitro models and applying drugs such as asiatic acid.To explore the possibility of preventing and treating AD and PD by protecting neuronal cell mitochondria and inhibiting microglia activation,and to reveal the possible mechanism of neurodegenerative diseases from mitochondria level,and to find suitable intervention targets.Methods: 1.Induction of mitochondrial defective cell oxidative damage model:Sodium azide(Na N3)was used as a specific inhibition of cytochrome c oxidase(COX)in the mitochondrial electron transport chain.Rotenone is used as an inhibitor of mitochondrial respiratory transport chain complex I,and combined with H2O2 to induce mitochondrial-deficient neuronal oxidative damage;direct application of Aβ-injured cells to mimic mitochondrial-deficient neuronal oxidative catalysis during AD and PD Stimulation,the process of triggering damage to nerve cells in specific brain regions.2.Analysis of cell activity and mitochondrial function: Cell viability was detected by MTT method;cell morphology was observed by microscope;mitochondrial membrane potential(MMP)was analyzed by flow cytometry.The above methods were used to observe the changes of mitochondria in nerve cells in this model,and the role of AA and andrographolide in regulating mitochondria,protecting neurons and regulating microglial inflammation.(1)In the mitochondrial mild injury model induced by sodium azide and rotenone(simulated AD,early PD),the number of mitochondria was analyzed by Mitotracker red fluorescent dye.The changes of mitochondria were detected,DCFHDA fluorescent dye was used to detect the production of ROS,and then the effect of AA on the cell survival rate of acute rotenone injury was detected by MTT assay.In addition,in the sodium azide-induced AD cell model,mitochondrial membrane potential were also examined.After adding AA,the same indicators were tested to evaluate the mitochondrial mechanism of AA neuroprotection.(2)In the PD in vitro experiment,the rotenone-injured SH-SY5 Y cells were used as a model to investigate the resistance of andrographolide to rotenone damage by detecting cell viability and apoptosis.Detection indicators include: oxidative stress and mitochondrial membrane potential,mitochondrial oxygen consumption,ATP content and mitochondrial morphology.The approximate process was as follows: SH-SY5 Y cells were treated with different concentrations of andrographolide for 3 h,and 30 μmol/L Rotenone was added for 6 h.The activity of the cells was detected by MTT assay;Apoptosis rate was detected by Annexin-V/PI double staining;DCFH-DA staining was performed.The intracellular ROS were analyzed.The changes of mitochondrial membrane potential were observed by JC-1 staining.The ATP content was detected by luciferase assay;the mitochondrial respiratory function was detected by Clark oxygen electrode;the MDA and SOD content of cells were determined by MDA and SOD kit.Analyze cellular oxidative stress.In addition,Mito Tracker green staining was used for flow cytometry to detect changes in mitochondria number.Through the above analysis,the mechanism of the protection of andrographolide against mitochondrial damage and the maintenance of mitochondrial morphology were investigated.3.Establishment and behavioral observation of mouse PD-like model: PD-like mouse model was constructed by MPTP to analyze the effect of andrographolide on behavior improvement of PD model of mice.Firstly,the behavioral changes of mice were detected by opening test,suspension test,climbing rod test,forced swimming test and runner test,and then the middle brain tissue was taken to detect oxidative stress index,inflammatory reaction,apoptosis and mitochondrial copy number.To explore the mechanism of andrographolide in improving the motor behavior of PD rats.(1)Modeling of Parkinson’s disease in mice: Model induction was induced by intraperitoneal injection of 25 mg/kg MPTP for 5 consecutive days.The intervention group was intraperitoneally injected with 2.5 mg/kg and 5 mg/kg andrographolide for 12 consecutive days.(2)In vivo test of mice: The changes of motor behavior were observed by opening test,climbing rod test,runner test,forced swimming and suspension test.Western Blot and immunohistochemistry were used to analyze the expression of tyrosine hydroxylase in the substantia nigra region;Nissl staining was used to detect brain protein synthesis;MDA,SOD and Catalase kits were used to analyze the oxidative stress level in the substantia nigra of mice;TUNEL staining was used to detect the apoptosis of neurons in the substantia nigra of mice.The expression level of inflammatory factors in peripheral blood was detected by ELISA.The m RNA expression level of inflammatory factors in mouse brain was determined by quantity Real-time PCR.The microglia was detected by immunofluorescence staining.Cell activation marker Iba-1.(3)In vitro test: SH-SY5 Y cells were treated with different concentrations of andrographolide for 3 hours,then 30 μmol/L Rotenone was added for 6 hours,and cell viability was detected by MTT assay;Annexin-V/PI double staining analysis Apoptosis rate;DCFH-DA staining method to detect intracellular ROS;JC-1 staining analysis of cell mitochondrial membrane potential changes;luciferase assay for ATP content;Clark oxygen electrode for detection of mitochondrial respiratory function.In addition,MDA and SOD kits analyze cellular MDA and SOD levels to understand cellular oxidative stress.After mitochondrial analysis by Mito Tracker green staining,the number of mitochondria was detected by quantity Real-time PCR.4.In the AD model: SH-SY5 Y cells were pretreated with AA at different concentrations(1,10,100 nmol/L)for 24 h,then Aβ1-42(10 μmol/L)was added for 24 h to induce cell damage and establish cells.model.Cell viability was detected by MTT assay,apoptosis was detected by flow cytometry and Hoechst 33342 staining,mitochondrial membrane potential was observed by JC-1 staining,intracellular ATP content was detected by luciferase assay,and cellular respiration function was analyzed by Clark oxygen electrode method.Mito Tracker Red staining was used to detect the number of mitochondria.The ROS content was detected by DCFH-DA fluorescent probe method.Bcl-2,Bax,cleaved-caspase 3,cyt C and PGC-1α(nuclear receptor peroxidase-proliferator were detected by Western blot).The expression level of the receptor γ coactivator 1α)is activated.In addition,we observed the effect of AA on the climbing ability and longevity of PD fruit flies,and examined the effect of AA on the levels of MDA and GSH in PD Drosophila.Results:1.Establishment of oxidative stress injury and mitochondrial damage model in SH-SY5 Y cells The results showed that for SH-SY5 Y cells,H2O2,rotenone and sodium azide began to produce significant damage at concentrations of 300μmol/L,10 nmol/L and 15mmol/L,respectively,and within a certain range,the degree of damage and drug presentation Dose dependence.Morphological observation also showed that the morphology of SH-SY5 Y cells changed significantly with the increase of drug concentration,which showed from the decrease of the protrusion to the disappearance,and the shrinkage of the cell body.2.AA has a significant intervention effect on rotenone-induced PD-like cell injury.(1)AA can exert neuroprotective effects by stabilizing the membrane potential of mitochondria.In rotenone-injured cells,the addition of AA counteracts the depolarization of mitochondrial membrane potential and the increase of ROS,and inhibits the up-regulation of BAX.The neuroprotective effects of AA were preliminarily observed by using SH-SY5 Y cell oxidative stress injury and mitochondrial injury model established in this study.The results showed that different doses of AA were able to resist 300 μmol/L H2O2 damage to cells 24 h before H2O2 stimulation of SH-SY5 Y cells.In addition,AA is also resistant to Na N3 or Na N3 + H2O2 induced SH-SY5 Y cell damage.Flow cytometry analysis of mitochondrial membrane potential revealed that AA slowed the decline of mitochondrial membrane potential in injured cells.This result suggests that the neuroprotective mechanism of AA may be related to the membrane potential of stable mitochondria.(2)AA exerts mitochondrial protective effects by inhibiting α-syn overexpression,mitochondrial translocation and Cyto C release,and down-regulating ROS and BAX.In the rotenone-induced mitochondrial mild injury model: 0.01–100 nmol/L AA can affect 100 nmol/L rotenone-induced PD-like cell damage.AA can counteract the mitochondrial membrane potential depolarization and ROS increase in rotenone-damaged cells and inhibit the up-regulation of BAX.In the PD-Drosophila model with α-syn overexpression: 0.5-2 mg AA /100 g medium can significantly improve the climbing ability of Drosophila and prolong its lifespan.The MDA content in PD Drosophila is significantly reduced,and the GSH content is significantly increased.This may be related to the antioxidant effects of AA.In α-syn-induced mouse brain mitochondrial injury model: AA can reverse the decrease of mitochondrial membrane potential induced by α-syn,stabilize membrane integrity and ATP synthesis,inhibit ROS production and apoptosis.The protective effect of AA is related to its prevention of α-syn mitochondrial translocation.In addition,in the mouse Parkinson’s disease model,we found that andrographolide can improve the motor function of PD-like mice,which can increase the distance of movement of PD-like mice in the opening test and the activity time in the central area after administration,shortening the climbing time and prolonging the balance time of the runner,improving the coordination ability of the PD-like mice during forced swimming and the grasping ability in the suspension test.At the same time,it can improve the level of oxidative stress and the expression level of inflammatory factors in the substantia nigra.Conclusion: This study used AD and PD models to detect mitochondrial function,Cyto C and α-syn expression,and explored the mitochondrial mechanism of AA or andrographolide against Aβ,α-syn or MPTP-induced neuronal injury,which is antioxidant.Centella asiatic acid can counteract cell damage induced by sodium azide and Aβ1-42 by regulating mitochondria-mediated apoptosis,protecting mitochondrial function,and increasing mitochondrial number.Andrographolide has the effect of protecting mitochondria against oxidative stress,indicating its potential to treat PD.The results suggest that mitochondria play an important role in the pathogenesis of AD and PD.The mitochondria-targeted drugs AA and andrographolide can be used as drug candidates for the prevention and treatment of AD and PD,and have further research and development value.Mitochondria can be used as an important target to study the mechanism of neurodegenerative diseases,and provide a new research idea for antioxidant and inflammatory factor injury,protection of nerve cells,regulation of microglia function and prevention of PD and AD.