Protective Effect And Mechanism Of Gansong On 6-OHDA-induced SH-SY5Y Cell Injury

Objective:Parkinson's disease(PD)is a progressive neurodegenerative disorder,the incidence of the aged is higher,the prevalence rate of the aged over 65 years old is 1%?2%.It has a serious impact on the health and quality of life of the patients.It is pathologically characterized by the progressive,generalized degeneration and selective loss of the dopaminergic neurons in the substantia nigra pars compacta(SNpc)and the appearance of alpha synuclein(alpha-synuclein)and Lewy body(LB).The loss of dopaminergic neuron can result in the decrease of dopamine content in the striatum.It is often accompanied by motor symptoms,such as tremor,gait disorders,rigidity and bradykinesia,and nonmotor symptoms(NMS)such as hyposmia,astriction and depression.The pathogenesis of PD is extremely complex,including genetic mutation,oxidative stress,mitochondrial dysfunction,immune system abnormalities,neuronal excitotoxicity,clumps of abnormal brain proteins,and so on,these factors can lead to cell apoptosis.The excessive apoptosis of dopaminergic neurons plays an important role in neurodegenerative diseases.Levodopa is the primary treatment for Parkinson's disease.However,its long-term use was limited by motor complications and drug-induced dyskinesia.It is an urgent need to find drugs that can prevent and delay the process of disease.Nardostachyos Radix et Rhizoma is the root and rhizome of Nardostachys jatamansi DC.It mainly contains terpenoids,which are mainly sesquiterpenoids.with a sedative,anticonvulsant,antiepileptic,antidepressant,regulating endocrine and other pharmacological effects.Studies showed that the ethanol extract of Nardostachyos Radix et Rhizoma has the ability to alleviate Parkinson's disease.Nardosinone is the main constituents of Radix et Rhizoma Nardostachyos which can promote the proliferation of primary cultured neurons and protect nerve cells against oxygen and glucose deprivation,it can also protect PC 12 cells against injury induced by corticosterone.It is evaluated that Radix et Rhizoma Nardostachyos plays a positive role in the prevention and treatment of Parkinson's disease.Therefore,the 6-OHDA-induced damaged SH-SY5Y neurocyte models were cultured outside of the body.We can evaluate the anti-Parkinson's activity of Nardostachyos Radix et Rhizoma and then investigate the active components and possible mechanisms.It provides some theoretical and experimental basis for the development of anti-Parkinson drugs.Methods1 The 6-OHDA induced damaged SH-SY5Y neurocyte models were cultured outside of the body.After treatment with different concentrations of 6-OHDA,the cell viability was determined by MTT assay,and the cell morphology was observed by microscope.And then the concentration of 6-OHDA was determined.2 Four fractions of Nardostachyos Radix et Rhizoma ethanol extract are Prepared.using SH-SY5Y cells damage induced by 6-OHDA as in vitro model,the protective effects of the four fractions are determined through MTT assay and the morphology of cells.3 The chemical constituents of petroleum of Nardostachyos Radix et Rhizoma was analyzed by using GC-MS method combined with HPLC method.4 First,we examined cell viability by MTT assay,Hoechst 33258 kit was used to stain the cells,LDH kit was used to detect the LDH activity of the supernatant,DCFH-DA fluorescent probe was used to detect the level of intracellular ROS generation.The purpose of the study is to investigate the protective effect of nardosinone on SH-SY5Y cells injured by 6-OHDA.5 Rhodamine123 was used to detect the change in mitochondrial membrane potential,and Western Blot analysis was used to detect the levels of protein expression related to mitochondrial apoptosis pathway and MAPK pathway,the purpose is to clarify the protective mechanism of nardosinone.ResultsThe main contents and results are as follows:1 The establishment of cell apoptotic model of Parkinson's diseaseThe viability of the cells was assayed by MTT method after incubation in different-concentration of 6-OHDA(25?M?50?M?100?M?150?M?200?M?300?M)for 24h.The viability of SH-SY5Y cells decreased with the increased dose of 6-OHDA,there was obvious dosage dependant.When the concentration of 6-OHDA was less than 50?M,the degree of cell damage was not enough.while cell survival rate was 50.87%when the concentration was 150?M,and the damage degree was appropriate.The cell morphology was observed by inverted microscope.The SH-SY5Y cells of control group had spindle-shaped,triangulate or polygonal cell body.Cellular body treated by 6-OHDA was corrugativus and even round.Therefore,150?M 6-OHDA was selected to induce SH-SY5Y cells to establish a PD cell model.2 Preparation of different fractions of Nardostachyos Radix et Rhizoma and screening of its anti-Parkinson activity2.1 Preparation of different fractions of Nardostachyos Radix et Rhizoma ethanol extractThe Nardostachyos Radix et Rhizoma were ultrasound extracted three times with 80%ethanol,and the filtrate was combined,reduced pressure concentration technology was used to condense to extract,and then suspended it with 10 times the amount of distilled water.Extracted to colorless by petroleum ether,ethyl acetate and N-butanol respectively.The extract was combined,and the extracts of various fractions are obtained by reduced pressure concentration technology.2.2 Screening the anti-Parkinson effect fractions of Nardostachyos Radix et RhizomThe extracts of Nardostachyos Radix et Rhizoma in Petroleum ether,ethyl acetate,N-butyl alcohol and 80%ethanol can protect SH-SY5Y cells from injured by 6-OHDA and improve the survival rates of SH-SY5Y cells.The petroleum extract has the strongest protective effect on SH-SY5Y cells,the inhibition rate is 49.53%,therefore,the petroleum extract of Nardostachyos Radix et Rhizoma was selected for further investigation.3 Analysis of the Chemical Constitution of Petrol extract of Nardostachyos Radix et RhizomaThe components and content of Petrol extract of Nardostachyos Radix et Rhizoma were identified by GC-MS and elucidated by the comparison with the standard mass spectral data.The relative contents in percentage were elucidated using the area normalization method.22 compounds were isolated and identified,mainly contains saturated alcohols,ketones,acid sand terpenoids.Among them,the content of sesquiterpene is the highest,accounting for 53.46%of the total amount.The main components are Ledeneoxide-(?)(23.91%),4a-7-Methano-4aH-naphth[1,8a-b]oxirene,octahydro-4,4,8,8-tetramethyl-,(1a?,4aa,7a,8aS)-(9 CI)(22.87%),-Epiglobulol(3.43%),(-)-Spathulenol(3.25%)and so on.It presumed that the active constituents of anti-Parkinson effects in Petrol extract of Nardostachyos Radix et Rhizoma may be sesquiterpenes.Nardosinone easily decomposed in high temperature,so HPLC method was used to determine the content of nardosinone in Petrol extract of Nardostachyos Radix et Rhizoma.The results showed that the content of nardosinone in Petrol extract of Nardostachyos Radix et Rhizoma was 30.21%,which was the main component.4 The protective effects of nardosinone on SH-SY5Y cells damaged by 6-OHDAThe cell viability was measured by MTT assay.6-OHDA decreased the viability of SH-SY5Y cells significantly(p<0.01),compared with the model group,nardosinone relived the cell damage induced by 6-OHDA.And there was no significant effect on cell viability by different concentrations of nardosinone.The result suggested that nardosinone protected SH-SY5Y cells from damaged by 6-OHDA.The cell morphology was observed by inverted microscope.The SH-SY5Y cells of control group had spindle-shaped,triangulate and polygonal cell body.Cellular body treated by 6-OHDA was corrugativus and even round.Compared with the model group,the morphology of cells protected by nardosinone tended to be normal and number of cells increased.The level of lactate dehydrogenase(LDH)in the cell supernatant was measured by LDH kit.We found that exposed to 6-OHDA markedly increased cell membrane permeability and LDH leakage from cells into culture medium,while treatment of SH-SY5Y cells with various concentrations of nardosinone inhibited 6-OHDA induced LDH leakage.The nuclear morphology was observed by fluorescence microscope after using the chromatin dye Hoechst33258.Cells injured by 6-OHDA obviously exhibited nuclear condensation,while the nuclear morphology of cells with the protection of nardosinone tended to be normal and the number of apoptotic cells decreased.The DCFH-DA fluorescence probe was used to detect the level of intracellular ROS generation,and we found that 6-OHDA elevated the levels of ROS,while treatment of SH-SY5Y cells with various concentrations of nardosinone reversed the levels of ROS.5 The mechanism of effects of nardosinone on SH-SY5Y cells damaged by 6-OHDAFirst the changes in mitochondrial membrane potential was detected by Rhodamine 123,the results showed that 6-OHDA reduced mitochondrial membrane potential,and nardosinone could increased the mitochondrial membrane potential.The levels of apoptosis-related protein Bax,Bcl-2,Cleaved caspase-3,Caspase-3,Cleaved caspase-9 and Caspase-9 in cells were detected by Western Blot analysis,and the results showed that compared with control group,6-OHDA elevated the ratios of Bax/Bcl-2,Cleaved caspase-3/Caspase-3 and Cleaved caspase-9/Caspase-9,and nardosinone could reduce three kinds of ratios,indicating that the nardosinone could inhibit mitochondrial apoptosis pathway.The levels of p-ERK,ERK,p-p38,p38,p-JNK and JNK were detected by Western Blot analysis,the results showed that compared with the control group,6-OHDA elevated the ratio of p-JNK/JNK and the ratio of p-p38/p38 has a rising trend.Nardosinone pretreatment could reduce the ratio of p-JNK/JNK,and the ratio of p-p38/p38 has a downward trend.Indicating that the nardosinone could inhibit the activation of JNK pathway,and had no influence on the ratio of p-ERK/ERK.The results showed that nardosinone could inhibit the activation of JNK pathway and inhibit apoptosis.Conclusion:In summary,the 6-OHDA-induced damaged SH-SY5Y neurocyte models were cultured outside of the body.The protective effect of Nardostachyos Radix et Rhizoma was investigated.The results showed that the extracts of Nardostachyos Radix et Rhizoma in Petroleum ether,ethyl acetate,n-butyl alcohol and 80%ethanol can protect SH-SY5Y cells from injured by 6-OHDA,and the Petrol extract of Nardostachyos Radix et Rhizoma has the strongest protective effect on SH-SY5Y cells.Among them,most of constitutents are sesquiterpenes,and nardosinone was the main component.The study showed that nardosinone has a significant protective effect on SH-SY5Y cells injured by 6-OHDA,through MTT assay,LDH release detection,Hoechst33258 staining and ROS detection.And the level of proteins was detected by Western Blot analysis.It is proved that the mechanism may be by inhibiting the activation of JNK signaling pathways,and then inhibiting cell apoptosis to achieve protective effect.Through the above research,it is clear that the protective effect and the mechanism of Nardostachyos Radix et Rhizoma and nardosinone on SH-SY5Y cells injured by 6-OHDA,which provides a new method and basis for the treatment of Parkinson's disease,and lays a foundation for the further development and utilization of Nardostachyos Radix et Rhizoma.