| China has an abundance ethnic traditional medicine.Mongolian medicine is an important part of traditional medicine.It has a relatively complete theoretical system,rich long-term clinical practice,and relatively complete literature records.Mongolian Gashun-Baoriqige,Sophora alopecuroides L,belong to the swallowtail subfamily Leguminous genus sophora alopecuroides,distributed in northwest desert and semi-desert areas of China.The grasses and seeds have been used to deheat and dehumidify for thousands of years.It has the functions of clearing away heat and detoxification,dispelling dryness,and dampness,relieving pain and killing insects.It is often used for the treatment of sore throat,acute dysentery,itchy skin,stomachache,and other diseases.In our previous study,we found that a new compound LY01 in Gashun-Baoriqige,which has the structure of alkaloid and isoflavone,could resist nerve injuries,reduce chronic neuroinflammation,affect the migration and differentiation of neural stem cells,and has the neuroprotective effect in anti-neurodegenerative disease,but the molecular mechanism is still unclear.Parkinson’s Disease(PD)is the second common neurodegenerative diseases in the world.It is estimated that there Parkinson’s patients in China will increase to 4.94 million in 2030,accounting for half of all Parkinson’s patients in the world.The pathogenesis of Parkinson’s disease is still unclear.In recent years,the role of exosomes in neurodegenerative diseases has attracted attention.Exosomes are a kind of membranous vesicles released from the cell into the external environment.They are composed of lipids containing transmembrane proteins and a core.Exosomes play an important role in information communication between cells and biomolecule transfer and are involved in many physiological and pathological processes.ObjectiveIn this study,the neuroprotective effect of LY01 on Parkinson’s disease was investigated in vivo and in vitro,the regulatory effect of LY01 on exosomes in Parkinson’s disease was studied,and the mechanism of the neuroprotective effect of LY01 in Parkinson’s disease model was explored from the aspects of behavioral pathology and molecular biology.This research is divided into four parts.Part I:Neuroprotective effect of LY01 on Parkinson’s disease model miceMethods1)According to the reported method established the C57BL/6 mice model of Parkinson’s disease,MPTP was intraperitoneally injected(20 mg/kg,every 2 h for a total of four doses).The mice were randomly divided into 6 groups LY01 was administered at concentrations of 0.067 mg/kg,0.2 mg/kg,and 0.6 mg/kg 1 hour before the first injection of MPTP,and once daily for 10 days thereafter;2)Rotarod tests were used to evaluate the motor coordination and balance ability of mice;3)Immunofluorescence,immunohistochemistry,and western blotting techniques were used to detect dopaminergic neurons and TH protein levels in the substantia nigra compact and striatum tissues;4)RT-PCR were used to detect the mRNA level of in the substantia nigra compact and striatum tissues;5)Biochemical method to detect serum superoxide dismutase and malondialdehyde levels;6)Western blotting techniques were used to detect Bcl-2 and Bax protein levels in substantia nigra compact and striatum tissues.Results1)LY01 significantly increased the residence time on the rotarod in Parkinson’s disease model mice(p<0.01);2)LY01 significantly decreased the loss of dopaminergic neurons in the substantia nigra compactus(p<0.05),increased the nerve fibers of dopamine neurons in the striatum tissue,and increased the level of tyrosine hydroxylase protein in the substantia nigra compactus and striatum tissue(p<0.05);3)LY01 significantly decreased the mRNA expression levels of IL-1β and IL-6 and increased the mRNA levels of IL-4 and TGF-β(p<0.05);4)LY01 significantly decreased the level of malondialdehyde(p<0.001),increased the level of superoxide dismutase(p<0.01);5)LY01 significantly reduced the level of Bcl-2/Bax in substantia nigra and the striatum(p<0.05).ConclusionsLY01 could alleviated MPTP-induced impaired motor coordination,reduce the loss of neurons in the substantia nigra and the striatum tissue,relieve oxidative stress injury,relieve neuroinflammation,and reduce apoptosis in mice.Part Ⅱ:Protective effect of LY01 on cell model of Parkinson’s diseaseMethod1)After pretreatment with 3 concentrations of LY 01(3.125 M,6.25 M,12.5 M)for 1 h,SH-SY5Y was induced by MPP+.The cells were treated together for 24 h;2)SH-SY5Y cell viability was detected by MTT assay;3)The levels of total SOD and MDA were determined by biochemistry;4)The anti-inflammatory evaluation model was established by using lipopolysaccharide(LPS)to induce mouse microglia BV2 cells.IL-1β,IL-6,TNF-α and iNOS were detected by RT-PCR after pretreatment with different concentrations of LY01(3.125,6.25 and 12.5 μM)for 1 h and then added into LPS medium.Results1)LY01 at 3.125,6.25,12.5 and 25 μM increased the viability of MPP+induced SH-SY5Y cells(p<0.05,p<0.01,p<0.05,p<0.01).2)LY01 at 3.125,6.25 and 12.5 μM significantly increased the T-AOC and SOD of SH-SY5Y cells(p<0.05,p<0.05,p<0.05),and decreased the MDA(p<0.01,p<0.01,p<0.05).3)The mRNA expression levels of IL-1β,IL-6,TNF-α and iNOS were significantly decreased by 3.125,6.25 and 12.5μL LY01(p<0.01,p<0.05).ConclusionsLY01 improved the decreased activity of SH-SY5Y cells induced by MPP+and alleviated oxidative stress injury.The inflammatory response of BV2 cells induced by LPS was reduced.Part III:Regulation of LY01 on exosomes in mice with Parkinson’s diseaseMethods1)The mice were randomly divided into three groups:normal control group,model control group,and LY01 high-dose group.The mice were intraperitoneally injected 0.6 mg/kg LY01 1 hour before the first injection of MPTP,and then once a day for 10 days;2)Widely targeted metabolomic analyses were used to assess differences in serum and brain exosome metabolites between healthy controls and MPTP-induced PD mice;3)Widely targeted metabolomic analyses were used to assess differences in serum exosome metabolites between MPTP-induced PD mice and LY01-mice.Results1)Metabolomics results showed that there were 69 significant differences in exosome metabolites in serum and 148 significant differences in exosome metabolites in brain tissue between PD model mice and control mice;2)There were 15 significant differences in exosome metabolites in serum between PD model mice and LY01 mice;3)Differentially expressed metabolites were significantly enriched in tyrosine metabolism pathways purine metabolism,niacin and nicotinamide metabolism,and phenylalanine tyrosine and tryptophan biosynthesis,which are associated with Parkinson’s disease.ConclusionsThe mechanism of LY01 in the treatment of Parkinson’s disease is related to the regulation of the exosome metabolite L-dopamine,which is related to the tyrosine metabolism pathway purine metabolism pathway,nicotinic acid and nicotinamide metabolism pathway,and phenylalanine tyrosine and tryptophan biosynthesis pathway.Part Ⅳ:Blood exosomes have neuroprotective effects in a mouse model of Parkinson1 disease.Methods1)Exosomes derived from blood of healthy volunteers were injected into MPTP-induced C57BL/6 mice to establish a Parkinson’s disease mouse model for 4 times;2)Rotarod tests were used to evaluate the motor coordination and balance ability of mice;3)Immunofluorescence,immunohistochemistry and western blotting techniques were used to detect dopaminergic neurons and TH protein levels in the substantia nigra compact and striatum tissues;4)IL-1β,IL-6,TNF-α,IL-4,IL-10 and TGF-β in the substantia nigra compact and striatum tissues;5)Biochemical method to detect serum SOD and MDA;6)Western blotting techniques were used to detect Bcl-2 and Bax protein levels in substantia nigra compact and striatum tissues.Results1)Exosomes significantly increased the residence time on the rotarod in Parkinson’s disease model mice(p<0.01);2)Exosomes significantly decreased the loss of dopaminergic neurons in the substantia nigra compactus(p<0.0001),increased the nerve fibers of dopamine neurons in the striatum tissue,and increased the level of tyrosine hydroxylase protein in the substantia nigra compactus and striatum tissue(p<0.05);3)Exosomes significantly decreased the mRNA expression levels of IL-1β and IL-6,and increased the mRNA levels of IL-4 and TGF-β(p<0.05);4)Exosomes significantly decreased MDA(p<0.001)and increased SOD(p<0.05);5)Exosomes significantly reduced the level of Bcl-2/Bax in substantia nigra and the striatum(p<0.05).ConclusionBlood derived exosomes from healthy volunteers alleviated MPTP-induced impaired motor coordination in mice,saved the loss of dopaminergic neurons in the substantia nigra and striatum of PD model mice,and restored homeostasis of oxidative stress,neuroinflammation and apoptosis in PD model mice.In conclusion,this study confirmed that LY01 has neuroprotective effects on Parkinson’s disease model mice and cells,and found that the mechanism is related to the regulation of exosomal metabolites by LY01,and further confirmed that exosomes are involved in the pathogenesis of Parkinson’s disease.This study provides a theoretical basis for the clinical use of the traditional ethnic medicine Gashun-Baoriqige,provides a research basis for the neuroprotective effect of LY01,and increases its research value. |
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