Protective Effect And Mechanism Of Astragaloside ? On Astrocytes Senescence

Parkinson's disease(PD),also known as tremor paralysis,is a type of age-related neurodegenerative disease,with more than 90%of patients over 50 years old.As the average life expectancy increases,the incidence of PD is increasing year by year.PD is characterized by the loss of nigrostriatal dopaminergic neurons and the deposition of Lewy body.The main manifestations are tremor,rigidity,slow movement and posture balance disorder.Cellular senescence refers to the phenomenon in which cells stop dividing due to irreversible cell cycle arrest.There are many reasons for cells aging,including oxidative stress,DNA damage,proto-oncogene activation,and the short chromatin telomere length.Recent studies have shown that cells aging,especially astrocytes aging,plays an important role in PD.Autopsy brain samples from patients with PD showed astrocytes aging more pronounced than non-astrocytes;the use of PD neurotoxin paraquat(PQ)can induce the human astrocytes senescence,and the soluble factors secreted by the induced human astrocytes can damage the function of neurons,and the aging cells can alleviate the neuropathological condition of PD mice induced by PQ.It also improved the behavior of model mice,suggesting that astrocyte senescence plays an important role in PD.China has a very long history of researching anti-aging natural products.Among them,the medicinal herb Astragalus,which was first recorded in the Shennong's Classic of Materia Medica in the Han Dynasty,which is recorded as a holy medicine for qi and solids.There is also a lot of research on anti-aging.Astragaloside ? is one of the important components of Astragalus membranaceus and it is the most biologically active triterpenoid saponin in the active ingredient of Astragalus.At present,astragaloside ? has been reported to have an effective protective effect against focal cerebral ischemia/reperfusion,cardiovascular disease,lung disease,liver fibrosis and diabetic nephropathy.Pre-laboratory work also confirmed the neuroprotective effect of astragaloside ? on PD model mice.In vivo,MPTP sub-acute model mice were prepared to observe the effect of astragaloside ?on the astrocytes aging of PD model mice.Natural senescent primary astrocytes were cultured in vitro,and PD-associated cell stimulation models(Lipopolysaccharide(LPS)model and(1-methyl-4-phenylpyridinium(MPP+)model)were applied.This study explored the protective effect and mechanism of astragaloside ? on aging of injury primary astrocytes.Object:To investigate the protective effect a nd the mechanism of astragaloside ? on senescent astrocytes.Methods:In vivo,4 months old mice with C57BL/6J genetic background were randomly divided into normal saline group,saline+astragaloside ? group,MPTP sub-acute model group,and astragaloside ?+MPTP sub-acute model group.1.To detect the expression of tyrosine hydroxylase(TH)in each group by Western Blot and detect the number of TH-positive neurons.2.To detect the co-localization of the astrocyte marker glial fibrillary acidic protein(GFAP)and aging related indexes p16Ink4a and Lamin B1 by immunofluorescence.Primary astrocytes were extracted from C57BL/6J newborn mice which born within 1 to 3 days.Aging primary astrocytes were obtained by increasing the number of days of culture and passage.After all cells were passaged for 2 times,the immunofluorescence co-localization confirmed that the positive rate of astrocytes was>95%,which was used for the experiment.The control group was administered to a young group of astrocytes in vitro for 20 days,and the experimental group was administered to an aged group of astrocytes for 40 days in vitro.The astrocytes of the young group and the aging group were administered with 0?M,1 ?M,10 ?M,50?M,100 ?M and 200 ?M of astragaloside ? respectively,and the medium containing the corresponding concentration of the drug solution was replaced on the 4th and 7th day of administration.1.The safe concentration range of astragaloside ? to primary astrocytes was detected by CCK-8.2.The expression of p16Ink4a protein in each group of astrocytes was detected by Western Blot.The Expression of Senescence-associated P-galactose(SA-?-gal)of each group was stained by SA-?-gal staining kit to observe the senescence-associated ?-galactose of each group.The expression of Lamin B1 in astrocytes by immunofluorescence co-localization.The expression of Senescence-associated secretory phenotype(SASP)in each group of astrocytes was detected by RT-PCR.Based on the above detection method,the optimal concentration of astragaloside ? against the injury of senescent astrocytes was selected.3.First,the astrocytes were pre-protected by astragaloside ?,and LPS and MPP+ stimulation were applied to the primary astrocytes to simulate astrocyte senescence.The protective effect of this concentration of astragaloside ? on the senescence of primary astrocytes under both stimuli was observed.4.To detect the mitochondrial damage and mitochondrial reactive oxygen species(ROS)production of primary astrocytes in each group by flow cytometry.5.Astragaloside ? protects against aging damage of astrocytes by promoting mitophagy.Compared with the young astrocytes,the expression of autophagy-related protein p62 was increased in the cytoplasmic proteins of astrocytes,and the ratio of LC3 ?/? was decreased.The expression of autophagy-related proteins Parkin and Pink1 in mitochondrial proteins increased and the expression of TOM20 decreased.It is indicated that the mitophagy function of aging astrocytes treated with astragaloside ? is promoted.6.The senescent astrocytes were divided into the senescent astrocytes group,the senescent astrocytes + autophagy inhibitor 3-Methyladenine(3-MA)group,the senescent astrocytes +administration group,the senescent astrocytes+administration+3-MA group.The expression of aging indicators in each group was examined by Western Blot,SA-?-gal staining and RT-PCR.Results:1 Astragaloside ? alleviated the loss of dopaminergic neurons in MPTP PD model mice.The expression of TH in the midbrain of the MPTP sub-acute model mice pre-protected by astragaloside ? increased.2.Astragaloside ? inhibits astrocytes senescence in MPTP PD model mice.MPTP treatment caused aging of mouse astrocytes,and the expression of pl61nk4a in astrocytes of substantia nigra pars compacta(SNpc)in mice with MPTP sub-acute pre-protected by astragaloside ? decreased,and the expression of Lamin B1 in astrocyte nuclear membrane increased.3.Astragaloside ? had no effect on the proliferation activity of primary astrocytes in the concentration range of 0-200 ?M.The primary astrocytes cultured for 20 days in vitro were treated with 0?M 1?M,10?M,50 ?M,100 ?M and 200?M of astragaloside,and the medium containing the corresponding concentration of the drug solution was replaced on the 4th and 7th day of administration.The survival rate of primary astrocytes in each group was measured by CCK-8 on the 10th day after administration.The results showed that astragaloside ? had no effect on the proliferation activity of primary astrocytes in the concentration range of 0-200?M.4.Astragaloside ? inhibits astrocytes senescence at 50 ?M.Astragaloside ? had no effect on the positive rate of SA-?-gal in primary astrocytes in young group.The expression of SA-?-gal in primary astrocytes was decreased at 50 ?M in the concentration range of 0-200 ?M.Astragaloside ? had no effect on the expression of primary astrocyte aging index p16Ink4a in the young group in the concentration range of 0-200 ?M.The expression of p16Ink4a in the aged astrocyte was reduced at 50 ?M.Astragaloside ? has no effect on the secretion of S ASP in primary astrocytes of young group,and can reduce the secretion of primary astrocyte SASP(such as IL-6,IL-1?,etc.)in aging group at 50?M.The young group of primary astrocytes SASP secretion has no effect,can reduce the secretion of primary astrocytes SASP(such as IL-6,IL-1?,etc.)in the aging group at 50?M.5.Astragaloside ? suppresses LPS/MPP+-induced astrocytes senescence at 50 ?M.LPS and MPP+ stimulation can cause aging damage of primary astrocytes,while 50?M pretreatment with astragaloside ? can reduce the expression of astrocyte aging index p16Ink4a and secretion of SASP caused by LPS and MPP+ stimulation.6.Astragaloside ? reduces mitochondrial damage in senescent astrocytes at 50 pM.The flow cytometry results of MitoSOX and JC-1 indicated that mitochondrial ROS production increased in astrocytes,mitochondrial damage increased,mitochondrial ROS production decreased and mitochondrial damage decreased significantly in 50?M aflatoxin-treated aging group.7.Astragaloside ? promotes mitophagy in senescent astrocytes.Compared with the young astrocytes,the expression of autophagy-related protein p62 was increased in the cytoplasmic proteins of astrocytes,and the ratio of LC3 ?/? was decreased.The expression of autophagy-related proteins Parkin and Pinkl in mitochondrial proteins decreased.Increased expression of TOM20 indicates that autophagy has occurred in aged astrocytes.The expression of autophagy-related protein p62 in astrocytes cytoplasmic protein of aging group treated with astragaloside ? decreased,the ratio of LC3 ?/? increased,the expression of autophagy-related proteins Parkin and Pinkl in mitochondrial protein increased,and the expression of TOM20 decreased.The autophagy function of aging astrocytes treated with astragaloside ? was promoted.8.Inhibition of mitophagy abolished the protective effect of astragaloside ? on astrocytes senescence.After autophagy was inhibited by autophagy inhibitor 3-MA pretreatment,scutellarin could not down-regulate the expressiol of astrocyte aging index p16Ink4a,decrease the positive rate of SA-?-gal cells and the secretion of SASP.Conclusions:1.Astragaloside ? alleviates the loss of dopaminergic neurons via inhibition of astrocytes senescence in MPTP sub-acute PD model mice.2.Astragaloside ? inhibits astrocytes senescence by promoting mitophagy.The major contributions of the present study lie in:.1.Astragaloside ? inhibits astrocytes senescence by promoting mitophagy.2.Astragaloside ? alleviates the loss of dopaminergic neurons via inhibition of astrocytes senescence in MPTP sub-acute PD model mice.