| Background:Parkinson’s disease(PD)is a common neurodegenerative disease with complex pathogenic factors and unclear pathogenesis.The major pathological change are interactions of aging,family genetics,environmental toxins,and cerebrovascular diseases,these factors lead to the injury of Dopaminergic(DA)neurons and then lead to the decrease of DA content in the brain,as well as the misfolding and abnormal aggregation of α-synuclein(α-syn).The pathogenesis of PD is still unclear,but current studies mainly focus on family inheritance,misfolding and abnormal aggregation of α-syn,neuroinflammation,mitochondrial dysfunction,oxidative stress,ubiquitin-proteasome system and autophagolysosome damage,and excitatory amino acid toxicity.Limb tremor is the first symptom in most of PD,followed by motor symptoms such as static tremor,movement disorders and myotonia.With the progression of the disease,non-motor symptoms such as abnormal mental activity,defecation dysfunction,cognitive function decline and sleep disorders will also occur.In modern medical treatment of PD,motor symptoms are mainly treated with DA supplement,while non-motor symptoms are treated with other drugs.However,long-term drug therapy may cause complications such as motor disorders,mental disorders and autonomic nervous symptoms,which seriously affect the prognosis of patients.After thousands of years of precipitation,Traditional Chinese medicine(TCM)takes "treatment based on pattern differentiation" as its main therapeutic policy.Clinical TCM believes that the pathogenesis of PD is "deficiency in origin and excess in superficiality",and the treatment mainly focuses on tonifying kidney and spleen and lung,which can play a good role in "enhancing effect and reducing toxicity".Scutellaria baicalensis is the main medicinal material in Chinese herbal compound Chaihu and Longgu Muli Decoction to treat PD,based on our previous study that this decoction could improve the motor symptoms and depression of PD rats.So we considered that Baicalein(BAI),the main flavonoid in Scutellaria baicalensis,also has a good anti-PD effect,and it has various pharmacological effects such as anti-oxidation,anti-inflammatory and anti-depression.Lipopolysaccharide(LPS)is a strong activator of immune cells,capable of acting on toll-like receptor 4(TLR4)on the cell surface.LPS can activate microglia in vivo,leading to the activation of ionized calcium-binding adaptor(Iba1)and inflammatory cytokines,and causing PD-related behavioral disorders in mice.The purpose of this study was to induce inflammatory activation of mouse BV-2 cells by using Lipopolysaccharide(LPS),and then used the activated BV-2 cell culture supernatan as the conditioned medium to culture SH-SY5Y cells to simulate the neuroinflammatory response in the brain of PD patients and induced dopaminergic neuron damage model.The protective effect and mechanism of BAI intervention on inflammation and neurons in this model were observed,and the related pathways and targets of BAI anti-PD were explored.Objective:To explore the protective effect and mechanism of different concentrations of BAI against injury of SH-SY5Y cells treated with conditioned medium to induce BV-2 cell activation.Methods:This study was conducted mainly from the following two levels.BV-2 cells used 1μg·mL-1 LPS to induced activation,establishing PD inflammatory response model in vitro,and set up the blank group,model group,BAI low,medium,high concentration group.BAI low,medium,high concentration group separately pretreated with BAI 4、8、16 μM for 4 h,then together with the model group used 1μg·mL-1 LPS to induced for 24 h.After being treated with BAI and LPS,BV-2 cells were observed by inverted phase contrast microscope and photographed to observe the effect of different concentrations of BAI on the morphology of LPS-activated BV-2 cells.Cell proliferation activity assay(CCK-8)kit was used to determine the cell viability of BV-2 cells in each group,and the effects of different concentrations of BAI on the proliferation activity of LPS-activated BV-2 cells were observed.The contents of tumor necrosis factor(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in the supernatant of BV-2 cells of each group were detected by enzyme linked immunosorbent assay(ELISA),and the effect of different concentrations of BAI on the secretion of LPS-activated pro-inflammatory factor in BV-2 cells were observed.BV-2 cells used 1μg·mL-1 LPS to induced activation,at the same time set up the blank group,model group,BAI low,medium,high concentration group(The final concentrations of BAI were 4、8、16 μM),and SH-SY5Y cells also set up the blank group,model group,BAI low,medium,high concentration group.SH-SY5Y cells were cultured with the supernatant of BV-2 cells which treated with BAI and LPS as conditioned medium for 12 h.After the SH-SY5Y cells were cultured in conditioned medium,the SH-SY5Y cells in each group were observed by inverted phase contrast microscope and photographed to observe the effect of LPS-activated BV-2 cell conditioned medium treated with different concentrations of BAI on the morphology of SH-SY5Y cells.The cell viability of SH-SY5Y cells in each group was detected by CCK-8,and the effect of LPS-activated BV-2 cell conditioned medium treated with different concentrations of BAI on the proliferation viability of SH-SY5Y cells were observed.Immunohistochemical staining(IHC)was used to observe the expression ofα-synapse nucleoprotein(α-syn)and tyrosine hydroxylase(TH)protein in SH-SY5Y cells to observe the protective effect of LPS-activated BV-2 cells conditioned medium treated with different concentrations of BAI on SH-SY5Y cells.The nuclear transfer of nuclear factor kappa-B p65(NF-κB p65,p65)in SH-SY5Y cells were observed by immunofluorescence(IF)method.Protein expressions of the toll-like receptor 4(TLR4),p65,phosphorylated nuclear factor kappa-B p65(p-NF-κB p65,p-p65),myeloid differentiation factor 88(MyD88)and phosphatase and tensin homolog deleted on chromosome ten(PTEN)in SH-SY5Y cells were detected by Western blot(WB)to observe the effect of LPS-activated BV-2 cell conditioned medium treated with different concentrations of BAI on TLR4/MyD88/NF-κB signaling pathways in SH-SY5Y cells.Results:In the test of LPS-activated BV-2 cells with different concentrations of BAI,morphological observation and CCK-8 results showed that compared with the blank group,cell body of BV-2 cells in the model group became larger,cell processes became longer and more,presented amoeba-like appearance,and cells vitality was significantly decreased(P<0.01),compared with the model group,in BAI low,medium and high concentrations groups BV-2 cells swelled the shorter and more decrease and cells vitality significantly increased(P<0.01).ELISA results showed that compared with the blank group,the contents of TNF-α,IL-6 and IL-1β in supernatant of BV-2 cells in the model group were significantly increased(P<0.01),Compared with the model group,in BAI low,medium and high concentrations groups TNF-α in the supernatant fluid were significantly lower(P<0.01),the content of IL-6 in the supernatant fluid reduced in the BAI high concentration(16 μM)(P<0.05)and the content of IL-1β in the supernatant fluid in BAI medium and high concentration decreased significantly(P<0.01).Results of SH-SY5Y cells treated with conditioned medium in each group showed,compared with the blank group,SH-SY5Y cells in the model group grew in clumps,crumpled,ruptured and damaged seriously,and cell vitality decreased significantly(P<0.01),compared with the model group,SH-SY5Y cells form of BAI low,medium and high concentrations groups gradually restore the spindle,damage reduction by decreases,and the boundary is clear,and the cell vitality were significant increase(P<0.01).The results of IHC method showed that compared with the blank group,TH protein expression in SH-SY5Y cell cytoplasm in the model group was significantly decreased(P<0.01),and α-syn expression was significantly increased(P<0.01),compared with the model group,in BAI low,medium,and high concentration groups,SH-SY5Y cells TH protein expression in the cytoplasm were significantly increased(P<0.01),α-syn protein expression significantly reduced(P<0.01).IF method results show that,compared with the blank group,the model group p65 protein expression in SH-SY5Y cells accumulated in the nuclear,and enhanced(P<0.01),compared with the model group,BAI low,medium and high concentrations groups p65 protein expression in SH-SY5Y cells gradually dispersed into the cytoplasm,and the expression level decreased gradually.WB method showed that compared with the blank group,the protein expressions of TLR4,MyD88 and p-p65 in SH-SY5Y cells of the model group were increased(P<0.05,P<0.01,P<0.01),while the protein expression of PTEN was decreased(P<0.05),compared with the model group,the BAI high concentration group of TLR4,MyD88,p-p65 protein expression reduced(P<0.05,P<0.01,P<0.05),the BAI medium concentration group of p-p65 protein and MyD88 protein expression reduced(P<0.05,P<0.05),the BAI low concentration group of MyD88 protein expression reduced(P<0.05),BAI low,medium and high concentrations PTEN protein expression were increased(P<0.05,P<0.05,P<0.01),while p65 protein expression there was no statistically significant difference in all groups.Conclusion:BAI can inhibit the activation of BV-2 cells caused by LPS,so as to inhibit the inflammatory response,and further inhibit the damage of inflammation to SH-SY5Y cells and protect SH-SY5Y cells.Its mechanism of action may depend on the regulation of TLR4/MyD88/NF-κB signaling pathway,so as to reduce the inflammatory response and play a neuroprotective role. |
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