Mechanism Of Jujuboside A On Lipopolysaccharide Induced Inflammation Of Microglia Cells And Neuroprotective Effect On Neuron

Neurodegenerative disease,such as Parkinson's disease(PD),Alzheimer's disease(AD),amyotrophic lateral sclerosis(ALS),etc.Most of them are caused by the damage of the normal function or structure of the specific neurons of the central nervous system.With the further exploration of the pathogenesis of the disease,the oxidative stress,excitatory toxicity,and so on,which have been imprisoned in the past,have been broken.It is considered that neuroinflammation permeates the pathogenesis of neurodegenerative diseases.one of these important participants is microglia.When stimulated by lipopolysaccharide(LPS)and other stimuli,microglia over-activate and release various inflammatory mediators.Jujuboside A is the active components of Semen Ziziphi Spinosae(SZS),which is commonly used to nourish the heart and soothe the mind.It can protect neurons and treat senile dementia mice,but whether Ju A can be used to treat neurogenic diseases by inhibiting neuroinflammation has not been reported.Objective: To explore the effect and mechanism of Ju A on LPS-induced inflammation on BV-2 cells,the effects of microglial activation on neuronal cells and the protective effect of Ju A on neuronal cells.Methods: BV-2 cells were treated with LPS for 24 h,and the neuroinflammatory model was established.The appropriate concentration of Ju A was used to pre-intervene,and the related indexes were detected.Detection of cell survival rate by MTT,Detection of NO content by Griess method,the expression of tumor necrosis factor(TNF-?)mRNA,interleukin(IL)-1?,IL-1? mRNA?iNOS mRNA?COX-2mRNA was detected by RT-PCR,Western blot was used to detect the expression level of iNOS?COX-2?nuclear factor kappa beta(NF-?B)?I?B??p-I?B??Akt?p-Akt?heme oxygenase 1(HO-1)? nuclear factor-E2-related factor 2(Nrf2).Immunofluorescence was used to observe the expression of NF-?B?Nrf2.Non-direct contact cell coculture system was established by using BV-2 cell conditioned mediumon HT-22 cells.The effect of BV-2 conditioned medium on survival rate of HT-22 cells was detected by MTT.Apoptosis of HT-22 cells was detected by Annexin V-PI double staining.The protein expression of cleaved-caspase-3 and Bcl-2/Bax was detected by Western blot.Results:(1)Through the MTT experiment,we can see that Ju A alone or combined LPS(1 ?g/mL)have no obvious influence on BV-2 cell survival rate within 0-10 ? M.Therefore,Ju A concentration of 2.5?5?10 ?M and LPS concentration of 1 ?g/mL are chosen as experimental concentration.(2)The Griess method showed that Ju A can inhibit LPS induced BV-2 cells NO generation;RT-PCR experiment demonstrated that Ju A ameliorated LPS induced the up-regulation of TNF-?,IL-1?,iNOS,COX-2 mRNA;Western blot analysis indicated Ju A pretreatment inhibited the release of iNOS,COX-2;confocal microscope was used to observe the morphological changes of BV-2 cell,compared with the normal group,cells stimulated by LPS become swollen and larger,while Ju A can reduce the variation of morphology,therefore the morphology tended to be normal.The results of these experiments showed that Ju A could inhibit the activation of inflammatory factors produced by LPS in BV-2 microglia.(3)Western blot analysis indicated that Ju A reduce the expression of p-I?B?/I?B??nuclear NF-?B p65 protein,up-regulated nuclear Nrf2 protein content,and immunofluorescence microscopy observation of NF-?B and Nrf2 into the nuclear situation,found that Ju A can inhibit the translocation of nuclear NF-?B p65,promote the translocation of nuclear Nrf2.The results showed that Ju A could inhibit the I?B?/NF-?B pathway and activate the Nrf2/ARE pathway to inhibit the inflammation of BV-2 cells.(4)Establishment of indirect contact Cell Co-culture system by BV-2 Cell conditioned medium,the results showed that Ju A-CM could reduce the HT-22 cell damage caused by L-CM detected by DAPI staining and Annexin V-PI double staining.The results showed that Ju A-CM could reduce HT-22 apoptosis.Westernblot assay showed that Ju A-CM could up-regulate the ratio of Bcl-2/Bax.The expression of cleaved-caspase-3 was decreased.Conclusion: In vitro experiments,It showed that Ju A can inhibit the neuroinflammation response induced by LPS-induced BV-2 microglial activation and protect HT-22 hippocampal neuron cells from toxic influence by activated microglia.These results indicated that Ju A could inhibit neuroinflammation by targeting the effects of anti-inflammation and anti-oxidation,Ju A may play a role in the treatment of neurodegenerative diseases caused by neuroinflammation.