Effect Of Hydroxychloroquine On Nerve Cell Injury Induced By Anti-ribosomal P Protein Antibody And Its Mechanism

Systemic lupus erythematosus(SLE)is a chronic autoimmune disease,which can involve a variety of tissues and organs.Nervous system involvement is called neuropsychiatric lupus erythematosus(NPSLE),which is one of the important reasons for the poor prognosis of SLE.At present,the etiology and pathogenesis of NPSLE are not very clear,and the therapeutic effect is limited.Therefore,it is an important task for rheumatology to explore the pathogenesis of NPSLE and find effective treatment methods.Anti-ribosomal P protein(Anti-P)antibodies in sera of patients with SLE are considered to be closely related to NPSLE.Anti-P antibodies can bind to the neuronal surface P antigen(NSPA)on the surface of neurons and mouse neuroblastoma-2a(N2a),which leads to the increase of intracellular calcium concentration and apoptosis.However,there are few reports about the damage of other functions of nerve cells caused by Anti-P antibodies,and the pathway of intracellular calcium concentration increase caused by Anti-P antibodies is not clear.Hydroxychloroquine(HCQ)is an antimalarial drug,which has been widely used in the treatment of SLE,but the preventive and therapeutic effect of HCQ on NPSLE is not clear.This study revealed the injury effect of Anti-P antibodies on nerve cells and whether HCQ could alleviate the damage caused by Anti-P antibodies in vitro.At the same time,the possible mechanism of HCQ in alleviating nerve injury was also discussed.In addition,in view of the fact that N2 a cells have the characteristics of nerve cells and the composition of the cell line is uniform,N2 a cell line was used as the research object of neurons in vitro.The specific research content is divided into the following sections:Part 1: The effect of anti-ribosomal P protein antibodies binding to N2 a cells on cell function and activity.Objective: To determine the damaging effect of Anti-P antibodies on N2 a nerve cells.Methods: N2 a cells were stimulated with 100 μg/mL Anti-P IgG and serumIgG from healthy controls for immunofluorescence staining.Intracellular calcium concentration was detected by confocal microscopy in extracellular medium with and without calcium,and the release level of ATP was detected by kit.Cell viability was detected by CCK-8assay and apoptosis was detected by flow cytometry after treating N2 a cells with Anti-P IgG of 100μg/mL and 200μg/mL and serumIgG of healthy controls respectively.Results:1.Anti-P IgG specifically bound to N2 a cells on the cell membrane.2.Anti-P IgG increased the level of calcium in N2 a cells.3.Anti-P IgG could increase the release of intracellular calcium pool and promote the influx of extracellular calcium ions.4.Anti-P IgG promoted the release of ATP from N2 a cells.5.Anti-P IgG decreased the proliferation and viability of N2 a cells.6.Anti-P IgG promoted apoptosis of N2 a cells.Conclusion: Anti-P antibodies could specifically bind to N2 a cells,and the combination of them induced N2 a cell injury by promoting intracellular calcium release and extracellular calcium influx,increasing intracellular Ca2+ concentration and promoting cell release of ATP.It was suggested that Anti-P antibodies could damage the function of nerve cells and had neurotoxicity.Part 2: The protecting effect of hydroxychloroquine sulfate on Anti-P IgG-induced N2 a cell damage.Objective: To investigate whether hydroxychloroquine can alleviate the damage of N2 a cells induced by Anti-P IgG.Methods: Cells were treated with different concentrations of HCQ(5,10,15,20μg/mL).CCK-8 assay and trypan blue staining were used to detect cell viability and proliferation.N2 a cells were stimulated with 200μg/mL Anti-P IgG,serumIgG of healthy controls and5/10μg/mL HCQ and 200μg/mL Anti-P IgG,respectively.Intracellular calcium concentration was detected by calcium imaging,ATP release was detected by kit,cell viability and proliferation were detected by CCK-8 assay and trypan blue staining,and apoptosis was detected by flow cytometry.Results:1.HCQ had no cytotoxicity to N2 a cells in a certain concentration range.2.HCQ inhibited the enhancement of intracellular calcium signal induced by Anti-P IgG in N2 a cells.3.HCQ inhibited the increase of ATP release from N2 a cells induced by Anti-P IgG.4.HCQ alleviated the inhibitory effect of Anti-P IgG on the proliferation and activity of N2 a cells.5.HCQ reduced the apoptosis of N2 a cells induced by Anti-P IgG.Conclusion: A certain concentration of HCQ could alleviate the increase of intracellular calcium and ATP release of N2 a cells induced by Anti-P antibodies,and then reduced the damage of N2 a cells caused by Anti-P antibodies.It was suggested that HCQ might have neuroprotective effect.Part 3: Study on the mechanism of protective effect of HCQ on N2 a cells.Objective: To explore the mechanism of protective effect of HCQ on N2 a cells.Methods: N2 a cells were treated withIgG,Anti-P IgG,HCQ combined with Anti-P IgG to detect the expression of apoptosis-related proteins(Bax,caspase-3,Bcl-2)by Western Blot.N2 a cells were pretreated with HCQ and serum-free medium respectively,and the changes of intracellular calcium concentration was continuously monitored after Anti-P IgG was added in calcium-free extracellular medium.EDTA was added to extracellular solution to chelate calcium ion.The expression of 1,4,5-inositol trisphosphate receptor(IP3R)in N2 a cells was detected by Western Blot after being treated withIgG,Anti-P IgG and the combination of and Anti-P IgG.And the binding of Bcl-2 to IP3R was detected by immunofluorescence staining and co-immunoprecipitation assay.Intracellular calcium concentration was detected by flow cytometry,cell viability was detected by CCK-8 assay,and apoptosis was detected by flow cytometry after treating N2 a cells with HCQ combined with Anti-P IgG,Venetoclax combined with HCQ and Anti-P IgG respectively.Results:1.HCQ could regulate the expression of apoptotic protein.2.HCQ reduced the intracellular calcium release induced by Anti-P IgG,but did not affect the extracellular calcium influx.3.HCQ did not affect the expression of IP3R.4.HCQ increased the combination of Bcl-2 and IP3R.5.Inhibition of Bcl-2 expression reversed the effect of HCQ on calcium signal and its neuroprotective effect.Conclusion: HCQ could regulate the apoptosis pathway protein in N2 a cells and alleviated the strong intracellular calcium signal induced by Anti-P antibodies by increasing the binding of Bcl-2 and IP3R,so as to exert its neuroprotective effect.