Study On The Function Of OCTN1 Transporter And Its Anti-Inflammation/Antioxidant Effect Using Bv2 Cell Model

Objective:In recent years,the physiological role of the OCTN1 transporter in the central nervous system has gradually attracted researchers’attention.A number of studies have found that its classic substrate ergothioneine has the effect of reducing oxidative stress and inhibiting inflammation in vitro.However,it is not clear whether OCTN1 plays a role in the progression of PD disease.In this study,a MPP⁺-BV2 cell model was constructed to explore whether the expression and function of the OCTN1transporter would change in PD,and whether the OCTN1 transporter substrate ergothione had a potential therapeutic effect in this model.Methods:（1）Construction of MPP⁺-BV2 cell model:BV2 cells were cultured with 100μM MPP⁺serum-free medium for 12 h after 24 h of seeding,optical microscope was used to observe cell morphology changes before and after modeling;flow cytometry to detect ROS fluorescence intensity;immunofluorescence staining to detect NF-κB p65 nuclear factor entering the nucleus;real-time fluorescent quantitative PCR to detect inflammatory factors（IL-6,IL-1β,TNF-α）and OCTN1 gene expression levels;Western blotting measures the expression levels of OCTN1 total protein,membrane protein,cytoplasmic protein and BV2 cell activation marker Iba-1 protein;ELISA kit measures IL-6、IL-1βand TNF-αprotein levels.（2）Experiment of ERGO uptake:In order to verify the changes in the uptake function of OCTN1 protein before and after modeling,the OCTN1 specific substrate ERGO was selected to investigate the changes in the uptake of ERGO by BV2 cells before and after modeling.Time-dependent assay:500μM ERGO uptake buffer was incubated for 0、0.25、0.5、1、1.5、2、4、6 h,samples were collected and LC-MS/MS was used to determine the concentration,and the protein concentration determined by BCA assay was used for correction;concentration-dependent assay:0、20、50、100、200、500、1000、2000μM ERGO uptake buffer was used to incubate for 1 h,and collected the samples for concentration determination and correction;the influence of the MPP⁺on the uptake:After incubating the BV2 cells with 0、25、50、100μM MPP⁺,then incubating with500μM ERGO uptake buffer for 1 h,the samples were collected and the concentration was measured and corrected;the inhibitor’s effect on uptake:After incubating BV2 cells with 100μM quinidine and 500μM ERGO uptake buffer for 1 h.After collecting the samples,the concentration is measured and corrected.（3）Knockdown of OCTN1 in BV2 cells:si-Octn1 was used to interfere with the Octn1 gene of BV2 cells,then measured the expression of OCTN1 protein by WB;incubated with 500μM ERGO for1 h to determine the uptake of ERGO followed by correction.（4）Study on ERGO anti-oxidative stress:0、75、150、300μM ERGO and 100μM MPP⁺were used to incubate for 12 h,detected the fluorescence intensity of ROS by flow cytometry;the concentration of GSH and NO products was determined by commercially available kit.（5）Study on ERGO inhibiting NF-κB and MAPK signaling pathways:0、75、150、300μM ERGO and 100μM MPP⁺were used to incubate for 12 h.WB was used to determine the expression levels of related proteins;ELISA kits were used to determine inflammatory factors（IL-6、IL-1β、TNF-α）level.（6）Molecular docking to study the mechanism of ERGO’s anti-inflammation effect:Autodock software was used to simulate molecular docking of ERGO and related proteins of inflammatory pathways to obtain binding energy,site of action,spatial structure and other information;To search for potential target proteins that can interact with ERGO,STRING database was used to search OCTN1-related proteins.Results:（1）Evaluation of MPP⁺-BV2 cell model:observation results of optical microscope demonstrated that BV2 cells showed obvious swelling in morphology after12 h of incubation with 100μM MPP⁺,and the cell antennae branches were shortened,which was in line with the morphological characteristics of successful modeling;100μM MPP⁺nuclear fluorescence showed a high degree of overlap with the fluorescence signal of NF-κB p65,which indicated that the treatment of 100μM MPP⁺to induce BV2 cells can effectively translate NF-κB p65 nuclear factor into the nucleus;WB experiments results showed that 100μM MPP⁺increased the activation marker of BV2cells（Iba-1）,OCTN1 total protein,OCTN1 membrane protein to 1.58、1.92、1.66 times;the mRNA and protein levels of IL-6、IL-1βand TNF-αin the model group were significantly increased;the ROS level in the model group was significantly increased;GSH level was significantly reduced.（2）Experiments of ERGO uptake:time-dependent experiments showed that linear uptake was observed with 1 h,concentration-dependent experiments showed that linear uptake was observed under the substrate concentration of 500μM;after incubating BV2 cells with 0、25、50、100μM MPP⁺for 12 h,the V_max value increased and the K_m value decreased,indicating that the uptake function of OCTN1 was also increased when the concentration of the model agent increased;inhibition experiments showed that 100μM quinidine can effectively inhibit the uptake of ERGO in both control and model cells,and the relative inhibition rates were 28.63%and 72.93%,respectively.The results indicated that the uptake of ERGO in the model group was more likely to be inhibited.（3）Model of Octn1 knockdown:si-RNA targeting Octn1 reduced the OCTN1 protein level to 32.77%.The ERGO uptake experiments showed that after knocking down OCTN1,the cellular uptake dropped to36.90%,indicating that the uptake function of OCTN1 is significantly reduced.The effect of OCTN1-knockdown is ideal.（4）ERGO anti-oxidative stress:the high-dose treatment group（300μM ERGO）can effectively reduce the average fluorescence intensity of ROS（from 171.35%to 124.94%）and increase the GSH concentration（from 44.43%to 60.74%）,reduce the concentration of NO products（from 191.19%to157.38%）and reduce the expression level of i NOS protein（from 191.53%to 158.18%）.After knocking down OCTN1,the above treatment effects were significantly weakened,indicating that the anti-oxidative stress effect of ERGO is mediated by OCTN1 uptake.（5）ERGO inhibits inflammation:the high-dose treatment group（300μM ERGO）can effectively reduce the inflammatory factors IL-6（from 155.28%to 112.92%）,IL-1β（from 148.85%to 106.07%）,TNF-αdecreased from（156.59%to 89.98%）,p-p65（from180.87%to 104.46%）,p-IκBα（from 181.60%to 101.59%）,p-p38（from 189.17%to108.87%）,p-ERK（from 168.66%to 107.01%）and p-JNK（from 195.69%to 101.89%）protein level.Immunofluorescence results showed that the overlap of the NF-κB p65fluorescence region and the nuclear fluorescence region after 300μM ERGO treatment decreased,indicating that the activation of the NF-κB pathway was inhibited.After knocking down OCTN1,the above therapeutic effects were significantly weakened,indicating that the anti-inflammatory effect of ERGO is mediated by OCTN1 uptake.（6）Molecular docking:The binding energy of ERGO and NF-κB-p50 is-5.75 kcal/mol,and the inhibition constant is 61.85μM,suggesting that ERGO has a certain inhibitory effect on the NF-κB signaling pathway;the binding energy of ERGO to the ERK1protein of the MAPK signaling pathway is-6.17 kcal/mol and the inhibition constant is30.16μM,suggesting that ERGO has a certain inhibitory effect on the MAPK pathway;the binding energy of ERGO to the AKT1-p H site is-5.74 kcal/mol,and the inhibition constant is 62.30μM,suggesting that ERGO may have an inhibitory effect on the PI3K/AKT signaling pathway;the binding energy of ERGO and SYK protein is-5.32kcal/mol,and the inhibition constant is 126.09μM,suggesting that SYK protein is a potential therapeutic target of ERGO.Conclusions:This study successfully used MPP⁺to induce BV2 cell to construct a PD model.The gene and protein levels along with uptake function of OCTN1 in the model group cells were significantly up-regulated.ERGO exerted an anti-oxidative stress effect by reducing the concentration of ROS and NO products,increasing the concentration of GSH;it exerted an anti-inflammation effect by reducing the secretion of inflammatory factors and inhibiting the activation of NF-κB and MAPK signaling pathways.After knocking down OCTN1,the above effects were significantly reduced,indicating that the therapeutic effect of ERGO is mediated by OCTN1 uptake.The molecular docking results showed that ERGO has an inhibitory effect on the NF-κB and MAPK signaling pathways,and may also have an inhibitory effect on the PI3K/AKT signaling pathway and SYK protein.