Study On The Mechanism Of Xiao Xuming Dection Cutting On BV-2 Cell Inflammation Through Exosomes

Purpose:Stroke is one of the leading causes of morbidity and mortality worldwide,and the inflammatory response is closely linked to a range of brain injuries including blood-brain barrier disruption,brain oedema,neuronal necrosis and apoptosis after stroke,of which microglia(B V-2)activation is central to post-stroke neuroinflammation.Since ancient times,Xiao Xuming Dection(XXMD)has been effective in the treatment of stroke,and its mechanism may be related to post-stroke inflammation.Based on the results of our preliminary experiments,we found that XXMD has anti-LPS-induced macrophage inflmmation effect,but there are 12 herbs in the XXMD formula,which has complex composition,Therefore,in this study,based on the anti-inflammatory effect of drugs,XXMD Chinese herbal medicines were cut to obtain a simpler Xiao Xuming Dection Cutting(XXMD-C).Since exosomes secreted by cells are also involved in the development of a series of brain injuries,Chinese herbal medicine or Chinese herbal compound prescriptions can interfere with the miRNA in exosomes to treat these injuries.Therefore,in this study,XXMD-C was used as the study subject to establish an inflammation model using LPS-induced BV-2 cells to simulate the inflammatory response after stroke,and to investigate the protective effect of XXMD-C on inflammatory injury This will provide a scientific basis for the application of the new compound in post-stroke neuroinflammation.Methods:1.Anti-inflammatory study:LPS-induced BV-2 cells were used to construct an inflammation model,with different concentrations of XXMD(10 mg·mL-1,5 mg·mL-1,2.5 mg·mL-1)and XXMD-C(5 mg·mL-1,2.5 mg·mL-1,1.25 mg·mL-1).Cell viability assay and analysis of interleukin 1β(IL-1β),nitric oxide(NO),tumour necrosis factor alpha(TNF-α)and interleukin 6(IL-6)expression in cells by inflammation kits and Western blot.2.Quality analysis study:The chromatographic analysis was performed on a Kinetex XB-C18(100 mm×21 mm,26 μm)column set at 30 ℃ with 0.1%formic acid water(A)-acetonitrile(B)solution as the mobile phase and a gradient elution at a flow rate of 250μL·min-1.The mass spectrometry analysis was performed in positive and negative ion scanning mode using an ESI ion source for the rapid qualitative analysis of the main chemical components of the four herbs in XXMD-C.3.Construction of a "self-interactive dialogue" model for microglia:Supernatants from normal BV-2 cell cultures and cell supernatants from XXMD-C interfered with LPS-induced inflammation in BV-2 cells were collected.Microglia-derived exosomes were isolated by ultra-high speed centrifugation and identified by nanoparticle tracking analysis,transmission electron microscopy(TEM)and Western blot for concentration particle size,appearance morphology and signature proteins of exosomes The exosomes were labelled with PKH26 dye(red)and the nuclei of BV-2 cells with DAPI dye(blue),and the access of BV-2 cell-derived exosomes to BV-2 cells was observed by laser confocal.4.Mechanisms study:After establishing the BV-2 cell inflammation model,cells were interfered with using XXMD-C(5 mg·mL-1),groups of exosomes were isolated using ultra-high speed centrifugation,and the exosomes were subjected to transcriptomic sequencing to screen for differential miRNAs.The cells were divided into 4 groups:miR-9-5p inhibitor group,miR-9-5p inhibitor NC group,ligand miR-9-5p mimic group and ligand miR-9-5p mimic NC group.miR-9-5p inhibitor was added to inhibit the expression of miR-9-5p on the basis of cellular inflammation model.The expression of miR-9-5p was increased by adding miR-9-5p mimic to the inflammation model of XXMD-C treated cells.Cellular proteins in well plates were collected using RT-PCR and Western blot to detect the expression of inflammatory factors in each group of cells to verify that XXMD-C regulates the inflammatory response through miR-9-5p.Results:1.The results of cell experiments showed that XXMD concentration at 10 mg·mL-1 and XXMD-C concentration at 5 mg·mL-1 had basically no effect on the viability of BV-2 cells.the results of Inflammation kit showed that the levels of NO,IL-6 and TNF-α increased significantly in the model group after LPS stimulation,which was statistically significantly different from that of the control group(P<0.01)The statistically significant difference(P<0.01)indicated that LPS could successfully induce BV-2 to form an inflammatory cell model.Compared with the model group,all dose groups of XX2D and XXMD-C were able to reduce NO,IL-6 and TNF-α levels with statistically significant differences(P<0.01).The results of Western blot method showed that NO,IL-1β and TNT-α protein expression levels were significantly increased in the model group after LPS stimulation,with statistically significant differences compared with the control group(P<0.01).Compared with the model group,all dose groups of XXMD-C could reduce NO,IL-1β and TNF-α protein expression levels,and all dose groups of XXMD could reduce NO and IL-1β protein expression levels except TNF-αprotein,with statistically significant differences(P<0.01).2.The results of ultra performance liquid chromatography coupled with quadrupole tandem time of flight mass spectrometry(UPLC-Q-TOF-MS)showed that seven chemical components were detected,namely ephedrine,pseudoephedrine,ginsenoside Rgl,baicalin,baicalin flavonoid I,baicalin flavonoid Ⅱ and cinnamaldehyde.3.Exosomes were successfully extracted using ultra-high speed centrifugation.Laser confoocal microscopy showed that the exosomes were absorbed by the cells and were present around the nucleus and that BV-2 cell-derived exosomes were absorbed by BV-2 cells.no red fluorescent signal was detected in the PBS control.4.Based on the sequencing results,the volcano plot showed that there were indeed differentially expressed miRNAs,and Te software detected a total of 126 significantly dysregulated miRNAs,including 11 up-regulated and 115 down-regulated miRNAs,in the miRNA differential analysis between the Control and model groups.266 significantly dysregulated miRNAs,including 152 up-regulated and 114 down-regulated miRNAs,were detected in the miRNA differential analysis between the XXMD-C and model groups The top 10 significantly up-regulated and significantly down-regulated miRNAs in the model group were screened for analysis compared to the control group.miR-9-5p,miR-3066-5p and miR-128-1-5p were found to be significantly up-regulated,while these miRNAs were significantly down-regulated in the XXMD-C group The relative levels of miR-9-5p,miR-3066-5p and miR-128-1-5p in the exosomes of each group were measured using RT-PCR,and the results showed that the expression of miR-3066-5p and miR-128-1-5p were not statistically different between the model group and the normal group;the expression of miR-9-5p was significantly elevated in the model group,and the results were statistically significantly different compared to the normal group(RT-PCR)The expression of miR-9-5p was significantly higher in the model group and statistically different from that of the normal group(P<0.001).miR-9-5p expression was significantly lower in the XXMD-C group and statistically different from that of the model group(P<0.01).Target gene validation was performed using RT-PCR and Western blot.Cell transfection inhibited miR-9-5p expression.Western blot and RT-PCR results showed that miR-9-5p inhibitor decreased the relative levels of IL-1β,IL-6,NO and TNP-α in cells.miR-9-5p mimic increased the expression of these inflammatory factors,while XXMD-C reversed the effect of miR-9-5p mimic and decrease d the inflammatory response.Conclusion:1.XXMD and XXMD-C can reduce the levels of inflammatory factors such as NO,IL-6,IL-1β and TNF-α,and both have better therapeutic effects on LPS-induced inflammation in BV-2 cells,and the anti-inflammatory effect of XXMD-C is more obvious.2.The main chemical components of the eperimental XXMD-C samples were detected using UPLC-Q-TOF-MS The main components were essentially characterised as a whole in the profiles and were representative of the overall and intrinsic quality of the experimental XXMD-C samples.3.Microglia are able to "Self-interactive dialogue".Sequencing of the exosome transcriptome has identified the differentially expressed gene miR-9-5p,which may be a key gene in the anti-inflammatory action of XXMD-C.4.XXMD-C ameliorates inflammation and reduces inflammatory factor levels in BV-2 cells after stroke,and the mechanism may be related to affecting miR-9-5p expression in BV-2 cell exosomes.