The Effect Of Catalpol On Schwann Cells Under LPS Injury And Its Mechanism Of Action

Objective:1 Rat Schwann cell(RSC)96 was cultured in vitro,to observe the biological characteristics of RSC 96.2 Observe the effect of lipopolysaccharide(LPS)on RSC 96 and the change of superoxide dismutase(SOD)expression.3 Study the protective action of catalpol extracted from rehmannia acting on RSC 96 under LPS injury and the change of SOD expression.4 Explore condition of the co-culture of RSC 96 and primary neurons.Methods:1 RSC 96 was cultured in vitro,authenticated by immunofluorescence staining of the collagen fibre acidic protein(GFAP)and S100,whose morphology was observed during different time.2 Choose different concentration-0?g/ml,40?g/ml,80?g/ml,160?g/ml,320?g/ml,640?g/ml of LPS,that injure RSC 96 for 24h,48h,72h,observe cell morphology,count the number of cell adherent,detect cell activity by cell counting kit-8(CCK-8),establish the model of gradient of injury time and concentration of LPS,select the appropriate concentration and time.Injury to RSC 96 by 160?g/ml LPS for 0h,24h,48h,72h.Establish normal control group,LPS injury group,LPS injury 24h group,LPS injury 48h group,and LPS injury 72h group.Fluorescence staining of SOD was performed,and Image J software was used to analyze the fluorescence intensity.Optical density(OD)value indicated the change of SOD expression.3 RSC 96 under normal and 160?g/ml LPS injury conditions were interfered by different concentrations of catalpol-0?mol/ml,25?mol/ml,50?mol/ml,100?mol/ml for 72h.By observing the morphological structure,number of adherent cells,and CCK-8 OD value,the optimal intervention concentration was selected and establish normal control group,model group,and catalpol intervention group,and observe the morphological structure,number of cell adherents,propulsion length,and CCK-8 OD vaule.Catalpol of different concentration-0?mol/ml,25?mol/ml and 50?mol/ml,100?mol/ml acting on RSC 96 under the condition of 160?g/ml LPS for 72h,set up the normal control group,model group and LPS+25?mol/ml catalpol alcohol group,LPS+50?mol/ml catalpol group,LPS+100?mol/ml catalpol group,performe fluorescence staining of SOD,and Image J software was used to analyze the fluorescence intensity.OD value indicated the change of SOD expression.4 Primary neurons were extracted from rat embryos and identified by immunofluorescence staining of microtubule-associated protein-2(map-2)of dendritic marker of neurons.Obtain the purification rate of primary neurons by the proportion of map-2 staining positive cells to DAPI staining positive cells.Observe the morphological changes of primary neurons at different time.The primary neurons and RSC 96 together were cultured in the co-culture medium at a ratio of 500:3,and observe the survival state of the two cell,conduct S100 and tubulin immunofluorescence staining.Results:1 RSC 96 was identified as SC by S100 and GFAP immunofluorescence staining,and its shape presented a spindle shape,the cell body was round and full,the nucleus was oval,and the process was elongated,according with the biological characteristics of SC.2 CCK-8 OD value of RSC 96 decreased with the extension of LPS injury time and the increase of LPS concentration.LPS injury concentration and time-160 ?g/ml,72h can form a semi-injury model,which is the best.SOD expression level decreased with the extension of LPS injury time.3 Catalpol of different concentrations interfered with RSC 96 under normal and 160?g/ml LPS injury conditions for 72h,when the concentration was 50?mol/ml,the number of adherents is the largest,and the CCK-8 OD value is the greatest,which was the optimal intervention concentration.Compared with the normal control group,the CCK-8 OD value of the model group was significantly reduced(P<0.01),the number of cell adherents was reduced(P<0.01),and the cell protuberances were significantly shortened.Comparison between the catalpol intervention group and the model group showed that catalpol could significantly increase the CCK-8 OD value(P<0.01),increase the number of adherent cells(P<0.01),and increase the cell process elongation.Under the condition of 160?g/ml LPS injury,the RSC 96 was interfered for 72 hours with different concentrations of catalpol.Compared with the model group,the expression of SOD was all increased(P<0.01,P<0.01,P<0.01).4 The extracted primary neurons were identified as neurons by map-2 with a purification rate more than 90%.In the condition of co-culture medium,primary neurons:RSC 96=500:3,two cells can coexist.The results of S100 and tubulin immunofluorescence staining were positive.Conclusions:1 RSC 96 was identified as SC by S100 and GFAP immunofluorescence staining,which was consistent with the biological characteristics of SC.2 The RSC 96 activity decreased with the increase of concentration and time of LPS injury.SOD expression level decreased with the extension of LPS injury time.3 Catalpol has the effect of protecting RSC 96 under LPS damage and increasing SOD expression level.4 Through cell co-culture,it was found that RSC 96 and primary neurons could coexist in vitro.