Correlation Between Geniposide And Scorpion Based On Monoclonal Antibody Specific Knockout Technique To Improve Oxidative Stress Injury In HUVEC Cells

ObjectiveThe specific knockout of geniposide?GE?in Gardenia jasminoides Ellis?GJE?extract was achieved by preparing immunoaffinity chromatographic column of geniposide monoclonal antibody to explore the correlation between geniposide and gardenia in oxidative stress injury.1.We synthesized the genomic antigens of geniposide,prepared the monoclonal antibody to geniposide,and purified the monoclonal antibody protein.2.We used geniposide monoclonal antibody to establish geniposide enzyme immunoassay.The precision and the recovery rate of the method were investigated,and the content of geniposide was determined.3.We prepared the immunoaffinity chromatographic column of geniposide monoclonal antibody,optimized the working conditions of the column,and realized the specific knockout of geniposide in gardenia extract.4.We investigated the efficacy of gardenia extract of geniposide and different geniposide in the protection of H2O2-induced oxidative stress injury in human umbilical vein endothelial cells?HUVEC?,and analyzed the effects of geniposide on gardenia The contribution of sub-pharmacodynamics.Methods1.We have chosen the efficient sodium acid oxidation method to synthesize geniposide artificial antigens.Animals were immunized to detect serum antibodies.We also performed a fusion of spleen cells with myeloma cells and screened positive hybridoma cells.After monoclonal culture,the antibody was induced by ascites,the antibody protein was purified by octanoic acid method,and the titer and specificity of the purified antibody were tested.2.We used the checkerboard method to detect the optimal working concentration of the original and primary antibody,and established the enzyme-linked immunoassay of the monoclonal antibody of geniposide.The precision and the recovery rate of the method were investigated.The correlation analysis was carried out to detect the content of geniposide in different prescriptions containing gardenia.3.We used the purified mononuclear antibody to geniposide and conjugated to the activated agarose gel to prepare the immunoaffinity chromatographic column of geniposide monoclonal antibody,optimized the screening incubation time,knockout buffer And the elution buffer and the flow rate of each buffer.The coupling rate and column capacity of the column were investigated.The fingerprints of geniposide knockout samples in gardenia extract were established under the following conditions:Agilent 1260 infinity,?A?-acetonitrile?C?,gradient conditions 0-37 min,95%?A??5%?C?;37-60min,85%?A?₁5%?C?;60-65min,70%?A?₃0%?C?;65-70min,95%?A?₅%?C?.4.We used HUVEC oxidative stress injury model induced by H202,and the effects of geniposide and different geniposides on the culture of HUVEC induced by H202 were studied by specific knockout and quantitative backfilling of geniposide.We investigated the efficacy of gardenia extract of geniposide and different geniposides on the oxidative stress injury induced by H202 in HUVEC and the related indexes of NO,NOS,SOD,GSH-px and other oxidative stress.Results1.Genomic assisted laser desorption/ionization time-of-flight mass spectrometry?MALDI-TOF-MS?was used to detect the binding ratio of geniposide to BSA by about 34:1.The results of SDS-PAGE showed that a large number of hybrid proteins in ascites were removed by purification and the higher purity of gardenia monoclonal antibody was obtained.The results showed that the hybridoma cell lines secreted with geniposide monoclonal antibody were successfully prepared by cell fusion.Antibody,before and after purification of ascites in the antibody titers are 1:60000 or so.Anti-antibody and its structural analogues,gardenia other major drug ingredients,often compatibility of traditional Chinese medicine compounds were no significant cross-reaction,with good specificity.2.We established the enzyme-linked immunoassay of the geniposide monoclonal antibody.The linear detection range was approximately lug/mL-50ug/mL,and the linear regression equation was Y =-0.1581n?x?+ 0.9392,R2 = 0.9912.The IC50 value?ie,the half inhibitory concentration?was 6.115 ug/mL and the minimum detection limit was about 258 ng/mL.The results were in good agreement with the results of HPLC.R2 = 0.9982.The difference coefficient between plate difference coefficient and plate difference coefficient is less than 15%,and the average recovery is 101.51%,which is in accordance with the requirement of biological sample detection.3.We prepared the immunoaffinity chromatographic column of geniposide monoclonal antibody with deionized water as knockout buffer,incubation time was set to 60 min,knockout fluid flow rate was 1 ml/min,0.1 M glycine-hydrochloric acid buffer Elution solution with eluent flow rate of 2ml/min as the optimal working condition of the geniposide immunoaffinity column.Antibody antibody coupling ratio of 87.3%,antibody protein and the coupling ratio of 5.74mg/ml,the cylinder capacity of about 3.25ug/ml.A method for knockout of geniposide was successfully established,and a sample of gardenia extract of genetically knocked out geniposide was prepared.4.The oxidative stress injury model of HUVEC12h was induced by 500?mol/1 H202,and GE and GJE containing 50%,100%,150%and 200%GE were respectively intervened.The results showed that the survival rate of HUVEC cells decreased to 54.037%,the NO content in cell culture medium decreased from 36.97?mol/1 to 19.86?mol/1,the activities of NOS,SOD and GSH-px were 3.07U/Mgprot,37.48 U/mgprot,217.40 U/mgprot to 1.39 U/mgprot,23.18 U/mgprot,110.87 U/mgprot.Compared with H202 model group,GE and 50%,100%,150%and 200%GE GJE extracts could improve the cell viability and increase the NO content and NOS,SOD,GSH-px activity in the culture medium,The difference was statistically significant?P<0.05?.The activity of NO and the activities of NOS,SOD and GSH-px increased with the increase of GE content in 50%,100%and 150%GE groups,and the difference was statistically significant?P<0.05?.Although the activity of NOS and SOD in 50%GE group was not significantly different from that in 100%GE group?P>0.05?,the activity of NOS and SOD was decreased.There was no significant difference in cell viability,NO content and NOS activity between the 200%GE group and the 150%GE group?P>0.05?.The activities of SOD and GSH-px were significantly lower than those in the 150%GE group?P<0.05?,and continue to increase the GE content can not further improve the protective effect of oxidative stress.ConclusionThe monoclonal antibody of the geniposide prepared by this study has strong specificity and affinity.The enzyme-linked immunosorbent assay?ELISA?based on the monoclonal antibody has good correlation with the results of the traditional high performance liquid chromatography method.Determination of Geniposide in Related Samples.On the basis of this,the immunoaffinity chromatographic column of geniposide monoclonal antibody could specifically knock out the geniposide composition in gardenia extract.The results of further pharmacological studies showed that gardenia extract and geniposide had different degrees of protection on oxidative stress injury,and with the increase of geniposide content,oxidative stress The protective effect is enhanced to varying degrees,but does not exhibit a strict dose-effect relationship.The results showed that geniposide had obvious effect on the efficacy of gardenia,and there was a certain correlation with the protective effect of gardenia in oxidative stress.