Experimental Study On Molecular Mechanism Of Baicalein Resistance To Type? Hypersensitivity

Objective:Immunoglobulin E(Ig E)and Compound48/80(C48/80)were used to induce rat basophilic leukemia cell line(RBL-2H3)to activate degranulation and establish Model of in vitro cell degranulation.To study the effect of Baicalein(BAI),a traditional Chinese medicine monomer,on cell degranulation model in vitro,and to explore its molecular regulation mechanism of inhibiting type I Hypersensitivity.Method:RBL-2H3 cell lines were cultured in vitro,and cell growth curves were drawn to determine appropriate culture conditions.Ig E and C48/80 were used to induce activation and degranulation of RBL-2H3 cells,and an in vitro cell degranulation model was established.CCK8 method and Annexin V/PI double staining method were used to detect the effect of BAI on cell proliferation activity in the concentration range of 3.125-100.000?M,and the concentration range of 50-200?M Effects of Cells on Cell-induced Apoptosis.After cells with different concentrations of BAI were applied,the cells were de-granulated according to the modeling conditions,and the cell morphology was observed under a light microscope.The ELISA method was used to detect the?-hexosaminidase,Histamine,IL-4 and TNF-?levels in the cell supernatant.The relative expression levels of intracellular IL-4 and TNF-?m RNA were detected by PCR;Western Blot was used to detect the key upstream and downstream proteins p-Lyn/Lyn,p-Syk/Syk and MAPK family p of Ig E/Fc?R?-mediated signaling pathway.-JNK/JNK,p-ERK/ERK,p-P38/P38 protein expression.Statistical methods were analyzed and graphed using Graph Pad Prism 5 Demo software.All experimental results were expressed as mean standard deviation(Mean SEM).The comparison between the two groups was performed using the Independent Samples T test,the comparison between multiple groups was performed using the One Way ANOVA,and the comparison between the two groups was performed using the Student-Newman-Keuls test,with P<0.05 as the comparison Statistically significant criteria.Morphological data are represented by pictures and described.Results:The growth curve of RBL-2H3 cells was drawn,and the optimal seeding density was determined to be 2×10~5/m L.RBL-2H3 cell degranulation model was established as follows:125ng/m L DNP-Ig E sensitized cells overnight,and the next day was stimulated with 500ng/m L DNP-HAS.The levels of HIS in the cell supernatant were detected when DNP-HSA was applied for 30 minutes,the?-HEX level was detected for 1 hour,and the inflammatory factors IL-4 and TNF-?were released for 4 hours.The C48/80 induced degranulation model of RBL-2H3 cells was used to determine the C48/80 modeling dose as 10?g/m L as the experimental working concentration,the stimulation time was 20 minutes and the morphological changes after cell activation were recorded under an inverted microscope.When the BAI concentration was 3.125-50.000?M for 24 hours,it was found that BAI had no significant change in the proliferation activity of RBL-2H3 cells(P?0.05),but when the concentration was greater than 50.000?M,it was found that BAI could induce RBL-2H3cell apoptosis,Where the apoptosis rate is 7.62%at a concentration of 100.000?M(compared with the control group P?0.01),and the apoptosis rate at a concentration of200.000?M is 10.86%(compared with the control group P?0.001),which shows that The pro-apoptotic effect was dose-dependent(comparison between each group P?0.05).After the cell degranulation model was established steadily(Ig E/Fc?R?pathway),the cell morphology observation revealed that BAI had a certain inhibitory effect on the activation of degranulation of RBL-2H3 cells at a concentration of 3.125-50.000?M;ELISA test results showed that?-HEX,HIS,IL-4 and TNF-?release in cell supernatants were significantly reduced compared with the control group,indicating that BAI has an inhibitory effect on the degranulation reaction of RBL-2H3 cells(P?0.05),Especially at 25.000 and 50.000?M(P?0.001);After 50.000?M BAI acted on RBL-2H3 cells,the relative levels of IL-4 and TNF-?m RNA were found to be low,indicating that BAI showed a good inhibitory effect on the production of inflammatory cytokines(P?0.001).At the same time,it was found that BAI also had a significant inhibitory effect on C48/80-induced degranulation of RBL-2H3cells at a concentration of 12.500-50.000?M(P?0.001).Further exploring the molecular mechanism by which BAI inhibits the Ig E/Fc?R?pathway to mediate cell activation and degranulation,it is found that the use of BAI before stimulating RBL-2H3 cells to degranulate can significantly reduce Ig E/Fc?RI-mediated key protein Lyn,Syk,ERK,JNK,p38 phosphorylation levels.Conclusion:1.Baicalin can inhibit the degranulation response of mast cells induced by Ig E pathway and C48/80 pathway in vitro.The mechanism may be related to the stabilization of cell membrane and inhibition of inflammatory factor production.2.The molecular mechanism of baicalein resistance to type?Hypersensitivity may be related to reducing the phosphorylation levels of Ig E/Fc?R?-mediated signaling pathway proteins Lyn,Syk and MAPK family.pathway transduction.