Study On Mechanism Of Mast Cell Degranulation Induced By Mast Cell Degranulation Peptide And Its Application In Chinese Medicine

Objective:The activation of mast cells degranulation process is closely related to the development of many allergic diseases.This research tries to discuss how mast cell degranulation peptide(MCDP)induced by mast cells activate mast cells sensitization model was constructed,and how LY294002(a kind of specificity of PI3K/Akt signaling pathway inhibitor)and Chinese medicine belvedere fruit saponins were respectively applied to the model.The purpose is,on the one hand,to explore MCDP regulation mechanism of mast cell degranulation;on the other hand,to observe whether belvedere fruit saponins will affect mast cell degranulation process by PI3K/Akt signaling pathways,thus to provides theory basis for the belvedere fruit's mechanism of anti-inflammatory allergy.Methods:1.Study about the mechanism of mast cell degranulation peptide induced by mast cell degranulation peptide.Mouse mast cell tumor cells(P815)were randomly divided into normal control group and MCDP(high,medium and low)dose group.(1)CCK8 method was used to detect the cytotoxic effects of MCDP.(2)ELISA method was used to detect the changes of amines in MCDP induced by cell degranulation.(3)rt-qpcr method was used to detect changes in mRNA expression levels of related genes in Pik3r3,Akt2 and gsk-3b in each group of PI3K/Akt signaling pathways.(4)flow cytometry was used to detect changes in protein expression of PI3K,Akt and gsk-3b in PI3K/Akt signaling pathway respectively.2.Based on PI3K/Akt signaling pathway,the effect of the saponin on MCDP induced by mast cell activation degranulation was discussed.The mouse mast cell tumor cells(P815)were randomly divided into the normal control group,the MCDP model group,LY294002 group,and the saponin group.(1)rt-qpcr method was used to detect changes in mRNA expression levels of related genes in Pik3r3,Akt2 and gsk-3 in each group of PI3K/Akt signaling pathways.(2)flow cytometry was used to detect changes in protein expression of PI3K,Akt and gsk-3b in PI3K/Akt signaling pathway respectively.Results:1.The toxic effect of MCDP on P815.The toxicity of MCDP to mast cells increased with the extension of intervention time,and shows positive correlation between 0.25?2.00 h,with significant toxicity to the cells after 1h(P<0.05).2.Effects of MCDP on the release quantity of P815 histamine and its release rate.The experimental results showed that the MCDP in the(low,medium and high)dose group could release the histamine of P815,which was positively correlated with the concentration of 0.01-1 M.Among them,the high dose group(1 M)could release the amine of P815 cells to 56.73%,and the release rate of histamine was significantly increased compared with the control group(P<0.05).3.RT-qPCR method's influence on the detection of MCDP Pik3r3 on PI3K/Akt signal pathway and Akt2,Gsk-3 beta gene mRNA expressionHigh dose group(including 1 M)of MCDP can significantly increase the Pik3r3,Akt2 gene transcription level of P815(P<0.05),while low dose groups(including 0.01 M)in the MCDP had no obvious effect on the Pik3r3,Akt2 gene transcription level of P815;High and low dose group of MCDP had no obvious change to the gene transcription level of GSK-3 beta.4.Flow cytometry detection MCDP's influence on PI3K,Akt,Gsk-3 beta protein expression on the PI3K/Akt signaling pathwayMCDP of high dose group(including 1 M)can make the P815 of Pi3k,and Akt protein expression level increase significantly(P<0.05),high and low dose group of MCDP had no obvious change in Gsk-3 beta protein expression level.5.RT-qPCR method in detecting the influences of Pik3r3,Akt2,Gsk-3 beta gene mRNA expression induced by LY294002,belvedere fruit saponins of PI3K/Akt signaling pathway.After the stimulation of MCDP,P815 expression Pik3r3,Akt2 gene transcription level increased significantly compared with controls(P<0.05);By using LY-294002 inhibitor and pretreatment of belvedere fruit saponins respectively,compared with model group,Pik3r3,Akt2 gene transcription level significantly decreased(P<0.05);LY-294002 inhibitor and belvedere fruit saponins,Pik3r3,Akt2 had no obvious effects in the change of gene transcription level;MCDP,LY-294002 inhibitor and belvedere fruit saponins had no obvious influence in Gsk-3 beta gene transcription level.6.Flow cytometry detection's influence of PI3K,and Akt Gsk-3 beta protein expression on the PI3K/Akt signaling pathway by LY294002 and belvedere fruit saponins ofAfter MCDP stimulation,P815 expression of Pi3k and Akt protein increased significantly compared with controls(P<0.05);With LY-294002 inhibitor and the pretreatment of belvedere fruit saponin,after MCDP stimulation,the expression of Pi3k,and Akt protein was significantly less than those MCDP stimulation expression(P<0.05);by comparison,we found that LY-294002 inhibitor and belvedere fruit saponins had no obvious effect on the expression of Pi3k,and Akt protein;MCDP,LY-294002 and belvedere fruit saponins had no significant effect on Gsk-3 beta protein expression.Conclusions:1.The mechanism of mast cell degranulation induced by MCDP is related to the activation of PI3K/Akt signal transduction pathway.2.The PI3K/Akt signaling pathway can inhibit the activation of P815 by belvedere fruit saponins,and its anti-inflammatory effect may be related to the inhibition of PI3K/Akt signaling pathway.