Study On The Mechanism Of Chuanjiao Recipe In The Treatment Of Allergic Conjunctivitis Based On IKK/I?B/NF-?B Signaling Pathway

ObjectiveTo observe the effects of serum containing Chuanjiao recipe on IKK/I?B/NF-?B signaling pathway,histamine release level and inflammatory factor IL-6 in allergic conjunctivitis,and to further explore the mechanism of Chuanjiao prescription in the treatment of allergic conjunctivitis.Methods1.Preparation of serum containing Chuanjiao recipe:Twenty normal SD male rats were divided into two groups,namely,Chuanjiaofang drug-containing serum group and blank serum group.The drug-containing serum group was in tragastrically administered with a medium dose of 20 ml/kg and a concentratio n of 1.24 g/ml of Chuanjiao.The drug was administered twice a day for 7 da ys;the blank group was given the same amount of normal saline.SD rats Ch uanjiaofang drug-containing serum and blank serum were prepared.2.RBL-2H3 cells were cultured in vitro,the cells were subcultured,cryo preserved and resuscitated.When the cells grew well and the number reached 1 ogarithmic growth phase,the expermiment was carried out.3.The effect of Chuanjiaofang drug-containing serum on the activity of R BL-2H3 cells was detected by CCK-8 method.Six concentrations(5%?10%?15%?20%?25%?30%)were set in the drug-containing serum group and the b lank serum group.4.After cell plating,anti-DNPIgE was added to stimulate RBL-2H3 cells to sensitize and incubate overnight in the incubator;RBL-2H3 cells were added with antigen DNP-BSA to establish a RBL-2H3 cell degranulation model.The experiment was divided into normal group,model group,negative control gro up,positive drug control group,Chuanjiaofang low-dose drug-containing serum group,Chuanjiaofang medium-dose drug-containing serum group,and Chuanjiao high-dose drug-containing serum group.5.Observation of cell morphology by toluidine blue staining.6.The level of histamine release and IL-6 in the supernatant of Chuanjia o Fangfang Granules were determined by enzyme-linked immunosorbent assay(ELISA).7.Detection of NF-?B,IKK-? and I?B-? gene(mRNA)expression in RB L-2H3 cells by real-time quantitative PCR(RT-PCR).Results1.The effect of each serum group on the activity of RBL-2H3 cells was detected by CCK-8 method.The results showed that 15%blank serum,10%,15%and 30%drug-containing serum had the least effect on the activity of RB L-2H3 cells,so this experiment 15%blank serum,10%,15%,30%drug-contai ning serum were selected as the experimental dose gradient of Chuanjiao.2.Toluidine blue staining:staining of normal RBL-2H3 cells and cell deg ranulation:normal cell cells are fusiform,cell membrane is smooth and shiny,contour is clear,cell cytoplasm is stained blue;when cell degranulation,RBL-2H3 cells have surface protrusions.When the cells are degranulated,the cells become larger and rounder,the intracellular inflammatory mediators are release d,the cytoplasm becomes sparse,the staining becomes shallow,the vacuoles a ppear in the middle.and the cell membrane edges are irregular.3.The amount of histamine and IL-6 secreted by ELISA:the amount of histamine and IL-6 secreted by the normal group was small,the antigen DNP-?gE and the antibody DNP-BSA were sensitized,and the histamine and IL-6 we re cleared by RBL-2H3 cells.The release was significantly increased,and the difference was statistically significant compared with the normal group(P<0.05),indicating that the antisen DNP-IgE and antibody DNP-BSA sensitization mod el can cause the release of histamine and inflammatory factor IL-6 from RBL-2H3 cells produce.The secretions of histamine and IL-6 increased in the nesat ive control group,the positive drug group and the Chuanjiao prescription.Com pared with the model group,the histamine and IL-6 in the supernatant of the drug-containing serum group and the positive drug control group.The expressio n levels of were significantly lower than the model group,and the difference was statistically significant(P<0.05),indicating that drug-containing serum and positive drugs could inhibit the release of histamine and IL-6 from RBL-2H3 c ells.Compared with the negative control group,the release rate of histamine a nd IL-6 in the positive drug group and Chuanjiaofang group decreased,the dif ference was statistically significant(P<0.05).Compared with the positive drug group,the ability of each treatment group of Chuanjiao to inhibit histamine an d IL-6 release rate was insufficient,and the difference was statistically signific ant(P<0.05).There was no significant difference in histamine and IL-6 secreti on between the two groups(P>0.05).4.PCR:Compared with the normal group,the expression of I?B-a mRN A,IKK-? mRNA and NF-?B mRNA in the RBL-2H3 cells of the model grou p was significantly increased,the difference was statistically significant(P<0.05),indicating that the antigen DNP-IgE,antibody DNP After BSA sensitization,the expression of I?B-?,IKK-?,and NF-?B increased.Compared with the mo del group,the mRNA levels of IxB-a,IKK-? and NF-?B in the positive drug control group and the drug-containing serum group decreased significantly,and the difference was statistically significant(P<0.05),indicating that the positive drug PP2 and drug-containing serum were available.Inhibition of I?B-?,IKK-?,NF-?B mRNA expression in RBL-2H3 sensitized cells.Compared with the posi tive drug control group,the ability of down-regulating I?B-?,IKK-? and NF-?B mRNA levels in each dose group of Chuanjiao was insufficient(P<0.05).Th e levels of IKK-? mRNA and NF-?B mRNA were not significantly decreased between the drug-containing serum dose groups,and the difference was not sta tistically significant(P>0.05).Conclusion1.Chuanjiaofang can inhibit the histamine and inflammatory factor IL-6 d uring mast cell degranulation.2.The mechanism of Chuanjiaofang in the treatment of allergic conjuncti vitis may be related to the regulation of the expression of I?B-?/IKK-?/NF-?B signaling pathway-related factors in stable mast cell degranulation.