Effect Of Yupingfeng San On Type Ⅰ Allergic Mice And Its Mechanism Of Inhibiting Mast Cell Degranulation

Objective: Yu-ping-feng-san(YPFS),as a commonly used anti-allergic compound in clinical practice,has the effects of strengthening Qi and strengthening the body,strengthening the body and eliminating evil.Allergic disease(Allergic disease)is an abnormal immune response of the body.Type I allergies are more common.The clinical symptoms are usually accompanied by skin itching,rash,and cough.Mast cells are important effector cells for allergic reactions,and degranulation of mast cells is the basis of pathological reactions such as allergic reactions and inflammation.Therefore,this project verifies the anti-allergic effect of Yupingfeng San by simulating type I allergy animal models in vivo,and simulating mast cell degranulation model in vitro to investigate the effect and mechanism of Yupingfeng San on mast cell degranulation,in order to develop more anti-allergic effects.Allergy Chinese medicine compound provides theoretical basis.Methods: Pharmacodynamic study of Yupingfeng San on type I allergy model mice:(1)Skin pruritus model: 48 KM mice,male and female,random blank group,model group,positive control group(loratadine),Yupingfeng San group,etc.The positive group and Yupingfeng San group were given loratadine and Yupingfeng San diluent saline respectively,and the blank group and model group were given equal volume of normal saline.One week later,except for the blank group Mice in each group were injected with dextran solution through the tail vein,and the incubation period of scratching,the number of scratches,and the time of itching in each group within 30 minutes were recorded;(2)Evans Blue experiment: the mice were given the same administration and grouping as(1),one week Afterwards,the middle of the abdomen of the mice in each group was depilated first,except for the blank group,the Evans blue dye was injected into the tail vein after intragastric administration for 1 hour,and then histamine phosphate was injected subcutaneously into the abdomen depilation area,30 minutes after bulging the skin hills Put to death,peel off the blue-stained skin of the abdomen and place it in a mixture of acetone and saline.After soaking for 24 hours,the absorbance of the soaking solution in each group was measured to calculate the locus coeruleus inhibition rate;(3)Serum total Ig E test: 48BALB/c mice were grouped in the same(1)Mice in the model group,positive drug group,and Yupingfeng San group were injected subcutaneously with OVA and aluminum hydroxide suspension on day 0 for primary sensitization,and on day 7,day 14,and 21 Suspension was repeatedly sensitized by subcutaneous injection,and intragastric administration began on the 19 th day.The administration of each group was the same as(1)for 7 consecutive days.After the intragastric administration on the 25 th day,except for the blank group,the other 5 groups were intraperitoneally injected 1 % OVA was used for final sensitization.After 3 hours,the mice in each group were removed from the eyeballs and blood was taken,the serum was separated,the spleen and thymus were taken out and weighed,the spleen index and thymus index were calculated,and the serum Ig E concentration was detected by ELISA.Study on the mechanism of Yupingfeng San in inhibiting degranulation of mast cells:(1)CCK-8 method to detect the survival rate of P815 mast cells:P815 cells in the logarithmic growth phase are evenly spread on a 96-well plate,and a blank group,a control group,In the Yupingfeng San group,after24 hours of culture,the Yupingfeng San group was added with final concentrations(1.5625μg/ml,3.125μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml)Yupingfeng San culture medium,add equal volume of culture medium to other groups and continue incubating for 24 hours,then replace each well with 10% CCK-8 medium,continue incubating for 1.5 hours,and measure the absorbance of each well at 450 nm wavelength;(2)ELISA method: P815 in the logarithmic growth phase is evenly spread on a 6-well plate,and the blank group,model group,and Yupingfeng San group are set.After 24 hours of culture,the other groups are replaced with equal volumes of culture medium,and the Yupingfeng San group is replaced differently.After 6hours of treatment,the model group and Yupingfeng San group were added with trypsin stimulation,the blank group was added with equal volume of PBS,and the cell supernatant and cells after stimulation for 1 hour were collected.The supernatant was tested by ELISA method.-Hexosaminidase,histamine,IL-4,IL-13,PAF concentration;(3)Western blot method: collect the above cells,WB method to detect PI3 K,Akt,m-TOR protein expression;(4)real-time fluorescence quantification PCR method: Collect the above-mentioned cells,and detect the expression changes of PI3 K,Akt,m-TOR in the cells at the m RNA level.Results: Pharmacodynamic study of Yiyupingfeng San on type I allergy model mice: The results show that Yupingfeng San can prolong the scratching latency of skin itching model mice,reduce the number of scratches,and shorten the time of itching(P<0.05);Inhibit the exudation of Evans blue dye on the skin of mice,and significantly reduce the absorbance(P<0.05);Yupingfeng San can significantly reduce the serum Ig E level in mice(P<0.05),and significantly increase the spleen index and thymus index(P<0.05);Study on the mechanism of Yupingfeng San in inhibiting degranulation of mast cells: The experimental results show that Yupingfeng San has no obvious inhibitory effect on cell activity when the concentration is below 25μg·m L-1,and at the concentration of 12.5μg·m L-1,25μg·m L At-1,Yupingfeng San can effectively inhibit the release of β-hexosaminidase,histamine and the secretion of IL-4,IL-13,PAF(P<0.05);Western blot results show that YPFS can inhibit PI3 K,Akt,Phosphorylation of m-TOR(P<0.05);q RT-PCR results showed that it can reduce the expression of PI3 K,Akt and m-TOR(P<0.05).Conclusion: Yupingfeng San can prolong the incubation period of scratching in mice,reduce the number of scratches and the time of itching.It can also inhibit the increase in capillary permeability and reduce the total serum Ig E content.The inhibitory effect on the activation and degranulation of mast cells may be through It is achieved by inhibiting PI3K/Akt/m-TOR signaling pathway.