Phellodendrine Inhibits C48/80-induced Allergic Reaction And Its Mechanism

Objective:Phellodendron amurense Rupr is one of the commonly used drugs for the treatment of allergic diseases,but its material basis and mechanism of action are rarely reported.In this paper,the main active ingredient of Phellodendron amurense Rupr,phellodendrine,was studied to clarify its inhibitory effect on the allergic reaction induced by compound 48/80（C48/80）at the animal and cell levels,and to determine the molecular mechanism of its anti-allergic reaction.It provides the experimental basis for the clinical application of Phellodendron amurense Rupr.Methods:（1）To establish a mouse model of allergic foot swelling,C48/80 was Injected into the hind paw of mice.Then,different doses of phellodendrine were injected into the hind paws of mice together with C48/80.The anti-allergic efficacy of phellodendrine at the animal level was evaluated by observing the degree of local swelling of the mouse’s paw and the degree of exudation of Evans blue dye.（2）The toxicity of C48/80 to rat basophils（RBL-2H3）was detected by thiazole blue colorimetry（MTT method）.Concentration of C48/80 without cytotoxic concentration was selected to act on RBL-2H3 cells for 20 min.Then it was detected by enzymatic methodβ-Expression level of hex protein.Finally,the concentration of C48/80,which is non-toxic to cells,is selected as the best model concentration.（3）The RBL-2H3 cells were treated with different doses of phellodendrine.The survival rate of RBL-2H3 cells was detected by MTT method to determine the safe dose of phellodendrine in RBL-2H3 cells.（4）Toluidine blue staining was used to detect the effect of phellodendrine on cell morphology.The release of specific intracellular granules in RBL-2H3 cell model to determine the anti-allergic efficacy of phellodendrine in the cell model.（5）Different doses of phellodendrine were used in RBL-2H3 cell model.The effects of phellodendrine on the release ofβ-HEX,HIS,IL-4 and TNF-αin RBL-2H3 cell model were detected by ELISA and enzymatic methods.To evaluate the effect of phellodendrine on the release of inflammatory mediators following mast cell activation.（6）RT-PCR and molecular docking were used to detect the effect of phellodendrine on MRGPRB2 gene expression in RBL-2H3 cell model and the binding site of the characterized protein to evaluate the effect of phellodendrine on MRGPRB2 channel in the cell model.（7）The cells were pre-incubated with different doses of phellodendrine for 1 h.Then C48/80 was added for 20 min.The effect of phellodendrine on phospholipase C（PLC）and phosphorylated protein kinase C/PK in RBL-2H3 cell model was detected by Western Blot.Changes in protein expression of protein kinase C（p-PKC/PKC）to evaluate the effect of phellodendrine on PLC/PKC signaling pathway in a cell model.（8）Flow cytometry and Western Blot were used to detect the effect of phellodendrine on intracellular Ca²⁺concentration and the expression of phosphorylated calmodulin kinase/calmodulin kinase（p-Ca MK/Ca MK）protein in RBL-2H3 cell model.To evaluate the effect of phellodendrine on the Ca²⁺/Ca MK signaling pathway in a cellular model.（9）Western Blot was used to detect the expression of p-JNK/JNK,p-ERK/ERK,p-p38/p38 of p-JNK/JNK in RBL-2H3 cell model by phellodendrine.The influence of MAPK signaling pathway in the model.The influence of the protein expression of IκBαand p-p65/p65 in the nuclear transcription factor NF-κB pathway family and the evaluation of the effect of phellodendrine on the NF-κB signaling pathway in the cell model.Results:（1）Phellodendrine can inhibit the degree of paw and the amount of exudation of Evans blue dye in a mouse model of foot swelling.This experiment proved that phellodendrine has anti-allergic effect on animal level.（2）When RBL-2H3 cells were stimulated by 20μg/m L C48/80 for 20 min,the release ofβ-HEX reached the maximum.This condition was defined as the film-forming condition.（3）The concentration of phellodendrine below 100μM has no effect on the survival rate of RBL-2H3 cells,so the following experiments were carried out with concentrations of0.5,5,and 50μM.（4）It was observed by toluidine blue staining that phellodendrine can inhibit the change of cell shape in RBL-2H3 cell model,and inhibit the release of specific granules from cells,thereby affecting the color of cytoplasm.Then,by ELISA and enzymatic methods,it was found that phellodendrine can inhibit the release ofβ-HEX,HIS,IL-4 and TNF-αin RBL-2H3 cell model,which proves that phellodendrine can inhibit the release of inflammatory mediators from mast cells.（5）Phellodendrine can inhibit the expression level of MRGPRB2 receptor gene in RBL-2H3 cell model,and can interact with MRGPRB2 receptor,indicating that phellodendrine may affect mast cell activation by affecting the expression of MRGPRB2 receptor.（6）Phellodendrine can reduce the expression levels of p-PLC/PLC and p-PKC/PKC proteins in RBL-2H3 cell model in a concentration-dependent manner,indicating that phellodendrine may regulate cell degranulation by inhibiting PLC/PKC pathway.（7）Phellodendrine can reduce the Ca²⁺concentration and the expression level of p-Ca MK/Ca MK protein in RBL-2H3 cell model in a concentration-dependent manner,indicating that phellodendrine may regulate cell degranulation by inhibiting the Ca²⁺/Ca MK pathway.（8）Phellodendrine can reduce the expression levels of p-JNK/JNK,p-ERK/ERK and p-p38/p38 proteins of MAPK signaling pathway in RBL-2H3 cell model.It can also increase the protein expression level of IκBαand decrease the protein expression level of p-p65/p65 in the NF-κB signaling pathway.Phellodendrine may affect mast cell activation by affecting MAPK signaling pathway and NF-κB signaling pathway.Conclusion:Phellodendrine has anti-allergic effects in both in vivo and in vitro experiments.The molecular mechanism of phellodendrine’s pharmacological effects may be related to the regulation of PLC/PKC,Ca²⁺/Ca MK,MAPK and NF-κB signaling pathways mediated by MRGPRB2 channel.