Chinese medicine has significant advantages in the treatment of skin toxicity such as EGFRIs-induced rash and has become an effective drug recommended by domestic guidelines.After years of clinical practice,Prof.Cui Huijuan,combined with the theory of TCM and developed the pure Chinese medicine topical preparation "Zhiyang Pingfu Liquid",which has been clinically applied for many years with remarkable effects and has been included in the domestic expert consensus and become a clinically recommended drug.However,the mechanism of "Zhiyang Pingfu Liquid" is not yet completely clear.Based on the previous network pharmacology research,PLA2G4A gene may be an important target of "Zhiyang Pingfu Liquid",and IL-6/JAK2/STAT3 signaling pathway may be its important action pathway,but the above conclusion has not been further verified.Based on the above research,this study further investigated the action targets and pathways of "Zhiyang Pingfu Liquid" through molecular docking,animal experiment and cellular experiments,aiming to provide more sufficient and complete data to support the clinical application of "Zhiyang Pingfu Liquid" and to provide new ideas for the treatment of EGFRIsrelated rash.Experiment 1:Validation of the main components and targets of "Zhiyang Pingfu Liquid" based on molecular docking and animal experimentObjective:To clarify the role of matrine and baicalin as important components of"Zhiyang Pingfu Liquid",and to explore their regulatory effects on the target gene PLA2G4A.Methods:1.Molecular docking:The 3D structures of four monomers were obtained from the TCMSP database and set as ligands.The 3D structure of PLA2G4A protein was obtained from the PDB database and set as receptors.Autodock software was applied to perform molecular docking simulation and PyMOL software was applied to visualize the data.2.Animal experiment:BN rats were randomly divided into control group,model group,Zhiyang Pingfu Liquid group(ZY),matrine middle dose group(Mm),matrine high dose group(Mh),baicalin middle dose group(Bm),baicalin high dose group(Bh),and PBS group.The control group was not intervened,and the remaining 7 groups were given gefitinib orally for 14 days.From day 15,all groups except the control group continued to apply gefitinib orally,and on top of that,different topical drugs were administered to each group for intervention.Drugs were applied continuously for 7 days.The skin phenotypic changes and skin pathological changes of rats were observed at the end of the experiment.The expression of PLA2G4 A protein in the skin of rats was detected by immunohistochemistry and Western Blot,and the concentrations of cytokines IL-6,TNF-α and IL-1β in the blood were detected by ELISA.Results:1.Molecular docking:The four main monomeric components were able to bind to PLA2G4A protein,and in terms of binding energy,the binding capacity of all four monomeric components to the target protein was less than-5 KJ/mol,and the docking results were plausible.2.Animal experiment:All rats showed different degrees of rash after the application of gefitinib.The rashes in the model group and PBS group gradually increased with the prolongation of time.The rashes of rats in the remaining groups were reduced to different degrees after 7 days of topical drug intervention.In terms of drug efficiency rates,Zhiyang Pingfu Liquid group,Mm group,Mh group,Bm group,Bh group and the PBS group were 83.33%,66.67%,83.33%,33.33%,33.33%and 16.67%,respectively.In terms of skin pathology,obvious inflammatory manifestations were seen in the model and PBS group,while the rest of the groups showed significantly better skin pathology.Immunohistochemistry showed that the expression of PLA2G4A protein in the skin of rats was not statistically different among the groups(P>0.05).Western Blot study showed that the expression of PLA2G4A protein in the Zhiyang Pingfu Liquid group tended to increase compared with the model group,but the difference was not statistically significant(P<0.05).The protein expression of all groups of matrine and baicalin was significantly higher than that of the model group,and the difference was statistically significant(P<0.05).In terms of the secretion of inflammatory factors,IL-6 levels were significantly lower in the Bh group compared with the Zhiyang Pingfu Liquid group,and the difference was statistically significant(P<0.05).No significant differences were detected between groups in terms of TNF-α and IL1β levels(P>0.05).Conclusions:Matrine and baicalin may be important components of "Zhiyang Pingfu Liquid".The expression of PLA2G4A protein can be regulated by "Zhiyang Pingfu Liquid".Experiment 2:Validation of the role of PLA2G4A gene in EGFRIs-related rashObjective:To clarify the role of PLA2G4A gene in the development of rash induced by EGFRIs.Methods:PLA2G4A gene overexpression lentivirus was applied to infect HaCaT cells to establish a PLA2G4A gene overexpression cell model,and HaCaT cells were infected with CON335 virus as a negative control group.TNF-α was applied as an inflammation inducing factor to continue the culture for 24,48 and 72 h.The cell viability and the secretion of inflammatory factor IL-6 were detected by CCK-8 method he ELISA method in both groups at 24,48 and 72 h.Results:The absorbance of PLA2G4A gene overexpression group was significantly lower compared with the negative control group at all time points,and the difference was statistically significant(P<0.05).After 72 hours of TNF-α intervention,the concentration of IL-6 in the PLA2G4A gene overexpression group decreased significantly compared with the negative control group,and the difference was statistically significant(P<0.05).Conclusions:PLA2G4A gene may be involved in the development of rash associated with EGFRIs by regulating cell viability and secretion of inflammatory factors.Experiment 3:Exploring the targets of "Zhiyang Pingfu Liquid" in the treatment of EGFRIs-related skin rash based on PLA2G4AObjective:To identify the PLA2G4A gene as a possible important target for the action of"Zhiyang Pingfu Liquid".Methods:The cells were divided into control group,model group,different concentrations of matrine or baicalin,PLA2G4A inhibitor group and DMSO group.Except for the control group,all groups were replaced with complete medium containing TNF-α 20 ng/mL and continued to be cultured for 72 hours.Thereafter,the model group continued to be replaced with complete medium containing TNF-α,the remaining groups were replaced with medium containing TNF-α and different concentrations of monomer,inhibitor or DMSO,and continued to be cultured for 24 hours.CCK-8 method was applied to detect cell viability,ELISA to detect IL-6 secretion,flow cytometry to detect cell cycle,and Western Blot to detect PLA2G4A protein expression.Results:In terms of cell viability,the model group decreased significantly compared with the control group,and the difference was statistically significant(P<0.05).The absorbance of matrine medium dose group,baicalin medium dose group,baicalin high dose group,and PLA2G4A inhibitor group increased significantly compared with the model group,and the difference was statistically significant(P<0.05).In terms of IL-6 concentration,the model group was significantly higher than the control group,and the difference was statistically significant(P<0.05).The IL-6 level in the matrine groups as well as the baicalin groups were not significantly changed compared with the model group,and the difference was not statistically significant(P>0.05).In terms of cell cycle,the percentage of S-phase cells in the model group decreased significantly compared with that in the control group,and the difference was statistically significant(P<0.05).The percentage of S-phase cells in the baicalin low dose group increased significantly compared with that in the model group,and the difference was statistically significant(P<0.05).There was no significant change in the percentage of S-phase cells in the low,middle and high dose groups of matrine and in the medium and high dose groups of baicalin compared with the model group,and the difference was not statistically significant(P>0.05).The expression of PLA2G4A protein was significantly decreased in the model group compared with the control group,and the difference was statistically significant(P<0.05).The expression of PLA2G4A protein was significantly increased in the middle and high dose groups of matrine compared with the model group,and the difference was statistically significant(P<0.05).The expression of protein was significantly decreased in the low and medium dose groups of baicalin compared with the model group,and the difference was statistically significant(P<0.05).Conclusions:PLA2G4A may be an important target of "Zhiyang Pingfu Liquid" in the treatment of rash induced by EGFRIs.Experiment 4:Exploring the pathway of "Zhiyang Pingfu Liquid" in the treatment of EGFRIs-related rash based on IL-6/JAK2/STAT3 signaling pathwayObjective:To make sure the IL-6/JAK2/STAT3 signaling pathway was identified as an important pathway of "Zhiyang Pingfu Liquid".Methods:The cells were divided into control group,model group,different concentrations of matrine or baicalin,IL-6 inhibitor group,JAK2 inhibitor group,STAT3 inhibitor group and DMSO group.Except for the control group,the cell culture medium was changed to complete medium containing TNF-α 20 ng/mL in each group and the culture was continued for 72 hours.At the end of the incubation period,the medium was changed again.The model group was replaced with complete medium containing TNF-α and the remaining groups were changed to medium containing TNF-α and different concentrations of monomer,inhibitor or DMSO,and continued to incubate for 24 hours.The cell viability was detected by CCK-8 method,the secretion of cytokines IL-8 and IL-17A was detected by ELISA,and the expression of pathwayrelated proteins was detected by Western Blot.Results:The absorbance of the model group decreased significantly compared with the control group,and the difference was statistically significant(P<0.05),The absorbance of baicalin high dose group was significantly higher than that of the model group,and the difference was statistically significant(P<0.05).The absorbance of the low,medium and high dose groups of matrine and the low and medium dose groups of baicalin did not change significantly compared with the model group,and the difference was not statistically significant(P>0.05).In terms of IL-8,the level was significantly higher in the model group compared with the control group,and the difference was statistically significant(P<0.05).The concentrations in the IL-6 inhibitor group and STAT3 inhibitor group decreased significantly compared with the model group,and the difference was statistically significant(P<0.05).In terms of IL-17A concentration,there was no significant change between groups,and the difference was not statistically significant(P>0.05)In terms of IL-6 protein expression,the expression in the low and high dose groups of baicalin decreased compared with the model group,and the difference was statistically significant(P<0.05).The IL-6 protein expression level of baicalin low,medium and high dose groups was significantly decreased compared with the control group,model group and IL-6 inhibitor group,and the difference was statistically significant(P<0.05).In terms of JAK2 protein expression,the low,medium and high dose groups of matrine was significantly higher than that in the control group,the model group and the JAK2 inhibitor group,and the difference was statistically significant(P<0.05).The protein expression in the baicalin high dose group was significantly decreased compared with the model group,and the difference was statistically significant(P<0.05).In terms of STAT3 protein expression,the model group was significantly decreased compared with the control group,and the difference was statistically significant(P<0.05).The expression in the matrine high dose group was significantly increased compared with the model group,and the difference was statistically significant(P<0.05).The protein expression of p-JAK2 and p-STAT3 in the matrine and baicalin groups was significantly lower than that in the model group,and the difference was statistically significant(P<0.05).Conclusions:The IL-6/JAK2/STAT3 signaling pathway may be an important pathway of"Zhiyang Pingfu Liquid" in the treatment of EGFRIs-related rash. |