Analysis Of Serum Proteins In Chronic Urticaria And Effect Of Vitamin D In Its Pathogenesis

BackgroundUrticaria is a localized dermal edema reaction with small blood vessel dilation and increased permeability which mainly caused by mast cell activation.It often accompanied by wheal,itch and angioedema of varying sizes.According to the duration and frequency of this disease,it is divided into acute urticaria?AU?and chronic urticaria?CU?.CU persists for more than 6 weeks,and the pathogenesis is not very clear.It may involve coagulation,inflammation and autoimmunity.Due to its recurrent characteristics,it often has a serious impact on the lives of patients.More than 30%of severe patients still recur after 5 years.Clinically,the diagnosis of CU mainly depends on the collection of medical history and judgment of doctors to symptoms.This retrospective diagnosis method often requires much clinical experience and is highly subjective and easily misdiagnosed resulting in treatment is not timely.But there is no effective biomarkers for diagnosis,activity evaluation and efficacy expectation.The treatment of CU is mainly to eliminate the cause and symptom control.Currently,drugs for urticaria including antihistamines,cyclosporine,glucocorticoids and so on also bury hidden dangers for the recurrence of CU.Proteomics,studies of protein characteristics on a large scale,can help to understand pathogenesis of diseases comprehensively and is one of the most effective methods for finding potential molecular biomarkers and drug targets.iTRAQ?isobaric tag for relative and absolute quantification?is a newly developed proteomics quantitative research technology in recent years.It has advantages of high sensitivity,strong separation ability,reliable results,wide detection range and low detection limit,and has been an important method of proteomics and widely used in medical research.In recent years,it has been found that vitamin D,as a necessary vitamin for human body,not only participates in the regulation of calcium salt,but also plays an important regulatory role in the process of immune response.In vivo,25-hydroxyvitamin D₃ and Vitamin D binding protein?VDBP?are transferred to the site of action and hydroxylated again,and its active form 1,25-dihydroxyvitamin D₃can bind with Vitamin D receptor?VDR?on the surface of cell membrane or in the nucleus to regulate the transcription of many genes.Studies have found that vitamin D has therapeutic effects on asthma,eczema and other allergies,and vitamin D deficiency is widespread in CU patients,suggesting that vitamin D plays a role in the pathological process of urticaria.Vascular endothelial growth factor?VEGF?is one of the strongest factors to the permeability of blood vessels.It can increase vascular permeability,promote the formation of blood vessels.Studies have found that the concentration of VEGF in serum of CU patients is significantly higher than that of healthy people,and it is mainly derived from mast cell and eosinophil.Therefore,in this study we conducted mass spectrometry screening for CU patients by iTRAQ and analyzed differentially expressed proteins to look for potential biomarkers.Then we established mast cell model to explore the mechanism behind,so as to provide certain theoretical basis for the diagnosis and treatment of CU.Objective1.To screen and analyze the differential expression of proteins in serum of CU patients using iTRAQ.2.To explore preliminarily the mechanism of vitamin D in the pathological process of CU.Materials and Methods1.Serum specimens of 80 patients with CU who visited the department of dermatology of the First Affiliated Hospital of Zhengzhou University from December2017 to June 2018 were collected as the case group,and 60 healthy subjects who underwent physical examination in the same period as the control group.There were no statistical differences in age and gender between two groups;2.iTRAQ was used for mass spectrometry analysis of serum proteins in case group and control group;3.ELISA?enzyme-linked immunosorbent assay,ELISA?was used to verify the differential expression of proteins in serum of CU and healthy population.4.20 CU patients with high level of IgE?IgE>200IU?,20 CU patients with low level?IgE<100IU?,and 20 healthy controls were selected,and their serum was mixed at the same volume in each group.Respectively mixed serum was used to stimulate mast cell lines LAD2 and HMC-1 for 36h,24h,48h in 1:5 proportion.Then using RT-PCR?real time polymerase chain reaction,RT-PCR?system to detect VEGF and VDR mRNA,and using ELISA to detect the contents of VEGF in the supernatant5.The expression levels of VEGF and VDR mRNA were detected by RT-PCR after treating LAD2 cells with 10^-9M,10^-8M,10^-7M,10^-6M 25-hydroxyvitamin D₃ at 12h,24h and 36h6.LAD2 cells were treated with 10^-8M 25-hydroxyvitamin D₃ for 12h and then stimulated with high IgE mixed serum for 36h to detect the mRNA and protein expression levels of VEGF7.The phosphorylation levels of Akt?p38 MAPK?ERK1/2 and NF-?B were detected by Western blot after stimulating LAD2 cells with high IgE mixed serum for0min,15min,30min and 60min.Transcription factor HIF-1?were detected after stimulating for 1h,3h,5h,and 8h.8.LAD2 cells were treated with Akt?p38 MAPK?ERK1/2?NF-?B and HIF-1?inhibitors respectively,then the expression levels of VEGF mRNA were detected after stimulating cells with high IgE mixed serum for 36h.9.LAD2 cells were treated with Akt?p38 MAPK and HIF-1?inhibitors respectively,then the phosphorylation levels of Akt and p38 MAPK were detected after stimulating with high IgE mixed serum for 15min.HIF-1?was detected after stimulating for 3h.10.LAD2 cells were treated with 10^-8M 25-hydroxyvitamin D₃ for 12h and then the phosphorylation levels of Akt and p38 MAPK were detected after stimulating with high IgE mixed serum for 15min.HIF-1?was detected after stimulating for 3h.11.Western blot was used to detect the phosphorylation of p38 MAPK,ERK 1/2,PI3K/Akt,and NF-?B,and the proteins expression of HIF-1?and VDR in mast cells stimulated by serum with or without 25-hydroxyvitamin D₃.And corresponding inhibitors were used to explore the relationship of these signal pathways.Results1.Compared with the control group,31 differentially expressed proteins were detected in the serum of CU patients,including 25 up-regulated proteins and 6down-regulated proteins.2.Compared with the control group,the level of VDBP in the serum of CU patients was significantly increased?P=0.0179?.3.The expressions of mRNA and protein of VEGF in the mast cell LAD2 and HMC-1 stimulated by high IgE serum were higher than those in other groups?P<0.05?.There was no significant difference in VDR mRNA expression between all groups?P>0.05?.4.The mRNA expression of VDR was higher in 10^-8M 25-hydroxyvitamin D₃treatment group than that in other groups?P<0.01?,and the mRNA expression of VEGF was lower than that in other groups?P<0.05?.5.The expressions of mRNA and protein of VEGF in high IgE serum group were higher than those in the other groups?P<0.05?,while those in the high IgE serum+10^-8M 25-hydroxyvitamin D₃ group were lower in the high IgE serum group,but higher in control group and 10^-8M 25-hydroxyvitamin D₃ group?P<0.05?.6.The phosphorylation of Akt,p38 MAPK,ERK1/2 and the NF-?B and the expression of HIF-1?protein in LAD2 cell stimulated by high IgE serum were higher than those in control group?P<0.05?.7.After treated with corresponding inhibitors,the mRNA expressions of VEGF in Akt,p38 MAPK and HIF-1?inhibitors groups were lower than that in high IgE serum group?P<0.05?,but there was no statistical difference when that in ERK and NF-?B inhibitors groups compared with high IgE serum group?P>0.05?.8.Akt inhibitor can inhibit both the phosphorylation of p38 MAPK and the expression of HIF-1?protein in LAD2 cell;P38 MAPK inhibitor inhibits the expression of HIF-1??P<0.05?but has no effect on phosphorylating Akt?P>0.05?.9.The phosphorylation of Akt and p38 MAPK in LAD2 cell stimulated by high IgE serum for 15min was significantly reduced after 12h preliminary treatment with10^-8M 25-hydroxyvitamin D₃,and the expression of HIF-1?protein stimulated for 3h was reduced similarly?P<0.05?.Conclusions1.Compared with control group,there are a variety of differentially expressed proteins in serum of CU patients,among which VDBP has the potential to become a new biomarker for diagnosis of CU.2.By activating PI3K/Akt/p38 MAPK/HIF-1?pathway,mast cells produced and secreted VEGF after stimulated by serum of CU patients,which may be dependent on the content of IgE and the existence of Fc?R I of mast cells.3.Proper concentration of 25-hydroxyvitamin D₃ can inhibit mast cell producing VEGF through inhibiting the activation of the PI3K/Akt/p38 MAPK/HIF-1?pathway.