The Study Of Satsuma-induced Inflammation And The Development Of Molecular Marker Related To Citrus Pro-inflammation

The functional components present in citrus fruits include carotenoids,flavonoids,vitamin C and limonoids,all of which are benefit for human health.It has long been observed that an intake of a certain amount of Satsuma(dose dependent,some people even eat one fruit)often caused symptoms,such as gum swelling,dry or sore throat,cheilosis,mouth ulcer,vomiting,diarrhea and so on,all of which is called shang huo in traditional Chinese public perception.The adverse reaction to Satsuma was defined as Satsuma-induced syndrome(SIS).To date,there are few studies that have investigated the pathogenesis of SIS.Volunteers were recruited and assigned to Satsuma challenge for three sequential years.A standard questionnaire scoring system was designed to evaluate SIS symptoms.Immunology assay and a comparative proteomic study were performed to investigate SIS.CitSHl protein was a main pro-inflammatory composition screened from Satsuma pulp in our laboratory,the preliminary research on CitSHl gene and its promoter was also conducted,a molecular marker was developed,which could successfully distinguish pro-inflammatory varieties from non pro-inflammatory ones and could be a useful tool for citrus breeding and variety improvement.The main innovation of this paper was as follows:(1)a standard questionnaire scoring system was designed to assess SIS symptoms;(2)it was the first time for volunteers to attend Satsuma challenge and their SIS symptoms were systematically and comprehensively analyzed,preliminary results indicate that SIS is an immune inflammatory response;(3)a molecular marker related to citrus pro-inflammatory effect was developed based on the sequence variation within the promoter region of CitSH1,which is important for citrus marker-assisted breeding.The main content and results were as follows:1.Satsuma-induced syndrome:an unrevealed immune inflammatory response by double-blind,placebo-controlled oral food challenge study1)Volunteers were assigned to oral Satsuma challenge for three sequential years in open manner(2009 and 2010)or double blind placebo-controlled manner(2011).Satsuma(Citrus unshiu Marc)was selected as the material and sweet orange(Citrus sinensis Osbeck)as the placebo control.We set up a dose of 1.5 kg citrus fruit for male and 1 kg for female volunteers each time.A standard questionnaire scoring system was designed to assess SIS symptoms,according to the severity scoring system,if a subject’s total score≥3 and then he/she was a SIS sufferer.After Satsuma challenge,the total rate of SIS volunteers was approximately 70%judged by the criteria and could be reproducible in different years.2)The levels of blood eosinophil and platelet were elevated significantly,whereas basophil and hemoglobin was reduced in SIS group after Satsuma challenge.Meanwhile,the serum total IgE and IgG levels did not differ significantly in SIS group.All the results show that SIS is quite different from IgE-mediated food allergy.Our preliminary results show that SIS is an immune inflammatory response induced by molecular "foreigner" to human body.2.Detection and identification of serum biomarker to understand inflammatoryresponse induced by Satsuma mandarin1)Among the 40 volunteers in 2010,15 were presented with severe SIS symptoms whose total score were greater than 6,and their sera were further divided into Pre-SC(before Satsuma challenge)and Post-SC(after Satsuma challenge)group.Pre-SC and Post-SC serum samples of 15 volunteers with severe SIS were analyzed by 2D gel electrophoresis and NanoLC-ESI-MS/MS peptide sequencing technology.10 proteins were identified to be differentially expressed(P<0.05).Of these differentially expressed proteins,levels of complement component C9 precursor increased significantly in the Post-SC serum samples,differential levels of which were further verified by enzyme-linked immunosorbent assay,C9 could serve as a potential biomarker for the diagnosis of SIS.2)The sera of SIS volunteers(severe SIS and mild SIS volunteers)were collected and further divided into Pre-SC and Post-SC group.All the serum samples were tested by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry(SELDI-TOF-MS)using the immobilized metal affinity capture(IMAC3)protein chip.A total of 50 biomarkers were detected to be different with statistical significance(P<0.05).A diagnostic model was constructed to distinguish the Post-SC from Pre-SC samples,using principal component analysis(PCA).3.The developing of a molecular marker related to citrus pro-inflammatory effect and utility testing of it in natural population1)Satsuma,’Succosa’ mandarin(Citrus Succosa hort.ex Tanaka),’Valencia’ sweat orange and ’Gaoban’ pummelo(Citrus grandis Osbeck)were selected as the material,and the first two can easily cause SIS,while the latter two do not induce SIS.Real-time quantitative PCR was used to study the expression of CitSH1 gene in different tissues of the four citrus varieties during fruit development.CitSH1 was slightly expressed in juice sacs of Satsuma(’Guoqing 1’)at the early stage of fruit development,but it increased sharply and reached the highest level at 185 days after flowering(DAF)(at ripening stage),then decresed to its former level,while,it was always slightly expressed in flavedo,albedo and segment membrane of Satsuma during fruit development.The CitSH1 was also always slightly expressed in juice sacs,flavedo,albedo and segment membrane of’Succosa’ mandarin,’Valencia’ sweat orange and ’Gaoban’ pummelo during fruit development(P<0.01).Meanwhile,the expression of CitSH1 gene in leaf of Satsuma,’Succosa’ mandarin and ’Gaoban’ pummelo was also studied.CitSH1 was relatively highly expressed in all leaf,and showed no statistical significance between three tested varieties,while,it was significantly different from CitSH1 gene in different tissues of four various citrus varieties during fruit development(except in juice sacs of Satsuma at 185 DAF,flavedo of Satsuma at 160 DAF and albedo of ’Valencia’ sweat orange at 160 DAF)(P<0.05).The most striking feature was that CitSH1 showed a high-level expression in leaf of three varieties and juice sacs of Satsuma just at ripening stage(at 185 DAF).2)Earlier work showed that there was little sequence variation and no difference in domain of CitSH1 between the four varieties.The sequence variation between varieties was studied in our laboratory,meanwhile,we used homologous cloning method to isolate the promoters of CitSH1 from four tested citrus varieties.The results showed that the promoters from each variety were highly heterozygous and eight sequences were obtained.Our isolated DNA bands revealed three sequence types of different sizes for the same region.The first sequence type was only found in ’Gaoban’ pummelo,with the insertion of a 45 bp sequence located～650 bases upstream of the translational start site.The second promoter type was present in all four varieties and showed limited variation between them.The third fragment type contained an insertion of～100 bp.This type was only found in the promoter region of ’Succosa’ mandarin and Satsuma(Guoqing 1).3)A molecular marker was developed based on the 100 bp insertion present in the third promoter type,which could successfully separate mandarin from sweet orange and pummelo.Total protein extracted from pulp of Satsuma,’Succosa’ mandarin and’Gaoban’ pummelo was used to stimulate mouse RAW264.7 cells,protein of Satsuma and’Succosa’ mandarin could significantly induce inflammatory response in mouse cells compared to that of ’Gaoban’ pummelo.Satsuma and ’Succosa’ mandarin are pro-inflammatory varieties while ’Gaoban’ pummelo is not.The molecular marker is well associated with citrus pro-inflammatory trait.4)PCR amplification of the CitSH1 promoter region from the 26 citrus varieties(both pro-inflammatory and non pro-inflammatory)showed a consistent genotype-phenotype relationship,i.e.,pro-inflammatory effect was associated with the insertion of～100 bp.Non pro-inflammatory varieties exhibited only the second promoter type,with no～100 bp inserted.5)Real-time quantitative PCR was used to study the expression of CitSH1 gene in mature fruit of ten various citrus varieties.CitSH1 was only relatively highly expressed in Satsuma,while there was a reduced expression in the other pro-inflammatory varieties(’Huang Lingmiao’ mandarin and ’Tangerine’etc)and non pro-inflammatory varieties(’Newhall Navel’ orange and ’Marsh grapefruit’ etc),indicating that the sequence variation(an insertion of～100 bp)in promoters does not completely determine the expression level of CitSH1,transcription factor may be also involved in this process.