Proteomic Screening Of Disease Specific And Treatment Monitoring Serum Biomarkers Of Inflammatory Bowel Disease

Background and ObjectivesInflammatory bowel disease is a chronic inflammatory disease mainly involves the bowel, with clinical manifestations of recurrent abdominal pain, diarrhea and bloody stool, it has serious impact on the quality of life of patients. Currently there is no effective diagnostic biomarker for IBD. Glucocorticoid can often significantly reduce inflammation and relieve symptoms quickly, however adverse side effects restrict its long-term use. While some patients do not respond to glucocorticoid therapy at all. Thus it is of great clinical significance to seek steroid treatment predictors. In recent years, the use of proteomic methods to find clinical markers has become one of the core applications of proteomics. In 2014, research group of Professor Li Zhili from Institute of Basic Medical Sciences found and confirmed novel circulating Immunoinflammation-Related Protein Complexes aspersonalized biomarkers of cancers using Native-PAGE, SDS-PAGE and MALDI-FTICR MS. Currently this method has not been used in the research of inflammatory diseases. In this study, we use Native-PAGE, SDS-PAGE and MALDI-FTICR MS method to find disease specific diagnostic serum biomarkers of IBD as well as treatment predictors through comparing the differences between serum proteins before and after glucocorticoid therapy.Part One:Proteomic Screening of Disease Specific Serum Biomarkers of Inflammatory Bowel DiseaseMethods:Serum protein complexes of 65 healthy controls,57 cases of colorectal cancer, 69 cases of UC and 67 cases of CD are isolated by native-PAGE. We quantify the gray values of the gel bands corresponding to the IIRPCs with Quantity One software. We then use SDS-PAGE separation to confirm the components of these complexes and MS to identify proteins.Results:According to the number and location of gel bands corresponding to the IIRPCs, all samples can be divided into pattern a, b, c, and others. Pattern a and b are the most common ones. Pattern a contains IIRPCsa1, a2, a3 and a4, pattern b contains IIRPCs b1, b2, b3, b4 and b5, while pattern c contains no IIRPCs. The levels of IIRPCs of UC, CD and Colorectal Cancer patients elevated significantly compared with healthy controls, with a p value less than 0.05. And the levels of IIRPCs of UC and CD patients are higher than Colorectal Cancer patients, also with a p value less than 0.05. However, there is no difference between the IIRPCs levels of UC and CD patients. The IIRPCs levels are higher in active UC patients than patients in remission (in moderate active patients b2, b4, b5, p<0.05; in severe active patients a3, b2, b5, p<0.05). The IIRPCs levels are higher in active CD patients than patients in remission, however with a p value over 0.05. The Pearson correlation coefficient between a3 and ESR are 0.576, p value<0.001; the Pearson correlation coefficient between a3 and hsCRP are 0.392, p=0.020; the Pearson correlation coefficient between b4 and ESR are 0.426, p=0.027; and the Pearson correlation coefficient between b4 and hsCRP are 0.375, p=0.054. The preliminary identification results of MS show that the main components of IIRPCs include IgG1, IgA1, haptoglobin protein, complement C3, complement C4A, complement factor H and others.Conclusions:1) Simple Native-PAGE can be used for the separation of disease-associated serum IIRPCs. And Based on the type of serum IIRPCs, IBD samples can be divided into four patterns:pattern a, b, c, and others. Pattern a and b are the most common ones.2) The main components of IIRPCs include IgG1, IgA1, haptoglobin protein, complement C3, complement C4A, complement factor H and others. All the proteins are related with immune system, inflammatory system and complement system.3) The levels of IIRPCs of UC and CD patients elevated significantly compared with healthy controls. It is a highly sensitive diagnostic biomarker for IBD. The IIRPCs levels are higher in active IBD patients than patients in remission, which implies that IIRPCscan be potential biomarkers to predict disease activity. IIRPCs also have a positive correlation with commonly used clinical inflammatory markers such as ESR and hsCRP.4) The levels of IIRPCs of UC and CD patients are higher than Colorectal Cancer patients. It can distinguish between IBD and colorectal cancer, however it can not distinguish between UC and CD.Part Two:Proteomic Screening of Glucocorticoid efficacy predictors in Inflammatory Bowel DiseaseMethods:Serum protein complexes of 45 groups of serum before and after glucocorticoid therapy are isolated by native-PAGE.We analyze the difference of serum proteins between each group to find Glucocorticoid efficacy predictors combing with clinical data.Results:Compared with serum before steroid treatment, the grey value of each gel band corresponding to the IIRPCs changes in the same trend. We divide patient into uptrend group and downtrend group. Statistical analysis showed that the rise or decline in the levels of IIRPCs are of statistical difference (p<0.05). The HGB levels and IIRPCs Ievels(a4、b5) before treatment are different between the uptrend and downtrend group (p<0.05). We also analyze the difference between 21 steroid effective and 7 steroid resistance UC patients, which showed that low albumin levels as well as CMV infection can be potential steroid resistance predictive factors. Because of limited number of serum samples after treatment, we can not conclude whether steroid efficacy-is related with uptrend or downtrend of IIRPCs.Conclusions:After steroid treatment, IIRPCs go either upward or downward. The HGB levels and IIRPCs levels (a4、b5) before treatment are different between the uptrend and downtrend group (p<0.05).