Investigate The Regulatory Effect Of Deficiency Of ERK Signaling Pathway In Lupus Epigenetics And The Role Of Osteopontin In SLE Patients With Renal Damage

Systemic lupus erythematosus(SLE) is a common autoimmune disease which damages multiple organs and lead to high mortality. In addition, Epigenetics defect with DNA methylation at the core is a principal element of SLE initiation.Studies found that, expression and enzyme activity of DNMT1 decline in T cells of SLE patients, in turn demethylation of the promoter of CD70 demethylated leads to excessive expression of related genes. Specific inhibitors of ERK signaling pathway would restrain DNMT1 gene expression and enzymatic activity in T cells and then arise immunologic disorder which similar to lupus T cells. Moreover, lower activity of ERK signaling pathway happened to exit in lupus T cells. Abnormality of cytokine secretion and complex regulatory network is the direct cause of the initiation of SLE immune disorders, chronic inflammation and damage of multiple organs and systems. Th17 cells (T helper cell 17) enhance inflammation of target organs of SLE by the main effector molecules interleukin-17, and further promote the development of their inflammatory injury. Abnormal activation of Thl7 cells and excessive secretion of IL-17 play an important role in SLE patients with renal damage especially. However, the exact mechanism of abnormal activation of Th17 cell and secretion of IL-17 in SLE with renal damage is still not clear. Recent studies found that osteopontin, as an major regulator of abnormal activation of Th17 cells and secretion of inflammatory factors, plays an important role in the initiation of SLE and inflammatory injury of target organs, especially in renal damage.Part 1 Investigate the regulatory effect of deficiency of ERK signaling pathway in SLE patients epigeneticsBackgroundEpigenetics defect with DNA methylation at the core is a principal element of SLE initiation.Our research finds that the DNMT1 gene expression in SLE patients’ T cells decreases and enzymatic activity declines, demethylation of promoters of CD70, IL-6 etc leads to corresponding gene overexpression. Recent studies report that specific inhibitors of ERK signaling pathway would restrain DNMT1 gene expression and enzymatic activity in T cells and in turn arise immunologic disorder which similar to lupus T cells. Moreover, lower activity of ERK signaling pathway happened to exit in lupus T cells, thus we infer that deficiency of ERK signaling pathway in Lupus T cell may be a key element of SLE epigenetic defects. We proposed to use flow cytometry, Western blot, RT-PCR to study the internal connection between ERK signal transduction pathway and epigenetic deficiency in lupus T cells. Meanwhile, we would treat normal T cells with specific inhibitor of ERK signal transduction pathway and DNMT1 to study the impact of ERK signal transduction pathway on lupus T cells epigenetics, thus improve the epigenetic theory of SLE pathogenesis and provide a new idea and a new target for the treatment of SLE.ObjectsTo explore the the regulatory effect of deficiency of ERK signaling pathway in SLE patients epigenetics with DNA methylation at the core through investigating the intrinsic connection of deficiency of ERK signaling pathway in SLE patient and T cells epigenetic defects and the impact of ERK signal transduction pathway in vitro on normal lupus T cells epigenetics, furthermore improve the epigenetic theory of SLE pathogenesis and fill the blank of domestic and foreign research, meanwhile provide new strategies and targets for the treatment of SLE.Methods1. According to the classification criteria for the diagnosis of SLE in 1997,10 cases of SLE patients were collected as experimental group and 10 cases of clinical data matched healthy check-ups as control.2. Glean peripheral blood of the experimental group and the control group, then separate CD4+T cells by immunomagnetic beads and test the percentage of CD4+T cells by flow cytometry;3. Collect peripheral blood of the experimental group and the control group, first separate mononuclear cells (PBMC) by Ficoll density gradient centrifugation, then separate human CD4+T cells by immunomagnetic beads, meanwhile extract total RNA, DNA, protein and store at appropriate conditions;4. Extract the total RNA, design primers of human ERK1, DNMT1 and CD70, amplify by reverse transcription and real-time quantitative PCR to analyze mRNA gene expression level;5. Detect the global DNA methylation level by Imprint Methylated DNA Quantification Kit (sigma, MDQ1, Amecica) using 5 methyl cytosine as DNA methylation biomarkers;6. Test the protein expression level of ERK1, DNMT1 and CD70 by Western blot;7. Human CD4+T cells Separated by 1MB were divided into ERK inhibited group (U0126), DNNT inhibited group (5-azacytidine), SLE and healthy control group, and specific inhibitors of ERK, DNMT1 were added respectively then co-cultured with human CD4+T cells for 72 hours, meanwhile CD4+T cells of the healthy control group were cultured without any treatment. Follow-up experiments were the same as step 4,5 and 6.Results1. Downregulation of DNMT and DNA hypomethylation were exist in lupus T cells. DNMT1 expression of T cells from peripheral blood in SLE group was significantly lower than that in healthy control group;2. Peripheral blood T cells in SLE patients overexpress CD70 which is methylation regulated gene. We find that the expression levels of CD70 in SLE patients increased significantly than healthy control group by western blot and RT-PCR;3. DNA methylation regulates the expression of a variety of methylation sensitive gene. Co-culturing of 5-azacytidine, a specific inhibitor of DNMT1 and human peripheral blood CD4+T cell would lead to declination of the level of DNA methylation and increasing of CD70 expression;4. Epigenetic regulating mechanisms in lupus T cells were associated with ERK1 signal transduction pathway. U0126, a selective inhibitor of the mitogen activated protein/extracellular signal regulated kinase could inhibit ERK1 signal transduction pathway in normal T cells, thus decrease mRNA and protein expression of DNMT1 and induce DNA demethylation, furthermore regulate the expression of methylation sensitive gene CD70.ConclusionsThe regulatory mechanism of epigenetics as DNA methylation at the core plays a principal role in SLE pathogenesis.Part 2 Investigate the regulatory effect of deficiency of ERK signaling pathway in MRL/lpr mice model epigeneticsBackgroundEpigenetics defect with DNA methylation at the core is a principal element of SLE initiation.Our research finds that the DNMTl gene expression in SLE patients’ T cells decreases and enzymatic activity declines, demethylation of promoters of CD70, IL-6 etc leads to corresponding gene overexpression. Recent studies report that specific inhibitors of ERK signaling pathway would restrain DNMT1 gene expression and enzymatic activity in T cells and in turn arise immunologic disorder which similar to lupus T cells. Moreover, lower activity of ERK signaling pathway happened to exit in lupus T cells, thus we infer that deficiency of ERK signaling pathway in Lupus T cell may be a key element of SLE epigenetic defects. We proposed to use flow cytometry, Western blot, RT-PCR to study the internal connection between ERK signal transduction pathway and epigenetic deficiency in MRL/1pr mice T cells. Meanwhile, we would treat normal T cells with specific inhibitor of ERK signal transduction pathway and DNMT1 to study the impact of ERK signal transduction pathway on MRL/1pr mice T cells epigenetics, thus improve the epigenetic theory of SLE pathogenesis and provide a new idea and a new target for the treatment of SLE.ObjectsTo explore the the regulatory effect of deficiency of ERK signaling pathway in MRL/lpr mice epigenetics with DNA methylation at the core through investigating the intrinsic connection of deficiency of ERK signaling pathway in MRL/lpr mice and T cells epigenetic defects and the impact of ERK signal transduction pathway in vitro on normal T cells epigenetics, furthermore improve the epigenetic theory of SLE pathogenesis and fill the blank of domestic and foreign research, meanwhile provide new strategies and targets for the treatment of SLE.Methods1.10 MRL/lpr mice were collected as experimental group and 10 healthy BALB/c mice as control.2. Glean splenic mononuclear cells of the experimental group and the control group, then separate CD4+T cells by immunomagnetic beads and test the percentage of CD4+T cells by flow cytometry;3. Collect splenic cells of the experimental group and the control group, first separate mononuclear cells (PBMC) by Ficoll density gradient centrifugation, then separate human CD4+T cells by immunomagnetic beads, meanwhile extract total RNA, DNA, protein and store at appropriate conditions;4. Extract the total RNA, design primers of mouse ERK1, DNMT1 and CD70, amplify by reverse transcription and real-time quantitative PCR to analyze mRNA gene expression level;5. Detect the global DNA methylation level by Imprint Methylated DNA Quantification Kit (sigma, MDQ1, Amecica) using 5 methyl cytosine as DNA methylation biomarkers;6. Test the protein expression level of ERK1, DNMT1 and CD70 by Western blot;7. Mouse CD4+T cells Separated by 1MB were divided into ERK inhibited group (U0126), DNNT inhibited group (5-azacytidine), SLE and healthy control group, and specific inhibitors of ERK, DNMT1 were added respectively then co-cultured with mouse CD4+T cells for 72 hours, meanwhile CD4+T cells of the healthy control group were cultured without any treatment. Follow-up experiments were the same as step 4,5 and 6.Results1. Downregulation of DNMT1 and DNA hypomethylation were exist in MRL/lpr mice T cells. DNMT1 expression of T cells from peripheral blood in SLE group was significantly lower than that in healthy control group;2. Peripheral blood T cells in MRL/lpr mice overexpress CD70 which is methylation regulated gene. We find that the expression levels of CD70 in MRL/lpr mice increased significantly than healthy control group by western blot and RT-PCR;3. DNA methylation regulates the expression of a variety of methylation sensitive gene. Co-culturing of 5-azacytidine, a specific inhibitor of DNMT1 and mouse peripheral blood CD4+T cell would lead to declination of the level of DNA methylation and increasing of CD70 expression;4. Epigenetic regulating mechanisms in lupus T cells were associated with ERK1 signal transduction pathway. U0126, a selective inhibitor of the mitogen activated protein/extracellular signal regulated kinase could inhibit ERK1 signal transduction pathway in normal T cells, thus decrease mRNA and protein expression of DNMT1 and induce DNA demethylation, furthermore regulate the expression of methylation sensitive gene CD70.ConclusionsThe regulatory mechanism of epigenetics as DNA methylation at the core plays a principal role in SLE pathogenesis.Part 3 The role of osteopontin in abnormol Th17 cell in SLE patients with renal damageBackgroundSystemic lupus erythematosus is an autoimmune disease characterized by target organ damage caused by a variety of inflammatory cytokines. Cell infiltration and inflammatory injury caused by immune complex formation, immunologic abnormalities such as immune cells and cytokines are key factors of the pathogenesis of renal damage in SLE.Th17 cells and multiple inflammatory cytokines secreted by Th17 cells have an important role in immunologic disorders and inflammatory injury in SLE. Scholars found that Th17 cells and IL-17 were highly expressed in peripheral blood and organ involved of SLE patients, especially in active SLE, yet declined after treatment. Other scholars found that the secretion of IL-17 and CD3 +CD4-CD8-T cells which secret IL-17 increased, furthermore Th17 cells secreting IL-17 exist in renal tissue with inflammatory injury in SLE patients.ObjectsIn order to investigate the regulatory role of osteopontin (OPN) in abnormal Th17 cells, we study the influence of OPN on serum IL-17 level and the rate of CD3+CD8-IL-17A+T cells from SLE patients.MethodsThe level of OPN and IL-17 in serum and the rate of CD3+CD8"IL-17A+T cell from 40 healthy controls and 50 SLE patients (25 SLE patients with renal damage and 25 SLE patients without renal damage) were detected by Enzyme-linked immunosorbent assay and flow cytometry methods.Results①The levels of OPN and IL-17 in serum, the rate of CD3+CD8-IL-17A+T cell in SLE patients with renal damage all obviously increased than SLE patients without renal damage and healthy controls(F=42.032,25.768,58.878, P<0.01), while there were no difference between SLE patients without renal damage and healthy controls (F=42.032,25.768,58.878, P>0.05);②Meanwhile, significant positive correlation were showed between serum OPN and serum IL-17 level, serum OPN level and CD3+CD8-IL-17A+T cell (correlation coefficients separately were 0.442、0.630, P<0.01).ConclusionOPN plays an important regulatory role in the abnormal Th17 cell of SLE patients with renal damage.