Author name:
صباح زيارة كاظم المالكي
Supervisor name:
عبد العظيم ياسين عبود البراك | متزي نكاركاتي
Abstract:
تعتبر اصابة الكبد الحاد احد الامراض ذو الاعراض السريرية التي تتصف بارتشاح الخلايا اللمفية الى الكبد وتجمع السوائل وارتفاع انزايمات الكبد مثل ناقل امين الاسبارتات Aspartate aminotransferase(AST) في مصل الدم. هنالك الكثير من الاسباب التي تؤدي الى حدوث ضرر | Acute Liver Injury is a vital clinical syndrome characterized by infiltration of lymphocytes in the liver, fluid accumulation, and elevation of liver enzymes such as Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum. There are many causes of liver injury, but in this particular study Staphylococcal Enterotoxin B (SEB) was used to induce acute liver injury in mice, it is previously known that (SEB) it act as superantigen that bind with T cell receptor variable region beta chain (VB8), and MHC II of Antigen Presenting Cells (APCs), which leads to activation of T - cells, and huge secretion of pro - inflammatory cytokines, all of these events cause an acute liver injury lead to liver failure, and death. In the last few decades the importance of some natural products appeared, in which these botanicals have an anti - inflammatory properties. Resveratrol is one of these botanicals with an anti - inflammatory effects, which was used as treatment in this study.This study was particularly intended to study the influence of Resveratrol on acute liver injury induced by Staphylococcus Enterotoxin B, lead to liver failure which includes estimation of aspartate aminotransferase levels (AST), histopathological analysis, immune cell counts of liver and spleen, in addition, study whether resveratrol has the ability to cause immunological changes on different immune cells that lead to suppress acute liver injury by using flowcytometry technique,these cells are inflammatory cells carrying CD3+ T cells, CD4+ T cells, CD8+ T cells, NK1.1+ T cells, CD44+ T cells, and suppressive cells such as Myeloid Derived Suppressor Cells (MDSCs), T regulatory cell (FOXP3), as anit - inflammatory markers. Furthermore evaluate the pro - inflammatory, and anti - inflammatory cytokines by using Bio - plex. Also to determine the molecular mechanism in which resveratrol can lead to induce changes in microRNAs (miRNAs) by microarray analysis. Finally validation (miRNAs) resulting from microarray analysis and genes of interest associated with these miRNAs by Real time polymearase chain reaction (RT - PCR).After sixteen hours blood samples were collected to determine AST levels in differents groups using Nanodrop, (Fisher - USA), after sixty hours animals sacrificing organs (liver, spleen) were collected afterward liver samples in 10% formalin were sent for histopathological study, liver immune cells were isolated for counts and molecular study (microarray analysis and quantitive (RT - PCR) total RNA was isolated from liver immune cells for microarray analysis then preparation of complementary Deoxyribonucleic acid (cDNA) by Reverse transcriptase PCR. Validation of the microarray analysis data was achieved by Real time PCR. Spleen immune cells were collected afterward for flowcytometric analysis of CD3+, CD4+, CD8+, NK1.1+, CD44+ T cells, MDSCs, and T regulatory cells using cell surface staining and intra cellular staining.This study showed that Resveratrol oppose the effect of SEB induced acute liver injury through decrease AST concentrations (P< 0.0001). Also Resveratrol 50mg/Kg body weight led to cause reduction in the size of the spleen, compared to SEB+ Vehicle group. Histopathology study reported that resveratrol led to decrease of hepatic necrosis up to 10% in comparison to vehicle group with 60% of hepatic necrosis and decrease in the inflammatory response represented by immune cells as in liver immune cell counts which showed a significant decrease (P< 0.014) after 50mg/ Kg body weight of Resveratrol treatment. Spleen immune cell counts result reported a significant decrease (P<0.013) in the total cells when Resveratrol was used as treatment compared to disease group. Regarding to Flowcytometric analysis, it was found that Resveratrol significantly decrease the absolute count of CD3+, CD4+, CD8+, NK1.1+, CD44+, Foxp3+ T cells in C57BL/6 mice (P< 0.0019, P< 0.001, P< 0.009, P< 0.002, P< 0.03, and P< 0.03 respectively). While MDSCs result reported significant increase with P< 0.01 in absolute cell counts after Resveratrol treatment. Depending on the Bio - plex data showed significant decrease in serum concentrations of inflammatory cytokines IL - 1?, IL - 1?, IL - 3, IL - 5, IL - 12p(40), IL - 12p(70), IL - 13, IFN?, MIP - 1?, MIP - 1?, KC and Rantes cytokines with significant P values (0.009, 0.004, 0.034, 0.004, 0.0009, 0.01, 0.0007, 0.009, 0.0037, 0.0001, 0.0035, 0.006 respectively), while anti - inflammatory cytokines IL - 6, IL - 10 and G - CSF c showed a significant increase in the serum concentration after Resveratrol treatment with a P values of, 0.012, 0.02 and 0.001 respectively. Microarray analysis results recognized 451 miRNAs with P< 0.05, according to fold change expression, 78 miRNAs have fold change greater than - 1.5 fold of expression in Resveratrol treatment group (downregulated), while 87 miRNAs have fold change greater than 1.5 fold of expression (upregulated). According to the ingenuity tools predicted target gene for some of these miRNAs by using miRNA.org database, the database showed that miR - 130a - 3p with - 2.28 fold expression have direct relation of binding with CSF1 gene, Real time PCR results confirmed the results obtained from microarray analysis, the result of miR - 130a - 3p showed a significant decrease of relative fold expression with P< 0.013, and Colony Stimulating Factor - 1 CSF1 (M - CSF) relative expression increased significantly after resveratrol treatment with P< 0.0001. From all of previous data, it can be concluded that Resveratrol can counteract acute liver injury induced by SEB, by decrease AST concentration. Resveratrol acts as an anti - inflammatory compound due to decrease of immune cell numbers, decrease of inflammatory markers, and increase of anti - inflammatory markers. miR - 130a - 3p with inflammatory properties downregulated after resveratrol treatment. Finally Resveratrol treatment increased relative fold expression of CSF1(M - CSF); gene which play a role in the MDSCs proliferation
