Study On The Effect Of Hypoxia/Reoxygenation And Cyclosporine A On Expression Of MIC And Yisheng Injection Intervention

Objective Chronic graft dysfunction is the most rigorous challenge to long-term graft survival although the immediate outcome in organ transplantation has been greatly improved. Hepatic ischemia/reperfusion injury(IRI) is an vital nonimmunologic factor and major stress factor, which remains a significant problem and limitation of liver transplantation and result in hepatic primary graft dysfunction, delayed graft function and chronic graft dysfunction.The human major histocompatibility complex class I chain-related antigen A, B (MICA, B) were deemed to "cell stress sensors", which encodes cellular stress-inducible surface proteins that binds to NKG2D, an activating receptor of NK, alphabeta and gammadelta T cells, and could function as a bridge between natural immune response and acquired immune response. Increasingdata demonstrate that MIC may play a role in allograft rejection, however, whether IRI affects the expression of MIC has not been documented. Our previous studies have found that hepatic ischemia/reperfusion mjury(IRI) increased RAE-1 and H60(MICA,B homologues) mRNA levels in mouse liver. One purpose of this study is to investigate whether hypoxia/reoxygenation (H/R) could upregulate expression of MIC A, Bin Human hepatocytes.Yisheng injection(YS) is a herbal preparation developed from traditional Chinese medicine. Our previous studies demonstrated that YS had protective effect on ischemia/reperfusion injury in vitro and in vivo . Based on the previous results, this study explored the effect of YS intervention on the expression of MICA and MICB gene in human hepatocytes suffered to hypoxia/reoxygenation.In addition, Cyclosporine (CsA) has improved patient and graft survival rates following solid-organ transplantation. However, the clinical use of CsA is often limited by acute and chronic toxicity, which remains a major problem. The toxicity of CsA is another violent stress factor also, so, this study want to explore whether CsA can effect the expression of MIC, which may be one of mechanisms underlying chronic CsA hepatic toxicity.Methods Use model of cell hypoxia/reoxygenation in vitro to mimicischemia/reperfusion injury of graft organ. There have three experimental groups: (1) hypoxia/reoxygenation group, (2) YS(lg7ml) treated group, (3) CsA treated(5ug/ml 20ug/ml) group.l.The cultured human hepatocytes were exposed to hypoxic condition for 15h and 22h under 1%O2,94%N2 and 5%CO2, then reoxygenation for 2ru 41k 6h,12h and 24h separately under normal condition. Total RNA was extracted from hepatocytes and quantified by UV spectrophotometer. RNA was reverse transcribed and amplified by semi-quantitative RT-PCR. Expression levels were calculated relative to /3-actin. All samples were detected in triplicate. On the other hand, the surface expressions of MIC protein were detected by immunofluorescence with a rabbit polyclonal antibody which is recommended for the detection of MICA and MICB of human origin.2. YS(lg/ml) was added to cells before hypoxia and a second dose was gived just before reoxygenation. The expression of MICA,B were measured by semi-quantitative RT-PCR and immunofluorescence.3. CsA with different doses(5,20/ig/ml) was added to culture medium for lh, 3h, 5h, 8h, 24h separately. Use immunofluorescence of cell to detect the expression of MIC protein .Results1.Compared with the mRNA level of normal group, Hpoxia/Reoxygenation increased MICA,B mRNA in hepatocytes. Undergoing 15h of hypoxia, MICA,B mRNA upregulated from 2h after reoxygenation compared with normal group (PO.05) and maintained the high level from 6h to 24h after reoxygenation. Undergoing 22h of hypoxia, MICA,B mRNA began to upregulate until 24h after reoxygenation (PO.05). Immunofluorescence also confirmed the results above.2. Administration of YS(l^ml) significantly reduced the mRNA and protein levels of MICA, B in human hepatocytes suffered to Hpoxia/Reoxygenation injury.3. CsA(5,20/ig/ml) up-regulated MIC protein expression in hepatocytes fromlh , peaked at 3h, and persisted until 5h, then went back to normal level after 8h.Conclusion The process of transplantation itself is a violent andpersistent stress including IRI, toxicity of imrnunosuppressor and so on. This study first reported that H/R induced a strong increase of MICA, B gene at mRNA and protein levels in human hepatocytes and administration of YS significantly reduced the mRNA and protein levels of MICA,B in hepatocytes subjected to hypoxia/reoxygenation though the mechanism was unknown. On the other hand, CsA can also up-regulate expression of MIC. MIC, as the "cellular stress biomarker" ,could play a significant role in activating NK , T lymphocyte responses and humoral immunity associated with transplantation. The rapid and delayed upregulations of MIC in hepatocytes might be one of reasons of primary graft dysfunction and delayed graft function in liver transplantation. So, these molecules might become the new interfering targets for prevention.