JNK Pathway Regulates LPS-induced PD-L1Expression In The Human Umbilical Vein Endothelial Cells

BackgroundCoronary artery disease(CAD), which was also one of the most common, serious cardiovascular disease affected daily life quality, has become one of the three major killer as a threat to human health worldwide. Studies have shown that atherosclerosis is an inflammatory disease, a large number of macrophages, monocytes, T lymphocytes and dendritic cell infiltration of inflammatory cells in atherosclerotic plaque, plaque inflammation the response was very obvious; In the development and progression of coronary heart disease, whether from fatty streaks to atherosclerotic plaque or fibrous plaque, or the generation of unstable plaque rupture, thrombosis, and always have the participation of a variety of inflammatory cells. Thus, inhibition of inflammatory cell activity, research and treatment of coronary heart disease has become a hot and difficult path blocking the inflammatory response by immune regulation. As we all know, the activation of the inflammatory response significantly correlated with T lymphocytes, in recent years, studies have shown that, the negative stimuli signal (PD-1/PD-L1) play an important role in the activation of T lymphocytes.Also known as c-Jun N-terminal kinase or stress-activated protein kinase (SAPK), JNK mitogen-activated protein kinase (MAPK) superfamily, is a like serine/threonine kinase, is located in the cytoplasm of the cell, the relative molecular weight of46×103and54×103, there is a lot of functional areas (Thr- Pro-Tyr), the gene encoding JNK1, JNK2and JNK3, wherein JNK1and JNK2are present in the body tissues, while JNK3is mainly exist in the brain, heart and testis. JNK activation is achieved through the amino-terminal residues of phosphorylated JNK activation can be initiated by stimulation of a variety of extracellular factors, including growth factors, cytokines (IFN-gamma, IL-1, TNF-alpha) stress stimuli (UV, high osmotic pressure of the cell, ischemia-reperfusion injury). JNK activation, JNK will be transferred to the cytoplasm to the nucleus, is widely involved in apoptosis, proliferation, metabolism and DNA damage repair a variety of biological responses, and its function disorders can cause neurodegenerative disease, ischemia-reperfusion injury, diabetes and cancer and other diseases.Programmed cell death factor ligand1(B7-H1, CD274or PD-L1) is a type â…  transmembrane protein consisting of290amino acids. PD-L1is an important member of the B7family molecules, it is neither receptor CTLA-4protein (CTLA-4) is not ICOS protein (inducible co-stimulator, ICOS) and CD28protein, its receptor programmed death factor1(CD279or PD-1). In the initial study found that PD-L1is highly expressed in tumor cells, therefore, it is speculated that PD-L1may be associated with tumor cell invasion; Recent studies show that, in addition to tumor cells, PD-L1expression in many inflammatory cells, such as T lymphocytes, B lymphocytes, Kupffer cells, macrophages, astrocytes, endothelial cells, dendritic cells, placental syncytiotrophoblast and so on; PD-L1function, except for significantly associated with the infiltration of tumor cells, also occur with a variety of diseases have a close relationship, such as graft immune response, microbial infections (viral infection), autoimmune diseases, recent studies also found with atherosclerosis plaque formation significantly. Found that the Gotsman with fluorescence immunoassay technology research coronary PD-L1expression in multiple atherosclerotic plaque. Lee et al study also found that patients with coronary heart disease than normal compared to T lymphocytes and dendritic cells PD-1/PD-L1expression were significantly reduced, but enhanced T lymphocyte proliferation, confirmed PD-L1/PD-1caused by atherosclerosis T lymphocyte immune regulation are closely linked, can be a negative signal control coronary plaque formation and development plays an important role in the occurrence of coronary heart disease. Affecting the expression of PD-L1cell signaling pathways may PI3K/Akt pathway, JAK/STAT signaling pathway, NPM/ALK path, MEK/ERK signaling pathway, and STAT-3and IRF-1transcription factor. As we all know, JNK signaling pathway is an important branch of the MAPK pathway, it is a variety of cell apoptosis induced by extracellular stimuli. However, little information on the relationship of the PD-L1protein expression and JNK pathway.In view of the above evidence, the subject of in vitro cultured human umbilical vein endothelial cells for the study of inflammatory cytokines (LPS)-stimulated umbilical vein endothelial cells simulation model of the invasion of pathogenic microorganisms, PD-L1phenotypic changes observed umbilical vein endothelial cells; JNK protein-specific inhibitor SP600125blocked JNK pathway to explore umbilical vein endothelial cells express PD-L1and JNK signaling pathway, to elucidate the molecular mechanisms of LPS-induced human umbilical vein endothelial cells express PD-L1protein, improve the negative synergy stimulate signal (PD-1/PD-L) mechanism of regulation of T lymphocyte activity artery atherosclerosis immunotherapy is expected to provide new ideas and theoretical basis.Objective:1. LPS can induce human umbilical vein endothelial cells express PD-L1.2. After blocking JNK protein-specific inhibitor SP600125JNK pathway, PD-L1phenotypic changes observed dendritic cells stimulated by inflammatory factors, to investigate dendritic cell expression of PD-L1and JNK signaling pathway.Subjects and Methods:1.Subjects:In vitro culture human umbilical vein endothelial cells.2. Methods:2.1Umbilical vein endothelial cell isolation, identification and subcultureUnder sterile conditions, the healthy maternal After Cesarean cleaning Fresh umbilical cord, phosphate buffered saline (PBS) buffer and digested with collagenase type I, centrifugation, adding M199culture medium containing20%fetal bovine serum, placed5%CO2incubator at37℃. Medium was changed after24hours, the covered bottom until the cells reached80%-90%,0.125%trypsin digestion subculture, and the inverted cell morphology observed under the microscope, the selective growth of4to5cells with in the experiment. The morphology and endothelial cells VIII related antigen were identified.Primary cells usually after4-5days of culture cells more than95%of the bottom covered with culture, cell fusion into a single layer cobblestone-like under the microscope, and this time could be passaged. Discard the culture flasks old culture medium to warm up for20minutes), washed2-3times with PBS (37℃, by adding1ml of0.125%trypsin (37℃to warm up for20minutes) at room temperature to digest the cell20s, the cells were observed under a microscope wrinkle reduction round, separated from each other can be sucked out of the trypsin, and then join the M199complete medium digestion was terminated straw gentle pipetting to a uniform cell suspension culture pass by1:2.2.2Program cooling method cryopreservation endothelial cells, rapid melting method recovery of cells2.2.1Endothelial cell cryopreservationWith a disposable pipette to absorb the culture bottle waste in the culture medium, the cells were washed with PBS twice.0.125%trypsin digestion for20seconds, under the microscope cell rounding, shrinkage. The disposable pipette suction trypsin frozen liquid culture flask digestion was terminated. Elbow straw wind and percussion bottle cell for a few minutes, the cells were observed under a microscope all off. And then the cell suspension was added to the frozen pipes, mark the date, cell type, sealed plastic sealing.Programmed cell cooling:4℃refrigerator for20minutesâ†'-20℃refrigerator overnight at-70℃refrigerator1hourâ†'next morning in liquid nitrogen tank.2.2.2The recovery of the endothelial cells Tweezers removed from the liquid nitrogen tank labeled endothelial cells frozen pipes, quickly placed in a37℃water bath Shake to thaw frozen pipes cells completely (about1minute). Into the freezing tube with an alcohol wipe clean benches. M199complete medium previously added3times in a15ml centrifuge tube cryopreserved cell volume, intracellular fluid is sucked out of the mix added to the centrifuge tube with a disposable pipette. Low speed centrifugation:300rpm centrifuged for5minutes or centrifugation at500rpm for3minutes. Supernatant, add culture medium. The flasks set incubating tank culture, passaged upon confluence.2.3Experimental groups (each with3holes, the experiment was repeated4times)Harvest the fifth generation of human umbilical vein endothelial cells, cell density was adjusted to2×107/ml were seeded in6-well plates. Depending on whether LPS and JNK protein-specific inhibitor SP600125were divided into three groups:negative control group, LPS group and SP600125+LPS group (LPS and SB203580in dimethyl sulfoxide (DMSO) was dissolved).The first group as the negative control group (NORMAL):No any drug the cells cultured for24hours was used as a negative control.The second group of LPS stimulation (LPS):DMSO (0.1%V/V) was added in the experimental cells for1hour, and then adding LPS (1.0μg/ml) cultured24hours (LPS per12h medium was changed).The third group SP600125+LPS costimulatory Group:First experimental cells SP600125(20micromol/L) for1h and then stimulated with LPS for24h (LPS12h medium was changed).2.4Western blot analysis of PD-L1proteinExtracted from human umbilical vein endothelial cells from the6-well plates, whole-cell protein sample was extracted with RIPA cell lysate protein concentration was determined by the BCA method, transferred to PVDF membrane after SDS-PAGE electrophoresis protein isolate,5%nonfat milk closed2h, polyclonal anti-PD-L1antibody overnight at4℃incubation, secondary antibody after washing in PBS film,37℃for2h after ECL light exposure in the darkroom, developing and fixing. Quantity One gel analysis software system to analyze the value of the optical density of the protein bands.2.5Western blot analysis of P-JNK, JNK proteinExtracted from human umbilical vein endothelial cells from the6-well plates, whole-cell protein sample was extracted with RIPA cell lysate protein concentration was determined by the BCA method, transferred to PVDF membrane after SDS-PAGE electrophoresis protein isolate,5%nonfat milk closed2h, to join the anti-p-JNK polyclonal antibody and anti-JNK polyclonal antibody overnight at4℃incubation, PBS washing the membrane after the second antibody,37℃for2h after ECL light exposure in the darkroom, developing and fixing. Quantity One gel software analysis system protein bands of the optical density of p-JNK/JNK protein optical density ratio represents the optical density of the p-JNK were calculated negative control group, LPS group, SP600125+LPS group light the ratio of the density.3. Statistical MethodsAll data using SPSS13.0software metering data as mean±standard deviation (±S), multiple statistical analysis was used to compare the single-factor analysis of variance (one-way ANOVA) for multiple comparisons using the SNK-q test, P<0.05was considered statistically significant.4. Result4.1MTT assay proliferation of HUVECsUmbilical vein endothelial cells OD value determination. The OD of the negative control group was0.32±0.003, the LPS group OD value of0.34±0.033, SP600125+LPS group OD value of0.31±0.018OD value of each group, P>0.05, the proliferation of cells in each group was no significant difference.4.2Western blot analysis of PD-L1proteinWestern blot exposure grayscale scan results (Z value), the LPS group1.12±0.72, SP600125+LPS group was0.66±0.36in the control group to0.59±0.30. SP600125+LPS group and the control group Z values were less than LPS group (P <0.05or P<0.05) the SP600125+LPS group Z value, and negative control group was no significant difference (P>0.05). 4.3Western blot analysis of JNK and P-JNK proteinWestern blot exposure grayscale scan results (Z value), the LPS group1.26±0.09, SP600125+LPS group and the control group to0.49±0.18and0.95±0.21. SP600125+LPS group Z value is less than the other two groups (P<0.05or P<0.05), the LPS group Z value was significantly greater than in the control group (P <0.05).5. Conclusion1. LPS can induce human umbilical vein endothelial cells PD-L1expression increased.2. JNK signaling pathway may be involved in regulation of human umbilical vein endothelial cells express PD-L1.