Effect Of Sesquiterpene Lactones On Inflammatory Factors In Rat Mesangial Cells Induced By AOPPs

Background and ObjectivesDiabetic nephropathy (diabetic ncphrophthy, DN) is one of the most prevalent and serious microvascular complications of diabetes. The pathology features of DN are characterized by expansion of the mesangial and matrix glomerular hypertroph and thickening of the glomerular basement membrane (GBM) which closely correlates with deterioration of renal function and therefore has long been considered as a crucial factor for progressive glomerulosclerosis in diabetes. With external stimuli, mesangial cells will rapidly phenotypic changes and then secrete a variety of growth factors such as platelet-derived growth factor, insulin-like growth factor, fibroblast growth factor, monocyte chemokines (MCP-1). These inflammatory cytokines to the recruitment of inflammatory cells, increased extracellular matrix accumulation. MCP-1has played a key role in this procedure, and is considered to be one of the main chemokines in renal disease pathogenesis.The pathogenesis of DN is not fully understood, many reasons may led to the DN, It is now generally believed that oxidative stress is the main reasons for damage to diabetic nephropathy. AOPPs are new uremic toxins reported by Witko-Sarsat in1996, which are the dityrosine-containing and cross-linking protein products formed during oxidative stress by reaction of chlorinated oxidants with plasma protein. The recent studies have found that chronic accumulation of AOPPs promotes inflammation in diabetic and non-diabetic kidney This suggest that AOPPs may be involved in the formation of diabetic nephropathy. Recent studies have shown that AOPPs can generate reactive oxygen (ROS) through NADPH oxidase and activation of protein kinase C in mesangial cells, and supr oxidative stress in mesangial cells. Mitogen-activated protein kinase (mitogen activated protein kinases, MAPKs), nuclear factor (nuclear factor NF-κB)-kappa B are both signal sensitive to oxidative stress, they mainly involved in cell proliferation, differentiation, transformation and apoptosis,and may occurr to the mechanism of inflammation, and the last contribute to renal inflammatory response.Parthenolide (parthenolide, PTL) is a purified sesquiterpene esters from the feverfew (Tanacetum parthenium), and is confirmed to be the best components of the pharmacological activity. It has been used to treat fever, migraine, asthma and joint pain. But its efficacy is far from these, many experiments show that parthenolide is a specific and strong effective inhibitor of NF-κB, directly blocking NF-κB and (or) aim to the IKK complex, showing a strong effectof antioxidant, antiinflammation, antitumor. In this experiment, we synthesized a series of sesquiterpene lactones and research the structure and function of sesquiterpene lactones.In this study, we researched mesangial cells in vitro, and observed advanced oxidation protein products whether or not activate the MAPKs/NF-κB signaling pathway and upregulation mcp-1expression, and sesquiterpene lactones whether or not suppress the NF-κB and mcp-1expression. More importantly, we observed the structure and function of sesquiterpene lactones in comparison with parthenolide.Contents and methods1. Preparation of AOPPsAOPPs was prepared in vitro according to the method described by Witko-Sarsat. AOPPs content was determined by measuring absorbance at340nm in acidic condition and was calibrated with Chloramines-T in the presence of potassium iodine.2. Cell cllllureEstablished rat glomerular MCs(HBZY-1)were obtained from life-Science Academy of wuhan uniwersity. Cells were cultured in DMEM supplemented with 10%fetal bovin serum,2mMglutamine,100u/ml penicillin and streptomycin at37℃,5%CO2. After confluence reached80%, mesangial cells were growth-arrested in1%FCS for overnight and treated with AOPPs for the indicated concentration and time.3. Determination of MCP-1protein expressionMesangial cells were seeded into12-well plates at3.5x104cells/ml in a volume of2ml medium. The cells were exposed to0,50,100,200(μg/ml) AOPPs and200(μg/ml), unmodified RSA after24hours incubation. Supematants were collected at the end of the experimental period and MCP-1in the media were determined by Enzyme-Linked immunosorbent Assays Kits.4. Determination of MCP-lmRNA by RT-PCRMesangial cells were treated with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after24hours incubation, respectively. Total RNA of RMCs was extracted with TRIZOL reagent. MCP-1mRNA was detected by RT-PCR according to the manufacturer’s protocol.5. P47phox Membrane TranslocationMesangial cells were treated with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hours incubation, extracted cytoplasm and cell membrane proteins according to the manufacturer’s protocol. The p47phox Membrane Translocation on RMCs was observed by Western blot.6. Effect of AOPPs on MAPKs signaling pathways in cellsMesangial cells were treated with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hour incubation, cells were collected and total protein was extracted according to the manufacturer’s protocol, the p-P38, P38, p-JNK, JNK, p-ERKl/2, ERK1/2protein expression was detected by Western blot.7. Effect of AOPPs on the intracellular signaling pathway of NF-κBMesangial cells were treated with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hour incubation, cells were collected and total protein was extracted according to the manufacturer’s protocol, the NF-icB/p65, p-of IκBa, IκBa, p-IKKa and IKKa protein expression and nuclear p-NF-icB/p65protein expression was detected by Western blot. 8. Effect of Sesquiterpene lactones compounds on AOPPs induced MCP-1The sesquiterpene lactones compound10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated24hours,Supematants were collected at the end of the experimental period and MCP-1in the media were determined by Enzyme-Linked immunosorbent Assays Kits-9. Effect of Sesquiterpene lactones on AOPPs-induced MCP-lmRNAThe sesquiterpene lactones compound10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated24hours, respectively. Total RNA of RMCs was extracted with TRIZOL reagent. MCP-1mRNA was detected by RT-PCR according to the manufacturer’s protocol.10. Effect of Sesquiterpene lactones on AOPPs-induced cell signaling pathway of NF-κBThe sesquiterpene lactones compound5,10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated1hours, cells were collected and total protein was extracted according to the manufacturer’s protocol, the total IκBα protein expression was detected by Western blot.StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0. The accepted level of significance was P<0.05.Result1.Characterization of AOPPsThe protein concentration of AOPPs and unmodified RSA were8.52mg/ml and10.3mg/ml, Respectively. AOPPs in the preparation of AOPPs and unmodified RSA were538.14μmol/1and1.56umol/1, respectively, which were63.16nmol/mg protein and0.15nmol/mg protein respectively after calibrated by protein concentration. The concentration of endotoxin in all preparations was lower than0.25EU/ml.2.1Effect of AOPPs on MCP-1expressionAfter the stimulation of RMCs with0,50,100,200(μg/ml) AOPPs or200(μg /ml) of unmodified RSA after24hour incubation, the Excretion of MCP-1increase gradually with the increase of doses of AOPPs (P<0.05). Unmodified RSA was without effect on these protein expression (P>0.05).2.2Effect of AOPPs on MCP-1gene expressionAfter the stimulation of RMCs with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after24hour incubation, the mRNA expression of MCP-1increase gradually with the increase of doses of AOPPs (P<0.05). Unmodified RSA was without effect on these gene expression (P>0.05).3. AOPPs induced intracellular p47phox membrane migrationAfter the stimulation of RMCs with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hour incubation, the cytoplasm of p47phox decrease gradually with the increase of doses of AOPPs (P<0.05), the cell membrane of p47phox increase gradually with the increase of doses of AOPPs (P<0.05). Unmodified RSA was without effect on p47phox membrane migration (p>0.05).4. AOPPs active MAPKs signaling pathways in cellsAfter the stimulation of RMCs with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hour incubation, then intracellular phosphorylation P38, JNK and ERK1/2expression gradually increased with the increase of doses of AOPPs (p<0.05), intracellular total of P38, JNK and ERK1/2protein expression levels unchanged (p>0.05). No significant changes of phosphorylation P38, JNK and ERK1/2with the unmodified RSA(p>0.05).5. AOPPs activation of NF-κB signaling pathways in cellsAfter the stimulation of RMCs with0,50,100,200(μg/ml) AOPPs or200(μg/ml) of unmodified RSA after1hour incubation, then intracellular phosphorylation IκBα, IKKα and nuclear p-NF-κB/p65protein expression gradually increased with the increase of doses of AOPPs (p<0.05), the total IκBα degradation is gradually increased with the concentration of AOPPs (p<0.05), intracellular total of NF-κB/p65, IKKa protein expression levels unchanged (p>0.05). No significant changes of phosphorylation IκBα, IKKα, NF-κB/p65with the unmodified RSA(p>0.05).6. Sesquiterpene lactones inhibit AOPPs induced MCP-1 The sesquiterpene lactones compound10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated24hours. MCP-1were significantly lower than AOPPs group (p<0.05).7. Sesquiterpene lactones inhibit the the AOPPs induction of cell MCP-1mRNAThe sesquiterpene lactones compound10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated24hours. MCP-1mRNA content in cells was significantly lower than AOPPs group (p<0.05).8. Sesquiterpene lactones inhibit AOPPs-induced cell signaling pathway of NF-κB activationThe sesquiterpene lactones compound5,10(μmol/L) after1hour incubation with the MCs, then AOPPs200(μg/ml) stimulated lhours. The cells within the overall degradation of IκBa was inhibited by SLs (p<0.05).ConclusionAOPP scan quickly induce generation of reactive oxygen species in rat mesangial cells, and active MAPK/NF-κB signaling pathway, eventually leading to the up regulation of MCP-1mRNA and protein expression. Sesquiterpene lactones can reveal AOPPs induced IκcBa protein ubiquitination degradation and NF-κB activation in mesangial cells, further inhibiting MCP-1mRNA and protein expression in mesangial cells. This suggests AOPPs may play an important role in the development process of DN, Sesquiterpene lactones inhibit AOPPs induced inflammation may be associated with the inhibition of NF-κB activity occurred in DN. Compared with PTL, The independent synthesis of Sesquiterpene lactones have a significant advantages in the preparation process, stability, bioavailability, toxicity, show a broad development prospects for clinical application.