Effects Of Erigeron On The Expression Of MyD88and TNF-α In Rat Peritoneal Mesothelial Cells Stimulated By High Glucose

Patients due to various causes which result in end-stage renal disease (End-Stage Kidney Disease ESKD) is increasing year by year, according to the researchers speculate. There is about one million ESKD patients who need peritoneal dialysis in China.Peritoneal dialysis (peritoneal dialysis PD) has become an important choice of these patients with end-stage renal disease.There is more than200thousand PD patients, accounting for the total number of dialysis14%. It has the advantages of simple operation, small, effects on cardiovascular protection of residual renal function, patients do not need to make a round trip in order to resieve hospital treatment (what means the patient can be on their own dialysis at home).PD can be divided into intermittent peritoneal dialysis (Intermittent Peritoneal Dialysis IPD), continuous ambulatory peritoneal dialysis (Continuouse Ambulatory Pritoneal Dialysis CAPD), continuous cyclic peritoneal dialysis (Automatic Peritoneal Dialysis, APD).Clinical observation found that peritoneal structure of chronic peritoneal dialysis is easy to damage and result in membrane transport function limiting.We name this ultrafiltration failure(UFF),which is the main reason made patient of PD give up such kind dislysis.Study found that the rate of ultrafiltration failure is high:4years after PD, the incidence of UFF is about35%.5%-10%PD of patients with ultrafiltration failure have to turn to hemodialysis. The peritoneum is a semipermeable membrane layer of physiology sex. Capillary wall, peritoneal interstitial, mesothelial cell layer, and dialysate and peritoneal mesothelial cells,endothelial cells in blood and fluid retention layer are the main barrier of peritoneal solute transport.Human peritoneal mesothelial cells(HPMC) is composed of peritoneal surface layer which is the mainly cell populations and mesenchymal transdifferentiation of HPMC is the initiating and reversible process caused in peritoneal fibrsis (PF) during the process of PD. The main physiological function of mesothelial cells is to reduce the friction of intra-abdominal visceral surface, provide a protective barrier between blood vessels and peritoneal solute, join in the exchange between vessel and peritoneal solute, the secretion of cytokines, impel inflammatory cells in the abdominal cavity to gather, secretion of vasodilators, regulate peritoneal microcirculation.Epithelial cell loss adhesion and change to the muscle phenotype transition fiber cells which is the key steps of mesenchymal transdifferentiation (epithelial mesenchymal transition,EMT).Induced factors med-iate inflammatory responses through the stimulation of mesothelial cells to produce cytokines.Many reasons can cause damage or apoptosis of mesothelial cells, leading to peritoneal transport function decline and resulting in UFF.The peritoneal dialysis mechanism is the use of high concentrations of peritoneal dialysis fluid and the function of peritoneal membrane’s semipermeable membrane to ultrafiltrate body water and medium molecular substance.According to the characteristics of the peritoneal membrane function, the UFF can be divided into4categories, namely the peritoneal permeability (type I), peritoneal lymphatic flow increased (type Ⅲ), the water channel protein (type IV) and peritoneal fibrosis (Ⅱ).The long-term repeated dialysis peritoneal mesothelial cells on carrier expression and glycosylation of peritoneal tissue, peritoneal ultrafiltration in peritoneal dialysis1years out of3%, in3years to10%in6years, often exceeding30%.Recent studies have found that Toll like receptor (TLRs) signal pathway is the bridge of mediating inflammatory response. The study also showed that the inflammatory reaction mediated by the important factors lead to peritoneal ultrafiltration failure. TLR4is the more mainly in-depth being studied one.TLRs is one of the main pathogen pattern receptors in innate immune system, which can identify endogenous ligand induced inflammatory process. Its activation and signaling pathways in open play an important role in the inflammatory reaction and the natural defense immunity.According to the signal conduction is dependent on MyD88, TLRs signaling can be divided into:a MyD88dependent pathway and the MyD88independent pathways,mainly in the first pathway.Therefore MyD88is a molecular key joint to downstream of Toll like receptor signal transduction.TNF-α is an important inflammatory factors release downstream of this signaling pathway.IL-2, IL-6and other cytokines mediate cytotoxicity and immune inflammatory responses,which lead to tissue damage.The expression of MyD88, TNF-αweak or strong can reflex the level of inflammatory reaction mediated by TLR4signaling pathway in peritoneal mesothelial cell.To sum up,this study provide theory evidence to reduce the injury of peritoneal mesothelial cell and improve the prognosis of peritoneal dialysis-related peritonitis through observing the effects of erigeron on the expression MyD88and TNF-α in rat peritoneal mesothelial cells stimulated by high glucose. The study also provide theoretical and experimental basis for the clinical application of breviscapine.The details are as follows:Chart I Effect of peritoneal dialysis fluid on the proliferation of RPMCS and the expression of MyD88. TNF-aObjective:To explore the effect of peritoneal dialysis fluid on the proliferation of RPMCS and the expression of MyD88> TNF-a.Methods:①To acquired of primary rat peritoneal mesothelial cellsSelected male SD rats with the weight of150±20g. Ether inhalation anesthesia were obtained.Got abdominal wall peritoneum, stripped vascular and adipose, steriled three times with PBS rinse, used0.125%trypsin to digest for15min with15ml.During the digestive process in37℃incubation, shock every5min, and observed the digestion under microscope.Collect the cells by centrifugation and used 1ml DMEM/F12culture medium containing20%fetal bovine serum (mixed with penicillin100u/ml, streptomycin was100μg/ml, hydrocortisone1μg/ml,0.5μg/ml insulin, transferrin,1μg/ml) suspend cells, and then inoculated in3ml complete medium, the bottom area of culture dish21cm in37℃5%CO2incubation box culture overnight after the first medium change.Passage the cells with complete medium which do not contain antibiotics every3days.The third generation cells were used for experiment. Morphological identification:dish of rat peritoneal mesothelial cells of fusiform culture under light microscope.About7days or so cells can reach fusion. The fusion cells were polygonal forming a cobblestone-like appearance.Use anti-keratin monoclonal antibody, anti vimentin monoclonal antibody to immunofluorescence identification.The third generation cells Jimusa smear, smear, cell morphology was observed under the microscope and the calculation of purity.︰sed the third generation of logarithmic phase growth of RPMCS to culture in96well plates according to the concentration of1X105for24hour.When the cells attached to the wall,cultured by serum-free medium ovemight.Cells were divided into control group (group a),1.5%glucose group (b group),2.5%glucose group (c group),4.25%glucose group (group d).each group was respectively stimulated by glucose of different concentration for4h,6h,8h. Add MTT solution20μL5mg/ml into6complex hole and cultured for4h. The cells aggregated to form a snowflake blue purple crystal under microscope.Then careful extract the liquid, add150p, L DMSO in shaker and shake10min. Use the wavelength of490nm to exam the value of OD.After the cells were adherent, culture supernatant with peritoneal dialysis fluid for6h.Got the liquid supernatant as sample.Examed according to ELISA kit instructions. Establish the standard curve under450nm UV detection. According the standard curve to calculate the concentration of MyD88, TNF-α in a sample to be tested (ng/ml).Results:1.Compared with the control group, the OD value of three glucose groups with different concentrations in RPMCS was significantly reduced (P<0.01) and was dependent on time and the glucose concentration.the longer and higher glucose concentration, the more significant value of OD decreased (P<0.05).2.After the cells were adherent,used peritoneal dialysis solution of1.5%,2.5%,4.25%stimulating for6h. Compare with control group, the expression of MyD88, TNF-α increased significantly (P<0.01), and the higher the glucose concentration is, the concentration of MyD88, TNF-a increases more obviously (P<0.01).Conclusions:1. Peritoneal dialysis fluid can inhibit the proliferation of RPMCS.This ability dependend on time and glucose concentration.The higher concentration and longer time are, the inhibition is more obvious.2. RPMCS is stimulated by peritoneal dialysis solution with the expression of MyD88, TNF-α and higher the glucose concentration is, the more significant the two expression is.Chart Ⅱ Effect of erigeron on the expression of MyD88, TNF-α and proliferation in rat peritoneal mesothelial cells stimulated by high glucoseObjective:To explore the effect of erigeron on the expression of MyD88and TNF-α and proliferation in rat peritoneal mesothelial cells stimulated by high glucose.Methods:1. Preparation of the drug serum:selected180+30mg SD male rats with the number of16.Injected erigeron into the rat tail vein.After7days,got the separation serum.2.The different concentration of glucose cultured cells for6h and added the breviscapine medicine serum to continue cultureing for8h.The concentrations of MyD88and TNF-a were detected in the supernatant and OD value was examed.Results: 1.Three groups of different concentration glucose were cultured for6h.Compared with groups not intervened by erigeron for8h,the OD value of the groups intervened were significantly different(P<0.05).2.The expression of MyD88,TNF-α in groups intervened by erigeron were significantly different compared with the not one(P<0.05).Conclusions:1. Erigeron can reduce the inhibition of glucose on the proliferation of RPMCS.2. Erigeron can lower the expression of MyD88、TNF-α in RPMCS stimulated by high glucose.Conclusions of the whole paper:After cultured by peritoneal dialysis solution containing glucose,the proliferation of RPMCS was inhibited and such inhibition depended on time and concentration.The expression of MyD88, TNF-α increased significantly.When intervened by erigeron, the inhibition on each group was weak and the expression decreased signifycantly.Erigeron has the effect of anticoagulant, anti-oxidation, can reduce inflammatory reaction product, inhibit the injury of glucose and AGEs on cells.Further more erigeron can alleviate inhibition of the high concentration glucose and decrease the expression of inflammatory factors.What meas erigeron can improve peritonitis and protect peritoneal function. Prolong the life of patients of peritoneal dialysis.