The Role Of P2X7 Receptor Via P38 MAPK In The Inflammation Microenvironment Present In Bovine Retinal Pericytes

Objective: To selective culture of microvascular pericytes from bovine retina and to comfirm the bovine retinal pericytes.Then to explore the effect of P2X7 expression and p38 MAPK pathway on pericyte IL-6 and ICAM-1 release under the pathological condition of high glucose.Method: a.We isolated the retinal microvessels pericytes by using collagenase I digestion. b.Using immunofluorescent staining to comfirm the origin of these cells by NG2 and Vimentin.c.The passage 2 and 3 Bovine retinal pericytes(BRPs) were used in the following study. The cells were routinely cultured in DMEM( low-glucose) supplemented with 10% fetal bovine serum(FBS). The cells were exposed to high glucose(final concentration of 30mmol/L glucose) in DMEM( low-glucose) containing 0.2%BSA for 24 h. Cells were incubated in the presence or absence of 100ummol/L Bz ATP,100ummol/L A438079-Hcl(P2X7 antagonist), and 10ummol/L SB203580(an inhibitor of p38), and to investigate the effects of high glucose on IL-6 and ICAM-1 release in pericytes, the BRPs were pre-treated with control(5.6m M glucose) and 30 m M glucose for 24 h and then stimulated with 100ummol/L Bz ATP for 1h. The A438079-Hcl and SB203580 were all added 1h or 24 h prior to subjecting the cells to high glucose. Cells were harvested and lysed with lysis buffer and the total RNA and/or protein was extracted. The m RNA and protein expression of P2X7 and ?-actin, the protein levels of phosphorylation of p38 MAPK in BRPs and the m RNA expression of IL-6 and ICAM-1 were detected by using the Real-time RT-PCR and Western blotting analysis methods.Results: a.The microvascular pericytes attached to the plate and growth well after one days in primary culture. After three to five days, there were numerous individual pericytes migrated from the capilliary fragments. The pericytes turn to be long shuttle-shaped adhered cells after five to seven days. After eight to eleven days, cells approached confluence and turn to be short fusiform shape. The cells attached to the plate within one hours after subcultured and the cell growth was influenced by a lot of things, they approached confluence for about five to seven days. The immunofluorescent staining results showed that the pericytes were possitive stained for NG2 and Vimentin, and the positive rate is more than 98%.b.Culture with high glucose increases the mRNA and protein expression of P2X7(P<0.05). Chronic glucose overload induces IL-6 and ICAM-1 release in BRPs(P<0.05). Blocking P2X7 attenuates high glucose induced IL-6 and ICAM-1 expression(P<0.05). High glucose increases the protein levels of phosphorylation p38 MAPK in BRPs(P < 0.05). Inhibition of P2X7 decreases high glucose promoted phosphorylation of p38 MAPK in BRPs(P<0.05). And Inhibition of p38 MAPK reduces high glucose induced up-regulated expression of P2X7,IL-6 and ICAM-1 in BRPs(P<0.05).Conclusion: a.By using collagenase I and different kinds of filter membrane digestion and sieving, bovine retinal microvascular pericytes can be cultrued successfully with high cell viabilities. And the BRPs can be transferred of culture serially. The passage 2 and 3 BRPs are the superior chose to be used in our next study.b.P2X7-dependent effects of high glucose is mediated by p38-MAPK, and both of P2X7 and p38-MAPK pathway participate in the conformation of inflammation microenvironment in BRPs, and they interaction with each other.