| Objective:To determine the effects of aging and LPS on the expression of P2X7 receptor and its splice variants in murine eyes in order to provide the basis for future research directed at the role of P2X7R in age- and inflammation-related diseases.Methods:Young (4 months old) and old (18 months old) C57BL/6 male mice were intraperitoneally injected with either 0.5 mg/kg Escherichia coli O111:B4 LPS (LPS groups) or saline (control groups) and sacrificed 0,3,24,48 or 72 hours later. The mRNA expression of inflammatory cytokines (IL-1β, IL-18, IL-6, TNF-a), and of P2X7R and its splice variants were detected by quantitative polymerase chain reaction analysis. The protein expression of P2X7R was determined by Western blot analysis.Results:1. The effect of aging on the mRNA expression of IL-1β, IL-18, IL-6 and TNF-a in mouse eyes. The mRNA expression levels of IL-1β and TNF-a at 24h and 72h, of IL-18 at 24,48 and 72h and of IL-6 at 3,24,48 and 72h in old control mice were higher than those in young control mice and the differences were statistically significant (P<0.05).2. The effect of aging on the expression of P2X7R-V1, P2X7R-V2, P2X7R-V3 and P2X7R-V4 in mouse eyes. The mRNA expression levels of P2X7R-V2 and P2X7R-V4 in untreated old mice were higher than those in young mice and the differences were statistically significant (P< 0.05). The mRNA expression levels of P2X7R-V1, P2X7R-V2, P2X7R-V3 and P2X7R-V4 at 3,24,48 and 72h in old control groups were higher than those in young mice and the differences were statistically significant (P< 0.05). The P2X7R protein expression level in old mouse eyes (0.118±0.034) was the 2.27 times that in young ones (0.052±0.004). The expression levels of P2X7R protein in old control groups were higher than those in young control groups at 3,24,48 and 72h and the differences for the P2X7R protein expression at 3h and 24h were statistically significant (P< 0.05).3. The influence of LPS on the mRNA expression of IL-1β, IL-18, IL-6 and TNF-a in young mouse eyes. LPS increased the mRNA expression of IL-1β at 72h and of IL-6 at 3h and the differences were statistically significant (P< 0.05). LPS had no effect on mRNA expression of IL-18 and TNF-a at each time point and the differences were not statistically significant (P>0.05).4. The effect of LPS on the expression of P2X7R-Vl, P2X7R-V2, P2X7R-V3 and P2X7R-V4 in young mouse eyes. LPS up-regulated the mRNA expression of P2X7R-V1 at 3h and 72h, of P2X7R-V2 at 48h and 72h and of P2X7R-V3 at 3h and 24h and the differences were statistically significant (P< 0.05). The differences of mRNA expression of P2X7R-V4 between LPS group and control group at each time point were not statistically significant (P> 0.05). P2X7R protein expression tended to increase at 24h after LPS treatment, but had little change at other points of time (P<0.05).5. The effect of LPS on the mRNA expression of IL-1β, IL-18, IL-6 and TNF-a in old mouse eyes. LPS down-regulated mRNA expression of EL-1β and IL-18 at 24h and of TNF-a at 72h and the differences were statistically significant (P<0.05). No statistically significant differences were found for IL-6 mRNA expression levels between LPS groups and control groups at all points (P> 0.05).6. The effect of LPS on the expression of P2X7R-Vl, P2X7R-V2, P2X7R-V3 and P2X7R-V4 in old mouse eyes. LPS down-regulated mRNA expression of P2X7R-V1 at 24h and of P2X7R-V3 and P2X7R-V4 at 3h and 24h and the differences were statistically significant (P< 0.05). LPS had no effect on mRNA expression of P2X7R-V2 and the differences was not statistically significant (P> 0.05). The expression levels of P2X7R protein in old LPS groups were higher than those in old control groups at all time point, but the differences were not statistically significant (P> 0.05).7. Comparison of the mRNA expression levels of IL-1β, IL-18, IL-6 and TNF-a between young LPS groups and old LPS groups. The mRNA expression levels of IL-1β at 48h, of IL-18 at 24h and of IL-6 at 24h and 48h in old mice were higher than those in young mice and the differences were statistically significant (P< 0.05).8. Comparison of the expression of P2X7R-V1, P2X7R-V2, P2X7R-V3 and P2X7R-V4 between young LPS groups and old LPS groups. The mRNA expression level of P2X7R-V1 at 24h, of P2X7R-V2 at 24 and 72h, of P2X7R-V3 at 24,48 and 72h and of P2X7R-V4 at 24,48 and 72h in old mice was higher than that in young mice and the differences were statistically significant (P< 0.05). The expression levels of P2X7R protein in old LPS groups were higher than those in young LPS groups at all time point, but the differences were not statistically significant (P> 0.05).Conclusions:1. Aging upregulates expression of ocular P2X7R and its splice variants in mice.2. LPS differentially regulates the expression of P2X7R and its splice variants in young and old mice. |
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