| Objective To analyze the safety and toxicity of peroxisome proliferators activated receptorγ(PPARγ) agonist by local application on ocular surface of rats.Methods PPARy agonist was prepared into 0.1%,0.5%,1.0% eye drops. And PPARγagonist eye drops were local used on ocular surface of rat right eyes. Clinical observation was made to evaluate the stimulation by the standard of stimulation reaction. Histopathology and immunohistochemestry were taken to detect the corneal pathologic changes. The concentrations of PPARy in rat squeous humor were measured by enzyme linked immunosobent assay(ELISA).Result The marks of rat eyes according to the standard of stimulation reaction were between 1 to 3 in all groups by locally using the PPARy agonist eye drops. There were no fluorescein stain on the corneal epithelium. The pathologic findings showed that there were no changes in cornea. ELISA revealed that there was significant increase in PPARy after using the 0.1%,0.5%,1.0% eye drops, but the differences had no statistical significance among 0.5%,1.0% two groups.Conclusion 1.0% PPARy agonist eye drops had no stimulation and toxicity by local application on ocular surface. Objective To evaluate the inhibitory effect of peroxisome proliferators-activated receptor y (PPARy) agonist on allograft rejection in the orthotopic allogeneic penetrating keratoplasty in rats.Methods Penetrating keratoplasties were performed, with SD rats serving as donors and Wistar rats as recipients. Group A was allogeneic control group and the rats of which received normal saline eye-drops. The rats in groups B, C were treated with PPARy agonist eye drops of different concentrations (0.1%,1.0% respectively). The rates in group D received the drug by mouth (5mg/kg-d). The rates in group E were treated with 1.0% cyclosporin A (CsA). Rats in group F served as Wistar/Wistar syngeneic controls. After the transplantation, the cornea grafts were examined for opacity, edema and neovascularization with slit-lamp microscope. Rejection index (RI) and corneal neovascularization index (CNI) were recorded and compared among the different groups. The real time PCR was taken to detect the changes of ICAM-1 mRNA in corneal grafts. Flow cytometrical detection of the expression of MHC-Ⅱand CD80 molecules on the surface of dendritic cells (DCs) in submandibular lymph nodes. The immunohistochemical examinations were performed to determine the levels of CD86 in submandibular lymph nodes. The concentrations of IL-10 in rat aqueous humor were measured by enzyme linked immunosobent assay (ELISA) at different time points after the operation.Results The mean survival time of grafts in A, B, C, D, E groups respectively was 9.2±1.5 d,10.1±1.8 d,18.3±1.1 d,20.1±1.6 d,18.1±1.5 d, and over 28 d in group F. Besides group B, the mean survival time of the grafts was significantly longer in groups C, D, E and F than in the group A (P<0.05). The mean survival time was significantly shorter in groups C, D and E than in group F (P<0.05). There existed no statistically significant differences in the mean survival time among groups C and E (P>0.05), and the group C had significant statistical difference with group D(P<0.05). At the 9th day after operation, the real time PCR showed that the level of ICAM-1 mRNA in corneal grafts of groups A, B were much higher than in groups C, D, E, F; flow cytometrical detected that the expression of MHC-Ⅱand CD80 of submandibular lymph nodes in groups C, D, E were lower than the group A, B (P<0.05); the immunohistochemical examinations found that the expression of CD86 was increased in groups A, B, and was not appeared in groups C to F. At the 18th postoperative day, the expression of CD86 was significantly increased in groups C, D, E. The concentrations of IL-10 had been increased after operation by ELISA.Conclusion Topical application of PPARy agonist could significantly prolong corneal allograft survival and inhibit corneal neovascularization after orthotopic allogeneic penetrating keratoplasty in rats. It is indicated that PPARy agonist could suppress the expression of ICAM-1 mRNA in corneal grafts, inhibit the immunological function of DCs in submandibular lymph nodes and regulate the secretion of IL-10. Objective To investigate the effect of peroxisome proliferators-activated receptor y (PPARy) agonist on the differentiation, maturation and function of rat bone marrow-derived dendritic cells (DCs) in culture.Methods DCs were isolated from Wistar rats bone marrow, and cultured with colony stimulating factor (GM-CSF) and Interleukin 4 (IL-4).①Two PPARy agonists of DK2 and rosiglitazone (ROS) were added to co-culture with 12-72 hours, the MTT method was used to assay the biologic activities of DK2 in different doses and time. The cytotoxicity effect was measured by LDH release assay.②The cultured DCs divided into 4 groups randomly: the control group, the LPS group, the low drug-treatment group and the high drug-treatment group. Observation the appearance of cells by the inverted microscope. The phenotype and functional properties of DCs were detected by flow cytometry and mixed lymphocyte reaction(MLR). The concentration of IL-10 in the supernatant was detected by ELISA. The expression of TLR4 was measured by immunofluorescence.Results①The IC50 of DK2 and rosiglitazone were 5.60±0.54μmol/L,53.8±1.94μmol/L. And DK2-treated cells did not release significant amounts of LDH compared with rosiglitazone (16.8% vs 27.4%).②The DC treated with high concentration of DK2 was shown low expressions of CD80,and MHC-Ⅱon cell surface(P<0.05). The drug decreased the concentration of IL-10 in the supernatant of the control and LPS group(P<0.05).The T cells proliferation inhibited with the relationship of stimulated cells(DCs) and PPARy agonist in the MLR(P<0.05). The real time PCR showed that the level of PPARy mRNA was increased when LPS and DK2 added into (P<0.05) ③Immunofluorescence examination found that DK2 could inhibit the expression of TLR4 on cells surface.Conclusion PPARy agonists could significantly suppress the differentiation, maturity and function of rat bone marrow-deprived DCs. Objective To study the effect of peroxisome proliferators-activated receptor y (PPARy) agonist on rats corneal neovascularization (NV) in vivo, and detect the level of vascular endothelial growth factor(VEGF) in cornea.Methods The 36 rats were randomly divided into 4 groups with 9 rats in each group. Group A was composed of normal rats. A piece of gelfoam(lmmxlmmxlmm) which soaked in the basic fibroblast growth factor (bFGF) was implanted into the corneal small pocket of each eye in groups B, C and D, and group B was treated with 0.9% sodium chloride, group C and D were given different concentrations of PPARy agonist eye drops (0.1%,1.0% respectively) 4 times daily during days 0-28. The development of corneal NV was observed by a slit lamp microscope. The level of VEGF in cornea in each group was examined by western-blot.Results The corneal neovascularization areas in group D was significantly smaller than that in group B and C in 3,7,14,21 and 28 days after operation(P<0.05). There was no significant difference in corneal neovascularization areas between physiologic saline group (group B) and 0.1% PPARγagonist group(group C) (P>0.05). The expression of VEGF was increased when the new vessels grown, and DK2 can inhibit the increase.Conclusions PPARy agonist can inhibit the growth of corneal neovascularization. Also, the suppression of VEGF may be one of the mechanisms to inhibit the neovascularization.
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