Experimental Study On The Effects Of Pioglitazone On Corneal Neovascularization And The High-risk Graft Rejection In Rat

BackgroundThough keratoplasty is the most successful organ transplantation,there is still a high rejection rate in the high-risk keratoplasty.The previous studies showed that the rejection rate in the low-risk cases is only 10%,while in the high-risk grafts is higher than 70%.How to suppress the high-risk graft immune rejection is an urgent task for the operation to be carried out.Piolitazone(PIO) is an thiazolidinediones medicine.It has been widly used as an insulin sensitizer for diabetes treatment.The biological effects of PIO is mediated by peroxisome proliferators-activated receptor(PPAR-γ),which is a member of the superfamily of "nuclear receptor".Recent studies suggest that PPAR-γhas most important effect on the inflammation and immune response.Previous study showed the function of PIO and its analogs in the treatment of cardiac transplatation,renal transplantation and autoimmune diseases.Howerver,there is fewer research on the effect of PIO in the high-risk keratoplasy.Therefore,studying the new effect of PIO can pave a new way for immunosupress the graft immune rejection.IL-2 is a critical multifunctional cytokine in immune response.And vascular endothelial growth factor(VEGF) is one of the important cytokine for accelerating the growth of corneal neovascularization.This experiment we first explore the effectivity of PIO eye drops when it is topically administrated on eyes.Furthermore,we establish a model of high-risk penetrating keratoplasty to study the effects of PIO on CNV and relationship among PIO and PPAR-γ,IL-2,VEGF,during the acute immune rejection.PartⅠHigh-performance liquid chromatographic method for determination of the concentration of pioglitazone in rats aqueous humorObjectiveTo develop a High-performance liquid chromatographic method for qualifying pioglitazone in aqueous humor,and explore the effectivity of PIO eye drops for its clinical use in ophthalmology.MethodFifty-four rats(one hundred eyes) was randomly assigned to three groups with 0.5%PIO eye drops,1%PIO eye drops and 2%PIO eye drops.Three concentration of PIO was topically administrated on rat eyes,and the aqueous humor samples were taken in six different time-points(1h,2h,4h,6h,12h,24h after administrate the PIO eye drops) in each group.High-performance liquid chromatography with Eclipse XDB-C18 colorimetric column as solid phase and acetonitrile:buffer phosphate(PH 6.5;40:60,v/v) as mobile phase was used to determine the PIO concentration of aqueous humor in 1h,2h,4h,6h,12h,24h times,after using the 0.5%,1%,2%PIO eye drops.And using SPSS 11.5 software for statistic analysis.Linear regression analysis the C and AUC.The data was recorded by mean±standard deviation. Factorial design data analysis of variance was carried out.Statistical tests were considered significant when P values were less than 0.05. ResultThe chromatograms of HPLC show that the PIO Peak can be separated from other materials,no interferential peak was seen.The PIO concentration of aqueous humor in these six time-pionts is significantly different(F=57.377P=0.000).After PIO eye drops was administrated four hours,the PIO concentration of aqueous humor was reach to peak value.The PIO concentration of aqueous humor between 0.5%,1%,2%PIO groups are significantly different(F=67.477P=0.000). From each time-points,the aqueous humor concentration of the 0.5%PIO group decreased significantly when compared with the 1%,2%PIO groups(P＜0.05);In the one hour time-point,the aqueous humor concentration of 1%PIO is markedly lower than 2%PIO group.In addition,these two groups showed no significant difference in the other time-points(P＞0.05).Conclusion1.The PIO showed an acceptable permeation via the cornea.2.After PIO was administrated four hours,the PIO concentration of aqueous humor was reach to peak value.3.The PIO concentration in aqueous humor of the 1%,2%PIO group is significantly higher than 0.5%PIO group.4.The PIO concentration in aqueous humor between 1%,2%PIO groups showed no significant difference.PartⅡInhibitory effect of pioglitazone on rat corneal neovascularizationObjectiveTo explore the inhibitory effect of pioglitazone on rat corneal neovascularization and test its effect on the expression of peroxisome proliferator-activated receptor(PPAR-γ) and vascular endothelial growth factor (VEGF).Method Twenty-four SD rats was used to induce comeal neovascularization(NV) by stitching comea.The rats were derided randomly into the pioglitazone treatment group and the normal saline(N.S) group.And compare with normal rats.The growth of corneal neovascularization(CNV) was observed by slit lamp microscope on each postoperative day and the area of CNV was recorded on 5^th,12^th,20^th,28^th days. On the 12^th,the cornea with NV was observed by histopathological method and the expression of PPAR-γand VEGF were detected by immunohistochemistry.And using SPSS 11.5 software for statistic analysis.The data was recorded by mean±standard deviation.Compare the area of CNV in 1%PIO treatment group and that in N.S group by factorial design data analysis of variance and the expression of PPAR-γand VEGF in these two groups were analysis by one-way ANOVA.Statistical tests were considered significant when P values were less than 0.05.Result①On the 5^th,12^th,20^th,29^th day,the neovascularization area in the pioglitazone treatment group was decreased markedly when compared with the normal saline group(P＜0.05).②On the 12^th postoperative day,by light microscope,there is a large amout of NV in cornea of group B and many lymphocytes and fibroblasts infilitrat the cornea.In contrast,the number of NV in cornea of group A is less than group B and with light inflammatory reaction.③The positive cell counting of PPAR-γin cornea with NV was increased significantly compared with the normal rats group(P＜0.05).④The positive cell counting of PPAR-γin PIO treatment group is significantly higher than normal saline group(P＜0.05).The positive cell counting of VEGF in PIO treatment group(P＜0.05)is significantly lower than normal saline group.Conclusion1%Pioglitazone eyes drop is effective in inhibition of rat CNV induced by sutures which may be associated with the activation of PPAR-γand suppression the expression of VEGF.PartⅢPioglitazone has effect on rat high-risk comeal allograft rejectionObjectiveTo study comparatively the immunosuppressive effect of 1%pioglitazone and 1%CsA on the high-risk comeal allograft rejection and the inhibitory effect of CNV after the keratoplasty.MethodHigh-risk corneal transplantation was performed orthopically from Wistar rats to SD rats of CNV.Thirty-six SD rats were devided randomly into the 1%PIO treatment group,1%CsA group and the N.S group.Rejection index,mean survival times and neovascular area on postoperative days were determined.On the 10^th postoperative day,the corneal graft was observed by histopathological method and the expression of PPAR-γ,IL-2 and VEGF were detected by immunohistochemistry. And using SPSS 11.5 software for statistic analysis.The data was recorded by mean±standard deviation.One-way ANOVA was carried out.Statistical tests were considered significant when P values were less than 0.05.Result①Compare with mean survival time of the corneal grafts,the1%PIO and CsA treatment groups were significantly longer than normal saline group(P＜0.05).the 1%PIO treatment group was increased significantly compared with CsA treatment group.②To qualify the area of CNV,there is no significant differences among all the groups,.on the 7^th postoperative day.On the 12^th postoperative day,the area CNV of 1%PIO group is significantly higher than that of all other groups,and the CsA treatment group increased markedly compared with the normal saline group.③ The expression of PPAR-γ,of 1%PIO group is increased significantly compared with the other two groups(P＜0.05).The expression of IL-2 of 1%PIO and CsA treatment group is significantly lower than that of the normal saline group.The expression of VEGF in 1%PIO group is significantly lower than the other two groups (P＜0.05),and the CsA treatment group is significantly lower than normal saline group(P＜0.05).ConclusionTopical administration with 1%PIO has a significantly positive effect in promoting corneal allograft survival and preventing the growth of CNV.It showed a better immunomodulatory effects than CsA in rat high -risk corneal allotransplants.The 1%Pioglitazone can activated PPAR-γ,and suppressed proteins expression of IL-2 and cytokines VEGF.That maybe related to the immunosupression of high-risk corneal transplantation rejection.