过氧化物酶体增殖物激活受体γ激动剂抑制角膜移植免疫排斥反应的实验研究
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摘要
目的
观察过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂局部应用对鼠角膜的影响,判断其药物毒性。
方法
将PPARγ激动剂DK2配制成滴眼液,将0.1%,0.5%,1.0% PPARγ激动剂局部滴用于Wistar大鼠右眼,观察眼局部改变和荧光素钠染色情况,并按照制定的眼部刺激反应评分标准,评判其对眼部刺激性,并行角膜病理组织切片染色和ELISA检测房水PPARγ浓度,综合评价药物安全性。
结果
药物各组鼠眼部刺激反应评分均为1-3分,角膜切片HE和免疫组化染色示角膜结构完整,细胞无损伤浸润。ELISA测得给药后房水中PPARγ浓度明显增加,且PPARγ的表达具有浓度依赖性,但0.5%,1.0%两个给药组之间差异无统计学意义(P>0.05)。
结论
1.0%浓度的PPARγ激动剂DK2滴眼液对鼠眼无刺激,具有安全性。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对大鼠角膜移植排斥反应的影响。
方法
SD大鼠为供体,Wistar大鼠为受体,建立SD-Wistar大鼠同种异体穿透性角膜移植模型。随机分A、B、C、D及E组,其中A组：对照组,术后予以无菌生理盐水点眼；B组：0.1%DK2滴眼液组；C组：1.0%DK2滴眼液组；D组：DK2口服灌胃给药组；E组：1.0%环孢霉素A(CsA))滴眼液组；F组：Wistar大鼠自体同基因移植对照组,术后生理盐水点眼。术后定期对各组角膜植片进行临床观察,以混浊、水肿、新生血管3项指标作为临床评估标准,记录排斥指数(RI)、新生血管指数(NI)、植片存活时间。实时定量荧光PCR检测鼠角膜内ICAM-1mRNA表达,流式细胞学检测各组大鼠颌下淋巴结MHC-Ⅱ、CD80的变化情况,免疫组织化学法检测大鼠颌下淋巴结CD86的表达。酶联免疫吸附试验(ELISA)检测鼠房水中IL-10的含量。结果
A-E组的角膜植片平均存活时间为9.2±1.5d、10.1±1.8d、18.3±1.1d、20.1±1.6d、18.1±1.5d,F组植片存活时间至观察期结束(>28.0d)。除B组外,其他用药组植片存活时间与A组比较,差异均有统计学意义(P<0.05)。C、E组植片存活时间两两比较差异无统计学意义(P>0.05),D组植片存活时间与C组相比较有统计学差异。术后第9d角膜植片ICAM-1 mRNA显示,A、B组相对表达量明显高于C、D、E、F组；颌下淋巴结免疫指标检测,流式细胞学结果显示第9 dC、D、E组DC表面MHC-Ⅱ及CD80单阳性表达率低于对照A,B组(P<0.05)；免疫组化染色显示,第9d时A、B组淋巴结高表达CD86,而C、D、E、F组未见有明显表达,至术后18d,C、D、E组CD86表达明显增加。ELISA检测术后第3、9、18天各组大鼠房水IL-10含量较低,各组平均含量无明显差异；第9 d后,随着排斥反应发生延迟,C、D、E组IL-10含量较A、B组增加,但变化幅度不大。结论
PPARγ激动剂能显著延长大鼠角膜植片的存活时间,其抑制角膜移植免疫排斥反应具有随浓度增加效应,并且全身用药效果强于局部滴用1.0%DK2滴眼液和1.0%环孢霉素A。PPARγ激动剂能有效抑制角膜植片炎性细胞浸润和ICAM-1 mRNA的表达,抑制局部淋巴结内树突状细胞的聚集和递呈功能,可能是其发挥抗排斥作用机制的一部分。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对体外诱导培养的大鼠骨髓来源树突状细胞(dendritic cells,DCs)分化成熟及免疫活性影响,探讨药物对DCs表达TLR4的影响。
方法
用GM-CSF和IL-4体外定向诱导Wistar大鼠骨髓细胞分化成树突状细胞,①分别加入两种不同的PPARγ激动剂DK2和马来酸罗格列酮,与细胞共培养12-72h后,MTT法检测树突状细胞生长抑制率,乳酸脱氢酶检测法(LDH)比较两种药物的药效；②细胞随机分成4组,即空白对照组,阳性对照组(脂多糖,LPS),低浓度药物组和高浓度药物组,倒置显微镜下观察DCs形态变化,流式细胞仪检测DCs表面免疫分子MHC-Ⅱ、CD80表达率,酶联免疫吸附试验(ELISA)检测DCs培养上清液中IL-10含量,混合淋巴细胞增殖反应(MLR)测定DCs刺激T淋巴细胞增殖能力的改变,实时定量PCR检测PPARγmRNA表达；③免疫荧光染色法检测药物对TLR-4表达的影响。
结果
①MTT结果显示两种PPARγ激动剂DK2和马来酸罗格列酮的IC50值分别为15.60±0.54μmol/L和53.8±1.94μmol/L,此时的LDH释放率分别为16.8%和27.4%。
②经DK2干预后,流式细胞仪检测DC表面MHC-Ⅱ、CD80阳性率降低,差异均有统计学意义(P<0.05)。ELISA显示LPS刺激后IL-10的含量增加,药物干预后表达量进一步增加,各组差异有统计学意义(P<0.05)。MLR实验显示随着刺激细胞(DC)浓度下降,反应T细胞增殖能力随之下降,并且药物能降低T细胞增殖能力,差异有统计学意义(P<0.05)。实时定量PCR结果显示脂多糖刺激后,DCs表达PPARγmRNA略有增加,加入DK2后,PPARγmRNA表达量随药物浓度增加而进一步增加,差异有统计学意义(P<0.05),PPARγ受体拮抗剂能抑制DK2的激动作用。免疫荧光检测示DK2抑制DCs表达TLR4。
结论
PPARγ激动剂DK2能抑制大鼠DC分化成熟及其免疫功能,抑制效率具有浓度依赖性。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对角膜新生血管的抑制作用及机制。
方法
36只SD大鼠随机分成A、B、C及D组,每组9只大鼠,右眼为实验眼。B、C、D组大鼠行角膜囊袋法制作角膜新生血管模型。A组：空白对照组,正常大鼠角膜；B：阳性对照组,制作模型后生理盐水滴眼；C、D组：建模后分别滴用0.1%、1.0% PPARY激动剂滴眼液,均4次／d,共28d。裂隙灯显微镜下测量新生血管面积,Western-Blot检测角膜血管内皮生长因子(vascular endothelial growth factor, VEGF)表达。
结果
D组大鼠角膜新生血管面积小于阳性对照B组和C组,差异有统计学意义(P<0.05),而C组与B组之间差异无统计学意义(P>0.05)。Western-Blot检测新生血管角膜表达VEGF明显增强,C、D组VEGF相对量有不同程度下降。结论
局部应用PPARγ激动剂能有效抑制角膜新生血管生成,抑制VEGF的合成和分泌可能是阻止角膜新生血管生长机制的一部分。
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.
观察过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂局部应用对鼠角膜的影响,判断其药物毒性。
方法
将PPARγ激动剂DK2配制成滴眼液,将0.1%,0.5%,1.0% PPARγ激动剂局部滴用于Wistar大鼠右眼,观察眼局部改变和荧光素钠染色情况,并按照制定的眼部刺激反应评分标准,评判其对眼部刺激性,并行角膜病理组织切片染色和ELISA检测房水PPARγ浓度,综合评价药物安全性。
结果
药物各组鼠眼部刺激反应评分均为1-3分,角膜切片HE和免疫组化染色示角膜结构完整,细胞无损伤浸润。ELISA测得给药后房水中PPARγ浓度明显增加,且PPARγ的表达具有浓度依赖性,但0.5%,1.0%两个给药组之间差异无统计学意义(P>0.05)。
结论
1.0%浓度的PPARγ激动剂DK2滴眼液对鼠眼无刺激,具有安全性。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对大鼠角膜移植排斥反应的影响。
方法
SD大鼠为供体,Wistar大鼠为受体,建立SD-Wistar大鼠同种异体穿透性角膜移植模型。随机分A、B、C、D及E组,其中A组：对照组,术后予以无菌生理盐水点眼；B组：0.1%DK2滴眼液组；C组：1.0%DK2滴眼液组；D组：DK2口服灌胃给药组；E组：1.0%环孢霉素A(CsA))滴眼液组；F组：Wistar大鼠自体同基因移植对照组,术后生理盐水点眼。术后定期对各组角膜植片进行临床观察,以混浊、水肿、新生血管3项指标作为临床评估标准,记录排斥指数(RI)、新生血管指数(NI)、植片存活时间。实时定量荧光PCR检测鼠角膜内ICAM-1mRNA表达,流式细胞学检测各组大鼠颌下淋巴结MHC-Ⅱ、CD80的变化情况,免疫组织化学法检测大鼠颌下淋巴结CD86的表达。酶联免疫吸附试验(ELISA)检测鼠房水中IL-10的含量。结果
A-E组的角膜植片平均存活时间为9.2±1.5d、10.1±1.8d、18.3±1.1d、20.1±1.6d、18.1±1.5d,F组植片存活时间至观察期结束(>28.0d)。除B组外,其他用药组植片存活时间与A组比较,差异均有统计学意义(P<0.05)。C、E组植片存活时间两两比较差异无统计学意义(P>0.05),D组植片存活时间与C组相比较有统计学差异。术后第9d角膜植片ICAM-1 mRNA显示,A、B组相对表达量明显高于C、D、E、F组；颌下淋巴结免疫指标检测,流式细胞学结果显示第9 dC、D、E组DC表面MHC-Ⅱ及CD80单阳性表达率低于对照A,B组(P<0.05)；免疫组化染色显示,第9d时A、B组淋巴结高表达CD86,而C、D、E、F组未见有明显表达,至术后18d,C、D、E组CD86表达明显增加。ELISA检测术后第3、9、18天各组大鼠房水IL-10含量较低,各组平均含量无明显差异；第9 d后,随着排斥反应发生延迟,C、D、E组IL-10含量较A、B组增加,但变化幅度不大。结论
PPARγ激动剂能显著延长大鼠角膜植片的存活时间,其抑制角膜移植免疫排斥反应具有随浓度增加效应,并且全身用药效果强于局部滴用1.0%DK2滴眼液和1.0%环孢霉素A。PPARγ激动剂能有效抑制角膜植片炎性细胞浸润和ICAM-1 mRNA的表达,抑制局部淋巴结内树突状细胞的聚集和递呈功能,可能是其发挥抗排斥作用机制的一部分。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对体外诱导培养的大鼠骨髓来源树突状细胞(dendritic cells,DCs)分化成熟及免疫活性影响,探讨药物对DCs表达TLR4的影响。
方法
用GM-CSF和IL-4体外定向诱导Wistar大鼠骨髓细胞分化成树突状细胞,①分别加入两种不同的PPARγ激动剂DK2和马来酸罗格列酮,与细胞共培养12-72h后,MTT法检测树突状细胞生长抑制率,乳酸脱氢酶检测法(LDH)比较两种药物的药效；②细胞随机分成4组,即空白对照组,阳性对照组(脂多糖,LPS),低浓度药物组和高浓度药物组,倒置显微镜下观察DCs形态变化,流式细胞仪检测DCs表面免疫分子MHC-Ⅱ、CD80表达率,酶联免疫吸附试验(ELISA)检测DCs培养上清液中IL-10含量,混合淋巴细胞增殖反应(MLR)测定DCs刺激T淋巴细胞增殖能力的改变,实时定量PCR检测PPARγmRNA表达；③免疫荧光染色法检测药物对TLR-4表达的影响。
结果
①MTT结果显示两种PPARγ激动剂DK2和马来酸罗格列酮的IC50值分别为15.60±0.54μmol/L和53.8±1.94μmol/L,此时的LDH释放率分别为16.8%和27.4%。
②经DK2干预后,流式细胞仪检测DC表面MHC-Ⅱ、CD80阳性率降低,差异均有统计学意义(P<0.05)。ELISA显示LPS刺激后IL-10的含量增加,药物干预后表达量进一步增加,各组差异有统计学意义(P<0.05)。MLR实验显示随着刺激细胞(DC)浓度下降,反应T细胞增殖能力随之下降,并且药物能降低T细胞增殖能力,差异有统计学意义(P<0.05)。实时定量PCR结果显示脂多糖刺激后,DCs表达PPARγmRNA略有增加,加入DK2后,PPARγmRNA表达量随药物浓度增加而进一步增加,差异有统计学意义(P<0.05),PPARγ受体拮抗剂能抑制DK2的激动作用。免疫荧光检测示DK2抑制DCs表达TLR4。
结论
PPARγ激动剂DK2能抑制大鼠DC分化成熟及其免疫功能,抑制效率具有浓度依赖性。
目的
探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对角膜新生血管的抑制作用及机制。
方法
36只SD大鼠随机分成A、B、C及D组,每组9只大鼠,右眼为实验眼。B、C、D组大鼠行角膜囊袋法制作角膜新生血管模型。A组：空白对照组,正常大鼠角膜；B：阳性对照组,制作模型后生理盐水滴眼；C、D组：建模后分别滴用0.1%、1.0% PPARY激动剂滴眼液,均4次／d,共28d。裂隙灯显微镜下测量新生血管面积,Western-Blot检测角膜血管内皮生长因子(vascular endothelial growth factor, VEGF)表达。
结果
D组大鼠角膜新生血管面积小于阳性对照B组和C组,差异有统计学意义(P<0.05),而C组与B组之间差异无统计学意义(P>0.05)。Western-Blot检测新生血管角膜表达VEGF明显增强,C、D组VEGF相对量有不同程度下降。结论
局部应用PPARγ激动剂能有效抑制角膜新生血管生成,抑制VEGF的合成和分泌可能是阻止角膜新生血管生长机制的一部分。
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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