The Amelioration Of Lonicerae Japonicae Flos On Diabetic Retinopathy And Its Engaged Mechanism

Objective: This study aims to observe the amelioration of Lonicerae Japonicae Flos aqueous extract(FL) and chlorogenic acid(CGA) on diabetic retinopathy(DR) in vivo and in vitro, to elucidate the engaged mechanism in their amelioration on PDR and NPDR by inhibiting retinal angiogenesis and inflammation. Thus, this study will provide experimental evidences for the application of CGA and Lonicerae Japonicae Flos(Jin-Yin-Hua) for the treatment of DR.Methods: 1. The process of pathogenesis of DR(PDR and NPDR) was observed in a mouse model of typeⅠdiabetes established by i.p STZ of 55mg/kg, and then the diabetic mice were given with FL and CGA. 2. The retinal neovascularization was evaluated by staining with cluster of differentiation 31(CD31) and histological assessment of retinas by H&E staining. 3. The blood-retinal barrier(BRB) damage in diabetic mice was evaluated by Evans blue leakage assay. 4. The m RNA expression of genes involved in regulating neoangiogenesis or inflammation was detected by Real-time PCR. 5. The expression of proteins involved in regulating neoangiogenesis or inflammation was detected by Western-blot. 6. The amounts of VEGF, IL-1b, IL-6 and TNFa in serum were detected by ELISA. 7. The contents of CGA, CA and Luteolin in FL were determined by using HPLC. 8. The effects of FL and CGA, CA, Luteolin on cell viability in RF/6A cells, and the effect of FL on VEGF-induced RF/6A cell proliferation were determined by using MTT assay. 9. The effect of FL, CGA, CA and Luteolin on VEGF-induced RF/6A cell tube formation was detected to find the effective compound in FL. The effect of CGA on VEGF-induced HREC tube formation and migration were detected. 10. Microglia BV-2 cells were pretreated with CGA(0.625、2.5μM), and then HG(25 m M) was added into cells. The effect of CGA on HIF-1α/VEGF/VEGFR2 signaling pathway was analyzed by Real-time PCR and Western-blot. 11. BV-2 cells were pretreated with CGA(0.625、2.5μM), and then HG(25 m M) was added into cells. The effect of CGA on the activation of inflammatory NFкB and Egr-1 were detected by Western-blot.Results: 1. Retinal angiogenesis was occurred at 5 month after the development of diabetes in STZ-induced diabetic mice. FL(100 mg/kg, 200 mg/kg) reduced the increased number of vessels in STZ-induced PDR mice, and also reduced the increased serum VEGF content. FL itself had no cytotoxicity in RF/6A cells, but obviously inhibited VEGF-induced RF/6A cell proliferation. FL also reduced VEGF-induced RF/6A cell tube formation. 2. The compounds including CGA, CA and Luteolin, contained by FL, all had no cytotoxicity in RF/6A cells. However, these three compounds all inhibited VEGF-induced RF/6A cell tube formation. Among which, the effect of CGA was the best, and the lowest effective concentration was 0.625 μM. The results of HPLC demonstrated that the contents of CGA, CA and Luteolin in FL were 14.73%, 0.17%, 0.061%, respectively. Of which, the content of CGA was the highest. 3. The results of STZ-induced PDR mice showed that CGA reduced the increased number of retinal vessels and serum VEGF content. CGA had no cytotoxicity on HREC cells, but CGA reduced VEGF-induced HREC cell proliferation, tube formation and cell migration. 4. Further, the results showed that HG(25 m M) induced the translocation of HIF-1α into nucleus in BV-2 cells, and then increased VEGF expression. However, CGA(0.625、2.5μM) obviously abrogated these above phenomena. 5. In STZ-induced NPDR mice, the results of Evans blue leakage detection demonstrated that FL(50、100 mg/kg) and CGA(1、5 mg/kg) abrogated BRB damage in NPDR mice in the dose-dependent manner. 6. Further, the results showed that FL(50、100 mg/kg) reduced the increased expression of retinal Iba-1in NPDR mice. FL reduced the increased retinal phosphorylated NFкBp65、IкB、IKK and the nuclear translocation of p65 in NPDR mice. In addition, FL also reduced the increased serum contents of IL-1b and IL-6 in NPDR mice. 7. Further results demonstrated that CGA abrogated the activation of NFкB signaling pathway in BV-2 cells, including the translocation of p65 into nucleus, the increased phosphorylation of NFкBp65, IкB, IKK, and the increased m RNA expression of some inflammatory cytokines such as IL-6 and TNFα. CGA also abrogated the translocation of Egr-1 into nucleus.Conclusions:1. FL and CGA ameliorate PDR via inhibiting retinal angiogenesis by reducing the expression of pro-angiogenic VEGF and inhibiting VEGF-induced retinal angiogenesis. The inhibition of CGA on HIF-1a-mediated VEGF expression in microglial BV-2 cells contributes to its inhibition on retinal angiogenesis. 2. FL and CGA ameliorate NPDR by inhibiting retinal inflammatory injury, and then attenuating BRB damage. Further results showed that CGA reduced the activation of microglial cells, and further attenuated retinal inflammatory injury by inhibiting the activation of NFkB and Egr-1, and thus reducing the expression of inflammatory cytokines. All those will contribute to CGA-induced amelioration on NPDR.