Effects Of Honokiol Liposome On Retinal Neovascularization In Oxygen-Induced Retinopathy Model And Its Mechanism

Part one Inhibitory Effects of Honokiol Liposome on Retinal Neovascularization in Oxygen-Induced RetinopathyPurpose:To explore the effects of honokiol liposome (HNK-L) on retinal neovascularizalion in oxygen-induced retinopathy (OIR) mice model.Methods:Seventy-two7-day-old (P7) C57BL/6J mice were divded into six groups randomly, including normal group, OIR model group, liposome control group and three dosages of HNK-L-intervented groups. The normal group was reared in normal air from PO-P17, other groups were incubated in an oxygen chamber under hyperoxic coudition (75%±2%O2) for5days (P7-P12), then were removed and kept in normal air condtion for a further5days (P12-P17) to establish the OIR model. Three HNK-L-intervented group mice received0.2ml injection of different dosages of HNK-L intraperitioneally (large-dosage HNK-L group:100mg/kg; middle-dosage:50mg/kg; small-dosage:25mg/kg) during P12-P17in normoxia. All mice were killed at P17. Retinal proliferative neovascularzations were quantified by counting the nuclei of neovacualr endothelial cell that protruding from the internal limiting membrane (ILM) into vetrious cavity in retinal cross sections (by HE staining and anti-CD105immunohistochemical staining).Results:There were massive retinal neovascmlarization in the OIR model compared with those in the normal mice (P<O.05), with CD105over-staining in the retina sections of OIR group. RNVs were reduced in the HNK-L treated groups comparing with OIR group. Compared with middle-dosage and small-dosage of HNK-L treatments, large-dosage HNK-L group showed obvious inhibition effect on RNV (P<0.05).Conclusion:RNV was inhibited by intraperitioneal-injections of HNK-L significantly. CD105in retina can be used as a specificity marker of RNV. Part Two Effects of Honokiol Liposome on Expression of VEGF-A, VEGFR-2and ES in Oxygen-Induced RetinopathyPurpose:To observe effects of HNK-L on VEGF-A, VEGFR-2and ES expressions in retina of oxygen-induced retinopathy model mice, exploring the possible mechanisms of HNK-L impact on RNV.Methods:Seventy-two7-day-old (P7) C57BL/6J mice were divided into six groups that include normal group, OIR model group, loposome control group and three dosages of HNK-L treated groups. OIR model were established as Part One described. HNK-L treated groups were received0.2ml intraperitioneal injections of three different dosages of HNK-L daily (large-dosge group:100mg/kg; middle-dosage group:50mg/kg; small-dosage group:25mg/kg) for5days (P12-P17). All mice were killed on P17. mRNA and protein levels of VEGF-A, VEGFR-2and ES in the retina were analysised by RT-PCR and western blot assays.Results:VEGF-A and VEGFR-2mRNA and protein expressions were up-regulated in OIR model group comparing with normal group (p<0.05), whereas expressions of ES showed no significant differences between these groups. VEGF-A and VEGFR-2mRNA and protein were reduced in HNK-L treated mice compared with OIR model and loposome control mice. HNK-L can reduce expressions of VEGF-A and VEGFR-2; however, expression of ES were upregulated in three HNK-L treated groups compared with normal group (P<O.05), interventional effects of HNK-L took on dose-dependent manners.Conclusion:HNK-L intraperitioneal injection could inhibit RNVs in OIR mice model significantly; it may be associated with HNK-L inhibitory effects on VEGF-A and VEGFR-2expressions, up-regulations on ES expressions. Part Three Effects of Honokiol liposome on MAPK Signal Pathway in Oxygen-Induced RetinopathyPurpose:Explore the possible mechanisms of HNK-L impacts on RNV in cellular signal pathway levels by observing effects of HNK-L on ERK1/2, p38MAPK and JNK belonged to MAPK family in OIR model retina.Methods:Thirty six P7C57BL/6J mice were divided into six groups that including normal group, OIR model group, liposome control group and three doseages of HNK-L treated groups. OIR modelings were established as Part One described. HNK-L treated groups received intraperitioneal injected with different dosages of HNK-L (described in Part Two) after OIR modeling from P12to P17. All mice were killed at P17. Western blot assays were used to evaluate p-ERK1/2, p-p38MAPK and p-JNK protein expressed in retinas.Results:P-ERK1/2, p-p38MAPK and p-JNK protein expression were increased in OIR model group than in normal group (P<0.05). HNK-L can reduced protein expressions of p-ERK1/2, p-p38MAPK and p-JNK (p<0.05) significantly.Conclusion:Protein members in MAPK signal pathway family are important which may involved in the RNV of OIR model. HNK-L can inhibit RNV significantly. This mechanism may be associated with of HNK-L inhibition effects on the expressions of p-ERK1/2, p-p38MAPK and p-JNK.