| Objective:Retinal neovascularization is one of the main causes of visual impairment and even blindness in various retinal diseases.Retinal laser photocoagulation and anti-vascular endothelial growth factor?VEGF?treatment have bottlenecks in clinical application.It is necessary to explore new interventions.The preliminary research results of our research group suggest that Panax notoginseng saponins?PNS?,the main component of Panax notoginseng,has anti-inflammatory,two-way regulation of angiogenesis and photoreceptor cell protection.Therefore,this study is to explore the role of PNS in retinal neovascularization.The intervention effect and possible molecular mechanism of neurovascular unit abnormalities in the course of disease provide partial experimental and theoretical basis for the clinical application of Panax notoginseng and Panax notoginseng saponins in the treatment of related retinopathy.Methods:This study used a mouse model of oxygen induced retinopathy?OIR?to investigate the effect and mechanism of PNS intervention on retinal neovascularization.The study was divided into two parts:1.The effect of PNS intervention on oxygen-induced retinopathy.Seven-day-old?postnatal 7,P7?C57/BL6J mice were randomly divided into normal control group?NC group?,OIR model group?OIR group?and PNS treatment group?PNS group?.OIR group and PNS group mice were placed in the oxygen-enriched box with oxygen volume fraction of 75%±2%from P7,and were placed in normal room air in P12.PNS group was peritoneal cavity according to PNS100mg/kg body weight.The drug was administered by injection,and the NC group and the OIR group were intraperitoneally injected with the same amount of physiological saline.The days of administration were adjusted according to different experimental contents,as follows:?1?Effect of PNS on body weight of OIR mice:After 21 days of administration,the body weight of the mice was measured at P7,P12,P19,P26 and P33respectively.?2?Experimental study of PNS intervention on retinal vascular leakage in OIR mice:After 7 days of drug administration,the mice were subjected to general anesthesia in P19,intraperitoneal injection of sodium fluorescein,images of retinal vessels were collected,and fluorescein leakage area was measured using Image J software.?3?Experimental study of PNS intervention on retinal artery tortuosity in OIR mice:After 21 days of drug administration,the mice were subjected to general anesthesia in P33,intraperitoneal injection of sodium fluorescein,images of retinal vessels were collected,and retinal artery tortuosity index?RAT index?was measured using the QUANT BV program in MATLAB software.?4?Effect of PNS on retinal function of OIR mice:After 21 days of drug administration,the mice were subjected to general anesthesia in P33,and the Ganzfeld ERG system was used to collect the waveforms of the mouse electroretinogram,for a wave and b wave amplitude is analyzed.?5?Experimental study of PNS intervention in retinal reactive gliosis in OIR mice:21 days after drug administration,the mice were sacrificed in P46 and P66,eyeballs were made into eye cup,and glial fibrillary was performed after frozen sectioning.Glial fibrillary acidic protein?GFAP?was for immunohistochemical analysis.2.Mechanism of PNS intervention in oxygen-induced retinopathy:mice were grouped,modeled and intervened in the same way as before,administered 1 day and 3 days respectively,mice were sacrificed at P13 and P15,and retinal tissue was taken for related gene expression.Real-time PCR was used to analyze vascular endothelial-cadherin?VE-cad?,neural-cadherin?N-cad?,VEGFA,and VEGF receptor 2?VEGFR-2?,erythropoietin?Epo?,angiopoietin-2?ang-2?,fibroblast growth factor-1?FGF-1?,fibroblast growth factor-2?FGF-2?,tumor necrosis factor-??TNF-??,interleukin-1??IL-1??,intercellular adhesion molecule-1?ICAM-1?and vascular cell adhesion molecule?VCAM-1?.Results:1.Effect of PNS intervention on oxygen-induced retinopathy.?1?Effect of PNS on body weight of OIR mice:There was no significant difference in body weight between the NC group,OIR group and PNS group at different ages during growth and development?P>0.05?.?2?Experimental results of PNS intervention in retinal vascular leakage in OIR mice showed that the leakage area of retinal fluorescein sodium in P19OIR group was significantly larger than that in NC group?P<0.05?,while the leakage area of PNS group was significantly smaller than OIR group?P<0.05?.?3?Experimental results of PNS intervention in retinal artery tortuosity in OIR mice showed that the RAT index of P33 OIR group was significantly larger than that of NC group?P<0.05?,while the RAT index of PNS group was significantly smaller than that of OIR group?P<0.05?.?4?The effect of PNS on retinal function in OIR mice showed that there was no significant difference in the amplitude of a wave between the P33 OIR group and the NC group?P>0.05?.The amplitude of the a wave in the PNS group was-2.0 log cd·s·m-2 and-0.8 log cd·s·m-2 were significantly higher than the OIR group?P<0.05?.The OIR group b-wave amplitude was significantly lower at 0.4 log cd·s·m-2and 1.6 log cd·s·m-2 in the NC group?P<0.05?,the b-wave amplitude of the PNS group was significantly higher than that of the OIR group?P<0.05?.?5?Experimental results of PNS intervention in retinal reactive gliosis in OIR mice showed that the GFAP immunoreactivity of retinal Müller cells in the OIR group of P46 and P66 mice was significantly enhanced,and reached the inner and outer nuclear layers.The GFAP immunopositive in the PNS group was similar to that in the NC group,and no immunopositive abnormality of GFAP was observed.2.The mechanism of PNS intervention on oxygen-induced retinopathy showed that the expression of VE-cad m RNA in P13 OIR mice was significantly lower than that in NC group?P<0.05?.There was no significant difference between PNS group and OIR group?P>0.05?.The expression of VE-cad m RNA in the retina of P15 OIR group was significantly lower than that in NC group?P<0.05?,while that in PNS group was significantly higher than that in OIR group?P<0.05?.The expression of N-cad m RNA in retina of mice in P13group there was no significant difference in the level?P>0.05?.The expression of N-cad m RNA in the retina of P15 OIR group was higher than that in NC group?P<0.05?,while PNS group was significantly higher than OIR group?P<0.05?.The expression of VEGFA m RNA in retina of P13 OIR group was significantly higher than that in NC group?P<0.05?.There was no significant difference between PNS group and OIR group?P>0.05?.The expression of VEGFA m RNA in retina of P15 OIR group was significantly higher than that in NC group?P>0.05?,while the PNS group was significantly higher than the OIR group?P<0.05?.The expression level of VEGFR-2m RNA in the retina of the P13 OIR group was significantly lower than that of the NC group?P<0.05?,and there was no significant difference between PNS group and OIR group?P>0.05?.The expression of VEGFR-2 m RNA in the retina of P15 OIR group was significantly lower than that in NC group?P<0.05?,while PNS group was significantly higher than OIR group?P<0.05?.The expression of Epo m RNA in the retina of P13 and P15 OIR mice was significantly higher than that in NC group?P<0.05?,there was no significant difference between PNS group and OIR group?P>0.05?.The expression of Ang-2 m RNA in retina of P13 OIR group mice was significantly lower than that in NC group?P<0.05?,there was no significant difference between PNS group and OIR group?P>0.05?.The expression of Ang-2 m RNA in retina of P15 OIR group and NC group had no significant difference?P>0.05?,but PNS group was significantly higher than OIR group?P<0.05?.The expression of FGF-1 m RNA in retina of P13 and P15 OIR group and PNS group was significantly lower than that of NC group?P<0.05?,but there was no significant difference between PNS group and OIR group?P>0.05?.The expression of FGF-2 m RNA in retina of P13 and P15 OIR group was significantly higher than that of NC group?P<0.05?,while there was no significant difference between the PNS group and the OIR group?P>0.05?.The expression of TNF-?m RNA in the retina of the P13 and P15 PNS groups was significantly lower than that in the OIR group?P<0.05?.The expression of IL-1?m RNA in the retina of P13 and P15 OIR group was significantly higher than that in NC group?P<0.05?,but there was no significant difference between PNS group and OIR group?P>0.05?.There was no significant difference in the expression of ICAM-1m RNA in the retina of P13 group?P>0.05?.The expression of ICAM-1 m RNA in the retina of P15 OIR group was significantly higher than that in NC group?P<0.05?.There was no significant difference between the OIR group and the PNS group?P>0.05?.The expression of VCAM-1 m RNA in the retina of the P13 OIR group was significantly lower than that in the NC group?P<0.05?,but there was no significant difference between the PNS group and the OIR group?P>0.05?.The expression of VCAM-1m RNA in the retina of P15 OIR group was significantly lower than that in NC group?P<0.05?,and PNS group was significantly higher than OIR group?P<0.05?.Conclusion:1.PNS significantly inhibited retinal vascular leakage in OIR mice,reduced retinal artery tortuosity,protected retinal function,and inhibited Müller cell reactive gliosis,suggesting that PNS has a certain intervention effect on retinal neurovascular unit abnormalities.2.The effective intervention of PNS intervention on retinal neurovascular unit abnormalities in OIR mice may be related to the following mechanisms:?1?PNS significantly up-regulates the expression levels of VE-cad and N-cad in the retina,possibly inhibiting the high permeability of retinal neovascularization.?2?PNS significantly down-regulated the expression level of TNF-?in the retina,suggesting that it has the effect of inhibiting the inflammatory response of retinal neovascularization.At the same time,the effect of PNS down-regulating TNF-?may also be one of the mechanisms for reducing vascular hyperpermeability and protecting neurons. |
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