| Objective1.To observe the feasibility and success rate of krypton laser induced choroidal neovascularization(CNV)models,and laying the foundation for the related experimental research of CNV.2.To observe the role of AKT/mTOR/HIF-la/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway in CNV formation.3.To observe the effects of modified Zhujing Formula and Conbercept on experimental CNV,and the effects of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway activityMethods1.The CNV models were induced by 659 nm krypton laser on Brown Norway(BN)rats.At 7 days,14 days and 21 days after photocoagulation,fundus fluorescein angiography(FFA)and indocyanine green angiography(ICGA)were used to observe the changes of CNV.After FFA and ICGA,the rats were sacrificed and the pathological specimens were made.The changes of CNV were observed by HE staining and CD 105 immunohistochemical staining.2.The CNV models were induced by 659 nm krypton laser on BN rats.At 7 days,14 days and 21 days after photocoagulation,the rats were sacrificed and the pathological specimens were made.Immunohistochemical method was used to observe the expression of AKT/mTOR/HIF-la/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway in CNV tissues.At 21 days after photocoagulation,rats were sacrificed for real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)research.The expression of mRNA of AKT/mTOR/HIF-la/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway were detected by RT-qPCR.3.The CNV models were induced by 659 um krypton laser on BN rats.After photocoagulation,the rats were randomly divided into model group,Chinese medicine group,western medicine group and combined treatment group.At 7 days,14 days and 21 days after photocoagulation,FFA and ICG A were used to observe the changes of CNV.After FFA and ICGA,6 rats in each group were sacrificed for making pathological specimens.CNV changes were observed by HE staining.The expression of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway were observed by immunohistochemistry method.At 21 days after photocoagulation,12 rats were sacrificed to obtain tissue samples in each group.The relative expression of mRNA and protein of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway were observed by RT-qPCR and Western blotting in each group.Results1.In model group,BN rats showed obvious fluorescein leakage after at 7 days after photocoagulation,and the leakage intensity increased gradually at 14 days after photocoagulation(P<0.05).There had no significant change was observed at 21 days after photocoagulation(P<0.05).The spots with fluorescein leakage were all filled in ICGA.The incidence of CNV was 77.08%,85.42%and 89.58%,respectively,at 7 days,14 days and 21 days after photocoagulation.And the difference was not statistically significant(P>0.05).Histopathological results showed that the central thickness of CNV increased gradually with the time increasing of photocoagulation(P<0.05).At 7 days after photocoagulation,CD 105 factor was found in CNV,and the colur of positive reactant was brownish yellow.The positive reactant was increased gradually at 14 days(P<0.05),and there was no significant change at 21 days(P>0.05).2.The expression of AKT,mTOR,HIF-1α,VEGF,TLR-4,NF-κB and IL-6 protein in the model group were strongly positive.A large number of yellow brown positive reactant were found in CNV.The expression of HIF-1α,TLR-4 and IL-6 protein were significantly increased at 7 days after photocoagulation,and reached a peak at 14 days,and decreased gradually at 21 days(P<0.05).The expression of AKT and mTOR protein reached the peak at 7 days after photocoagulation,and decreased slowly at 21 days(P<0.05).The expression of VEGF protein reached the maximum at 7 days after photocoagulation,and then decreased gradually(P<0.05).The expression of NF-κB protein was significantly increased at 7 days after photocoagulation,and reached a peak at 14 days,which was not significant change at 21 days(P<0.05).The results of RT-qPCR showed that the relative expression of AKT.mTOR,HIF-1α,VEGF,TLR-4,NF-κB and IL-6 mRNA in model group were significantly higher than that in control group(P<0.05).3.From 7 days to 21 days after photocoagulation,the formation rate of CNV between model group and treatment groups had no significant difference(P>0.05),which was more than 70%.The leakage intensity of FFA was the strongest at 7 days after photocoagulation,and the change was not obvious in the following time in all the treatment groups.The light spot was filling in ICGA.At 7 days after photocoagulation,the leakage intensity of fluorescein in the Chinese medicine group and the model group were the highest,while that of the combined treatment group was the lowest(P<0.05).At 14 days and 21 days,the intensity of fluorescein leakage in each treatment group was weaker than that in the model group.The treatment effect of combined treatment group was the best,and the effect of the western medicine group was better than that of the traditional Chinese medicine group(P<0.05).At 7 days after photocoagulation,the central thickness of CNV in the western medicine group and the combined group was significantly lower than that in the model group and the traditional Chinese medicine group(P<0.05).At 14 days and 21 days after photocoagulation.the central thickness of CNV in each treatment group was lower than that in the model group.The reduction of CNV central thickness from strong to weak were the combination group,western medicine group and Chinese medicine group(P<0.05).Immunohistochemistry results showed that the expression of AKT,mTOR.HIF-1α,VEGF,TLR-4.NF-κB and IL-6 protein were positive in retinal photocoagulation area of each treatment group.A large amount of brown positive reactant was found in CNV.With the prolongation of treatment time,the interest protein expression of each treatment group was lower than that of the model group(P<0.05).From 7 days to 21 days after photocoagulation,the expression of AKT,mTOR,HIF-1α and VEGF in Chinese medicine group,western medicine group and the combination group decreased successively(P<0.05).The expression of TLR-4 and NF-κB were highest in western medicine group,the lowest in combined group(P<0.05).At 7 days and 14 days after photocoagulation,the expression of IL-6 in combined group was lower than that in western medicine group(P<0.05).At 21 days after photocoagulation,the expression of IL-6 both in Chinese medicine group and combined group was lower than that in the western medicine group(P<0.05).RT-qPCR results showed that the relative expression of the target gene mRNA in the model group and the treatment group were significantly higher than that in the control group(P<0.05).In addition to mTOR mRNA,the relative expression of interest mRNA in each treatment group was lower than that of model group(P<0.05).And there was no significant difference of mTOR mRNA relative expression between Chinese medicine group and model group(P>0.05).The relative expression of AKT,mTOR,VEGF and NF-κB in each treatment group was Chinese medicine group,western medicine group and combination group,from high to low in the order.The difference was statistically significant(P<0.05).The relative expression of HIF-1αmRNA in Chinese medicine group was higher than that in western medicine group and combination group(P<0.05).There was no significant difference between western medicine group and combination group(P>0.05).The relative expression of TLR-4 mRNA was the lowest in combined group(P<0.05).There was no significant difference between western medicine group and Chinese medicine group(P>0.05).The relative expression of IL-6 mRNA was the lowest in Chinese medicine group(P<0.05),the western medicine group and combination group had no significant difference(P>0.05).The results of Western blotting showed that the relative expression of target protein in experimental group was significantly higher than that in the control group(P<0.05).In addition to AKT protein,the relative expression of interest protein in each treatment group was lower than that of model group(P<0.05).The relative expression of AKT and HIF-la protein in Chinese medicine group were higher than that in western medicine group and combination group(P<0.05).The relative expression of mTOR and VEGF protein was Chinese medicine group,western medicine group and combination group,from high to low in order(P<0.05).The relative expression of TLR-4 protein in western medicine group was higher than that of Chinese medicine group and combination group(P<0.05).The relative expression of NF-κB protein in Chinese medicine group was higher than that in western medicine group and combination group(P<0.05).The relative expression of IL-6 protein in Chinese medicine group was lower than that in western medicine group and combination group(P<0.05).Conclusion1.659 nm krypton laser induced BN rat CNV model with high success rate,and has a similar pathological processes and cell composition of human CNV.2.FFA combined with ICGA can dynamically observe the changes of CNV,and the AOD value of fluorescence leakage intensity can be used to semi quantitatively evaluate the formation of CNV.3.AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway are activated in CNV animal model.The activation of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway promoted the formation of experimental CNV.4.The effect of inhibiting CNV in combination group was better than that of traditional Chinese medicine group and Western medicine group.Combined treatment of Chinese and Western medicine has obvious inhibitory effect on the activity of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway.5.Modified Zhujing Formula can inhibit the formation of experimental CNV.Modified Zhujing Formula can reduce the activity of AKT/mTOR/HIF-1α/VEGF signaling pathway and TLR-4/NF-κB/IL-6 signaling pathway.Modified Zhujing Formula may be through the above signal pathway to play the role of multi target inhibition of CNV. |
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