The Protective Effect Of Quercetin Phospholipid Complex Solid Dispersion On Dry Age-related Macular Degeneration Model Mice Was Studied Based On Nrf2 Signaling Channel

Objective:Prepare a solid dispersion of quercetin phospholipid complex(Q-PC)and PVP K30 to improve the physical and chemical properties of quercetin(QT),its solubility and dissolution rate in vitro were increased;evaluate the protective effect of Q-SD on dry age-related macular degeneration model mice,and study its possible mechanism of action.Methods:Using lecithin and PVP K30 as the carrier,Q-PC and Q-SD were prepared by solvent method,and the phase was characterized by infrared spectroscopy(FTIR)and ultraviolet spectrum.The solubility and dissolution rate in vitro of the drug were measured,and the blood concentration of the drug was detected in rats.A high-fat diet combined with hydroquinone was used to make a mouse retinal oxidative damage model in Nrf2 WT and Nrf2 KO mice.84 Nrf2 WT mice were randomly divided into age control group,model control group,and drug treatment group,intervention drugs were QT 200mg/kg/d,Q-PC 200mg/kg/d and Q-SD(three groups of high-dose 200mg/kg/d,medium-dose 100mg/kg/d,and low-dose 50mg/kg/d).36 Nrf2 KO mice were divided into age control group,model control group and Q-SD 200mg/kg/d(high-dose)group.Both model control group and drug treatment group were given intragastric administration once per day for 3 months.At the end of the observation period,the animals were routinely sacrificed.After the eyeballs were removed,the anterior segment was removed.The mouse retina was observed by electron microscopy,and the deposition severity of the sediments under RPE and the Bruch film thickness were analyzed.Colorimetric method was used to determine the levels of ROS,MDA and the activities of CAT,SOD,GSH-Px in serum and retinal tissue.Western blot and fluorescent quantitative PCR were used to detect the mRNA transcription and protein expression of Nrf2 and its downstream antioxidant enzymes(HO-1,NQO-1 and GCL)in the mouse retinal tissue of each group.Result:1.The water solubility and lipid solubility of quercetin in Q-SD were significantly improved,which was far better than that in QT and Q-PC groups(P<0.01).The cumulative dissolution rates of QT,Q-PC and Q-SD were 31.4%,46.1%and 80.2%,respectively.The dissolution rate and degree of QT from Q-SD were significantly faster than that of Q-PC and QT.FT-IR and UV spectra:PVP K30 interacts with all active hydrogens on Q-PC.The Cmax of QT,Q-PC and Q-SD were 1.517 μg/mL,2.523 μg/mL and 4.143 μg/mL,respectively,and the difference was statistically significant(P<0.05).AUC 0→∞ of QT,Q-PC and Q-SD were 5.461 μg·h/mL,8.074 μg·h/mL and 12.015 μg·h/mL,and the difference was statistically significant(P<0.05).2.Compared with the model control group,there was no statistical significance in the decrease of Bruch film thickness and sediment severity score in the QT and Q-PC groups,but the results in the Q-SD group were dose-dependent,and the effect in the high-dose Q-SD group was the most prominent.3.The activities of CAT,SOD and GSH-Px in serum and retinal tissue of Nrf2 KO mice were lower than those of Nrf2 WT mice(P<0.05),and the levels of ROS and MDA were higher than those of Nrf2 WT mice(P<0.05)in age control and model control.Q-SD 200 mg/kg group significantly decreased ROS and MDA levels in Nrf2 WT mice(P<0.05),but had no significant effect on Nrf2 KO mice(P>0.05).Q-SD 200 mg/kg group significantly increased the activities of CAT,SOD and GSH-Px in serum and retinal tissue of Nrf2 WT mice(P<0.01),but had no significant effect on Nrf2 KO mice(P>0.05).4.Compared with the age control group,the mRNA level of Nrf2 in Nrf2 WT mice was significantly increased(P<0.05),while the mRNA level of Nrf2 KO mice was almost undetectable.Compared with the model control group,Q-SD 200 mg/kg group significantly increased the level of Nrf2 mRNA in Nrf2 WT mice(P<0.01).In addition,compared with the age control group,the nuclear abundance of Nrf2 in the Nrf2 WT mouse model was significantly increased(P<0.05).Compared with the model control group,Q-SD 200 mg/kg group significantly stimulated Nrf2 nuclear translocation(P<0.01).However,there was no significant change in the cytoplasmic abundance of Nrf2 in the model control group and Q-SD groups(P>0.05).No expression of Nrf2 protein was detected in the nucleus or cytoplasm of Nrf2 KO mice.5.Real-time PCR results showed that the expressions of HO-1,NQO-1 and GCL mRNA in nrf2 WT mice and Nrf2 KO mice were significantly different between age control and model control group(P<0.01).Compared with the age control group,the transcription levels of HO-1,NQO-1 and GCL in Nrf2WT mouse model were significantly up-regulated(P<0.01).Q-SD 200 mg/kg group significantly up-regulated HO-1,NQO-1 and GCL transcription levels(P<0.01).Compared with the age control group,mRNA expressions of HO-1,NQO-1 and GCL in Nrf2 KO mouse model control group and Q-SD 200 mg/kg group were not increased,and the results were not statistically significant(P>0.05).Western blot results showed that the protein expression of NQO-1 and GCL in retinal tissue of Nrf2 WT mouse model was increased(P<0.05),and the protein expression of HO-1 was significantly increased(P<0.01),compared with the age control group.Q-SD 200 mg/kg group significantly up-regulated the protein expressions of HO-1,NQO-1 and GCL(P<0.01).The protein abundance of Nrf2 KO mice was significantly lower than that of Nrf2 WT mice.The protein expressions of HO-1,NQO-1 and GCL in Nrf2 KO mice were significantly lower than that of Nrf2 WT mice in age control group and model control group(P<0.01).Compared with the age control group,the protein expressions of HO-1,NQO-1 and GCL in Nrf2 KO mouse model control group and Q-SD 200 mg/kg group were not statistically significant(P>0.05).Conclusion:1.Q-SD prepared by QT can significantly improve the solubility and dissolution rate of QT.2.Q-SD prepared by QT can significantly improve the bioavailability of QT.3.QT can reduce the pathological damage of retina in dry age-related macular degeneration model mice.4.QT can up-regulate the expression of Nrf2 downstream antioxidant enzymes and the activity of antioxidant enzymes by regulating the Nrf2 signaling pathway.5.The results of this study initially suggest that Q-SD is a potential drug for the treatment of dry AMD.