Inhibitory Effects Of Matrine And Matrine-loading Nanoparticles On The Cytotoxicity Of Amyloid-? Protein

The formation of ?-amyloid protein 1-42(A?42)oligomers and the cytotoxicity of A?42 oligomers are two important pathological characteristics of Alzheimer's disease(AD),which play a key role in the pathogenesis of AD.Selective inhibition of the formation of A?42 aggregates and the cytotoxicity of A?42 oligomers provide the best target for AD therapy.Some natural compounds from Chinese herbal medicine(CHM)have the effects of anti-A?42 aggregation and inhibiting the neurotoxicity of A?42,which can protect nerve cells from the damage of the cytotoxicity of A?42 aggregates.In our early studies and literatures by other scholars,we have found that matrine(Mat)can inhibit the toxicity of A?42 oligomer to human neuroblastoma cells(SH-SY5Y).In our previous study,we further found that the inhibitory effect of Mat on the cytotoxicity of A?42 oligomer is not completely proportional to the concentration of Mat,which suggests that the inhibitory effect of Mat on A?42 oligomer may not be single,but may have diversity,or may not be unidirectional.However,the content of these aspects has not been further revealed and reported before.In this study,we found that the inhibitory effect of Mat on the toxicity of A?42oligomer was not parallel in concentration,in order to further reveal the(multiple)mechanism of Mat on the target of A?42 oligomer,and to reveal its dose-response relationship,and we further studied the low-dose and high-efficiency inhibition of Matrine on the cytotoxicity of A?42 oligomer.We used nanotechnology to study and determine the optimal preparation system of Mat nanoparticles by using safe and biodegradable polymer compounds.With the help of the sustained-release characteristics of Mat nanoparticles,we used App/PS1 transgenic mice(AD model mice)to determine the sustained efficacy of Mat nanoparticles in vivo.The results are as follows:?.For the first time,it was found that Mat could maintain or even enhance the cellular nutritional efficacy of A?42 monomer by inhibiting the aggregation of A?42 monomer and cooperating with A?42 monomer.1.Mat can selectively bind to A?42 oligomer and A?42 monomer at a concentration far below its IC50 value(such as 10 ?M),rather than A?42 fiber.2.When Mat is directly combined with A?42 monomer or the new A?42 oligomer formed by A?42 monomer,Mat can not only reduce or even eliminate the cytotoxicity of A?42 oligomer,but also promote the proliferation or increase the survival rate of SH-SY5 Y cells in vitro.At the same time,it was found for the first time that there was no absolute linear correlation between the protective effects of Mat on SH-SY5 Y cells and the concentration of Mat used.Only at a certain concentration(1.0-50?M)can it play a better role.Based on the results of molecular docking of Mat with A?42 monomer or newly formed A?42 oligomer,it was revealed for the first time that Mat can enhance the cellular nutritional function of A?42 monomer,which may be realized by the positive synergistic effect of Mat with A?42 monomer.It is speculated that there may be Mat-like metabolites in the human brain,which can act as a molecular chaperone of A?42 monomer to stabilize A?42 monomer.The functional properties of Mat to A?42 or the dose-response relationship of Mat to A?42 have not been reported before,which should be related to the amphotericity and integrated conformation of A?42.?.It was found for the first time that the cytotoxicity of Mat to A?42 oligomer also showed the characteristics of regional concentration.The interaction between Mat(1.0-50 ?M)and A?42 oligomer not only inhibited the cytotoxicity of A?42 oligomer,but also promoted the depolymerization of A?42 oligomer into A?42 monomer,which played the role of cell nutrition and cell protection on SH-SY5 Y cells.However,higher concentrations of Mat(100 ?M)only inhibited the cytotoxicity of A?42 oligomer.According to the docking results of Mat and A?42 oligomer,it is proposed for the first time by conformational analysis that these differences in the efficacy of Mat are due to the interaction of Mat and A?42 oligomer at different molecular ratios,and also point to the same speculation that there may be Mat-like metabolites in human brain,which can be used as molecular chaperones of A?42 monomer.However,in the absence of this chaperone,A?42 monomer will gradually aggregate to form A?42 oligomer with cytotoxicity.?.Mat can effectively achieve brain transport,and its nanoparticle forms are conducive to maintaining its effective concentration in the brain.The results of animal experiments showed that although Mat could be rapidly absorbed into the brain after oral administration,the level of Mat in the blood and brain tissue continued to decline with the extension of time.In order to make Mat continuously inhibit the cytotoxicity of A?42 oligomer,and cooperate with A?42 monomer to exert the effect of cell nutrition,this paper adopts nanotechnology,using the poly(lactic-co-glycolic acid)(PLGA)and Polyethylene glycol-poly(lactic-co-glycolic acid)(PEG-PLGA),which have biological safety and degradability to be prepared Mat loaded nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)respectively,At the same time,the effective brain transport and sustained release in brain of Mat were realized.1.Use emulsion solvent evaporation method,and base on single factor analysis and orthogonal test analysis,and be detected by high performance liquid chromatography(HPLC),dynamic light scattering(DLS)and scanning electron microscope(SEM)to determine the optimum conditions for the preparation of two kind of Mat nanoparticles.The optimum conditions for the preparation of Mat-PLGA-NPs were as follows: the dosage of PLGA was 50 mg,the concentration of Polyvinylalcohol(PVA)was 2%,the stirring time was 3 h,the ultrasonic time was 8min,and the volume of Mat solution was 200 ?L;The optimum conditions for the preparation of Mat-PEG-PLGA-NPs were as follows: the dosage of PEG-PLGA was 50 mg,the concentration of Polyvinylalcohol(PVA)was 1%,the stirring time was 3 h,the ultrasonic time was 8 min,and the volume of Mat solution was200 ?L.The preparation conditions of the two kinds of Mat-NPs are very similar,but the weight of each factor is different.The optimized conditions are the most suitable for the corresponding Mat-PLGA-NPs or Mat-PEG-PLGA-NPs,and the conditions and processes are simple,easy to operate and feasible.2.Ten batches of Mat-PLGA-NPs or Mat-PEG-PLGA-NPs were prepared under their respective optimum conditions,and their important performance indexes were analyzed: the encapsulation efficiency of Mat-PLGA-NPs was 80.67±2.5%,drug loading was 3.24±0.06%,and the average particle size was 190.5±2.43 nm;the encapsulation efficiency of Mat-PEG-PLGA-NPs was 81.75±3.96%,drug loading was 3.27±0.16%,and the average particle size was 190.5±2.43 nm The diameter is 121.2±3.15 nm.Mat-PLGA-NPs and Mat-PEG-PLGA-NPs were observed by SEM(50.00 KX).The particles are well dispersed and their average particle size was controlled within 200 nm(the average particle size of Mat-PEG-PLGA-NPs was 121.2 ± 3.15 nm,while the average particle size of Mat-PLGA-NPs was 190.5 ± 2.43 nm).The appearance is spherical,the surface is smooth and the size is uniform.The above results show that PEG not only introduces its efficacy,but also reduces the size of nanoparticles,which is not only conducive to the absorption and transportation of nanoparticles in vivo,but also helps to improve the brain transport efficiency of nanoparticles and improves the drug loading of nanoparticles.3.Sustained release is one of the most important purposes of nanoparticles as drug delivery system.Drug release performance of drug loaded nanoparticles in vitro is one of the important indicators to evaluate the quality of the prepared nanoparticles.In vitro release test showed that the effective release of Mat-PLGA-NPs lasted for 9 d,and the cumulative release rate was 93.2±0.26%;the effective release of Mat-PEG-PLGA-NPs lasted for 10 d,and the cumulative release rate was 92.4±0.69%.According to the release results,the release equation fitted by Mat-PLGA-NPs was Q=33.956t(?)-4.849(R2 = 0.9958);the release equation fitted by Mat-PEG-PLGA-NPs was Q=31.795t(?)-5.166(R2 = 0.9978).Therefore,we prepared two kinds of Mat nanoparticles are in line with the Higuchi equation,which belongs to the sustained-release drug delivery system,in line with the expected preparation of sustained-release Mat nanoparticles.The in vitro release process of Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can be roughly divided into the first two days of sudden release process and 3-9/10 d of sustained release process.It is speculated that the sudden release in the first two days is mainly due to the direct release of Mat carried on the surface of the prepared nanoparticles into the medium.The slow release in the next 3-9/10 d was due to the gradual degradation of the polymer(PLGA or PEG-PLGA)skeleton and the release of Mat into the medium.This is in line with the basic requirements of drug release mode of drug loaded nanoparticles.In addition,compared with Mat-PLGA-NPs,Mat-PEG-PLGA-NPs has better sustained-release effect,longer release duration and better bioavailability of Mat.4.Two Mat nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)were similar to free Mat in general,and could inhibit the cytotoxicity of A?42 in a certain level range.However,compared with free Mat,the effect of Mat released from the two kinds of Mat nanoparticles is similar to that of the interaction of medium concentration level Mat(1.0-50 ?M)with A?42 oligomer.More importantly,when the cumulative release of Mat from Mat-NPs was higher than that of free Mat,they could not only inhibit the cytotoxicity of A?42 oligomer,but also enhance the nutritional effect of A?42 on cells.These results indicate that the inhibition effect of two Mat nanoparticles on cytotoxicity of A?42 oligomer is better and lasts longer,which fully reflects the significance and application advantages of sustained-release nanoparticles.?.Mat and its nanoparticles can effectively reduce the load of A?42 deposition in the brain of AD model rats and alleviate the histopathological changes in the hippocampus.1.The blood-brain barrier(BBB)model in vitro was established,and it was proved that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs could effectively transport in the brain.Compared with free Mat,the two kinds of Mat nanoparticles maintained the level of Mat across the BBB more persistently.The cumulative release rates of Mat in 24 h were15.0±0.53% and 14.3±0.78%,respectively.2.Animal experiments of Mat and its nanoparticles Mat-PLGA-NPs and Mat-PEG-PLGA-NPs were carried out in AD model rats.(1).HE staining showed that Mat and its nanoparticles Mat-PLGA-NPs and Mat-PEG-PLGA-NPs could alleviate the histopathological changes in hippocampus of AD model rats,and Mat nanoparticles were more effective than free Mat.In the hippocampus of AD model rats treated with Mat-PLGA-NPs and Mat-PEG-PLGA-NPs,the cells were arranged in order,most of them had obvious nucleolus,clear nuclear membrane and abundant chromatin,only a few cells had pyknosis in cytoplasm and nucleus,and the staining became deeper and smaller,and the overall situation was closer to that of negative control rats.(2).The results of immunohistochemistry showed that Mat and its nanoparticles Mat-PLGANPs and Mat-PEG-PLGA-NPs could reduce the number and volume of amyloid plaques in the hippocampus of AD model rats,and could effectively reduce the burden of A?42 plaques in the brain of AD model rats,and the effect of the latter was more obvious.These results suggest that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can inhibit the deposition of A?42in hippocampus of AD rats continuously and effectively,thereby alleviating the pathological changes in hippocampus.(3).Dot blotting results showed that Mat and its nanoparticles(Mat-PLGA-NPs and Mat-PEG-PLGA-NPs)could reduce the level of amyloid protein in the hippocampus of AD model rats,and the effect of the latter was more significant.These results show that Mat-PLGA-NPs and Mat-PEG-PLGA-NPs can reduce the total level of amyloid protein in hippocampus for a long time by releasing Mat from its nanoparticles.The results of in vitro and in vivo studies show that Mat and its nanoparticles can positively enhance the beneficial physiological effects of A?42 monomer,negatively inhibit the aggregation of A?42 and the neurotoxicity of A?42 aggregates and the formation of senile plaques.Under the optimal conditions,the prepared Mat nanoparticles can exert these positive and negative effects more persistently and effectively than free Mat.These results lay a foundation for the clinical use of Mat and provide a strong basis.