Research On The Mechanism Of Hydroxy-α-sanshool Against Alzheimer’s Disease And Nasal Liposome Preparation

Zanthoxylum bungeanum Maxim.,is derived from the dried mature pericarp of Zanthoxylum bungeanum,a plant of Rutaceae family.Because of its unique aroma,it can be used for both medicine and food.The main effect of Zanthoxylum bungeanum Maxim.is calefaction to relieve pain,killing roundworms and relieving itching.In modern research,it is found that the active component hydroxy-α-sanshool（HAS）is the amide compound with the highest content in Zanthoxylum bungeanum Maxim.It is not only the basis of the taste of Zanthoxylum bungeanum Maxim.,but also can improve the learning and memory ability of Alzheimer’s disease.However,due to the instability of HAS,it will undergo chemical changes under normal temperature and oxygen conditions,and its anti-AD mechanism is not clear,which limits the clinical application of HAS.Therefore,this paper chooses HAS as the research object to study its anti-AD mechanism and related preparations.Objective:The cell model of AD was established by stimulating PC12 cells with H₂O₂ to explore the mechanism of anti-AD of HAS.HAS was made into liposome through related process screening,and then it was applied to AD model mice to study the pharmacological activity and related metabolic kinetics of HAS liposome against AD,so as to provide scientific basis for HAS used in clinical treatment of AD,and lay a foundation for the next drug development of HAS.Methods:（1）The in vitro cell model of AD,PC12 cell model induced by H₂O₂,was established to study the mechanism of anti-AD effect of HAS.The cytotoxic effect of HAS on PC12 cells were evaluated by CCK-8 assay.The protective effect of HAS on apoptosis induced by H₂O₂ was investigated by mitochondrial membrane potential detection,DAPI staining and flow cytometry.The inhibitory effect of HAS on oxidative stress was verified by reactive oxygen species and related oxidative stress kinases like SOD,CAT and GSH-Px.Finally,the protective mechanism of HAS was studied by Western blot test and pathway inhibitor.（2）After methodological testing,the content of HAS was detected by HPLC method.With the entrapment efficiency of liposomes as the target,the optimal preparation process of HAS-LP was selected.The morphology,particle size and potential of HAS-LP were investigated,and then the release rate was detected to explore the stability of HAS-LP under the optimal conditions.（3）The prepared HAS-LP were directly used in nasal administration of normal rats to study their toxic effects on nasal mucosa,HAS-LP were administered intranasally after the establishment of D-galactose-injection AD model in mice,and the anti-AD activity of HAS-LP was verified by Morris water maze test,passive avoidance test and pathological tissue sections such as hematoxylin-eosin staining,Nissl staining and Tunel staining.Finally,after nasal administration of HAS-LP and HAS solution,the HAS content was determined by HPLC in plasma and brain tissue of AD mice,and the main pharmacokinetic parameters were calculated with pharmacokinetic software to analyze the targeting ability of HAS-LP.Results:（1）The CCK-8 assay showed that HAS had no obvious cytotoxic effect on PC12cells and had a protective effect on the viability of PC12 cells induced by H₂O₂.What’s more,in further experiments,the results of MMP,DAPI staining and flow cytometry showed that HAS pretreated PC12 cells could significantly inhibit the apoptosis induced by H₂O₂,while the decrease of intracellular reactive oxygen species（ROS）and malondialdehyde（MDA）content and the increase of oxidative stress kinase indicated that HAS could inhibit the oxidative stress induced by H₂O₂.In addition,the protective effect of HAS on PC12 cells,namely in vitro anti-AD effect is mainly achieved through the PI3K/Akt signal pathway.Once the PI3K/Akt signal pathway is blocked by the pathway inhibitor,the protective effect of HAS will almost disappear.（2）The entrapment efficiency of HAS liposome prepared by thin film evaporation method was the highest when the mass ratio of drug:cholesterol:soybean phospholipid was 1:4:16 and the volume of hydrated medium was 15 m L.Under these selected conditions,the particle size of HAS liposome was 162.3 nm,and the surface charge-33.2m V.What is more important,the HAS lipospme had a more sustained release effect.（3）HAS solution can slightly stimulate the nasal mucosa of rats and make some of the blood vessels in the lamina propria hyperemia,while the effect of HAS liposome on rat nasal mucosa is similar to that of normal saline which means that HAS liposome has no obvious effect on nasal mucosa.In the Morris water maze experiment,HAS-LP can significantly reduce the escape latency of mice,while the time in the target quadrant and the number of passing through the platform were also increased by HAS-LP,while in passive avoidance test HAS liposome can also increase the escape latency and error times of mice.In addition,it was also observed in the pathological sections that HAS liposomes could improve the nerve damage of CA1 and CA3 in the hippocampus of AD mice.The results of pharmacokinetic experiments showed that HAS liposomes had stronger targeting and easier to target brain tissue than HAS solution.Conclusion:In this study,H₂O₂-stimulated PC12 cells are imitated to establish a cell model of AD in vitro,and it was found that PI3K/Akt signaling pathway play a key role in HAS’s effects of antioxidant stress and anti-apoptosis,thus protecting cells from H₂O₂stimulation.Then HAS was prepared into HAS liposome by thin film evaporation method,which made HAS more stable.Finally,the nasal administration of HAS liposome in rats showed almost no toxicity to the nasal mucosa,while in the nasal administration of AD mice,HAS liposome could significantly improve the learning and memory impairment of mice,inhibit the nerve injury of mice,and enhance the brain targeting of HAS.