Study On Anti-dementia And Anti-tumor Effects Of Piper Wallichii

Objective:Alzheimer’s disease and cancer are major threats to human health.Discovery of lead compounds with pharmacological activities and study of their mechanisms from natural products such as traditional Chinese medicine(TCM)has been one of the important ways and contents of the related drug development.Piper wallichii,is a common kind of Chinese traditional medicine for promoting blood circulation and removing blood stasis and eliminate the wind-damp,strong waist and knee,invigorate the circulation of analgesic effect and so on.This doctoral thesis embarks from the commonly used traditional Chinese medicine(TCM)for promoting blood circulation and removing blood stasis.The anti-dementia and anti-tumor effects of Piper wallichii were found.A variety of pharmacological experiment models including animal models,cell and molecular levels and technical means such as molecular biology and cell biology were used to carry out the experiments.The material bases and relevant mechanisms of Piper wallichii against Alzheimer’s disease and cancer were clarified.Methods:1.Established a dementia model in mice for lateral ventricle injection Aβ25-35.With indicators of Morris water maze escape latency and the distances looking for a platform,we observed a variety of commonly used TCM that promoting blood circulation and removing blood stasis to improve the ability of learning and memory in mice of the AD model.2.Within SOD,MDA,AChE and MAO in mice brain as indexes,we discussed the effects of Piper wallichii on oxidative stress system of the AD mice.Using the navigation experiment,space exploration test and avoid dark test,observed the influence of Piper wallichii extracts from different parts and flow on the ability of learning and memory in AD mice.3.By classical chemical separation method of purification for Piper wallichii active site,we established Aβ25-35 PC 12 cell model,applicated the MTS and LDH method to detect monomer compound effects on cell survival rate.4.Using the SOD,MDA and GSH-Px content as indexes,we observed the effects of Wallichinine and Burchellin C for Aβ25-35 PC 12 cell model on the oxidative stress research.5.By RT-PCR and Western blot method,we discussed the effects of Wallichinine and Burchellin C for Aβ25-35 PC 12 cell model on the P21、CDK4、E2F1、NF-κB P65、Caspase-3mRNA and proteins.6.By using MTT method on KB/KBV200 cells to screen and track Piper wallichii extract and flow of the anti-tumor activity,we examined the cytotoxicity of Wallichinine in two ABCB1-overexpressing cells KBV200 and its parental cells KB by MTT assay.Tested the cytotoxicity of combination of Wallichinine with two ABCB1 substrates Vincristine and Doxorubicin and one non-ABCB 1 substrate Cisplatin at the various concentrations.7.Estimated the effects of Wallichinine in combination with chemotherapeutic agents in the ABCB 1-overexpressing cells,cell apoptosis and the related proteins were also detected by FCM and Western blot.8.Measured the intracellular levels of two ABCB1 substrates Rhodamine 123 and doxorubicin in the presence or absence of Wallichinine.9.The predicted binding mode showed the hydrophobic interactions of Wallichinine within the large drug binding cavity of ABCB1Results:1.Morris water maze experiment results showed that the Piper wallichii can improve the ability of learning and memory in mice learning memory impairment.Chloroform part from Piper wallichii total extract most can improve learning and memory ability in AD mice model.2.Piper wallichii could improve SOD activity and reduce MDA content of AD mice brain tissue,reduce MAO and AChE activity,also could improve the antioxidant capacity,reduce the oxygen free radical damage to the body,improve the learning memory abilities.3.The MTS and LDH experimental results of the PC 12 cell model showed that,Wallichinine(10μM、25μM)and Burchellin C(20μM、25μM)can significantly increase the cell survival rate.10μM Wallichinine and 20μM Burchellin C could significantly increase SOD content,reduce the GSH-Px and MDA content.4.Different concentrations of Wallichinine and Burchellin C can inhibit P21 mRNA and protein expression decreased,CDK4,E2F1,NF-κB P65,Caspase-3 mRNA and protein expression increases of the PC 12 cell model accordingly.5.KB/KBV200 cell anti-tumor experiments showed that,Wallichinine potentiates the effects of Vincristine and Doxorubicin on inhibition of growth,arrest of cell cycle and induction of apoptosis in ABCB1 over-expressing cancer cells.Wallichinine blocks the drug efflux activity of ABCB1 to increase the intracellular accumulation of Rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1.6.To understand the mechanism of binding of Wallichinine to ABCB1 at a molecular level,docking studies were performed.Wallichinine is stabilized through specific interactions such as hydrogen bonding to large drug binding cavity of ABCB1 and nonspecific interactions such as hydrophobic interactions with residues in the hydrophobic cavity of ABCB1.Conclusion:Piper wallichii has anti-dementia and anti-cancer pharmacological effects and its chloroform fraction of crude extract is relative active part.Three active neolignans including Wallichinine were isolated.Their activities of improving the learning and memory ability of AD model mice and the level of oxidative stress and cholinergic system were found in AD mice model and PC 12 cell model.The activity of inhibiting apoptosis of PC 12 cell was also found,which may include the inhibition of the activation of apoptosis factors NF-κB,cutting of Caspase-3 involved in the activation of apoptosis cascade and the inhibition of the expression of cycle regulatory proteins such as CDK4 and E2F1.Wallichininecan reverse the ABCB1-mediated cancer multidrug resistance by potentiating the effects of two ABCB 1-substrate chemotherapeutic agents Vincristine and Doxorubicinon inhibition of growth,arrest of cell cycle and induction of apoptosis in ABCB1 over-expressing cancer cells.