Biosafety Evaluation Of Nanoparticles From 'Ershiwuweishanhu' Pill

Background:'Ershiwuweishanhu' Pill was recorded in the "Chinese Pharmacopoeia" in 2010,which was used by the Tibetan people for the treatment of "Baimai disease".It was used to treat epilepsy,stroke,migraine and other nervous diseases in modern times.Its biological safety has been brought into sharp focus since it contains cinnabar,aconite and other highly toxic substances,on which many previous paper focus.Recently,some researchers found that the nanoparticles could be extracted from ESP by using modern technology,and expected that it could be applied to clinical meidicine.However,the biosafety of ESP has not been tested.By taking fluorescent tracing approach,this experiment respectively intervened with cells and SD rats,observing HE staining,Western blot,animal behavior to analyze and evaluate the biological safety of ESP nanoparticles,which can provide theory basis for the further development of new formulation of'Ershiwuweishanhu' pill and improve the clinical efficacy.Objective:To evaluate the biological safety of the 'Ershiwuwei shanhu' Pill nanoparticles.Methods:This experiment applied granularmetric analysis and zeta potential analysis to ESP nanoparticles.And the nanoparticles were co-cultured with SH-SY5Y cells for 8 h.Then fluorescence tracer and transmission electron microscopy(TEM)were used to observe the cell uptake and distribution.Rats were randomly divided into control group and ESP-treated group.The rats were sacrificed by intravenous injection of saline or ESP nanoparticles.The open field test was used to evaluate the changes of excitability in rats.Immunofluorescence was used to observe the expression of nanoparticles in the brain and distribution in the liver,and HE staining were used to observe the changes of brain and liver morphology.Western blot was used to detect the expression of GFAP and tau protein.The.levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in plasma were detected by TBA,and Q-pcr detected the expression level of mir-7.Results:Granularmetric analysis reveals that nanoparticles exists in ESP.The results of fluorescence microscope and transmission electron microscope points out ESP nanoparticles can diffuse across cell membrane,which leads to nuclear fragmentation.Compared with physiological saline group,the rats in ESP-treated group have a significant increasing in their movement distance(P<0.05).Observation under fluorescence microscope shows that ESP nanoparticles appears in hippocampus and liver.The results of HE stain indicates that rats in ESP-treated group occur inflammatory reaction in hippocampus region and liquefaction of hepatocytes.WB shows that the expression of GFAP and tau protein increase in the brain of rats after ESP nanoparticles treatment.TBA points out that T-SOD in blood decrease while MDA increase.Results of q-pcr reveals the expression level of mir-7 decrease after ESP nanoparticles process SH-SY5Y cells.