Effects Of Sialic Acid On Anti-alzheimer's Disease And Its Mechanisms

Objective:To investigate the potential acute toxicity of sialic acid by single dose toxicity test in mice,the protective effects of sialic acid on acute cerebral ischemia injury in mice,the effects of Sialic acid on activating blood circulation to dissipate blood stasis in mice,the effects and mechanisms of Sialic acid on the alzheimer's disease?AD?rats induced by D-galactose?D-gal?and Aluminium chloride?AlCl₃?,and to provide the experimental basis for clinical treatment of the alzheimer's disease.Methods:In the single oral dose acute toxicity test,after three days of adaptation,seventy mice were randomly divided into seven groups:Control group and six Sialic acid groups(6.0,7.2,8.7,10.4,12.5 and 15.0 g·kg^-1),with 5 males and 5females per group.The mice were fasted,but allowed water,for 14¹6 h before the treatment.Mice in Control group were oral treated with 0.5%Carboxy methylcellulose(0.5%CMC-Na,30 mL·kg^-1),while mice in Sialic acid groups were oral treated by 30 mL·kg^-1 with different concentrations of sialic acid,the acute toxicities were observed and recorded after single treatment for14 days.In the pharmacodynamic test of sialic acid on acute cerebral ischemia injury,after three days of adaptation,one hundred mice were randomly divided into five groups:Control group,Nimotop group(18 mg·kg^-1)and three Sialic acid groups(28,55,110 mg·kg^-1),with 10 males and 10 females per group.Mice in Control group were oral treated with pure water(20 mL·kg^-1),mice in Nimotop group and Sialic acid groups were oral treated by 20 mL·kg^-1 with different concentrations of nimotop or sialic acid,all the mice were oral treated for seven consecutive days.The mice were fasted,but allowed water,for 14¹6 h,and 1h after the last treatment,5 males and 5 females per group were selected,the decapitation method was used to establish the acute global brain ischemia model in mice,the open-mouth times and gasping time were recorded;The other ten mice per group were selected,the bilateral common carotid artery and parasympathetic nerve occlusion method was used to establish the acute incomplete cerebral ischemia model in mice,the survival time and life span-prolonging rate was recorded;the water content in small intestine and colon was recorded.In the pharmacodynamic test of sialic acid on activating blood circulation to dissipate blood stasis,after three days of adaptation,fifty mice were randomly divided into five groups:Control group,Aspirin group(18 mg·kg^-1)and three Sialic acid groups(28,55,110 mg·kg^-1),with 5 males and 5 females per group.Mice in Control group were oral treated with pure water(20 mL·kg^-1),mice in Aspirin group and Sialic acid groups were oral treated by 20 mL·kg^-1 with different concentrations of aspirin or sialic acid,all the mice were oral treated for thirty consecutive days.The mice were fasted,but allowed water,for 14¹6h,and thirty minutes after the last treatment,the tail tip of all the mice were cut for three millimeters long,and the bleeding time was recorded.After the test of tail tip cut,glass-tube method and slide method were used at the same time,the coagulation time of each mouse was recorded.In the pharmacodynamic test of sialic acid on the AD rats induced by D-gal and AlCl₃,after seven days of adaptation,sixty six male SD rats were randomly divided into six groups:Control group,Model group,Piracetam group(504mg·kg^-1)and three Sialic acid groups(19,38,76 mg·kg^-1),with eleven males per group.In the morning,rats in Control group were intraperitoneal injected of0.9%saline by 1 mL·kg^-1,and oral treated of pure water by 10 mL·kg^-1,the other rats were intraperitoneal injected of D-gal(120 mg·kg^-1)by 1 mL·kg^-1,and oral treated of AlCl₃(15 mg·kg^-1)by 10 mL·kg^-1 once per day for sixteen consecutive weeks to establish model of the AD rats.While in the afternoon,rats in Control group and Model group were oral treated with pure water(10mL·kg^-1),and rats in Piracetam group and Sialic acid groups were oral treated by 10 mL·kg^-1 with different concentrations of piracetam or sialic acid,all the rats were oral treated for sixteen consecutive weeks.On D₁₁₂,the cataract formation rate of rats in each group was calculated.On D₁₁₃,rats were trained with freestyle for 90 seconds.On D₁₁₄^D₁₁₈,the place navigation test was run,a platform with a diameter of 10 cm was placed 1cm below the water surface.Rats were trained in the water maze to find a hidden platform for five consecutive days,four trials per day per rat were run,if a rat failed to find the platform within 90 seconds,it was guided to the platform and kept there for 10 seconds.The escape latencies of each rat in four trials were recorded,and the mean escape latencies of each rat per day was calculated.On D₁₁₉,the spatial probe test was run,and the platform was removed,the indexes in the spatial probe test as First time crossing the target?s?,Time in target quadrant?s?,Target crossing?n?and Total distance?cm?were recorded.On D₁₂₀,the blood samples were collected,the brains and hippocampuses were removed.The serum levels of Albumin?ALB?,Total protein?TP?,Aspartate transaminase?AST?,Alanine transaminase?ALT?,Total bilirubin?TBIL?,Creatinine?CREA?,Blood urea nitrogen?BUN?,Total cholesterol?TC?,Triglycerides?TG?,High density lipoprotein cholesterol?HDLC?,Low density lipoprotein cholesterol?LDLC?,Maleic dialdehyde?MDA?and Superoxide dismutase?SOD?were determined by biochemical estimation,Tumor necrosis factor-??TNF-??,Transforming growth factor-??TGF-??,Interleukin-6?IL-6?and Interleukin-1??IL-1??were determined by the double-antibody sandwich enzyme-linked immunosorbent assay?ELISA?,and histopathological changes of brains and hippocampuses were examined by hematoxylin and eosin?HE?staining.Results:In the single oral dose acute toxicity test,mice in control group had noting difference in the clinical signs,no death.The main clinical signs induced by sialic acid in 6 h after single treatment were spontaneous activity reduction,quiet,lie prostrate,ochrodermia,body cool.The death time of the mice in Sialic acid groups was concentrating on 20 min⁵ h after single treatment.The LD₅₀ of sialic acid in the single acute toxicity test was 8823.07 mg·kg^-1 and the 95%confidence limit space was 8078.78⁹625.18 mg·kg^-1,LD₅ was 6832.20 mg·kg^-1,LD₉₅ was 11394.07 mg·kg^-1.In the pharmacodynamic test of sialic acid on acute cerebral ischemia injury,compared with control group,after treated with sialic acid(28,55,110 mg·kg^-1)for seven days,the open-mouth times and gasping time were significantly increased in global brain ischemia mice?P<0.01?,the prolonging rates of gasping time were 33.54%,35.98%,56.71%,the survival time was significantly prolonged in acute incomplete cerebral ischemia mice?P<0.01?,the life span-prolonging rates were 41.24%,47.45%,94.35%,the water small intestine and content in colon was significantly increased?P<0.01?.In the pharmacodynamic test of sialic acid on activating blood circulation to dissipate blood stasis,compared with control group,after treated with sialic acid(28,55,110 mg·kg^-1)for thirty days,the bleeding time of tail tip cut were obviously prolonged,the bleeding time-prolonging rates were 26.24%,28.14%,29.28%,the coagulation time of slide method were significantly prolonged?P?0.05 or P?0.01?,the coagulation time-prolonging rates were 21.48%,34.07%,42.22%,the coagulation time of glass-tube method were significantly prolonged?P?0.05?,the coagulation time-prolonging rates were 27.74%,20.51%,32.90%.In the pharmacodynamic test of sialic acid on the AD rats induced by D-gal and AlCl₃,after treated with sialic acid(19,38,76 mg·kg^-1)for sixteen consecutive weeks,compared with the Model group,the cataract formation rate of rats were obviously decreased,and the cataract formation rate were 40.0%,9.09%,0.The bodyweight gain was significantly increased?P?0.05 or P?0.01?.The escape latencies were obviously decreased in the place navigation testand,the First time crossing the target and Total distance were obviously decreased in the spatial probe test,the Target crossing numbers were significantly increased?P?0.05 or P?0.01?,but Time in target quadrant was no significantly changed?P>0.05?.The liver functions in serum as ALB and TP were obviously increased,AST,ALT and TBIL were significantly decreased?P?0.05 or P?0.01?.The renal functions in serum as CREA and BUN were significantly decreased?P?0.05 or P?0.01?.The lipids in serum as TC and LDLC were obviously decreased,but TG and HDLC were significantly changed?P>0.05?.The serum levels of SOD were obviously increased,and the serum levels of MDA were significantly decreased?P?0.01?.The serum levels of IL-1?,IL-6 and TNF-?were significantly decreased?P?0.05 or P?0.01?,and the serum levels of TGF-?were significantly increased?P?0.05?.The histopathological changes of brains and hippocampuses were obviously alleviated.Conclusions:Sialic acid is low toxicity substance according to our study refer to the toxicity classification standard of World Health Organization?WHO?,and has obvious actions of anti-cerebral ischemia,increasing water content in small intestine and colon and blood-activating and stasis-transforming.Sialic acid has obvious actions of ameliorating the spatial learning and memory impairment in AD rats induced by D-gal and AlCl₃,which could be associated with decreasing the serum levels of inflammatory factors?IL-1?,IL-6 and TNF-??,improving the antioxidant capacity,decreasing serum lipids,having good hepatoprotective and renal protective effects,protecting the neurons in brain.