Protective Effects Of Fucoidan On Injury Of PC12 Cells And Aging Mice Induced By A?25-35 And D-gal

Objective:Alzheimer's disease(AD)is a progressive neurodegenerative disease,clinical manifestations include loss of memory and other cognitive functions.Modern medical research shows,AD pathogenesis is caused by many factors,including the abnormal remove of ?-amyloid,cholinergic neurons damage,oxidative stress,nerve inflammation and gene mutation induced by ?-amyloid25~35(A?25~35)abnormal deposition in brain tissue and dysfunction of neurovascular unit.Fucoidan,isolated from brown algae and marine invertebrates,compared to other sulfated polysaccharides,the fucoidan extracted from Sporophyll of Undaria pinnatifida has higher sulfate and L-fucose content.It has recently been reported that fucoidan have wide variety of biological activities,including anticoagulant,antivirus,anti-angiogenesis,immuno-modulation and antitumor activities.Obtain the aging cell model by culturing the PC12 cells with combination of ?-amyloid and D-gal.At the same time,obtain the aging animal model induced by D-gal.Then test the protective effect of fucoidan on aging animal and PC12 cells aging model.Method:1.The fucoidan was purified by alcohol precipitation and the contents of total carbohydrate was detected by the phenol-sulfuric acid reaction,the sulfate radical and uronic acid were measured by BaCl2-gelation and sulfuric acid-carbazole colorimetric method,respectively.Meanwhile,the molecular weight of the sample was evaluated by size exclusion chromatography using a TSK-gel G3000 PWXL.The optical rotation was tested at 589 nm by the WZZ-1 polarimeter at 20?.2.A?25~35,which is the most toxic peptide fragment derived from amyloid precursor protein,was dissolved in deionized distilled water and incubated with constant oscillation at 37? for 3 days to induce its aggregation.After aggregation,the solution was stored at-20? until use.The stock solution was diluted to desired concentrations immediately before use and added to cell culture medium.0.1816 g D-gal was dissolved in 100 mL deionized distilled water,and after that,the solution war 10 mM D-gal solution.The next step was we filtered the solution by using ultra filtration membrane.The solution was stored at 4? until use.3.Cell viability was estimated by the MTT reduction assay,which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases.Cell viability was normalized as relative percentages in comparison with untreated controls.Following incubation with fucoidan and A? & D-gal for 48 h at 37?and 5% CO2.4.The cells treated with different concentration A? & D-gal were stained with Hoechst 33258(4?g/m L)for 30 min,fixed for 10 min in 4% para-formaldehyde(PFA),and then observed by DMI-4000 B inverted fluorescence microscopy.5.PC12 cells were seeded into cell culture flask,allowed to adhere for 24 h,and treated with 100,200 and 400?g/mL fucoidan and A? & D-gal(25?M & 10mM)for 24 h and 48 h respectively.The cells were collected,washed twice with chilled PBS and stained using annexin V-FITC labeling solution(annexin V-fluorescein in binding buffer containing PI)according to the manufacturer's protocol.Apoptotic cells were analyzed by using a FACSCalibur flow cytometry.6.Cells treated with different concentration A? & D-gal were collected,washed twice with chilled PBS and lysed in RIPA buffer according to the manufacturer's instructions.Equal amounts of protein extract were subjected to 12% SDS-PAGE gels and transferred to nitrocellulose(NC)membranes.Cells treated with fucoidan and A? & D-gal were collected,washed twice with chilled PBS and lysed in RIPA buffer,and the next processes like the methods mentioned above.The proteins were isolated from cells,and then put it into ice.The proteins were used for the assays of SOD and GSH levels,according to the manufacturer's instructions,respectively.7.On each trial,mice were allowed to swim freely until they found the platform and left for an additional 10 s on the platform after they found the platform.However,the mice failing to find the platform within 120 s were placed on the platform manually for 10 s and escape latencies were considered as 120 s.8.The global cerebrals were isolated and blotted dry.The homogenate was centrifuged and the supernatant was used for the assays of GSH,Ach,AchE and ChAT levels,according to the manufacturer's instructions,respectively.The blood was centrifuged and the serum was used for the assays of GSH and SOD levels according to the manufacturer's instructions,respectively.Nissl staining of hippocampus CA1 neurons of mice after D-gal injection.Results1.The fucoidan sample consisted mainly of carbohydrates(79.14 %),sulfates(21%)and uronic acid(10.895 %)with fucose and galactose constituting the monosaccharide composition.The molecular weight of fucoidan was about 10.44×104 Da.The optical rotation of the fucoidan(0.6mg/mL)showed a value of 0.99° at 20°C.2.Compared with control group,viability of the others group decreased significantly,and viability of the 25?M group was about 50% after 48 h treatment,so the 25?M group was a good choice.3.Just treated PC12 cells with fucoidan(100,200 and 400?g/mL)had no different in the viability compared with the control group.4.With the increase of fucoidan concentration,the length of the nerve filament and density of the cells were recovered.PC12 cells gradually showed diffuse homogeneous low fluorescence intensity,appeared the characteristics of normal cells.These results showed that fucoidan inhibited cells apoptosis effectively.5.With the increase of fucoidan concentration,the percentage of apoptosis cell decreased obviously.6.When used fucoidan protect cells for 24 h,western blot results showed that apoptosis factors were significantly decreased,but on the contrary anti-apoptosis factors were significantly increased.Meanwhile,the vitality of SOD and the content of GSH within the cells increased.7.D-gal-injected rats showed a spatial learning and memory impairment in Morris water maze test,as indicated by a significant longer escape latencies compared to relevant data for the control group.However,administration of fucoidan(100 and 200mg/kg)caused a significant improvement in the longer escape latencies.8.Fucoidan at doses of 100 and 200mg/kg increased the blood serum level of SOD,and that of GSH.Fucoidan at doses of 100 and 200mg/kg increased the brain tissue level of GSH,Ach,ChAT,but decreased the brain tissue MDA level.Conclusions Fucoidan showed excellent neuroprotective effects against D-gal induced learning and memory impairment in AD model mice and induced cell injury in PC12 cells with A?25~35.The mechanisms might be attributed to regulating the cholinergic system,reducing oxidative stress,inhibiting caspase family protein translocation and preventing CytC activation.These results suggested that fucoidan is a candidate neuroprotective agent against AD.