| Objective: To investigate the protective effect of taurine on improving the learning and memory deficits and the apoptosis of hippocampal neurons in STZ-induced T2D rats,and explored the anti-apoptosis mechanism of taurine.Methods: Sixty SD male rats(140-180 g)were purchased from animal center.These rats were divided randomly into two major groups:Control group(n=12)and experimental group(n=48).Control group was given normal diet and normal water for whole study period while experimental group was given high fat diet(25% surcose +15% cholesterol + 1% bile acid + 57.55% normal diet).After four week period they were injected with streptozocin(STZ)at 25mg/kg of body weight.Blood glucose was checked after 72 hours(h),to confirm it above ≧ 16.7 mmol/L considered diabetes.Diabetic rats were kept on normal diet and provided normal water and then divided into four groups i.e.T2D= type 2 diabetic rats given normal water,T1= given 0.5% taurine solution,T2= given 1% taurine solution and T3= given 2% taurine solution for a period of eight weeks while normal diet was provided to all groups.After eight weeks,rats from each group were tested for their spatial learning and memory ability using Morris Water Maze(MWM)test for one week.Then animals were sacrificed and hippocampus tissue were preserved.Some brain tissue from each group were acquired then used the4% PFA to fix them,after that put them into 15%,20% and 30% surcose solution for future studies.In vitro,we used the HT-22 cell,the hippocampal neurons line,the cells were divided into five groups,control(Con)group: cells were exposed to the normalmedium,high glucose(HG)group: cells were exposed to the normal medium with 150 m M glucose,taurine treatment groups: 10 m M,20 m M and 40 m M taurine were added into the normal medium with 150 m M glucose as T1,T2 and T3 groups.We used the TUNEL Assay and immunofluorescence stain of Neu N to check the apoptosis of hippocampal neurons,Western blot was used to check the level of relative proteins(NGF,Trk A/p-Trk A,Akt/p-Akt,Bad/p-Bad),the cell activity was tested by MTT and we also checked the LDH activity by LDH Assay Kit.Result: 1.The effect of taurine on the learning and memory impairment in T2D rats There were two parts in this test,one was spatial learning ability test for four days and another was spatial memory ability test for one day.During four days of training test,the swimming speed of rats did not show significant differences between the groups.On the other hand,compared to the Con group,the rats in the T2D group took more time to find the platform(P <0.05)and the distance of swimming significantly increase(P <0.05).While taurine treatment(T2,T3)groups significantly reduced the escape time and swimming distance of rats compared with T2D group(P<0.05).In the last day,the T2D rats took more time to cross the platform area for the first time compared with Con group(P <0.05)while the number of crossing platform area and the percentage of time spent in target quadrant were less than that in Con group(P <0.05),on the contrary,taurine supplementation changed the index above.2.The effect of taurine on the apoptosis in hippocampal neurons in T2D rats and HG treated HT-22 cells The TUNEL results showed that the number of apoptotic neurons in hippocampal CA1 area and caspase-3 activity significantly increase in T2D group compared with Con group(P <0.05).However,taurine treatment mitigated the increased neurons apoptosis in T2D group(P <0.05)and the activity of caspase-3 was lower than that in T2D group(P <0.05).In vitro,the apoptosis and caspase-3 activity in HG group was evidently higher than that in control group with significant difference(P <0.05).However,these indicators in T2D group were significantly mitigated by taurine(P<0.05).3.The effect of taurine on the expression of NGF in hippocampus of T2D rats and HG treated HT-22 cells The western blot result showed that the expression ofNGF and p-Trk A in T2D group showed significantly lower expression than that of control(P <0.05).However,the expression of NGF and p-Trk A in taurine groups was significantly higher than that of T2D group(P <0.05).Moreover,the effect of taurine in the HG treated HT-22 cells was blocked in the presence of a NGF neutralizing antibody ab16161(ab)(P <0.05).4.The effect of taurine on the Akt/Bad and mitochondria-dependent pathway in hippocampus of T2D rats and HG treated HT-22 cells The western blot results showed that Akt and Bad were not significantly different among the groups.But the contrary,the expression levels of p-Akt and p-Bad in T2D group were significantly low as compared to control(P <0.05).However,taurine changed these index above(P <0.05).The expression of Cyt-C in mitochondria was lower in T2D group than that in control(P <0.05),while the expression of Cyt-C in cytosol was higher in T2D group than in control group(P <0.05).However,taurine treatment dose-dependently reversed the results by T2D(P <0.05).The same results were showed in vitro.But the protective roles of taurine were blocked in the presence of ab and Akt inhibitor Perifosine(P).5.Taurine protected against the apoptosis of HG-treated HT-22 cells via NGF/Akt/Bad pathway The TUNEL results showed that HT-22 cells apoptosis in HG treated cells was significantly higher than that in control group(P <0.05).But the indicator in the HG group was significantly mitigated by taurine(P <0.05).Nonetheless,the effect of taurine was blocked in the presence of ab or P(P <0.05).Moreover,data of caspase-3 activity in HT-22 cells were also accordant with the results in above.6.Taurine prevented the apoptotic death of HG-treated HT-22 cells via NGF/Akt/Bad pathway The MTT and LDH results showed that the cells treated with HG showed lower cell viability but higher activity of LDH compared to control group(P <0.05).On the contrary,taurine significantly increased cell viability and decreased the activity of LDH compared to HG group(P <0.05).However,the protection of taurine was blocked in the presence of ab or P.Conclusion:1.Taurine improved the cognitive deficits in T2D rats;2.Taurine ameliorated the apoptosis of hippocampal neurons in T2D rats via NGF/Akt/Bad pathway;... |
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