| Epidemiological data show that China has become a big country of diabetes,the number of patients is on the rise.Diabetes is one of the most common metabolic diseases in clinical,characterized by chronic persistent hyperglycemia,long-term improper treatment or even no treatment,can induce multiple system complications,including diabetic nephropathy,vascular disease and neurological disease.Cognitive impairment is an important complication of the diabetic nervous system.Its clinical features are slow cognitive impairment,including learning,memory,orientation,thinking,ability to understand the decline and personality changes,unconsciousness.At present,the pathogenesis of diabetic cognitive dysfunction is unclear.It is generally believed that inflammation,oxidative stress and apoptosis and other factors involved in the occurrence of diabetes cognitive impairment,in particular,the pathways of apoptosis.PI3 K / AKT / GSK-3β pathway regulates cell proliferation,survival,metabolism and apoptosis in mammalian cells.Endogenous or exogenous stimuli can activate this pathway,allowing the mitochondria to pass through the open pore(MPTP),promote Cyt c release to the cytoplasm,stimulate caspase cascade,and initiate mitochondrial apoptosis.Sulforaphane(SFN)is widely found in cruciferous plants such as cauliflower,broccoli and kale.With a variety of present,SFN is widely used in anti-cancer by regulating susceptibility,apoptosis,cell cycle,angiogenesis and invasion and metastasis and other tumor-related events to play its anti-tumor effect.SFN has a variety of pharmacological effects,including lowering blood sugar,regulating lipid metabolism and improving heart,brain,kidney and muscle damage.In this study,SD rats were used as experimental materials to construct diabetic rats by intraperitoneal injection of STZ.The learning and memory ability and hippocampal neuron morphology of diabetic rats were observed by morphological and morphological changes.To investigate the phosphorylation of PI3 K / AKT / GSK-3β and the mechanism of mitochondrial apoptosis-related protein(caspase-3),and to explore the mechanism of SFN to improve the cognitive impairment of diabetes mellitus by interfering with diabetic rats with cognitive impairment.Part one Establishment of diabetic cognitive impairment model and brain protection of sulforaphaneObjective: To observe the changes of behavior and morphology of SFN in diabetic rats with cognitive impairment,and to clarify the neuroprotective effect of SFN on it.Methods: 120 rats were randomly divided into normal control group(CON group)(n = 53)and diabetic model group(DACD group)(n = 67).According to diabetic rats modeling method,after one week of adaptive feeding,STZ was injected intraperitoneally(fasting for 12 h before injection)at a dose of 60 mg / kg.Injection method: STZ solution in the dark environment,the whole operation on the ice,STZ solution concentration of 2.5%.The rats were divided into two groups according to the same volume of DMSO.After 3 days,the rat blood glucose was greater than 16.7mmol / L,which was regarded as successful in diabetic rats.Blood glucose and body weight were measured before and 2,4,6,7,8,9 and 10 weeks after modeling.Five rats were selected at each time point and subjected to behavioral and HE staining experiments.To confirm whether diabetic rats have cognitive impairment.Diabetic cognitive function rats were randomly divided into diabetic cognitive dysfunction group(DACD group)(n = 8),sulforaphane group(SFN group)(n = 24).The SFN group was divided into three groups: SFNL group,SFNM group and SFNH group(n = 8).8 rats were selected as the normal control group.Morris water maze was used to observe the escape latency before and after the intervention,and the change of the neurons in the hippocampal CA1 region was detected by HE staining.The experimental results were analyzed by Image Lab5.1 and Image J.The data were analyzed by SPSS 20.0 statistical software,with mean ± standard deviation,P <0.05 was statistically significant.Results:1 72 hours after STZ injection,the blood glucose of experimental animals was ≥16.7mmol / L,and the diabetes model was established successfully.The blood glucose of diabetic rats was significantly higher than that of CON group at the 2th,4th,6th,7th,8th,9th and 10 th week after the model was established.With the course of the disease gradually extended,diabetic rats were lower than the same time point of the CON group rats(P<0.05);2 Y maze test results showed that DM rats did not significantly reduce the cognitive level at the 4th and 6th week after the onset of DM rats.Diabetic rats at the end of the 8th week,observed a slight cognitive changes,but compared with CON there was no significant difference(P> 0.05).Diabetic rats at the end of 9-10 weekend after the onset of DM rats,there was learning and memory loss,the model of cognitive impairment in diabetic rats was successfully established,and the difference compared with the CON group was statistically significant(P <0.05);3 The results of HE staining showed that the neurons in the hippocampal CA1 region of the CON group were neat and compact,and the cytoplasm and nucleus were full and clearly visible.At the end of the 8th week,the number of cells in the hippocampal CA1 region of the hippocampus was found to be morphologically changed.At the end of the 9th to the 10 th week after the onset,the neurons in the hippocampal CA1 region were disturbed,loose,Shrinkage,chromatin aggregation,and cytoplasm reduction;4 After 4 weeks of SFN intervention,the monitoring results showed that the weight of diabetic rats was significantly higher than that of DACD group(P<0.05),the blood glucose of rats was significantly lower than that of DACD group after SFN intervention,(P<0.05).The blood glucose and body weight of SFNM group and SFNH group were significantly different from those of DACD group(P<0.05),and the difference was statistically significant(P<0.05).It was suggested that SFN could regulate the blood glucose and body weight of diabetic rats.The regulation of metabolism of SFN increased with the increase of concentration.However,the concentration of 25 mg / kg,to further increase the dose,SFN hypoglycemic effect is no longer increased;5 The effect of SFN on the behavioral behavior of SFN in diabetic rats was investigated by Morris water maze.The escape latency of rats was shorter than that of DACD group(P<0.05),among which SFNM group and SFNH group were the most obvious(P<0.05).After the removal of the platform,the rats in the DACD group at the target quadrant dwell time was shorter than those in the CON group(P<0.05),but still longer than the CON group,the SFNM group and the SFNH group had no significant difference(P>0.05).SFNM group and SFNH group at the target quadrant dwell time were longer than the DACD group,but still shorter than CON group(P<0.05),and there was no significant difference between SFNM group and SFNH group(P> 0.05);6 HE staining showed that the neurons in the hippocampal CA1 region of the CON group were neat,compact,and the cytoplasm and nucleus were clear and visible.The neurons in the hippocampal CA1 region of the brain tissue of diabetic rats were disturbed,loose,and the cells became smaller,and the nuclear pyknosis and chromatin aggregation occurred,and the cytoplasm was decreased.The number of normal neurons in hippocampal CA1 region of SFN group was higher than that of DACD group after SFN intervention.The number of normal neurons in hippocampal CA1 region of SFNM group and SFNH group was the highest,there was no difference between SFNM group and SFNH group(P>0.05),suggesting that the optimal dose of SFN was 25 mg / kg,and then increase the dose of SFN,the brain protection is no longer increased.Part two The neuroprotective mechanism of PI3 K / AKT / GSK3β pathway in the intervention of SFN in diabetic ratsObjective:To investigate the changes of PI3 K / AKT / GSK3β in signal transduction pathway in SFN,and to determine whether the neuroprotective effect of SFN on diabetic rats with cognitive impairment is achieved by activating PI3 K / AKT / GSK3β.Methods: The experimental animals were randomly divided into five groups: CON group,DACD group,SFN group,SFN + SY294002 group and SFN + Lcil group.Western blot was used to detect the phosphorylation of AKT,GSK3β and the expression of MCL-1 protein.TUNEL was used to detect the apoptosis of hippocampal neurons.Results:1 Western blot detection results showed that P-AKT expression levels of DACD group were significantly reduced.Compared with DACD group,SFN made P-AKT protein levels significantly increased,while the inhibitor LY294002 offset the effect of SFN.After Licl(GSK-3β specific inhibitors)intervention,p-AKT expression level was higher than those of DACD and SFN group.The total level of AKT expression in each group had no significant change(P>0.05);2 Western blot detection results showed that the expression of phosphorylated GSK-3β(ser9)in DACD group was significantly lower than that in CON group(P<0.05)Compared with the DACD group,SFN increased the level of p-GSK-3β(ser9)protein,while the inhibitor LY294002 counteracted this effect of SFN.The expression level of p-GSK-3β(ser9)in SFN + Licl group was higher than that in SFN group(P<0.05).There was no significant difference in GSK-3β(ser9)expression between the two groups(P>0.05);3 Western blot detection results showed that the expression of MCL-1 in DACD group was significantly lower than that in control group(P<0.05).SFN significantly increased the MCL-1 protein level in the DACD group,and the inhibitor LY294002 counteracted the intervention of SFN.After Licl(GSK-3β specific inhibitor)administration,the expression level of MCL-1 was higher than that of DACD group and SFN group(P<0.05);4 TUNEL results showed that t TUNEL results showed that there was a small amount of apoptotic neurons in the CON group,DACD group and SFN + LY294002 group was the highest,the difference was statistically significant compared with CON group(P<0.05).Compared with DACD group,the number of neurons in SFN group and SFN + Licl group was significantly decreased,the difference was statistically significant(P<0.05).Part three Sulforaphane through PI3 K / AKT / GSK-3β inhibition of mitochondrial apoptosis pathwayObjective: Animals were randomly divided into five groups: CON group,DACD group,SFN group,SFN + SY294002 group and SFN + Lcil group.Western blot was used to detect the expression of caspase-9,caspase-3 and Cyt c protein.The degree of MPTP in hippocampal neurons was detected by spectrophotometry.The ultrastructure of hippocampal neurons was observed by transmission electron microscopy.Results:1 Compared with DACD group,the expression of mitochondria(maxA520-minA520)was significantly higher than that of DACD group(P <0.05),(DACD vs CON = 0.096 ± 0.018 vs 0.021 ± 0.004,P<0.05).Compared with the DACD group,(maxA520-min A520)increase was alleviated by SFN(SFN vs DACD = 0.059 ± 0.020 vs 0.096 ± 0.018,P<0.05);and the inhibitor LY294002 counteracted the effect of SFN;Licl,GSK-3β inhibitor,superimposes the effect of SFN;2 The expression of caspase-9 in CON group was only a small amount.Compared with DACD group,the expression of caspase-9 in DACD group was significantly lower than that in DACD group(P<0.05).The expression of caspase-9 in SFN group was significantly lower than that in DACD group(P<0.05).The expression of caspase-9 was significantly decreased in the group of SFN + Licl(GSK-3β specific inhibitor)compared with DACD group and SFN group(P<0.05);3 Western blot results showed that CON group caspase-3 protein only a small amount of expression.Compared with DACD group,the expression of caspase-3 in DACD group was significantly lower than that in DACD group(P<0.05),and the expression of caspase-3 was decreased by LY294002(P <0.05).The expression of caspase-3 was significantly decreased in the group of SFN + Licl(GSK-3β specific inhibitor)compared with DACD group and SFN group(P<0.05);4 The results of Cyt c showed that there was only a small amount of Cyt c protein in CON group.Compared with DACD,the expression of Cyt c in DACD group was significantly increased(P<0.05),and the expression of Cyt c was decreased by SFN(P<0.05),while LY294002 reduced the effect of SFN on the expression of Cyt c(P< 0.05).The expression of caspase-3 was significantly lower in Licl(GSK-3β specific inhibitor)compared with DACD group and SFN group(P<0.05);5 Rat brain tissue electron microscopy results showed that hippocampal neurons organelles of rats in CON group arranged closely,complete shape,mitochondria and endoplasmic reticulum was clearly visible.Neurons of the DACD group chromatin a large number of edge set,mitochondrial swelling,ridge disappeared,the pulp found more vacuoles.The above-mentioned ultrastructural damage was significantly reduced after SFN intervention,but LY294002 counteracted the effect of sulforaphane.Licl(GSK-3β specific inhibitor)superimposes the effect of SFN.Conclusions: SFN can improve the cognitive function of diabetes mellitus,and its molecular mechanism may be through the activation of PI3 K / AKT / GSK3β signaling pathway to inhibit the opening of MPTP and inhibit mitochondrial apoptosis. |
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