| Objective: With the change of lifestyle and the aging population, theincidence of diabetes is rising sharply. Diabetes can lead multiplecomplications, especially the study of cognitive dysfunction in diabetesmellitus has become an important research domain. Oxidative stress is animportant mechanism in the development of diabetic cognitive dysfunction.Nuclear factor E2-related factor2(Nuclear factor E2a related factor2, Nrf2)is the core transcription factor of the anti-oxidative stress. Early preventionand treatment of diabetic cognitive dysfunction can reduce the incidence ofdementia and improve the quality of life of diabetic patients. Therefore, byanalyzing the relationship between Nrf2and cognitive dysfunction of diabeticrats, we intended to explore the evidence of using Troxerutin in the earlypreventive stage.Methods: Forty healthy male (weighing140-160g) Sprague-Dawley (SD)rats were randomly divided into normal control group (NC group, n=10rats),diabetic group (n=30rats). After fasting on diabetic rats overnight, they wereinjected by STZ (60mg/kg) at one time and tested blood glucose on their tailvein after72h. Those whose blood glucose≥16.7mmol/L were deemed as asuccessful model. The diabetic rats which are successful models wererandomly divided into diabetic control group (DC group, n=15) and diabetictroxerutin intervention group (DT group, n=15).After this, DT group were injected Troxerutin (60mg/kg), DC group andNC group were injected with physiological saline once a day, which lasted12weeks.Observe and record the mental state of rats, eating, drinking, urine, etc.In general, test and record the change of blood glucose and body weight. The rats would have the Morris water maze test after12weeks. Theywould have the navigation test to record the escape latency at the first fivedays and the space exploration experiments recording frequency of crossingthe platform at the6th day.At the day after the Morris water maze day, the rats were killed and theirhippocampus were quickly removed to measure SOD activity and MDAcontent. The mRNA level and protein level which conclude total protein andnucleoprotein of Nrf2in hippocampus were detected by Real-time polymerasechain reaction (R-T PCR) and Western blotting. Immunohistochemistrystaining was used to detect the expression level of Nrf2.Data was dealt and analyzed by statistical software packages SPSS19.0,which includes one factor analysis of variance with completely random design.In the results of Morris water maze, we use repeated-measure analysis ofvariance. The method of Bonferroni should be used to do pairwisecomparisons of the repeatedly measured data in different measurement time ofeach treated group. With multivariate ANOVA, data in different treated groupof each measurement time could be compared pairwise. As the data passed thenormality test (P＞0.10), he results were expressed as the mean±standarddeviation. Statistical significance was set at0.05.Results:1General ConditionAfter the diabetic model was established, the rats of DC group appearedpolyuria, polydipsia, polyphagia and the blood glucoses maintained at highlevels (＞20.0mmol/L). Then, with the progression of the experiment, thediabetic rats gradually appeared gloomy sparse fur and dull, weight lose, slowin response and so on. While the NC group rats were in good shape with purewhite and glossy fur and gain steady growth. After intervention, DT groupappeared faster reaction ability than DC group, but still had high levels ofblood glucoses (＞20.0mmol/L), weight lose, which had no significantdifference with DC group.2Results of Morris water maze Basic swimming speed: There was no statistical difference in swimmingspeed between each group.Navigation test: The escape latency of DC group was prolongedcompared to NC group (P<0.01). The result showed that diabetic rats hadcognitive dysfunction after having been mold for12weeks. DT group wasshorter than DC group in the escape latency (P<0.01). Pretreatment oftroxerutin for12weeks could reduce cognitive dysfunction.There was no significant difference of escape latency in the NC, DC andDT group at the first day (P>0.05); Compared with the NC group, the escapelatency of DC rats were prolonged during the last4days (P<0.01); In thesecond day, the escape latency of the DC group and DT was not significantlydifferent (P>0.05); At the third to the fifith days, the escape latency of DTgroup was significantly shorter than the DC group (P<0.01).The escape latency variations from the fifth day to the first day: NCgroup was significantly shorter (P <0.01); DC group, no significant changes inthe escape latency (P>0.05); DT group was shorter (P<0.05). This resultsuggest that pretreatment of troxerutin for12weeks could reduce cognitivedysfunction.Probe test: For the number in60s of crossing platform, NC group weremore than DC group and DT group (P<0.01) and DT group were more thanDC group (P<0.01).3Results of HE stainingAfter observing a series of histopathologic alterations in hippocampusneurons of DC group, the number of normal neurons was appearingsignificantly decreasing. On the contrary, the majority of the CAl neurons inDT and NC group remained normal morpholog and neurons showedintegrated structures and lined up in order. Moreover there were also lessneurons depletion．4Results of Immunohistochemistry stainingThe OD value of the positive cell of Nrf2in hippocampus of rats in groupNC was higher than that in the DC group and the DT group (P＜0.01). Compared with the DC group, the The OD value of the positive cell ofhippocampal Nrf2in the rats of group DT was increased (P＜0.01).5Results of Rt-PCRThe expression of hippocampal Nrf2mRNA in the rats of group DC andDT were decreased comparing to the NC group (P＜0.01). There wasstatistical significance (P＜0.01) between group DC and DT, compared withDC group, The expression of hippocampal Nrf2mRNA in the rats of DT groupwas increased.6Results of Western blotTotal Nrf2protein: The expression of total Nrf2protein in hippocampusof rats in group DC and DT were lower than that in the NC group (P＜0.01).There was statistical significance (P＜0.01) between DC group and DT group.The total Nrf2protein expression of DT group were increased compared withDC group (P＜0.01).Nuclear Nrf2protein: The expression of nuclear Nrf2protein inhippocampus of rats in group DC and DT were lower than that in the NCgroup (P＜0.01). There was statistical significance (P＜0.01) between groupDC and DT. The expression of nuclear Nrf2protein of the rats in DT groupwere higher than that in DC group (P＜0.01).7SOD activity and MDA contentSOD activity: The SOD activity of NC group was higher than DC group(P＜0.05). There was no statistical significance (P＞0.05) between group NCand DT. The SOD activity of DT group were enhanced compared with DCgroup (P＜0.05).MDA content: The MDA content of NC group appeared lower than DCgroup (P＜0.01). There was no statistical significance (P＞0.05) betweengroup NC and DT. The MDA content of DT group were decreased comparedwith DC group (P＜0.01).Conclusion:1STZ-induced diabetic rats with cognitive dysfunction had lower SODactivity, higher MDA content and less expression of Nrf2in rat’s hippocampus.2Prophylactic using troxerutin could upgrade the expression of Nrf2inSTZ-induced diabetic rats and improve cognitive impairment in diabetic rats.It also could delay the occurrence of cognitive dysfunction in diabetic rats. |
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