As diabetes mellitus can cause cognitive dysfunction, it has become an important research domain. However, the exact mechanisms of diabetic encephalopathy are yet to be identified. Nevertheless, increasing evidence indicates that oxidative stress is an important mechanism in the development of diabetic cognitive dysfunction. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity are classic indicators in assessing the balance between oxidative and antioxidant systems. Nrf2 is considered to be the main transcription factor of antioxidative stress [
Troxerutin is a flavonol (a type of flavonoid) or, more accurately, a hydroxyethylrutoside. Troxerutin has been reported to possess strong antioxidant and anti-inflammatory properties [
There was no statistical difference in swimming speed between the three groups (Table
The basic swimming speed of the rats in different groups (cm/s,
Group | The swimming speed | |
---|---|---|
NC | 10 | 30.67 ± 2.58 |
DC | 10 | 28.32 ± 1.65△ |
DT | 10 | 30.06 ± 2.58△▲ |
Note: compared to NC,
Repeated measures ANOVA was used to compare the data between each group. The effect of time did not result in significant changes among the groups (
The escape latencies of the rats in different groups in place navigation test (s,
Group | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | |
---|---|---|---|---|---|---|
NC | 10 | 22.08 ± 12.22 | 11.01 ± 4.19 | 7.82 ± 3.84 | 7.52 ± 3.06 | 5.81 ± 2.33 |
DC | 10 | 29.22 ± 14.10△ | 32.56 ± 15.66△ | 28.58 ± 12.21△ | 28.72 ± 3.59 |
24.68 ± 8.02△ |
DT | 10 | 25.94 ± 15.03△▲ | 25.38 ± 16.21△▲ | 16.00 ± 8.33△▲ | 14.72 ± 6.29△▲ | 15.04 ± 10.20△▲ |
Note: compared to NC,
When comparing the number of times the rats crossed to the platform in 60s, the number of the NC group was greater than those of the DC group and DT group. Moreover, the number of the DT group was greater than that of the DC group (
The times of crossing platform in 60s in different groups in spatial probe test (
Group | Times of cross the platform area | |
---|---|---|
NC | 10 | 16.00 ± 1.83 |
DC | 10 | 3.00 ± 0.91△ |
DT | 10 | 13.00 ± 1.83△▲ |
Note: compared to NC,
After observing a series of histopathologic alterations in the hippocampal neurons of the DC group, the number of normal neurons significantly decreased. On the contrary, the majority of the CAl neurons in the DT and NC groups maintained normal morphology, showing integrated structures and being lined up in order. Moreover, there was less neuron depletion in the DT and NC groups compared to the DC group (Figure
Representative photomicrographs of the hippocampal CA1 area showing the histological changes in different groups (HE, ×200). NC: normal control group; DC: diabetic control group; DT: diabetic troxerutin intervention group.
The optical density (OD) value of the Nrf2-positive cells in the hippocampus of rats in the NC group was higher than that of rats in the DC group and the DT group. Compared with the DC group, the OD value of the Nrf2-positive hippocampal cells of rats in the DT group was increased (
The optical density values of Nrf2 in hippocampal CA1 area of each group (IHC, ×200). Results shown are mean ± SD (
When compared with the NC group, mRNA expression of Nrf2 decreased in the DC group and the DT group. Moreover, there was a statistically significant difference between the DC group and the DT group, the Nrf2 mRNA level being higher in the latter (
(a) The expression of total Nrf2 mRNA in different groups. Results shown are mean ± SD (
Total Nrf2 protein: The expression level of total Nrf2 protein in the hippocampus of rats in the DC and the DT groups was lower than that of rats in the NC group. Moreover, the total Nrf2 protein expression of the DT group was significantly higher than that of the DC group (
Nuclear Nrf2 protein: The expression level of nuclear Nrf2 protein in the hippocampus of rats in groups DC and DT was lower than that of rats in the NC group. Furthermore, the expression level of nuclear Nrf2 protein of rats in the DT group was higher than that of rats in the DC group (
SOD activity: The SOD activity of the NC group was higher than that of the DC group. There was no statistical significance between groups NC and DT. However, the SOD activity of the DT group was increased compared with that of the DC group (
(a) The activity of SOD in different groups. Results shown are mean ± SD (
MDA content: The MDA content of the NC group was lower than that of the DC group. There was no statistical significance between the NC group and the DT group. However, the MDA content of the DT group was decreased compared with that of the DC group (
Modeling is crucial during an experiment, because if an animal model fails, the experimental results will not be reliable.
To successfully develop the rat model of type 1 diabetes mellitus, rats with blood glucose level ≥ 16.7 mmol/L at 72 h after an STZ injection (60 mg/kg) were included in the model.
Rats with blood glucose level < 16.7 mmol/L were rejected once a week [
Troxerutin is 3-hydroxy-rutin and soluble in water [
Furthermore, the dose administered to the rats was equivalent to that previously administered to mice, and the dose administered intraperitoneally was 0.3 to 0.5 times greater than the dose administered intragastrically.
Therefore, in this experiment, rats in the DT group were injected with 60 mg/kg/d (i.e., 150 × 0.4 mg/kg/d) of troxerutin.
In the present study, the Morris water maze test was proven to be a credible test for cognitive dysfunction related to hippocampal synaptic plasticity [
In this experiment, hippocampus tissues were stained with HE, and CA1 hippocampal nerve cells of rats in the NC group were eumorphic, structurally integral, and ordered; cells in the DC group were disordered, with shrunken cell bodies and red cytoplasm, and the number of nerve cells with normal morphology was significantly reduced; cells in the DT group were arranged in order, structurally integrated. These results suggest that the hippocampal damage in the DC group is morphological, which affects the cognitive function. The degree of hippocampal impairment in the DT group was lower than that in the DC group; thus, the preventive use of troxerutin could alleviate damage to the hippocampus and reduce cognitive impairment, to a certain extent.
In this experiment, the blood glucose level of rats in the DT group was not significantly different from that of rats in the DC group. This illustrates that troxerutin cannot decrease blood glucose level, which agrees with the results of Geetha et al., who tested 36 rats weighing 25~30 g by administrating a high-fat, high-carbohydrate diet. The results suggested that troxerutin reduces body weight, blood pressure, and insulin resistance [
All procedures involving animals were in accordance with the
Rats in the DT group were intraperitoneally injected with troxerutin (60 mg/kg, 1 mL/kg, Jingchun Biochemistry Technology Corporation, Shanghai, China), while those in the DC and NC groups were intraperitoneally injected with physiological saline once daily for 12 weeks. Finally, the blood glucose levels were measured after 12 weeks of troxerutin treatment.
The rats were tested in the Morris water maze at the end of the treatment period (12 weeks). Basic swimming speed of the rats was assessed the day prior to the test. Rats were pretested to determine their treadmill running willingness and those rats which refused to run were excluded from the water maze experiments. The maze consisted of a circular pool (150 cm in diameter and 60 cm in height) that contained a hidden platform. The apparatus was used to assess the learning and memory performance of rats. Rats were trained to locate the hidden platform using only distal extra maze cues.
For the navigation test, the tracks of rats were recorded and analyzed by using a camera connected to a video recorder and the EthoVision tracking system (Zhenghua Bio-instruments Co. Ltd., Huaibei, Anhui). Each rat was allowed to search for the platform for 60 seconds, and rest on the platform for 30 seconds once found. Then, the rats were placed back in their respective cages. Rats that failed to find the platform within 60 seconds were manually guided to the platform and placed on the platform for 30 seconds before being returned to their cages. Each rat received four learning trials in the four quadrants (N, S, E, and W) per day for five consecutive days. The times spent searching for the platform and the mounting latency were calculated.
On the sixth day, the rats were given the probe test, in which the platform was removed from the pool. Rats were randomly placed into a quadrant and were allowed to swim freely for 60 s. The number of times the rats swarm across the platform in 60 s was recorded [
On the seventh day, the rats were sacrificed, and their left and right hippocampus was removed promptly. The right side of the hippocampus was used for hematoxylin and eosin (HE) staining and immunohistochemistry. The left side of the hippocampus was transferred to a 4°C ice-cold board for dissection and then used to measure SOD activity and MDA content and to detect the mRNA (reverse transcription polymerase chain reaction, RT-PCR) and protein (Western blot analysis) levels of Nrf2 in both cytoplasm and nucleus.
The syringe containing the sample was kept vertically and still for 10 to 20 minutes, with the needle end pointing downward. The glass slides were cleaned, disinfected with 75% ethanol, and then dried. One droplet of test solution was added to the glass slide and dried at room temperature. The sample was then fixed in methanol solution for 30 minutes. The methanol solution was removed, and the sample was hydrated with distilled water and subjected to conventional HE staining.
Sections were dehydrated in xylene and graded alcohol series. The sections were then rinsed for 5 minutes with tap water and incubated for 30 minutes in 0.3% H2O2 in methanol. After, they were washed for 5 minutes with buffer and incubated for 20 minutes in diluted normal serum from the species in which the secondary antibody was obtained. Excess serum from sections was blotted. Then, the sections were incubated for 30 minutes in primary antiserum diluted with buffer. Slides were subsequently washed for 5 minutes with buffer. Sections were incubated for 30 minutes in diluted biotinylated secondary antibody solution, followed by a 5-minute washing step in buffer. Sections were then incubated for 30 minutes in VECTASTAIN ABC Reagent and washed for another 5 minutes with buffer. Next, sections were incubated in peroxidase substrate solution until the stain developed to the desired intensity. Lastly, sections were rinsed with tap water, counterstained, cleared, and mounted for observation.
Four rats from each group were randomly selected. Total RNA from the hippocampus was isolated using Trizol (Invitrogen Co.), according to the manufacturer’s instructions. Reverse transcription was performed using the DEPC (USA Co.). The expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard. PCR was performed for 30 cycles in an Eppendorf Mastercycler. Denaturing, annealing, and extension reactions were performed at 94°C for 30 minutes, 60°C for 30 seconds, and 72°C for 1 minute, respectively. Then, the PCR products were separated by 1% agarose gel electrophoresis, stained with GoldView, photographed under ultraviolet illumination, compared with a known standard ladder, and quantified by densitometry using the Bio-1D system. The levels of Nrf2 mRNA were expressed as their respective ratios to GAPDH mRNA.
Approximately 500–600 mg of tissue homogenate were added to 1 mL of extraction solution. The mixture was then centrifuged at 12,000 rpm for 5 minutes, at 4°C, and the supernatant was collected. An equal volume of sample buffer was added to the supernatant on
SOD activity is measured by testing the capacity of pyrogallol to autoxidize. The inhibition of autoxidation of this compound occurs when SOD (Jian Cheng Technology Corporation, China) is present, and the enzymatic activity can be assayed indirectly using a temperature-controlled double-beam spectrophotometer at an absorbance of 420 nm. A 50% inhibition of pyrogallol autoxidation is defined as 1 U SOD. For testing MDA (Jian Cheng Technology Corporation, China) levels, the samples were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid and then heated in a boiling water bath for 30 minutes. Malondialdehyde equivalents were detected in both tissue and submitochondrial particles of the rats’ brain using a spectrophotometer at an absorbance of 532 nm.
Data were collected and analyzed by the statistical software package SPSS 19.0, which includes one-factor ANOVA with completely randomized design. Repeated measures ANOVA was used to analyze the results of the Morris water maze test. The method of Bonferroni was used to perform pairwise comparisons of the repeatedly measured data from the different measurement times of each treated group, as previously described [
This research states that all animal experiments conformed to institutional standards.
The authors declare no financial or other conflicts of interest.