Fish oil supplementation attenuates cognitive impairment by inhibiting neuroinflammation in STZ-induced diabetic rats

FO suppressed diabetes-induced behavioral deficit

The MWM test was carried out to assess whether FO improved spatial learning and memory ability in DM rats. During the five day test, there was no obvious difference in swimming speed between the groups (Figure 1B). In the orientation navigation test, rats in the DM group spent more time finding the hidden platform than the rats in the CON group on day 90, 91 and 92 (Figure 1A); FO administration reduced the escape latency of DM rats on day 90, 91 and 92 (Figure 1A). In the probe test, as shown in Figure 1C, the number of crossings in DM group was much less than that in the CON group; after FO treatment, we observed a significant improvement in the DM+FO group. The DM group, spent significantly less time in the target quadrant than the CON group; after FO treatment, rats spent markedly more time in the target quadrant than did rats in the DM group (Figure 1D).

Figure 1. The spatial learning and memory ability of rats in different groups. (A) Escape latency in navigation training. (B) Swimming speed in the probe test. (C) The number of platform quadrant crossings. (D) Time spent in the target quadrant. Data were analyzed using repeated-measures ANOVA with post hoc Bonferroni tests. Data are shown as the mean ± SEM (n=7). ^*P<0.05, ^{* *}P<0.01 and ^***P<0.001 vs. the CON group; ^# P<0.05, ^##P<0.01 and ^###P<0.001 vs. the DM group.

FO changed morphology of the cortex and hippocampus

H&E staining was used to observe the change in morphology in the cortex and hippocampus under an optical microscope. Few abnormal neurons were observed in the CON and CON+FO groups. In the DM group, the neurons were abnormally and disorderly arranged, the staining was clouded and the number of neurons was decreased. FO treatment alleviated the abovementioned abnormal morphological changes (Figure 2).

Figure 2. Morphological changes in cortical and hippocampal neurons. (A) Morphological changes in cortical nerve cells in rats from different groups, CON, CON+FO, DM and DM+FO; (B) Morphological changes of hippocampal neurons in rats from different groups, CON, CON+FO, DM and DM+FO. (n=8).

Effects of FO on neuroinflammation

We used an ELISA kit to determine whether DM could stimulate a neuroinflammatory response. As hypothesized, in the brain region of the cortex, the levels of IL-1β, IL-6, and TNF-α were much higher in the DM group than in the CON group, while the levels of IL-4 and IL-10 were lower in the DM group than in the CON group (Figure 3C). FO supplementation suppressed pro-inflammatory cytokine levels (IL-1β, IL-6, and TNF-α) and enhanced anti-inflammatory cytokine levels (IL-4 and IL-10) in the cortex (Figure 3C). In the hippocampal region, IL-1β, IL-6, and TNF-α levels were increased, and IL-4 and IL-10 levels were decreased in the DM group compared with the CON group (Figure 3D). FO supplementation inhibited the increase in pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and the decrease in anti-inflammatory cytokines (IL-4 and IL-10) in hippocampus (Figure 3D).

Figure 3. Proinflammatory cytokine in different groups. (A) Expression of mIL-1β, mIL-6, mTNF-α, mIL-4 and mIL-10 in the cortex. (B) Expression of mIL-1β, mIL-6, mTNF-α, mIL-4 and mIL-10 in the hippocampus. (C) Content of IL-1β, IL-6, TNF-α, IL-4 and IL-10in the cortex. (D) Content of IL-1β, IL-6, TNF-α, IL-4 and IL-10 in the hippocampus. Data are shown as the mean ± SEM (n=7) ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the CON group; ^# P<0.05, ^##P<0.01 and ^###P<0.001 vs. the DM group.

To further understand the effect of fish oil on inflammatory cytokines, we measured the mRNA expression of the five inflammatory cytokines in cortex and hippocampus, using RT-PCR. The findings showed that the transcription levels of the pro-inflammatory cytokines IL-1β and TNF-α were obviously increased, and IL-4 and IL-10 were significantly decreased in the cortex (Figure 3A), whereas FO administration markedly inhibited the diabetes-induced upregulation of IL-1β and TNF-α and downregulation of IL-4 and IL-10 (Figure 3A). Rats in the DM group had clearly higher expression of IL-1β and TNF-α and lower expression of IL-4 and IL-10 than rats in the CON group. The rats that received FO had obviously lower expression of IL-1β and TNF-α and higher expression of IL-4 and IL-10 than the rats in the DM group (Figure 3B). However, the mean expression of IL-6 in the cortex and the mean expression of TNF-α in the hippocampus were not significantly different among the groups.

Effects of FO on oxidative stress

To identify the mechanisms underlying the neuroprotective effect of FO, we also measured the levels of ROS and oxidative stress markers including MDA, CAT, SOD and GSH in the cortex and hippocampus. ROS and MDA were dramatically increased, and SOD and GSH were dramatically decreased in the cortex and hippocampus of rats in the DM group compared to the CON group (Figure 4A, 4B, 4D, 4E), CAT was only observably decreased in the hippocampus of rats in the DM group compared with that of the CON group (Figure 4C). Treatment with FO reduced ROS and MDA levels both in the cortex and hippocampus (Figure 4A, 4B), increased the GSH level in the cortex and hippocampus (Figure 4E) and enhanced CAT in hippocampus and SOD activities (Figure 4C, 4D) in the cortex and hippocampus of the rats in DM group.

Figure 4. Oxidative stress measurements in different groups. (A) ROS levels in the cortex and hippocampus. (B) MDA level in the cortex and hippocampus. (C) CAT activities in the cortex and hippocampus. (D) SOD activities in the cortex and hippocampus. (E) GSH levels in the cortex and hippocampus. Data are shown as the mean ± SEM (n=7) ^*P<0.05, ^{* *}P<0.01 and ^{* * *}P<0.001 vs. the CON group; ^# P<0.05, ^##P<0.01 and ^###P<0.001 vs. the DM group.

Effects of FO on protein expression of Nrf2 and HO-1

To further reveal the mechanism underlying the neuroprotective effect of FO, Nrf2 and HO-1 protein expression were measured by Western blot analysis. As shown in Figure 5, the protein expression of Nrf2 and HO-1 was decreased in the rats with diabetes compared to the rats in the CON group. FO treatment increased the protein expression of Nrf2 and HO-1 in both cortex and hippocampus.

Figure 5. Effects of FO on the expression of Nrf2 and HO-1. (A) Expression of Nrf2 and HO-1 in the cortex. (B) Expression of Nrf2 and HO-1 in the hippocampus. Data are shown as the mean ± SEM (n=7) ^*P<0.05, ^{* *}P<0.01 and ^{* * *}P<0.001 vs. the CON group; ^# P<0.05, ^##P<0.01 and ^###P< 0.001 vs. the DM group.

Treatment

Diabetes was established with a high-fat diet (10% cholesterol 10 ml/kg) for two weeks. After overnight fasting, rats were injected intraperitoneally with 40 mg/kg streptozotocin (STZ, Sigma, USA) [26]. Control rats received a citrate buffer injection. Before high-fat diet consumption, all the animals were divided into four groups. The animals in the control group (CON group, n=15) and diabetes mellitus group (DM group, n=15) were fed standard rat feed. The animals in the control +FO group (CON+FO group, n=15) and diabetes mellitus + FO group (DM+FO group, n=15) were fed a standard containing 4% FO (1.5 g/kg/d, 34% EPA, 24% DHA, Sheng Tianyu Biotechnology, China). The chemical structure of EPA and DHA are shown in Figure 6. Seventy-two hours after STZ administration, rats with a blood glucose level more than 16.7mmol were identified as DM models. A timeline of the test procedures is shown in Figure 7.

Figure 6. The structure of EPA and DHA.

Figure 7. Timeline of the experimental procedures.

Morris water maze test

Ten weeks after FO treatment, the learning and memory abilities of the rats were assessed using the MWM [27]. On day 89, all the animals were subjected to trainings trials that lasted 4 days. Rats were trained to find hidden platform submerged 2 cm below the water surface. If the rats failed to find the platform in 60 s, they were gently placed onto the platform and allowed to stay for 10 s. The time spent finding and climbing onto the platform was recorded as the escape latency. On day 93, the hidden platform was removed, and a probe trial test was conducted to evaluate the spatial memory of the rats, which were given 60 s to swim freely in the pool. The time the rats spent in the target quadrant, the number of times the rats crossed the platform, and the swimming speed was recorded.

Histopathological examinations

After the MWM test, morphological alterations in the cortex and hippocampus (CA1) were analyzed by hematoxylin and eosin (H&E). Rats were anesthetized with sodium pentobarbital (60 mg/kg) and the head was cut off to remove the brain tissue in an ice tray. The brain tissue was fixed by perfusing 200–300 ml of 4% paraformaldehyde. The tissue was collected, postfixed, and embedded in paraffin. The paraffin tissue blocks were cut into 5-mm-thick sagittal sections for H&E staining. Cortex and hippocampal cells were counted by light microscopy.

Quantitative real-time PCR analysis

The transcription levels of IL-1β, IL-6 and TNF-α, IL-4 and IL-10 were measured using quantitative real-time PCR. Cortex and hippocampal tissues were prepared to extract total RNA using RNA-Solv Reagent (Omega). Reverse transcription was conducted with 2 μg of RNA using ReverTra Ace (Toyobo) and Oligo(dT)18(TaKaRa). qRT-PCR was performed on SYBR Premix Ex Taq (TaKaRa) using a ViiA 7 RealTime PCR System (Thermo). All procedures were performed following the manufacturer’s instructions. The primers were shown as the following: IL-1β fwd 5′-AGGTCGTCATCATCCCACGAG-3′, rev 5′-GCTGTGGCAGCTACCTATGTCTTG-3′; IL-6 fwd 5′-CACTCCGGAGAGGAGACAAG-3′, rev 5′-ACAGTGCATCATCGCTGTTC-3′; TNF-α fwd 5′-GAGAGATTGGCTGCTGGAAC-3′, rev 5′-TGGAGACCATGATGACCGTA-3′; IL-4 fwd 5′-CTCAGGACGGTAAGTGTCCCTG-3′, rev 5′-GTTCAGGCGCGCATGTTGTCG-3′; IL-10 fwd 5′-GGCTTCCTTACTCCGCGTTGG-3′, rev 5′-TCCTAGAACTGGAACCACGGCT-3′; β-actin fwd 5′-CAGCCGCGAGATTACCTACAA-3′, rev 5′-CTTTGCATGCCTCCGCTCCGT-3′.

Measurement of ROS and oxidative stress

Tissue from the cortex and hippocampus were homogenized and prepared for cytokine assays. The levels of ROS and MDA, and the activities of CAT, SOD and GSH were evaluated using ROS assay kit, lipid peroxidation (MDA) assay kit, CAT assay kit, SOD assay kit and GSH assay kit (Nanjing Jiancheng Bioengineering Institute, China), respectively. All the procedures followed the manufacturer’s instructions.

Western blot analysis

Total protein was extracted from cortex and hippocampus tissues. BCA kit was used to determine the concentration of the total protein. Equal quantities of protein (20 mg/lane) were loaded on a 10% SDS-PAGE gel and transferred to a PVDF membrane (200 mA, 2 h). The membranes were blocked for 2h in 5% skim milk at 37°C, and then incubated at 4°C overnight with primary antibodies, against Nrf2 (1:1000, ab31163, Abcam, USA), HO-1(1:500, ab13243, Abcam, USA) and β-actin (1:4000, Proteintech, USA). The membranes were incubated with secondary antibodies for 40 min. The bands were analyzed using Image J software.

Data analysis

Data analysis was conducted using SPSS 25.0 (IBM software) and Sigma Plot10 (Sigma Plot software). The data are shown as the means ± SEM. Bonferroni means separation test was performed after ANOVA. Results were only reported when significant differences (P<0.05, P<0.01 or P<0.001) were observed.

Ethics statement

The rat experiments were approved by the Animal Ethics Welfare Committee of North China University of Science and Technology.