3.1 DORA significantly prolonged sleep duration with little residual effect
After drug injection at 600 a.m., the mice in the DORA group fell asleep faster and fell asleep at 60 mg > TGN + 30 > 30 mg > 10 mg > TGN > CON group. The total sleep time of 24 hours in the 10mg, 30mg and 60mg groups was 12 hours 6 minutes 14 seconds, 14 hours 27 minutes 40 seconds and 16 hours 57 minutes 40 seconds, respectively, which were significantly different (P < 0.001) from those in the control group, while DORA prolonged sleep time significantly improving sleep duration.
During the next 12 h after injection, the mice in each group had a large difference in sleep duration and exhibited a significant dose-response graph. In the following 12h, except for the 60mg group of mice sleeping up to 29 min 5 s at 4 h, the other groups of mice sleeping for less than 4 h and the differences were less pronounced, as shown in Fig. At 3-6 am the following day, which was the active phase of the mice, the camera observed that the activity, sensitivity, and alertness of mice in each group injected with DORA were almost different from those of the control group. This seems to indicate that DORA, this hypnotic drug, effectively promotes sleep and does not produce the day-to-day activities of the following day.
3.2 DORA and TGN have little effect on body weight, food intake, and drinking water in mice
After 28 days, the body mass of mice in each group decreased, and the TGN + 30 group (2.65 ± 1.27) and the TGN group (1.89 ± 2.62) showed the greatest decrease compared with the control group (0.95 ± 0.82), and the results were statistically significant; (P = 0.074 > 0.05) in the TGN group. The body weights of mice in the 10 mg / kg, 30 mg / kg, and 60 mg / kg groups also decreased, with changes of 0.93 ± 2.66, 0.87 ± 2.75, and 1.21 ± 1.78, respectively, and different doses of DORA and TGN did not significantly affect the body weights of mice. The least amount of food intake was 4.9 ± 0.61 in the 60 mg / kg group and 7.5 ± 0.66 and 7.2 ± 0.79 in the 10 mg / kg and 30 mg / kg groups, respectively. The group with the least amount of food intake was 8 ± 0.63.60 mg / kg, P < 0.01, which was statistically significant compared with the control group. Mice in the 60 mg / kg group and the TGN + 30 group had decreased water intake, 12.0 ± 1.38 and 10.8 ± 1.27, respectively. The water intake of mice in the TGN group was also reduced to 10.8 ± 1.27. The water intake of mice in the 10 mg / kg and 30 mg / kg groups was 17.5 ± 1.41 and 18.1 ± 1.61, respectively. The differences between the 10 mg / kg group (P = 0.3 > 0.05) and the 30 mg / kg control group (P = 0.646 > 0.01) were not significant. 60mg mice and tgn-020 + 30 mice appeared to have anorexia and decreased water intake, possibly related to their increased amount of sleep and decreased activity time as shown in figure1,2,3.
3.3 DORA and TGN are well tolerated in mice with fewer adverse effects
In addition to the delayed response rate of 6%, other adverse reactions such as convulsions, hematochezia, and paralysis occurred in 1.5%.
Around 5 s after the completion of the injection for the 60 mg / kg group on day 7, the mice experienced sudden loss of muscle tone, sudden collapse of the head drooping body toward the right side, visible limb curvature, and ophthalmoplegia. One hematochezia mouse appeared in the control group on day 22 Paralysis the mice were hemiplegic with decreased limb muscle strength, crawling activity but insufficient muscle strength to support the mice to stand. The mortality rate of mice in this experiment was 7.9%, after dissection, two mice died from intestinal obstruction due to improper injection manipulation such that the small intestine adheres and the intestinal contents pass through the obstruction. The remaining three mice likely died for reasons such as intolerance to the drug, startle, bacterial viral infection, etc.
3.4 Y-maze treatment with DORA ameliorates working memory impairment in APP / PS1 mice
The Y-maze test reflects the animals' short-term spatial working memory and reference memory. The time ratio of the novel arm recognition experiment the time ratio of the mice in the 10 mg / kg and 30 mg / kg groups to enter the novel arm was 0.48 ± 0.30 and 0.42 ± 0.31, respectively. * P < 0.05, compared with the con group. 0.17 ± 0.07 and 0.20 ± 0.15 in the TGN group and TGN + 30 group, respectively. Mice in the 10 mg / kg and 30 mg / kg groups took significantly longer to enter the novel arm and were more inclined to explore the novel arm; The number of novel arm recognition experiments was lower than that mice in the 60 mg / kg group entered the novel arm, and the mice showed decreased exploration, 0.25 ± 0.07; Spontaneous alternation experiments compared with the control group, the percentage of free alternation experiments was decreased in mice of the TGN group and increased in mice of the TGN + 30 group. The experimental percentages of free alternation in the TGN and TGN + 30 groups were 0.31 ± 0.10 and 0.35 ± 0.16, respectively, P = 0.601 > 0.05 in the TGN group and P = 0.899 > 0.05 in the TGN + 30 group, as shown in Fig. 4.
3.5 Morris water maze treatment with DORA rescues spatial learning and memory deficits in APP / PS1 mice
MWM can test the ability of mice hippocampus dependent long-term spatial learning and memory[17], in the water maze test, there was no statistical difference in the total distance traveled by the mice in each group, the speed of locomotion showed that there was no significant difference in the activity of mice in each group, indicating that drug injection did not obviously affect the body strength of mice. The percentage of the target quadrant in the 60 mg / kg group, TGN group, and TGN + 30 group was 0.17 ± 0.53, 0.181 ± 0.04, and 0.183 ± 0.43, respectively. The P values were p = 0.517 > 0.05, P = 0.455 > 0.01, which were not statistically significant. In the spatial exploration experiment, mice in the 10 mg / kg and 30 mg / kg groups could cross the platform several times and actively attempted to find the platform in the target quadrant; However, mice in the 60 mg / kg group were unable to accurately recall and find the original platform location, showed a marginal trajectory of the platform, and the number of platform crossings was significantly reduced; Compared with the control group, mice in the 30 mg / kg group were able to find the original platform location, explore less, and show increased length of stay in the target quadrant.
In the directional navigation experiment, the escape latencies of mice in each group decreased as the training time increased, and the differences between the 30 mg and 60 mg groups were more significant. On the third day, the escape latencies of mice in the 10 mg / kg and 30 mg / kg groups were 29.02 ± 9.02 and 24.95 ± 6.18, P < 0.01, respectively. The escape latencies of mice in the 60 mg / kg, TGN, and TGN + 30 groups were 53.59 ± 6.77, P = 0.512 > 0.05; 40.03±5.93,P=0.051>0.05; 43.72±5.77,P=0.232>0.05。 There was no statistical difference between the two groups. On day 4, the values in the 30 mg / kg, 60 mg / kg, and TGN groups were 16.74 ± 4.84, 44.96 ± 6.03, and 36.03 ± 6.60, respectively, compared with the control group (20.03 ± 5.09), all P values < 0.01, with statistically significant differences. The other two groups (10 mg / kg and TGN + 30 groups) were 18.41 ± 7.36 and 34.98 ± 7.86, respectively. P = 0.243 and P = 0.721, respectively, without significant differences, as shown in Figure4,5.
3.6 Application of DORA reduces aβ deposition, the TGN can increase a β deposition
A β Plaque deposition is an important step in the pathological process of AD, and in this experiment, immunohistochemical staining was used to quantify a β with a high propensity to aggregate β 42 plaque. A) in the hippocampus of 10 mg / kg and 30 mg / kg mice (Fig aβ 42 plaque deposition rates were 13.51 ± 0.55 and 11.03 ± 0.52, respectively. Compared to controls, a β 42 compared with plaque deposition (15.38 ± 0.57), P = 0.053 > 0.05, not statistically significant. 30 mg / kg group (P < 0.01). Versus control (15.38 ± 0.57, P < 0.01), a β 42 increased plaque deposition (18.39 ± 0.52); In the hippocampus of TGN and TGN + 30 mice aβ 42 increased plaque deposition, 19.71 ± 0.58 and 16.32 ± 0.50, respectively. Immunohistochemistry showed that 10 mg / kg and 30 mg / kg DORA reduced deposition of a β 42, whereas TGN increases a β 42 deposition, as shown in Figure 6,7,8,9.