An interventional study of baicalin on neuronal pentraxin-1, neuronal pentraxin-2, and C-reactive protein in Alzheimer’s disease rat model

2.1 Experimental drug

Baicalin (purity >99%) was supplied by Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Amyloid β-protein, Fragment1-40 (Aβ1-40) (Abcam, St Louis, MO, USA) was attenuated to 5 g/L with sterile double-distilled water, and then incubated in an incubator (37°C) for 7 days. After that, Aβ1-40 was stored at −20°C.

2.2 Animals and AD rats model preparation

A total of 30 healthy Sprague Dawley (SD) male rats (weighing 250–300 g) were obtained from the Animal Center of First Hospital affiliated to Harbin Medical University (Harbin, China). The SD rats were raised under the barrier system of room temperature and humidity (40–50%), and experimental conditions were kept according to the SPF standard.

The 30 SD rats were randomly classified into the control group, the AD model group, and the AD + baicalin group (n = 10 per group) for anesthesia without signs of peritonitis. Rat brains were fixed on a stereoscopic instrument, and the hair and scalp were removed to make the cranium expose. The hippocampus was injected unilaterally with 2 μL (10 µg) Aβ1-40 solution using a micro-syringe in 5 min. The control group was treated with an equal volume of sterile normal saline. After surgery, dental clay was used to seal the holes and the scalp was sutured and treated with a disinfectant to avoid infection [10]. The rats were kept in a single cage until fully awake. After obtaining the AD model, the AD + baicalin group was given 10 mg/kg of the drug via gavage for 30 days. The control group and the AD group were given an equal volume of saline via gavage.

2.3 MWM

The MWM (sansbio, China), was first applied by Morris (1984) to assess memory function and spatial learning which normally used visual cues [15]. We used the MWM to perform the place navigation test as follows. The rats were first placed into the water in southeast, northeast, northwest, and southwest, randomly [16]. The experimental device could track the movement of the rats in the water through the video system. The time from initial entry into the water to their first arrival at the platform was recorded as the escape latency. If the rats did not find the platform within 90 s, the rats were guided to the platform to rest for 15 s and the incubation period was recorded as 90 s. After 60 s, we proceeded to the next test. The rats were tested four times a day for 6 days, and the escape incubation period was recorded in detail using the video system.

The spatial probe test was used to detect changes within the spatial memory of the rats. This test was conducted on the second day after the place navigation test, during which time the platform was removed. The rats were placed into the water at a random entry point within the quadrant corresponding to the platform. The rats were allowed to swim freely in the water for 90 s. Then, we recorded the time that rats stayed in the quadrant corresponding to the platform.

2.4 Tissue preparation and hematoxylin–eosin (HE) staining

Chloral hydrate was used to anesthetize animals at a dose of 300 mg/kg. After fixation, the brains were removed, and then the rats immediately were euthanized with an injection of sodium pentobarbital at a dose of 150 mg. When the rat’s breathing stops and the heart stops beating, it is determined to be dead. The brain was dehydrated by gradient alcohol, and embedded in paraffin for immunohistochemical analysis, HE staining (HE, Wanleibio, China), and immunofluorescence. The paraffin-embedded tissue was cut into 3 µm-thick sections and paraffin was removed by gradient ethanol. Brain sections were stained with hematoxylin for 1–2 min and eosin for 30–60 s. After that, the sections were dehydrated with ethanol, sealed with neutral gum, and cleared with xylene [17]. After finding the hippocampus area under the low power microscope, we randomly selected five high power fields and counted the number of neurons in each high power field with Image J.

2.5 Immunohistochemistry and immunofluorescent staining

Sections of paraffin-embedded brain slices were deparaffinized and rehydrated before blocking non-specific antigen binding with 5% bovine serum albumin (Boster Biological Technology, China). The following primary antibodies were used: NPTX-1 (1:50; Abcam, Cambridge, UK), NPTX-2 (1:200; Abcam, Cambridge, UK), and CRP (1:100; Abcam, Cambridge, UK). Goat anti-rat/rabbit (Beijing Zhongshan Jinqiao Biotechnology, China) polymer enhancer and goat-resistant rabbit/rat IgG-conjugated polymer were added to each tissue section. Afterward, the immunostained sections were developed in diaminobenzidine (Beijing Zhongshan Jinqiao Biotechnology, China) and counterstained with a weak solution of hematoxylin solution. The stained tissue slices were visualized on a microscope (DMI6000B, Leica, USA).

The immunofluorescence staining protocol on the first day was the same as the immunohistochemical staining protocol, which was performed to evaluate the localization of NPTX-1, NPTX-2, and CRP. The luciferin antibody (100 µL) was added to each stained section. After incubating the sections in the dark at room temperature for 1 h, a drop of 4′,6-diamindino-2-phenylindole (Beijing Zhongshan Jinqiao Biotechnology, China) staining agent was added to each tissue section. An anti-quenching agent was added to seal the sections. The staining results were observed under a fluorescence microscope.

2.6 Western blot

The radioimmunoprecipitation assay buffer was used to isolate the total protein, and then it was quantified using bicinchoninic acid assay (Thermo Invitrogen, Waltham, MA). The total protein (20 μg) was added into every well, and then transferred onto polyvinylidene fluoride membrane (IPVH00010, Millipore, USA). The primary antibody including Bax (1:1,000, Proteintech), caspase-3 (1:1,000, Proteintech), and actin (1:1,000, Proteintech) were incubated with membrane at 4°C overnight. Secondary goat anti-rabbit antibody (1:5,000, Jackson ImmunoResearch, USA) and goat anti-mouse IgG (1:5,000, Jackson ImmunoResearch, USA) were incubated with membrane at room temperature for 2 h. The protein bands were visualized and the images were analyzed through chemiluminescence meter (4600, Tanon, Shanghai, China).

2.7 Statistical analysis

The experimental data positioning navigation and space exploration time from MWM were recorded and analyzed using one-way ANOVA method in SPSS software 22.0 (SPSS Inc., Chicago, IL, USA). The data were exhibited as mean ± standard deviation. Then, the immunohistochemical staining results of NPTX-1, NPTX-2, and CRP were semi-quantitatively analyzed, and ten fields of view in and around the hippocampus were selected for high-magnification imaging through an optical microscope, and the integrated optical density (IOD) value of each image was measured using ImageJ digital software. The average value of the 10 IOD values obtained for each slice represents the average IOD value of the slice. The IOD values obtained from the brain slices of mice in different groups are expressed as mean ± SD. Meanwhile, the immunofluorescence results to evaluate the gene localization were measured using ImageJ digital software. The formula was as follows: average fluorescence intensity (Mean) = sum of fluorescence intensity in this area (IntDen)/area of this area (Area). The difference between the groups was analyzed using one-way analysis of variance. A P value <0.05 was deemed statistically significant.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. Approval was obtained from the Ethics Committee of The First Hospital of Harbin Medical University (Approval No. SYXK (black) 2017-003) and conducted in strict accordance to the standard of the Guide for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People’s Republic of China in 2006.

3.1 MWM test

The MWM test was used to assess the memory and spatial learning of rats. It is on the sixth day that the memory and spatial learning of rats was improved in AD + baicalin group when compared with AD group (Figure 1a–c). In addition, as shown in Table 1, with the extension of training days, the time required by different groups of rats to find the platform showed gradually different travel characteristics. On the sixth day, the escape latencies of the AD + baicalin group rats were significantly shorter than those from rats in the AD group (Figure 1d). In addition, we used a space exploration experiment to investigate whether the spatial memory of rats changed. We observed and recorded the time the rats spend in the target quadrant of the platform as well as the number of times the animals crossed the platform. As indicated in Figure 1 and f, baicalin treatment could significantly improve the number of times the animals crossed the platform and time spent in the target quadrant of the platform (Tables 2 and 3). These results indicated that baicalin could improve the memory and spatial learning of AD rats.

Figure 1

The swimming paths of the MWM test of each group: (a) control group, (b) AD group, (c) AD + baicalin group, (d) escape latencies on the sixth day, (e) time spent in the target quadrant, and (f) number of passes across the platform. Abbreviation: AD, Alzheimer’s disease.

3.2 Effect of baicalin on neuronal cell numbers of AD rats

In the controls, no obvious cell deletion of the pyramidal cells was found. The morphology of the cells was complete, the nucleus was clear, and the cells were round or quasi-round (Figure 2a). However, in the AD group, the cell structures were observed to be disordered, the pyramidal cells were anomalous, and the cell membranes were narrowing. The nuclei of the damaged cells showed pyknosis, the nuclear membranes were not clear, and the nucleoli had disappeared (Figure 2b). The arrangement and morphology of cells in the AD + baicalin group was improved compared with the AD group (Figure 2c). The average number of hippocampal neurons in the AD group was significantly lower than that of the control group (2.896 × 10⁴ cell/mm³ vs 4.132 × 10⁴ cell/mm³, P < 0.05). However, the mean number of hippocampal neurons in the AD + baicalin group was significantly higher than the AD model group (3.498 × 10⁴ cell/mm³, P < 0.05) (Figure 2). Moreover, we validated the apoptosis of neurons through examining the expression of bax and caspase-3. As shown in Figure 3, the results indicated that baicalin could effectively inhibit the expression of bax and caspase-3 (P < 0.01; P < 0.001), which were consistent with the results of HE staining.

Figure 2

Effect of baicalin on the number of hippocampal neurons: (a) HE staining in the control group, (b) HE staining in the AD group, and (c) HE staining in the AD + baicalin group. Abbreviation: HE, hematoxylin–eosin; AD, Alzheimer’s disease.

Figure 3

Effect of baicalin on neuronal apoptosis: (a) protein band and (b) gray-scale analysis of the protein band. **P < 0.01, ***P < 0.001, control vs AD model; ##P < 0.01, ###P < 0.001, AD model vs AD + baicalin. Abbreviation: AD, Alzheimer’s disease.

3.3 Effect of baicalin on NPTX-1, NPTX-2, and CRP expression of immunohistochemistry and immunofluorescence staining

The expression of NPTX-1, NPTX-2, and CRP in rats’ brain was examined using immunohistochemistry. Then, we evaluated the localization of NPTX-1, NPTX-2, and CRP using immunofluorescence staining. The results indicated that the expression of NPTX-1 in the AD + baicalin group was significantly reduced compared to the AD group (0.05953 ± 0.00603 vs 0.07076 ± 0.00788, P = 0.000) (Figure 4a–d). The fluorescence intensity in the AD model group was stronger than that of the controls and the AD + baicalin group (Figure 4e–h; Table 3).

Figure 4

Effect of baicalin on NPTX-1 immunohistochemical (a–c) and immunofluorescence (d–f) staining: (a) immunohistochemical staining in the control group, (b) immunohistochemical staining in the AD group, (c) immunohistochemical staining in the AD + baicalin group, (d) quantification for immunohistochemical staining, (e) immunofluorescence in the control group, (f) immunofluorescence in the AD model group, (g) immunofluorescence in the AD + baicalin group, and (h) quantification for immunofluorescence. Abbreviation: AD: Alzheimer’s disease; NPTX-1: neuronal pentraxin-1.

Besides, the expression of NPTX-2 in the AD + baicalin group was significantly improved compared with the AD group (P = 0.033) (Figure 5a–d; Table 3). Meanwhile, the fluorescence intensity of the AD model group was stronger than that of the controls and the AD + baicalin group (Figure 5e–h). In addition, the expression of CRP in the AD + baicalin group (0.02034 ± 0.00397) was significantly reduced compared to the AD model group (P = 0.021) (Figure 6–d). The fluorescence intensity in the AD model group was stronger than that of the controls and the AD + baicalin group (Figure 5e–h; Table 4).

Figure 5

Effect of baicalin on NPTX-2 immunohistochemical (a–c) and immunofluorescence (d–f) staining: (a) immunohistochemical staining in the control group, (b) immunohistochemical staining in the AD model group, (c) immunohistochemical staining in the AD + baicalin group, (d) quantification for immunohistochemical staining, (e) immunofluorescence in the control group, (f) immunofluorescence in the AD model group, (g) immunofluorescence in the AD + baicalin group, and (h) quantification for immunofluorescence. Abbreviation: AD: Alzheimer’s disease; NPTX-2: neuronal pentraxin-2.

Figure 6

Effect of baicalin on CRP immunohistochemical (a–c) and immunofluorescence (d–f) staining: (a) immunohistochemical staining in the control group, (b) immunohistochemical staining in the AD model group, (c) immunohistochemical staining in the AD + baicalin group, (d) quantification for immunohistochemical staining, (e) immunofluorescence in the control group, (f) immunofluorescence in the AD model group, (g) immunofluorescence in the baicalin group, and (h) quantification for immunofluorescence. Abbreviation: AD: Alzheimer’s disease; NPTX-2: neuronal pentraxin-2; CRP: C-reaction protein.