Regulation of the NF-κB/NLRP3 signalling pathway by Shenghui Yizhi decoction reduces neuroinflammation in mice with Alzheimer’s disease

SHYZD consists of 12 herbs (all botanical drugs that make up SHYZD are listed in the Supplementary Material Table 1↗). The medicinal materials were purchased from Beijing Tongrentang Pharmaceutical Co., Ltd. prepared by the Pharmacy Department of China-Japan Friendship Hospital. Assumed the body weight of an adult man is 70Kg, and thus the dose of SHYZD is 2 g (raw herb)/Kg. In accordance to body surface area conversion between humankinds and mice, the dose of SHYZD is 15.6 g (raw herb)/Kg. The medicinal materials were decocted three times using the traditional water decoction method Specific protocol was as follows: a package of SHYZD was soaked in 1200 g of distilled water for 4 h and decocted for 60 min and finally filtered. The filtrate was decocted in 1200 g of distilled water for 30 min and then filtered through gauze. The step was repeated again. The three filtrate was mixed and concentrated to 115.4 mL. Each 1 mL of the liquid medicine contains 1.04 g of SHYZD.

Donepezil hydrochloride is a long-acting symptomatic treatment for AD and can be used as a positive control for improving memory and cognitive impairment. Donepezil hydrochloride tablets (batch no. 1702010, 5 mg/tablet) was purchased from Eisai China pharmaceutical Co., Ltd. Interleukin-1β (IL-1β) ELISA kits (CRE0006), Interleukin-6 (IL-6) ELISA kits (CRE0005), and Tumour necrosis factor-α (TNF-α) ELISA kits (CRE0003) were purchased from 4 A Biotech. NF-κB p65 antibody(ab8227), NLRP3 antibody(ab16502), Caspase-1 antibody(ab214185), and IL-1β antibody(ab1872) were purchased from Abcam. Reverse Transcription System (A3500) was purchased from Promga. SYBR^® Green Realtime PCR Master Mix(QPK-201) was purchased from TOYOBO.

SAM-P8 is a kind of senescence-accelerated mouse, whose characters manifest themselves in age-related impairment in memory and the ability to learn and obvious degenerative changes of the central nervous system [11]. Twenty-four SPF SAMP8 male mice and 8 SAMR1 male mice aged 3 months, with body weight of 30 ± 2 g were used in this study. The animals were provided by the Department of Experimental Animal Science, Peking University Health Science Center (No. SCXK, Beijing, 2016-0010). Animals were housed at a temperature of 22 ± 2 °C, humidity of 40%∼70%, and with a 12-hour light-dark cycle. The mice were given unrestricted access to water and food. All animal procedures and protocols were approved by the China-Japan Friendship Hospital Animal Care Committee (zryhyy-21-21-9-21) and conducted in accordance with the “Guiding Principles in the Care and Use of Animals” published by the American Physiological Society.

After acclimating the SAMP8 mice for one week, they were randomly divided into 3 groups (n = 8 in each group): the model group, the positive control group (donepezil hydrochloride), and the SHYZD group. Eight SAMR1 mice served as a control group. The equivalent dose for mice was converted according to the body surface area of humans and mice. The dose administered to mice was 9.1 times that of humans. Assumed the body weight of an adult man is 70Kg, and thus the dose of SHYZ is 1.7 g (raw herb)/Kg. The dose of SHYZ donepezil hydrochloride is 0.1 mg/Kg. The SHYZD group was administered 15.6 g/kg SHYZD, the positive control group was administered 1 mg/kg donepezil hydrochloride solution, and the model and the control groups were administered equal volume of double distilled water. The doses were administered via gavage at 15 ml/kg once a day for 90 days, and the mice were weighed every 4 weeks.

The whole experimental process consisted of positioning navigation and spatial exploration. On the first day, the pool was filled with water until the visible platform rose above the water in the second quadrant, and mice were placed gently in the water facing the pool wall. The tracking device was initiated simultaneously. If the mouse discovered the platform within 60 s, it was allowed to stay on the platform for 5 s; Otherwise, the mouse was placed on the platform for 20 s. The second to fifth day, the experiment focused on positioning navigation: water was added to the pool to submerge the platform to 1 centimeter below the surface. The mice were placed in the water facing the pool wall from one of four center points of the different quadrants. After locating the platform, the mouse rested on the platform for 15 s and then continued the next training from a different location; if the mouse did not find the platform within 60 s, they were guided to the platform by the experimenters and remained on the platform for 15 s; the escape latency was recorded as 60 s. Training was repeated four times for each mouse, with each mouse being put into the water from four starting points of different quadrants in each time. The tracking device recorded escape latency, swimming path, the time spent in each quadrant and other parameters of the mouse in locating the platform. The average latency of four times was considered as the learning performance of the mouse. On the sixth day, the experiment focused on spatial exploration: the platform in the second quadrant was removed, and the mice were placed in water facing the pool wall from the same starting positions. The swimming path, the number of times of crossing the platform position, and the time spent in the second quadrant within 60 s were recorded to measure the spatial positioning ability of the mice. The mice were monitored by a fixed camera directly above the pool and Ethovision 3.0 software was used to measure the parameters.

After the last behavioural test, mice were anesthetized with isoflurane. The brain was extracted through craniotomy following cardiac perfusion and fixation. The brain tissue was subsequently encased in paraffin and sectioned into slices measuring 5 μm in thickness. These sections were subsequently subjected to deparaffinization and hydration processes. High-pressure antigen retrieval was carried out using a citrate buffer (pH = 6.0). The sections were treated with 3% hydrogen peroxide in methanol for 15 min to inhibit the endogenous peroxidase, followed by an incubation with normal serum to prevent any non-specific staining. At 4 °C, sections were incubated overnight with rabbit anti-amyloid β1-42 (Aβ1-42). After washing, the tissue sections were treated with the biotinylated anti-rabbit secondary antibody, followed by additional incubation with the streptavidin-horseradish peroxidase complex. After staining with the diaminobenzidine kit, the sections were counterstained with hematoxylin. A negative control was treated simultaneously with the same procedure, but phosphate-buffered saline was used instead of the primary antibody. In the CA1 region of the hippocampus, three successively distinct visual fields were chosen from each slice. The integral optical density and area of each image were captured and analyzed by the Image-ProPlus 6.0 software, and the mean optical density (MOD) was calculated by dividing integral optical density by area.

The mice were sacrificed after the behavioural tests. The hippocampal tissue was quickly removed from each mouse and stored in a freezer at −80 °C for further analysis. Expression levels of IL-1β, IL-6, and TNF-α in the hippocampal tissue were determined using ELISA kits based on the protocol provided in the kit. The concentration was set based on the manual of the kit and was calculated with reference to the standard curve.

TRIzol was used to extract total RNA from hippocampal tissue. In accordance with the manufacturer’s instructions, RNA was reverse-transcribed into cDNAs using the TransScript RT kit. Real-time PCR was used to conduct quantitative PCR. In order to amplify DNA, the reaction conditions were 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. Based on the 2 − ΔΔCt method, the PCR data was normalized to the level of β-actin gene expression and the value was calculated. Real-time quantitative PCR primers were acquired from Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. The primers are exhibited in Supplementary Material Table 2↗.

The hippocampal tissue was homogenized in radioimmunoprecipitation assay lysis buffer (RIPA lysis buffer, containing protease inhibitor), then centrifuged at 12,000 ×g for 15 min, and the protein concentration was determined using Coomassie-blue reagent. Subsequently, protein extracts from different groups were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk at room temperature for 2 h before being incubated overnight at 4 °C with the respective primary antibodies. After washing with Tris Buffered Saline Tween three times, the PVDF membrane was incubated with the HRP-conjugated secondary antibodies. The primary antibodies used were IKKα, IκBα, NF-κB, NLRP3, Caspase-1, and IL-1β. Subsequently, the developed bands were visualized by enhanced chemiluminescence advanced kit and gel imaging system. β-acting was used as internal reference. The grayscale values of the bands were analyzed using Image J software (National Institutes of Health).

Graphs were prepared and the statistical data were analyzed using GraphPad software (version 8.0, GraphPad Software, Inc., La Jolla, CA, USA) and SPSS Statistics software(version 25.0, IBM., Armonk, NY, USA). Continuous variables were presented as the mean values ± standard deviation (SD). If data showed a normal distribution, Student’s t-test was used to assess the differences between the two groups; if not, the Mann–Whitney test was utilized. P-value <0.05 was considered to indicate a statistically significant difference, n = 3.

The escape latency of mice in the model group was prolonged when compared with the control group (p < 0.05). The escape latency of mice in the positive control group (donepezil hydrochloride) and SHYZD group was shortened from the 3^rd day when compared with the model group (p < 0.05 or p < 0.01) (Figure 1a, Supplementary Material Table 3↗).

The mice in the model group spent less time in the target quadrant, and the times of crossing the platform decreased when compared with the control group (p < 0.05). Mice in the positive control group (donepezil hydrochloride) and SHYZD group took longer to cross the platform and remain in the target quadrant when compared with the model group (p < 0.05, Figure 1b, Supplementary Material Table 4↗).

The expression of Aβ1-42 protein in the hippocampus of the model group was increased when compared with the control group (p < 0.01). The expressions of Aβ1-42 in the positive control group (donepezil hydrochloride) and SHYZD group were reduced, and the differences were statistically significant when compared with the model group (p < 0.01, Figure 2).

The levels of IL-1β and IL-6 in the model group were significantly increased when compared with the control group (p < 0.01). The IL-1β, IL-6, and TNF-α content in the positive control group and SHYZD group were significantly reduced when compared with the model group (p < 0.01 or p < 0.05, Figure 3a, Supplementary Material Table 5↗).

The expressions of NLRP3, Caspase-1 and IL-1β mRNA in the hippocampus of the model group were elevated when compared with the control group (p < 0.01). The expressions of hippocampal NLRP3, Caspase-1, and IL-1β mRNA in the positive control group and SHYZD group were reduced when compared with the model group (p < 0.01 or p < 0.05, Figure 3b, Supplementary Material Table 6↗).

The expressions of hippocampal NLRP3, Caspase-1, and IL-1β proteins in the model group were elevated when compared with the control group (p < 0.01). The expressions of hippocampal NLRP3, Caspase-1, and IL-1β proteins in the positive control group and SHYZD group were decreased when compared with the model group (p < 0.01 or p < 0.05, Figure 4a-b, Supplementary Material Table 7↗).

Similarly, related marker proteins were identified in the NF-κB/NLRP3 pathway. The expressions of hippocampal NF-κB, IκBα, and IKKα proteins in the model group were increased when compared with the control group (p < 0.01 or p < 0.05). The expressions of hippocampal NF-κB, IκBα, and IKKα proteins in the positive control group and SHYZD group were reduced when compared with the model group (p < 0.01 or p < 0.05, Figure 4c-d, Supplementary Material Table 8↗). In addition, phosphorylated protein expression was measured, and a consistent pattern emerged (Figure 4c).