Male C57BL/6 mice (7 weeks old weighing 20–22 g) were used in this study. The mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Jiangsu, China, SCXK (Su) 2020-0009). The mice were housed with the Animal Experiment Center of Yan’an University and given free access to food and water under temperature- and humidity-controlled conditions with a 12 h light/dark cycle. Experiments started after 7 days of adaptive feeding. All procedures were approved by the Animal Ethics Committee, Yan’an University, China, and complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Mice were randomly divided into nine groups ( Table 1 ). The AD mouse model was established by injecting Aβ_1-42 oligomers (Aβ_1-42Os) (10 µg, GenScript Probio Co., Ltd., Nanjing, China) into the lateral ventricle (AP −0.2, ML ±1.0, DV −2.5) as previously described. Before Aβ_1-42O injection, mice were continuously treated with zunyimycin C (14 mg/kg, nasal instillation) for 21 days to study the preventive effect of zunyimycin C on AD. Other groups were continuously treated with different doses of zunyimycin C, donepezil, benfotiamine, or vehicle for 21 days. Donepezil and benfotiamine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Behavioral tests were assessed 21 days after intervention. Mice were sacrificed after behavioral testing, and the brains of mice were collected. The left brain was used to measure Il-1β, Tnf-α, IFN-γ, Iba1, Creb, Pparγ, Mek1, Akt, and Gapdh by reverse transcription polymerase chain reaction (RT-PCR). The right brain was used to measure the glial fibrillary acidic protein (GFAP), CD163, interleukin (IL)-6, IL-1β, and GAPDH by Western blot.

The Morris water maze (MWM) was performed with reference to the previous literature (Othman et al., 2022). An MWM analysis system (Zhongshi DiChuang Technology Co., Ltd., Beijing, China) was used for this experiment. The MWM was carried out in a circular pool (120 cm in diameter and 40 cm high) filled with water (the height of the water was 30 cm). The pool was divided into four quadrants. A platform with a diameter of 8 cm was located in the third quadrant, 1 cm underwater, and the water temperature was controlled at 22°C. Mice were trained four times a day at the same time period, with each time from a different quadrant, for 5 consecutive days. Mice were placed in the pool for 60 s during training. If the mouse successfully found the platform, it was allowed to rest on the platform for 10 s. If the mouse did not successfully find the platform, it was guided to the platform and rested for 10 s. On the 6th day, the platform was removed, and the mice were placed into the pool from the diagonal quadrant of the original platform and allowed to swim in the pool for 60 s. The animal behavior analysis system was used to record the animal’s on-stage latency, distance before on-stage, and swimming speed.

Twenty four mice were randomly divided into three groups, namely, blank (KB), model group (CTX), and zunyimycin C group. Mice were intraperitoneally injected with CTX (100 mg/kg) once a day for 3 days to build an immunosuppressive mouse model, whereas mice in the KB group were intraperitoneally injected with normal saline (NS) as control. From the fourth day, mice were given normal saline by gavage in the KB and CTX groups, and other mice were given the drug for 15 days. The feces were collected with sterile centrifuge tubes and stored at −80 °C. SCFA was determined by Shanghai LuMing Biotechnology Company via LC-MS-MS.

SCIEX OS-MQ software (Sciex, USA) was employed to identify and integrate MRM transitions with default parameters. Abscissa of the extracted ion current diagram (XIC) of each substance was the retention time (RT) of metabolite detection, and the ordinate was the ion strength (count per second, cps) of ion detection, generating the map of each metabolite. The peak area of the metabolite was interpolated into the regression equation fitted by the standard curve to obtain the concentration of the metabolite. On the basis of the parameters such as sampling and/or dilution ratio, we calculated the concentration of metabolites and obtained the absolute content of each metabolite in the actual sample.

16S rRNA amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Total genomic DNA was extracted using a DNA Extraction Kit following the manufacturer’s instructions. The quality and quantity of DNA were verified with NanoDrop and agarose gel. Extracted DNA was diluted to a concentration of 1 ng/μL and stored at 20°C until further processing. The diluted DNA was used as the template for PCR amplification of bacterial 16S rRNA genes with the barcoded primers and Takara Ex Taq (Takara). For bacterial diversity analysis, V3-V4 variable regions of 16S rRNA genes were amplified with universal primers 343F and 798R. For eukaryotic diversity analysis, variable regions of 18S rRNA genes were amplified with universal primers 817F and 1196R. Amplicon quality was visualized by gel electrophoresis, purified with AMPure XP beads (Agencourt), and amplified for another round of PCR. After purifying with the AMPure XP beads again, the final amplicon was quantified using the Qubit dsDNA assay kit. Equal amounts of purified amplicon were pooled for subsequent sequencing.

Raw sequencing data were in FASTQ format. Paired-end reads were then preprocessed using cutadapt software to detect and cut off the adapter. After trimming, paired-end reads were filtered for low-quality sequences, denoised, merged, and detected. The chimaera reads were cut off using DADA2 with the default parameters of QIIME2 (2020.11). Finally, the software produced the representative reads and ASV abundance table. The representative read of each ASV was selected using the QIIME2 package. All representative reads were annotated and blasted against the Silva database Version 138 (or Unite) (16s rDNA) using q2-feature-classifier with the default parameters.

Eight mice were randomly divided into three groups, namely, KB, CTX, and zunyimycin C group. The KB group was intraperitoneally injected with NS, whereas all mice in the six other groups were intraperitoneally injected with CTX (100 mg/kg) once a day for 3 days. On the fourth day, all mice were intraperitoneally injected with 0.2 mL of 6% (v/v) chicken red blood cells. Meanwhile, mice in the experimental groups were given drug by gavage once a day for 15 days; otherwise, the KB and CTX groups were given normal saline by gavage for 15 days. Mice in each group were euthanized by taking blood from orbit, and the amounts of hemolysin, IL-2, IL-6, Interferon(IFN)-y, and Tumor necrosis factor (TNF)-α were measured. On the fourth day, all mice were sensitized by intraperitoneal injection of 0.2 mL of 6% chicken red blood cells, and mice blood samples were collected from orbit. After resting at room temperature for 1 h, blood samples were centrifuged at 4°C and 2000 rpm for 20 min, and the supernatant was collected and incubated for 1 h at 37°C with 250 µL of 6% (v/v) chicken red blood cell and 250 µL of 10% guinea pig serum. The reaction was stopped at 15 min in ice and then centrifuged at 4°C and 2000 rpm for 20 min. About 200 µL of supernatant was collected in 96-well plate, and the optical density value was measured at 540 nm.

After the behavioral experiment, the mice were anesthetized and perfused with normal saline, and the brain tissue was removed, immediately placed in liquid nitrogen, and transferred to a −80°C freezer for cryopreservation. The brain tissue was thawed from the −80°C freezer during testing. Total RNA was extracted from mouse brain tissue using Trizol reagent (TaKaRa, Osaka, Japan). The concentration and purity of total RNA were detected using a microplate reader (MOLECULAR DEVICES, CA, USA). PrimeScript™ RT Master Mix (Perfect Real Time) kit (TaKaRa, Osaka, Japan) was used to reverse transcribe RNA to cDNA. In q-PCR, PCR was performed in Cobas z 480 (Roche Applied Science, Basel, Switzerland). cDNA was analyzed using TB Green Premix Ex Taq™ (Tli RNaseH Plus) kit (TaKaRa, Osaka, Japan). GAPDH served as an endogenous control. The Ct value of each group was derived, and the relative level of mRNA was analyzed using the 2^-ΔΔCt method. The primer sequences used in this experiment are shown in Table 2 . The primers were designed using the online primer design website Primer3Plus (http://www.primer3plus.com), and the online primer analysis tool NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyze the specificity of the primers. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

The mouse brain tissue was removed from the −80 °C freezer, thawed, and added with 500 μL of RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to 100 mg of brain tissue. The samples were homogenized with a glass homogenizer and placed at room temperature 10 min. The samples were then centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant was collected. The protein concentration was detected using a BCA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Equal amounts of proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes, which were blocked in 5% nonfat dry milk (dissolved in TBST) for 2 h. They were then mixed with primary antibody GFAP (1:1000, BOSTER Biological Technology Co., Ltd., Wuhan, China), CD163 (1:1000, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), IL-1β (1:1000, BOSTER Biological Technology Co., Ltd., Wuhan, China), IL-6 (1:1000, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA) and incubated overnight at 4°C. After washing the membranes three times with TBST (5 min/time), the membranes were incubated with secondary antibody with HRP diluted in TBST containing 5% nonfat milk powder for 2 h. The membranes were completely immersed in HPR substrate luminescent solution (ThermoFisher LLC. Waltham, MA, USA), and the bands were exposed to a chemiluminometer (GBox Chem3, Syngene, the UK). The exposure results were saved for subsequent analysis.

The data of each group were expressed as the mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8 software. One-way ANOVA was used for comparison between multiple groups, and t-test was used for comparison between two groups. P<0.05 indicated that the difference was statistically significant.

Zunyimycin C was not detected in the blood of mice administered intranasally, whereas zunyimycin C was detected in the blood of mice administered by gavage and tail vein. Thus, both gavage and tail vein injection facilitated the drug’s entry into the blood circulation, but nasal administration did not. In the chromatogram of the blood extract from the gavage mice, the target peak RT was 1.498, the peak height (PH) was 0.13, and the peak area (PA) was 0.445. The concentration of zunyimycin C obtained by the standard curve was 1.1904 mg/mL, and the total amount was 0.23808 mg. In the chromatogram of the blood extract of mice injected with tail vein, the target peak RT was 1.507, PH was 0.08, and PA was 0.237. The zunyimycin C concentration was 1.1903 mg/mL, and the total amount was 0.23806 mg. Zunyimycin C was detected in the brain tissue of mice administered nasally. However, no zunyimycin C was detected in the brain tissue of mice injected by gavage and tail vein. In the liquid chromatogram of the chloroform-extracted extract of mouse brain tissue after nasal administration, the target peak RT was 1.298, PH was 0.11, and PA was 0.830. The concentration of zunyimycin C was 1.1907 mg/mL. These results suggested that intranasal administration of zunyimycin C enabled zunyimycin C to exert its effects in the brain via entry into the olfactory or trigeminal nerves (rather than into the blood and then across the blood–brain barrier).

The escape latency of training for 5 consecutive days in each group and the results ( Table 3 and Figure 2 ) showed that the escape latency of the benfotiamine intervention group was higher than that of the Vehicle group but lower than that of the model group (P<0.05), indicating that the cognitive ability of the benfotiamine intervention group was in the recovery period. The positive control was donepezil, their escape latency also shows the same trend as benfotiamine, i.e. lower than that of the model group (P<0.05) after three days. The escape latency of Zun-Pre group, Zun-Int L group, Zun-Int M group and Zun-Int M +benfotiamine goup are all lower than that of the model group (P<0.05).

The stay time of the mice in each group in the quadrant of the original platform ( Figure 3A ) showed that the stay time of the mice in the model group in the quadrant of the original platform was 18.08 ± 4.52 s, which was shorter than the stay time of the mice in the Vehicle group (28.65 ± 3.71 s). The difference was statistically significant (P<0.05). The residence times of the Zun-Int H group, donepezil intervention group, and combination group in the quadrant of the original platform were 15.88 ± 6.50, 15.19 ± 5.93, and 9.93 ± 1.30 s, respectively, which were all lower than that of the model group. The mice in the Zun-Pre group, Zun-Int L group, Zun-Int M group, and benfotiamine intervention group stayed longer than those in the model group in the quadrant where the original platform was located but shorter than those in the Vehicle group. These mice were in the recovery phase.

The platform crossing times of the mice in each group in the space exploration experiment ( Figure 3B ) showed that the platform crossing times of the Zun-Int L group were 4 ± 1.78, whereas that of the Zun-Int H group was 3.42 ± 0.97. These values were all higher than those of the model group. The number of platform crossings was 1.14 ± 1.06, and the difference was statistically significant (P<0.05).

From the movement trajectories of mice in the space exploration experiment ( Figure 4 ), activities in the original platform location were more frequent in the Vehicle group, Zun-Pre group, Zun-Int L group, Zun-Int M group, and benfotiamine intervention group. Mice in the model group, donepezil intervention group, Zun-Int H group, and combination group demonstrated irregular movements in the pool, and they did not stay in the quadrant where the original platform was located for a long time.

Differential expression of mRNAs of inflammatory factors and pathways in brain tissue of the AD mouse model after different interventions was compared with the model group ( Figure 5 ). The model group fold change was defined as 1. The target gene expression fold change was greater than 2 as upregulation and less than 1 as downregulation. The results showed that the mRNA expression levels of the Il-1β, IFN-γ, Cd163, Iba1, Mek-1, Creb, Akt, and Pparγ genes in the brain tissue of the mice in the Zun-Pre group were all upregulated. The mRNA expression levels of IFN-γ, Mek-1, Creb, and Akt genes in the brain tissue of mice in the low-dose intervention group were upregulated. The mRNA expression of the IFN-γ gene in the brain tissue of the mice in the medium-dose zunyimycin intervention group was upregulated. The mRNA expression levels of Il-1β, Iba1, Mek-1, Akt, and Pparγ in the brain tissue of the mice in the Zun-Int H were all downregulated. The mRNA expression of the Tnf-α gene in the brain tissue of mice in the donepezil group was downregulated, whereas the mRNA expression of IFN-γ and Creb gene was upregulated. The mRNA expression of the Creb gene in the brain tissue of the mice in the benfotiamine group was upregulated. The mRNA expression levels of the Tnf-α, Iba1, and Mek-1 genes in the brain tissue of the mice in the combination group were downregulated, and the mRNA expression of the Creb gene was upregulated.

The difference in the expression of inflammation-related proteins in the brain tissue of AD mouse models after different interventions ( Figure 6 ) showed that the relative expression of GFAP in the brain tissue of the AD mouse model in the Zun-Pre group was higher than that in the donepezil group, benfotiamine group, Zun-Int M combined benfotiamine group, and Vehicle group; the difference was statistically significant (P<0.05). There was no significant difference in the relative expression of GFAP among different doses of zunyimycin C intervention groups. The relative expression of GFAP in the donepezil group, benfotiamine group, and combination group was lower than that in the zunyimycin C intervention group, but the relative expression of GFAP in these intervention groups was lower than that of the model group and higher than that of the Vehicle group. The relative expression of IL-6 protein in the brain tissue of mice in the Zun-Int L group was significantly lower than that in the Zun-Pre group, Zun-Int H, donepezil group, benfotiamine group, combination group, model group, and Vehicle group. The difference was statistically significant (P<0.05).