Mouse microglia BV2 cells were purchased from the Cell Bank of Chinese Academy of Sciences; Dulbecco’s modified Eagle medium (12100046), fetal bovine serum (10099141C), and penicillin/streptomycin (10378016) were purchased from Gibco; Aβ protein fragment 1–40 (A1075) was purchased from Sigma-Aldrich; Lipofectamine 2000 Transfection Reagent (ab51243) was purchased from Abcam; CD38 siRNA (AM160089) was purchased from Invitrogen; CD38 CRISPR activating plasmid (sc-419548), CD38 antibody (SC374650), and M-IgGκ BP-HRP (sc-516102) were purchased from Santa Cruz Biotechnology; anti-rabbit IgG HRP-linked antibody (5490S) and β-actin mouse monoclonal antibody (3700) were purchased from Cell Signaling Technology; P21 rabbit antibody (AF5252), P16 rabbit antibody (AF1069), Iba1 rabbit antibody (AF7143), Cell Proliferation and Cytotoxicity Assay Kit (C0009), Enhanced ATP Assay Kit (S0027), Reactive Oxygen Species Assay Kit (S0033S), Mitochondrial Membrane Potential Assay Kit (C2006), and Mouse amyloid beta peptide 1–42 ELISA Kit (MU30114) were purchased from Bioswamp; Mouse TNF-α ELISA Kit (PT512), Mouse IL-6 ELISA Kit (PT312), Mouse IL-1β ELISA Kit (PT512), Senescence β-Galactosidase Staining Kit (C0602), Alexa Fluor 488 goat anti-rabbit IgG (a0423), DAPI-staining solution (C1006), phosphate buffered saline (C0221A) and bovine serum albumin (ST025) were purchased from Shanghai Beyotime Biotechnology; and NAD/NADH Quantitation Colorimetric Kit (K337) was purchased from Biovision.
Fourteen 30-week-old C57BL/6J mice were used as controls, and forty-two 30-week-old APP/PS1 mice were selected as AD model mice. The AD model mice were randomly divided into the model group, the CD38 inhibitor group, and the NMN group. The CD38 inhibitor and NMN were injected intraperitoneally once a day for 4 week, with 0.2 ml each time. The CD38 inhibitor was dissolved in 10% DMSO + 90% distilled water. NMN was also dissolved in distilled water to a concentration of 0.093 mg/ml, and CD38 inhibitor and NMN solution were intraperitoneally injected at 30 weeks old (0.0186 mg/d) once a day for 4 weeks. All of the administration operations and tissue extractions were authorized by the Ethics Committee of Shanghai University of Traditional Chinese Medicine, according to the animal protection law and experimental method (No. PZSHUTCM191025004).
Senescence β-Galactosidase staining
According to the instructions of the Senescence β-Galactosidase Staining Kit, we first prepared the cells and brain slices in a 24-well plate. Next, 400 μl phosphate buffered saline (PBS) was added to the plate and washed three times (× 5 min per wash). Subsequently, 400 μl β-galactosidase staining fix solution was added, discarded after 20 min, and further washed three times with PBS (× 10 min per wash). After washing, 400 μl staining solution was added. The plate was wrapped to prevent evaporation before placing in a 37 °C incubator overnight. Following incubation, the slides were observed under an ordinary optical microscope (BX63, Olympus Corporation, Japan).
Three brain slices from each group were placed in 24-well plates, 400 μl PBS was added, and the slices were washed three times (× 5 min per wash). Next, 400 μl 1% Thioflavine-S solution was added and incubated in the dark at room temperature with shaking for 10 min. After staining, the Thioflavin-S dye solution was discarded, and 600 μl distilled water was added to each well before washing three times (× 5 min per wash). The brain slices were sealed and observed under a fluorescence microscope (BX51, Olympus Corporation, Japan).
Protein samples were heated for 5 min at 95 °C, separated by SDS polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was sealed with 10% bovine serum albumin (BSA) for 1 h, and the primary antibody was added at a 1:2000 dilution before incubating overnight at 4 °C. After incubation, the membranes were washed with TBST (10 min × three washes). Next, the corresponding secondary antibody was added at a 1:1000 dilution before incubating at 37 °C for 1 h, followed by further washing with TBST (three times × 15 min per wash). ImageJ (V22.214.171.124) was used to analyze the protein bands to integrate the absorbance (IA = mean OD × Area). The relative level of the target protein was normalized to β-actin (target Protein IA/β-actin IA).
After the intervention, the BV2 cells were fixed with 4% paraformaldehyde for 15 min. Then BV2 cells and brain slices were washed with PBS for 10 min and then penetrated with 1% Triton for 10 min. A 10% BSA-blocking solution was sealed for 1 h, and the primary antibody was added at a ratio of 1:1000, incubated at 37 °C for 1 h and 4 °C overnight. After washing with PBS (three times × 10 min), the fluorescent secondary antibody was diluted with PBS at a ratio of 1:500, and DAPI was added at a ratio of 1:500. After incubation at 37 °C for 1 h, the cells were washed further with PBS.
Aβ oligomer (AβO) preparation
The Aβ1-40 fragment was prepared as described above . Briefly, 0.5 mg of Aβ1-40 lyophilized powder was placed at room temperature for 0.5 h. Then, PBS containing 0.05% sodium dodecyl sulfate was used to dissolve Aβ1-40, which was then mixed at a concentration of 100 µmol/L. Subsequently, Aβ1-40 was incubated at 37 °C for 7 days and stored at 4 °C for further use in the following experiments.
Cell culture and treatments
BV2 cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin in 6-well plates. The cells were passaged when the fusion rate reached 80–90%. The cells were divided into the control, Aβ1-40, Aβ1-40 + CD38 siRNA, and Aβ1-40 + CD38 overexpressed groups. The control group was cultured with DMEM without any treatment for 24 h; the Aβ1–40 group was cultured with DMEM containing 1 μM Aβ1-40 for 24 h; and the Aβ1-40 + CD38 siRNA group was cultured with DMEM containing 1 μM Aβ1-40 for 24 h. Then, CD38 siRNA was transfected into the BV2 cells. The Aβ1-40 + CD38 overexpressed group was treated with 1 µM Aβ1-40 for 24 h, then the CD38 CRISPR plasmid was transfected into BV2 cells.
According to the instructions of the MTT Cell Proliferation and Cytotoxicity Assay Kit, each well of cells was supplemented with 100 µl MTT solution (0.5 mg/ml) and incubated at 37 °C for 4 h. The supernatant was discarded, and 100 µl dimethyl sulphoxide was added to fully dissolve the crystals. Subsequently, absorbances were measured at a wavelength of 570 nm using a PowerWave XS microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
An enhanced ATP assay kit was used to determine the ATP levels as previously described. Briefly, the sample and lysate were mixed, and, after full pyrolysis, was centrifuged at 12,000 g for 5 min at 4 °C. Next, the supernatant was collected, and 100 μl of supernatant was added to each well of a 96-well plate and placed at room temperature for 5 min, before adding 20 μl working solution for ATP detection. The relative light unit (RLU) value was measured at 450 nm with a PowerWave XS microplate reader.
NAD + /nicotinamide adenine dinucleotide hydride (NADH) measurements
First, 400 µl NADtotal was extracted according to the kit instructions. Subsequently, 200 µl NADtotal samples were transferred and heated at 60 °C for 30 min, and then centrifuged at 12,000 g for 30 s at 4 °C. The supernatants were labeled as NADHsamples for further assays. The standard curve was configured, 40 µl samples were loaded into the wells, and NAD + /NADH extraction buffer was added to a total volume of 50 µl. Finally, 100 µl enzyme-reaction mix and 10 µl NADH developer were added. After 4 h, the absorbance at 450 nm wavelength was measured using a PowerWave XS microplate reader.
Mitochondrial extraction and mitochondrial membrane potential (MMP) measurements
Mitochondria were extracted from cells and brain according to the instructions of the Mitochondrial Isolate Kit. The MMP was measured using the mitochondrial membrane potential assay kit with JC-1. The prepared JC-1 staining-working solution was diluted five times, and 0.9 ml JC-1 staining-working solution was added to 0.1 ml purified mitochondria with total protein of 10–100 µg. The change in MMP was detected by the change in fluorescence color using a PowerWave XS (BioTek Instruments, Inc.) microplate reader at 490 nm excitation wavelength and 530 nm emission wavelength.
Detection of reactive oxygen species (ROS)
Changes in intracellular ROS levels were determined by measuring the fluorescent dichlorofluorescein (DCF). DCFH-DA was diluted with serum-free DMEM (1:1000) and incubated at 37 °C for 20 min in the dark. Then, the cells were washed in serum-free DMEM to remove redundant DCFH-DA. The fluorescence intensity of DCF was measured with 488/525 nm excitation/emission wavelengths via a PowerWave XS microplate reader.
Detection of CD38 enzyme activity
To detect CD38 enzyme activity, sucrose buffer was added to an appropriate amount of cells. Next, ultrasonic treatment was conducted on ice, with 30–50 W power and 20 kHz frequency, 5 s each time for a total of three times. After ultrasonic treatment, the sample was centrifuged at 4 °C, 13.8 g for 10 min, and the supernatant was collected. When preparing the reaction mixture, 1 ml of total reaction mixture contained 5 μl 10 mM ε- NAD and 40 μl 10 mM NGD. The reaction mixture was added to an opaque 96-well plate, and 100 μl sucrose buffer was added to each well (including the blank well). The values were read at an excitation wavelength of 300 nm and an emission wavelength of 410 nm using a power wave XS plate reader.
Detection of neuroinflammatory factors (IL-1β, IL-6, TNF-α)
According to the instructions of the kit, six concentrations of standard (2000, 1000, 500, 250, 125, and 62.5 pg/ml) and samples were prepared and added to the corresponding wells at 100 μl/well. After incubation at room temperature for 2 h and subsequent washing, 300 μl cleaning solution was added to each well, followed by 100 μl horseradish peroxidase-labeled streptavidin. After incubation for 1 h, the plates were washed and 100 μl TMB solution was added to each well before incubating at room temperature in the dark for 20 min. After incubation, 50 μl termination solution was added to each well. After mixing, the absorbance was measured at 450 nm with a PowerWave XS microplate reader.
Morris water maze experiment
The Morris water maze test was used to test the spatial learning and memory ability of mice in each group. The water pool was divided into four quadrants (I, II, III, and IV), and the platform was placed 1 cm below the water surface. In the quiet state, the mice were put into the water from the center of the four quadrants. The time taken for the mice to find the platform within 70 s was calculated. If the mice failed to find the platform, the time was calculated as 70 s. The experiment lasted for 5 days; on the sixth day, the space exploration experiment was conducted, and the number of times that the mice crossed the original platform in 70 s without placing the platform was calculated.
New object recognition experiment
We further tested the cognitive ability of the mice using a new object recognition experiment. At the beginning of the training, two objects (A and B) were placed on the test box, and the mice were put into the field. After 10 min, the contact between the two objects was recorded. Then, object B was replaced by Object C. The mice were put into the field in the same way. After 5 min, the exploration of Object C by the mice was recorded. The new object recognition index (NOI) was calculated as the time of exploring new objects/(time of exploring new objects + time of exploring on-site objects) × 100%.
ELISA for the determination of Aβ
The Aβ1-42 concentration was measured using the Mouse amyloid-beta peptide 1–42 ELISA Kit using a standard curve to determine the Aβ1-42 level. A 40 μl sample and 10 μl biotin labeled anti-Aβ1-42 antibody was added to the sample well. Finally, 50 μl enzyme-labeling reagent was added and incubated at 37 °C for 30 min. Following incubation, the wells were washed and 50 μl of reagents A and B were added separately. The absorbance values of the microplate were read at 450 nm using a PowerWave XS microplate reader.
Data analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). The data are expressed as mean ± standard error. Normality and variance homogeneity were determined using the Shapiro–Wilk and Levene’s tests, respectively. Variance analyses were completely randomized. The Bonferroni t-test and Wilcoxon rank-sum test were used for pairwise comparisons. Meanwhile, the Kruskal–Wallis test was used for the analysis of nonparametric data. P-values < 0.05 were considered to indicate a statistically significant difference.