Drosophila melanogaster strains and genetic cross

Wild type (w1118) and Arc2E arc2E/arc2E; +/+red/upstream active sequence (UAS)-Aβ42 mutant strains of D. melanogaster was purchased from Shanghai Chinese Academy of Sciences. elav-galactose-regulated upstream promoter element 4 (GAL4) line driving pan-neuronal expression was from the State Key Laboratory of Medical Genetics, Central South University. The GAL4/UAS system [29, 30] was used to express Aβ42 pan-neuronally in fly brains, male progenies that were generated by crossing elav-GAL4 female flies and w1118 male flies were used as the control group, while male progenies that were produced by crossing elav-GAL4 female and Aβ42 male flies were used as the experimental group.

Preparation of TPPU-treated fly culture medium

The TPPU stock solution was prepared by dissolving 5 mg TPPU (GC15387, GLPBIO, USA) in 200 µl DMSO. TPPU working solutions were then prepared at concentrations of 1µM, 10µM, 15µM and 20µM. The final concentration of DMSO was carefully maintained below 0.1% to avoid causing toxic damage to the flies. A total of 20.87 g culture medium (ingredients: 81.5 g/l sucrose, 9.2 g/l yeast powder, 1.3 g/l sodium benzoate, 108.5 g/l corn flour, 8.2 g/l agar) was dissolved and boiled in 110 ml distilled water, and then 0.65 ml propionic acid and TPPU working solutions were added and mixed well. Media for rearing larval flies containing different concentrations of TPPU were prepared, and the media were divided into sterilized culture vials while hot. We stored the culture medium in a refrigerator at 4 °C for use. The media that were used for the control and experimental groups were supplemented with the same concentration of DMSO to ensure that the two groups can be compared.

The culture condition of flies

Fly cultures were maintained at a density of twenty adults per vial and were placed vertically in flies’ incubator. Flies were maintained at a constant temperature of 25 °C, in controlled humidity of 50%~60%, and under 12-h light and 12-h dark light cycles. Fly media was replaced with fresh media every 3 days.

To assay the lifespan of flies [29]

We measured the lifespan of adult flies to study the effect of Aβ toxicity on the adult fly’s lifespan and to determine whether TPPU can improve the lifespan of flies with Aβ deposits. The newly emerged adult first generation males were collected within 48 h, placed in a culture vial (twenty flies/vial, five vial), and incubated in an incubator at 25 °C and 50%~60% humidity. The medium was changed every 3 days, and each vial was labeled with the date and strain. The number of dead flies was counted when the medium was changed. If there was contamination, the medium was replaced. It should be noted that only the survival time of flies that died naturally were counted; the survival times of flies that escaped, drowned, were exposed to excessive anesthetization, adhered, or experienced other unnatural deaths were not recorded. The assay ended when all the flies had died.

The crawling ability of flies [30]

The crawling ability of the adult flies were performed to observe the effect on the physiological ability to crawl against gravity as well as disorders of muscle movement [30]. After hybridization, the male offspring were collected, twenty adult flies were placed in each vial, and the medium was changed every 3 days. A clean culture vial with a height of 12 cm and an inner diameter of 2.5 cm was selected as the crawling vial, and it was divided into three sections: the top, middle, and bottom sections. A total of one hundred adult flies were collected from each group and divided into five crawling vials. After CO2 anesthesia, the adult flies were placed into the crawling vial and maintained for 15 min, and their crawling ability was assessed. During the test, the flies were shaken down to the bottom of the vial at the same time and then allowed to crawl freely. After 15 s, the numbers of flies in the top, middle, and bottom sections were counted. After each measurement, the flies were allowed to recover their strength for 15 min and were assessed again. The crawling ability of flies at 7, 14 and 21 days was investigated. The average number and percentage of flies in each section were calculated.

To assay the olfactory memory of flies

The flies olfactory memory defect experiment in adult flies was conducted to observe the effect on the olfactory sense [31]. Each group of adult flies (twenty flies/group) was starved for 2 h in an empty culture vial and then transferred to a 1000 ml beaker. Two 50 ml centrifuge tubes with holes were placed in the beaker, and one tube was directly placed in a banana. The flies in this experiment can smell bananas, and the adult flies in this experiment could directly eat the banana once they entered the centrifuge tube through the hole. The other tube contained banana wrapped with gauze, and the flies could smell this banana but could not eat it. The number of flies that entered the barrier-free centrifuge tube was recorded every hour for 12 h.

Malondialdehyde (MDA) content in brain tissues of flies

The effect on lipid peroxidation in brain tissues of flies was determined by measuring the MDA content. The brain tissues of flies were homogenized with normal saline at a 1:10 (weight: volume) ratio. The MDA content in brain tissues were measured using a commercially available kit (A003-1-1, Nanjingjiancheng, China) according to the manufacturer’s instructions. Determine the absorbance of MDA at 532 nm with enzyme-labeled instrument (SpECTRAMAX-M5, USA). The data are expressed as absorbance after protein content normalization.

Superoxide dismutase (SOD) activity in brain tissues of flies

SOD activity was measured to observe the effect on the anti-peroxidation damage capacity of flies. SOD activity was measured by ice bath homogenization at the ratio of flies’ brain tissue mass (g): extraction liquid volume (ml) (1:10). We measured the SOD activity in flies brain tissues with commercially available kits (BC0175, Solarbio, Beijing, China) according to the manufacturer’s instructions. SOD absorbance was measured at a wavelength of 560 nm. The data are expressed as absorbance after protein content normalization.

Aβ (25–35) and TPPU preparation

Amyloid β-protein (25–35) amide (GA20745, GLPBIO, USA) was dissolved in sterile distilled water at a concentration of 2.5 mM. To obtain a higher solubility, the tube was heated at 37 °C and shaken for a while by ultrasonication. Then, the Aβ(25-35) solution was incubated in a 37 °C incubator for 72 h and lightly mixed to promote aggregation.

The TPPU stock solution was prepared by dissolving 5 mg TPPU (GC15387, GLPBIO, USA) in 200 µl DMSO, and then the stock solution diluted into 0.1 µM, 1 µM, and 10 µM TPPU working solutions in cell experiments.

Human cell cultures

SH-SY5Y cells (CL0278, Fenghuishengwu, China) are human neuroblastoma cells. These cells were cultured in medium (MEM) (C11095500BT, Gibco, USA) supplemented with 10% FBS (BC-SE-FBS06 C, Bio Channel, Nanjing, China) and 1% penicillin-streptomycin solution (S110 JV, Basal Media, China).

HMC3 cells (ATCC CRL-3304, Pricella, China) are human microglia. These cells were cultured in MEM (including NEAA) (PM150410, Pricella, China) supplemented with 10% FBS (BC-SE-FBS06 C, Bio Channel, Nanjing, China) and 1% penicillin -streptomycin solution (S110 JV, Basal Media, China).

During coculture, HMC3 cells were pretreated with 1 µM TPPU for 3 h and then incubated with 30 µM Aβ(25–35) for 48 h. The medium was replaced with fresh MEM (including NEAA), and the MEM was collected after 24 h. The collected MEM was centrifuged, added to SH-SY5Y cells, and incubated for 48 h for further analysis. All the cells were cultured at 37℃ in 5% CO2.

CCK-8 cell viability assay

Cell viability was determined by CCK-8 (Cell Counting Kit-8) assay. The cell density of SH-SY5Y cells or HMC3 cells was adjusted to 4 × 103 cells/well, and 100 µl/well was seeded in 96-well plates. Intervene after cell adhesion, 10 µl CCK-8 solution (GK10001, GLPBIO, USA) was added to each well and then incubated for 4 h. The OD value of each well was measured at 450 nm. Cell viability = (OD experimental group-OD blank group)/(OD control group-OD blank group).

Crystal Violet staining to test cellular morphology in SH-SY5Y cells

To observe the changes in cell number and axon length, SH-SY5Y cells were stained with crystal violet. After counting the cells, SH-SY5Y cells were seeded in 6-well plates at a cell density of 3 × 105 cells/well (2 ml/well). After the cells adhered, the treatment group was pretreated with medium containing 0.1 µM TPPU for 3 h. The control group and Aβ(25–35) group were pretreated with medium containing 0.1 µM DMSO. After incubation for 3 h, the Aβ(25–35) group and treatment group were treated with 25 µM Aβ(25–35) solution, and incubation was continued for 48 h. The medium in the 6-well plates was discarded, the cells in each well were gently rinsed 3 times with 1× PBS, and then 4% paraformaldehyde (1 ml) was added to each well to fix the cells for 20 min. Then, the formaldehyde solution was discarded, and 1× PBS was added to wash the cells 3 times for 5 min each. Then, 1% crystal violet solution (G1063, Solarbio, Beijing, China) was added in a volume of 500 µl/well, the cells were stained for 10 min, the crystal violet solution was discarded, the cells were washed with 1× PBS 3 times for 5 min each, and finally, the cells were viewed under a microscope and photographed.

Hoechst 33258 staining to test cell apoptosis in SH-SY5Y cells

Hoechst 33258 staining was used to analyze changes in apoptosis. SH-SY5Y cells or cocultured SH-SY5Y cells were seeded at a cell density of 3 × 105 cells/well in 6-well plates on a slide, and the cells were fixed in 4% paraformaldehyde for 20 min after treatment. After washing with 1× PBS 3 times for 5 min each, 10 µg/ml Hoechst 33258 staining solution (C0021, Solarbio, Beijing, China) was added and incubated in a 37 °C incubator for 20 min. After washing with 1× PBS 3 times for 5 min each, a drop of anti-fluorescence attenuation mounting medium (S2100, Solarbio, Beijing, China) was added, and the stained cells were observed under a fluorescence microscope. At least 200 cells from three fields were randomly selected for detection and quantification in each experiment, and the percentage of apoptotic cells was calculated with the following formula: apoptotic cells/total number of cells ×100.

MDA content and SOD activity in the cells

The MDA content and SOD activity in the cells were determined. After centrifugation, the medium was discarded, 1 mL of extraction solution was added for every 5 million cells, the cells were homogenized by ultrasonication and centrifuged at 8000 g and 4 °C for 10 min, and finally the supernatants were collected for analysis. We used a commercially available Malondialdehyde (MDA) Content Assay Kit (BC0025, Solarbio, Beijing, China) and Superoxide Dismutase (SOD) Activity Assay Kit (BC0175, Solarbio, Beijing, China) to measure the MDA and SOD levels in cells according to the manufacturer’s instructions.

ROS to test oxidative stress in cells

The ROS Assay Kit - Highly Sensitive DCFH-DA- (R252, Dojindo, China) was used to detect oxidative stress. Cells (2 × 104 cells/well) were seeded in a 24-well plate. After the cells had adhered, they were pretreated with 1 µM TPPU for 3 h and then incubated with 30 µM Aβ(25–35) for 48 h. The culture medium was removed, the cells were washed with HBSS buffer twice, Highly Sensitive DCFH-DA Dye working solution was added, and cells were cultured for 30 min at 37 °C and 5% CO2 in an incubator. The working solution was removed, the cells were washed with HBSS buffer twice, HBSS buffer was added again, and the cells were observed with a fluorescence microscope.

Interleukin-1β (IL-1β), 11,12-epoxyeicosatrienoic acid (11,12-EET), and 14,15-EET levels were determined by ELISA

The homogenized cells were centrifuged to remove cell debris. According to the manufacturer’s instructions, IL-1β levels were measured using the Human IL-1β High Sensitivity ELISA Kit (EK101BHS-AWI, MULTI SCIENCES, China). The optical density (450 nm and 570 nm) of experimental wells was compared with the standard curve.

The medium of homogenized cells was collected, repeatedly frozen at −20 °C and thawed 3 times, and then filtered by glass fiber for analysis. An 11,12-epoxide eicosatrienoic acid (11,12-EET) ELISA kit (MM 927114O1, Meimian, Jiangsu, China) and a 14,15-epoxidized eicosatrienoic acid (14,15-EET) ELISA Kit (MM 927107O1, Meimian, Jiangsu, China) were used to measure the levels of 11,12-EET and 14,15-EET according to the manufacturer’s instructions. The absorbance (OD value) was measured at 450 nm, and 11,12-EET and 14,15-EET levels in the samples were calculated according to the standard curve.

Western blot

Human cells were washed 3 times with 1× PBS, and the cells were lysed with RIPA lysis buffer (R0010, Solarbio, Beijing, China) supplemented with protease inhibitor mixture (P6730, Solarbio, Beijing, China) and PMSF (P0100, Solarbio, Beijing, China). The lysed cells were collected and centrifuged at 12,000 rpm and 4 °C for 20 min. After the protein concentration was determined with a BCA protein detection kit (P0010, Beyotime, China), the protein was denatured in loading buffer at 100 °C for 10 min. An equal amount of protein was resolved with a 10–12% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Immobilon, USA). After incubation with 5% skim milk (P1622, Applygen, China) for 2 h, the PVDF membrane was incubated with primary antibodies overnight at 4 °C. Antibodies include rabbit anti-TNF-α (ER65189, HUABIO Antibodies, China, 1:1000), rabbit anti-EPHX2 (HA721056, HUABIO Antibodies, China, 1:500), rabbit anti-TLR4 (AF7017, Affinity Biosciences, China, 1:1000), rabbit anti-MyD88 (ET1610-81, HUABIO Antibodies, China, 1:1000), rabbit anti-p38 MAPK (#8690, Cell Signaling Technology, USA, 1:1000), rabbit anti-P-p38 MAPK (#4511, Cell Signaling Technology, USA, 1:1000), rabbit anti-NF-κB p65 (ET1603-12, HUABIO Antibodies, China, 1:500), rabbit anti-P-NF-κB p65 (#3033, Cell Signaling Technology, USA, 1:1000), and rabbit anti-β-actin (#4970, Cell Signaling Technology, USA, 1:1000) antibodies. The PVDF membranes were washed with 1× TBST 3 times and incubated with HRP-conjugated goat anti-rabbit IgG antibody (HA1001, HUABIO Antibodies, China) for 2 h. The protein bands were detected by an Ultra High Sensitivity ECL Kit (GK10008, GLPBIO, USA). β-actin (#4967, Cell Signaling Technology, USA, 1:1000) used as loading control. ImageJ software was used to quantify the protein expression levels.

Real-time PCR for gene expression analysis in cells

Real-time PCR was used to measure the mRNA expression of inflammatory markers in human cells. Total RNA was extracted from human cells by the TRIzol method (T9424, SIGMA, USA). RNA concentrations were measured using a spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was performed using Evo M-MLV RT Premix for qPCR (AG11706, AG, China), and cDNA was synthesized according to the manufacturer’s instructions. SYBR®Green Premix Pro Tag HS qPCR kit (AG11701, AG, China) was used to perform quantitative PCR amplification of the designated genes on a fluorescence quantitative PCR instrument (A25619 CN, Thermo Fisher Scientific, USA). After initial denaturation (95 °C, 30 s), amplification was performed 40 times at 95 °C for 5 s and 60 °C for 30 s. GAPDH was used as the internal reference. The relative gene expression was calculated by the 2-ΔΔCt method. The human cell primer sequences are shown in Table 1. 

Table 1 Primer sequences to assay gene expression in human cellsStatistical data analysis

All the experiments were repeated n ≥ 3 times, and all the data were analyzed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA) with Student’s t test or one-way analysis of variance (ANOVA) and Bonferroni post event test. The data are expressed as the mean ± SEM. GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used to generate the graphs. Differences were considered statistically significant when P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001).