Animals and Treatments
The experiment was performed on C57BL/6 mice (male, 8 weeks, 18-22 g). These mice were obtained from the Qinglongshan Animal Breeding Farm (Nanjing, China), Jiangning District, Nanjing City, Jiangsu Province and kept in standard experiment room conditions. The mice were randomly housed in cages with a light/dark cycle of 12 hours (light on at 7:00 in the morning), a humidity of 60%, and a temperature of 24 ± 1°C, and they could drink and eat freely. All procedures were strictly in accordance with the regulations and general recommendations of China's laboratory animal management regulations.
To establish a chronic MPTP/p PD mouse model: MPTP (20 mg/kg, dissolved in normal saline (NS), 0.1ml/10g; Selleck, S4732) was injected subcutaneously into mice, and probenecid (250mg/kg, dissolved in dimethyl sulfoxide (DMSO), 10μl/10g; Macklin, P822732) was injected intraperitoneally one hour later to delay metabolism of MPTP, twice a week for 5 weeks. Control mice were treated with NS only. Part of the experimental group and control group mice were randomly subjected to behavior testing and sample collection within 7 days after 1/2/3/4/5 weeks of administration.
Using TLR2 inhibitor CU-CPT22 to interfere in subacute MPTP PD mouse model: The mice were randomly divided into 4 groups: Saline group, MPTP group, CU-CPT22 group and MPTP+CU-CPT22 group. The saline group was injected with NS (0.1ml/10g) subcutaneously, the MPTP group was injected with MPTP (30mg/kg, dissolved in NS, 0.15ml/10g) subcutaneously, the CU-CPT22 group was injected intraperitoneally with CU-CPT22 (3mg/kg, dissolved in DMSO, 40μl/10g; Selleck, S8677) [17] and in the CU-CPT22+MPTP group, 30 minutes after intraperitoneal injection of CU-CPT22 (3mg/kg, dissolved in DMSO, 40μl/10g), MPTP (30mg/kg, dissolved in NS, 0.15ml /10g) was injected subcutaneously. Once a day for 5 consecutive days. Sample collection was performed within 7 days after the last dose.
Behavioral Analysis
The Y-maze was a "Y"-shaped three-arm labyrinth made of polyvinyl chloride (PVC) board. The angle of each arm was 120 degrees. The size of each arm was 30cm×8cm×15cm (length × width × height). Each arm had a movable partition in the center. The three arms were randomly defined as: Starting arm, Novel arm and Other arm. Each arm was marked with different geometric figures, allowing mice to form visual differences. The Y-maze experiment consisted of a training period and a test period, with an interval of 2 hours. During the training period, each Novel arm was blocked with a movable baffle with the same color, and the mouse was placed in the Starting arm with its head facing the wall and allowed to explore the Starting arm and the Other arm freely for 5 minutes. During the test period, the baffle was removed, the mouse was placed in the Starting arm with its head facing the wall, and the mouse was free to explore the three arms for 5 minutes. Before and after the experiment, the device was wiped with 75% ethanol to avoid the interference of residual odor to the experiment. A camera captured and recorded the movement of the mouse at 1.5 meters above the Y-maze. The analysis system (TopScanLite Version 2.00 Animal Routine Experimental Behavior Analysis System) connected to it can analyze the percentage of bouts and the percentage of duration the mouse entered the Novel arm. All tests were carried out by two independent experimenters who did not understand animal handling methods.
Tissue Section Preparation
The mice were anesthetized with sodium pentobarbital and perfused with NS and 4% paraformaldehyde (PFA) in sequence. The brains were harvested and fixed in 4% PFA.
Paraffin section preparation: After dehydration, the tissue was embedded in paraffin and cut into 5μm coronal sections. Bake, dewax and rehydrate the paraffin slices.
Frozen section preparation: The tissue was dehydrated in 20% sucrose- phosphate buffer saline (PBS) and 30% sucrose-PBS and fixed with opti-mum cutting temperature (OCT) compound, and cut into 25μm coronal sections. The brain slices were immediately rinsed three times with 0.01M PBS.
Immunohistochemistry Staining (IHC)
Treat the slices with 3% hydrogen peroxide (H2O2) and let them stand for 20 minutes in the dark at room temperature to quench the endogenous peroxidase activity. After washing with PBS, citrate buffer was used for antigen retrieval. The sections were blocked with PBST (PBS containing 0.3% Triton X-100) containing 5% bovine serum albumin (BSA) at room temperature for 1 hour. Subsequently, the sections were incubated overnight at 4°C with the following primary antibody: anti-p-α-syn (1:1,000; Abcam, ab51253). The sections were washed with PBS and incubated with the corresponding secondary antibody for 1 hour at room temperature: Peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (1:500; Yeason, 33101ES60). Rinse the samples and visualize the results by Diaminobenzidine (DAB) Chromogenic Kit (KeyGEN BioTECH, KGP1045) and Hematoxylin-Eosin Staining (Solarbio, G4070 and G1100). After dehydrating to make each part transparent, use neutral gum to mount the slide. Use Olympus BX51 microscope and MicroBrightField Stereo Investigator stereology system to observe and analyze images.
Immunofluorescence Staining (IF)
The tissue sections were blocked with PBST containing 5% BSA for 1 hour at room temperature. Subsequently, the sections were incubated overnight at 4°C with the following primary antibodies: anti-Tyrosine hydroxylase (TH) (1:1,000; Sigma, T1299), anti-p-α-syn (1:1,000; Abcam, ab51253), anti-ChAT (1:200; Abcam, ab181023), anti-GFAP (1:500; Abcam, ab33922), anti-TLR2 (1:250; Abcam, ab209216), anti-p62 (1:250; Proteintech, 18420-1-AP) and anti-IL-1β (1:200; Santa Cruz, sc-52012). Wash the sections with PBS, then incubate with the corresponding secondary antibody for 1 hour at room temperature: Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1,000; Invitrogen, A-21202) ), Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (1:1,000; Invitrogen, A-21422), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1,000; Invitrogen, A-11008) and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (1:1,000; Invitrogen, A-31572). Use Hoechst (1:1,000; Cell Signaling Technology, 4082) to stain and mount the slides. Use ZEISS AXIO fluorescence microscope and MicroBrightField Stereo Investigator system to observe and analyze images.
Western Blotting (WB)
The mice were sacrificed after the behavioral test. The midbrain and hippocampus were quickly removed from the entire brain and cooled in liquid nitrogen. Tissue samples were homogenized in radio immunoprecipitation assay (RIPA) lysate (containing protease inhibitor and phosphatase inhibitor mixture), then centrifuged at 16,000g at 4°C for 15 minutes, and the supernatant was collected. The protein concentration was determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher, 23225). Use sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 8-12%) electrophoresis to separate 30 µg protein aliquots of each sample, and transfer them to polyvinylidene fluoride (PVDF) membrane (Millipore, ISEQ00010) by electrophoresis and transfer. Block with TBST (Tris buffered saline (TBS) containing Tween 20) containing 5% skimmed milk powder for 2 hours. Then, the membrane was incubated overnight at 4°C with the following primary antibodies: anti-TH (1:5,000; Sigma, T1299), anti-α-syn (1:2,000; Abcam, ab1903), anti-p-α-syn (1:2,000; Abcam, ab51253), anti-p-tau (1:500; Invitrogen, MN1020), anti-Aβ1-42 (1:800; Abcam, ab12276), anti-IL-1β (1:200; Santa Cruz, sc-52012), anti-NF-κB (1:800; Bioss, bs-0465R), anti-ChAT (1:2,000; Abcam, ab181023), anti-GFAP (1:2,000; Abcam, ab33922), anti-TLR1 (1:200; Santa Cruz, sc-514399), anti-TLR2 (1:1,000; Abcam, ab209216), anti-TLR3 (1:200; Santa Cruz, sc-32232 ), anti-TLR4 (1:200; Santa Cruz, sc-293072), anti-TLR7 (1:200; Santa Cruz, sc-57463), anti-NALP1 (1:100; Santa Cruz, sc-390133), anti-AIM2 ( 1:200; Santa Cruz, sc-515514), anti-NLRP3 (1:1,000; AdipoGen, AG-20B-0014-C100), anti-ChAT (1:2,000; Abcam, ab181023), anti-GFAP (1:2,000; Abcam, ab33922), anti-LC3 (1:1,000; Proteintech, 14600-1-AP), anti-p62 (1:1,000; Proteintech, 18420-1-AP) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:2,000; Proteintech, 60004-1-Ig). Wash the membrane with TBST and incubate with the corresponding secondary antibody for 1 hour at room temperature: Rhodamine Red-X AffiniPure Goat Anti-Mouse IgG (H+L) (1:5,000; Yeason, 33210ES60), Peroxidase-Conjugated Goat Anti -Rabbit IgG (H+L) (1:5,000; Yeason, 33101ES60). SuperSignal™ West Femto Maximum Sensitivity Substrate kit (Thermo Fisher, 34096) and Tanon 5200 automatic chemiluminescence imaging analysis system were used for detection. Use ImageJ software (National Institutes of Health) to quantify the band intensity. The protein level was determined by normalization to the level of GAPDH and was presented relative to the control.
Statistical Analysis
Use GraphPad Prism 7 to analyze the results. The t-test was used for single factor analysis. In all studies, P <0.05 was considered statistically significant. The data graph was plotted as Mean ± SD, and “n” represents the number of samples in each group.