Salidroside (purity: 99%, CAS: 10338-51-9) was purchased from Mansite Bio-technology (Chengdu, China). Metformin, rosiglitazone, GW9662, and Compound C were purchased from Sigma-Aldrich (St. Louis, MO, United States). Antibodies for IBA-1 and PPARγ were purchased from Abcam (Cambridge, United Kingdom). Antibodies for AMPK, p-AMPK (Thr172), NF-κB p65, CD86 were purchased from Cell Signaling Technology (MA, United States). Antibody for CD163 and GAPDH, and all second antibodies were purchased from Proteintech (Wuhan, China). HE staining kit, DAB kit, and hypersensitive ECL chemiluminescence kit were purchased from Solarbio (Beijing, China). Other reagents were purchased from Sinopharm Reagent Group Co. Ltd (Shanghai, China).
Schrödinger (2018) was used to conduct molecular docking between AMPK and salidroside to explore the binding mode. Firstly, crystal structure of AMPK from the (PDB:4CFE) was get from https://www.rcsb.org/. The structure of AMPK was imported into the Protein Preparation Wizard module to optimize protein structure, for removing all crystal water molecules, correcting side chains with missing atoms, adding hydrogen atoms. Then using the OPLS3 force field to distribute protonated states and partial charges. The crystal structure energy was minimized until the root mean square deviation (RMSD) of the non-hydrogen atoms reached 0.3 Å. After that, the LigPrep module was used to optimize 11 small ligand molecules to generate ionization and tautomerism states between pH 6.8 and 7.2. The OPLS3 force field was again used to minimize the energy of small molecules, the maximum number of stereoisomers was set to 32. Finally, the Receptor Grid Generation module was used to generate lattice files for docking. Glide module was used for molecular docking, and Extra Precision (XP) was used for analyzing. The docking score was recorded to assess the docking effect between salidroside and AMPK.
Animals and orthopedic surgery model
Adult male C57BL/6 mice (15 months of age) were provided by the Experimental Animal Center at Nanjing Medical University, Nanjing, China. All experimental protocols and procedures were approved and licensed by Nanjing Medical University Animal Care and Use Committee in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.
Orthopedic surgery was imitated by the model of tibial fracture with intramedullary fixation . The mice were anesthetized with sodium pentobarbital (35 mg/kg). The left hind limb of the mouse was shaved and disinfected. After the tibia was exposed, a 0.3 mm pin was inserted into the tibial medullary cavity. Then the fracture occurred, and the skin was sutured with 4.0 nylon. Lidocaine cream was used locally for analgesia. The mice in sham group received all perioperative operations, except the tibial fracture and intramedullary fixation. The mice in surgery + salidroside group were intraperitoneally injected with different concentrations of salidroside (20, 40, 80 mg/kg, once a day) for 5 days before surgery.
Morris water maze (MWM) test
The MWM test was performed as described previously . Briefly, a transparent cylindrical escape platform with a diameter of 10 cm was placed in one quadrant of 1.25 m-diameter circular water pool filled with water, and the platform was 1 cm below the water surface. The latency was recorded, which is the time it takes for the mouse to enter the water and climb onto the platform. If the mouse did not find the platform within 60 s, it would be guided to the platform, stay for 20 s, and then be taken out. The experiment was carried out at the same time every day for 5 consecutive days. On the 6th day, the platform was removed to start the probe test, and the time for the mouse to enter the target quadrant and the crossing number of the position of the platform within 60 s was recorded.
Section and staining of brain tissue
After the mice were sacrificed, their brains were taken out and fixed in 4% paraformaldehyde for 24 h. After the fixed mouse brain tissue was dehydrated, it was soaked in xylene and placed in paraffin melted at 65°C for 4 h. Then, the mouse brain tissue was prepared into a wax block using a paraffin embedding machine. Mouse brain tissue was sliced into 5 μM slices using a paraffin rotary microtome. After the paraffin sections were baked, they were deparaffinized and then soaked in xylene for 10 min. After washing and soaking with absolute ethanol, the slices were soaked in ethanol solution of decreasing concentration until placed in water.
As for hematoxylin-eosin (HE) staining, mouse brain tissue slices were covered with hematoxylin staining solution, washed after 10 min, differentiated in 1% hydrochloric acid-ethanol solution, and fully turned blue in dilute ammonia. The slices were washed again and covered with eosin staining solution for 1 min. The excess staining solution on the brain tissue was washed away. After the slices were rapidly dehydrated, they were soaked in xylene for 5 min, and then mounted with a neutral resin slicing solution. The slices were placed under an optical microscope for observation and photographs were taken.
As for immunohistochemical and immunofluorescence staining, mouse brain tissue slices were soaked in 3% hydrogen peroxide solution for 20 min. After washing with PBS, the slices were immersed in a 0.01M sodium citrate solution with a pH of 6.0 after heating to 95°C for 20 min. After natural cooling, the slices were covered with 5% BSA. After 20 min, the BSA solution was removed.
As for immunohistochemical staining, the slices were incubated with the primary antibody, IBA1 (1:100), CD86 (1:100), CD163 (1:100), and incubated overnight at 4°C in the refrigerator. The next day, after washing with PBS, the slices were incubated with diluted HRP-conjugated secondary antibody (1:100) for 2 h. After washing again, the slices were developed with DAB color reagent, and washed with water immediately after full color development. The brain tissue was covered with hematoxylin staining solution, washed after 10 min, and then fully returned to blue in dilute ammonia after differentiation with 1% hydrochloric acid-ethanol solution. The slices were washed again, and after rapid dehydration, the slices were soaked in xylene for 5 min, and then mounted with a neutral resin slicing solution. The slices were placed under an optical microscope for observation and photographs were taken.
As for immunofluorescence staining, the slices were incubated with the first antibody, goat-IBA1 (1:100) and rabbit-AMPK (1:100), or goat-IBA1 (1:100) rabbit-PPARγ (1:100), overnight at 4°C. On the second day, after the slices were washed with PBS, they were incubated with TRITC-labeled rabbit-anti-goat IgG and FITC-labeled goat-anti-rabbit-IgG for 2 h. After washing, the slices were incubated with DAPI staining solution for 5 min. After washing, the slices were mounted with glycerin gelatin mounting solution. The expression of IBA1 and AMPK or PPARγ was observed with a fluorescence microscope, photographed, and analyzed.
Cell culture and viability measurements
BV-2 cells were cultured in DMEM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in a 37℃ incubator containing 5% CO2. The cells were treated with LPS (1 μg/ml) with or without salidroside (25, 50, and 100 μM) for 12 h. Cell extracts and supernatants were collected and analyzed.
As for cell viability, after the cells were treated with or without salidroside (25, 50, and 100 μM) for 12 h, LPS (1 μg/ml) was used to induce the BV-2 cells for 24 h, after that the CCK-8 reagent (10 ul) was added to each well. After 4 h, the absorbance value of each well was measured at 450 nm using a microplate reader. According to the formula, cell viability= (OD value of detection well/mean OD of control wells) *100.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR)
For the brain tissues of the sacrificed mice and the cells collected after treating with or without salidroside for 12 h followed by LPS for 24 h, Trizol reagent was used to obtain the total RNA. After the addition of isopropanol, the supernatant was collected, and the RNA was precipitated with ethanol, and then the mRNA of mouse brain tissue and BV-2 cells was reverse transcribed into cDNA using a reverse transcription kit. The primers of IL-1β, IL-4, IL-6, IL-10, IL-12, TNF-α, IFN-γ, CCL5, CXCL1, CXCL10, CD16, TGF-β, Arg1, YM1, and GAPDH were provided in table 1. These primers were synthesized and provided by Biotech Bioengineering (Shanghai) Co., Ltd. The method was carried out with reference to the instructions of One Step TB Green® PrimeScript™ RT-PCR Kit II (Cat. No. RR086A/B, Baori Biotechnology (Beijing) Co., Ltd., Beijing, China), and the gene expression was analyzed using the ABI7500 system.
Enzyme-linked immune sorbent assay (ELISA)
After the cells were treated with or without salidroside for 12 h, LPS (1 μg/ml) was used to induce the BV-2 cells for 24 h, then the supernatant of the cells was collected. The levels of TNF-α, IL-1β, IL-6, and IL-10 in these supernatants were detected using mouse TNF-α, IL-1β, IL-6, and IL-10 ELISA detection kits, referring to the instructions. TNF-α, IL-1β, IL-6, and IL-10 ELISA test kits were purchased from Solarbio (Beijing, China).
For the brain tissues of the sacrificed mice and the cells collected after treating with or without salidroside for 12 h followed by LPS for 24 h, after lysis with Protein Lysis Buffer, the supernatant was collected after centrifugation at 12000 g/min for 10 min. After the sample was prepared, it was added to the SDS-PAGE gel and the proteins were separated by electrophoresis. The protein was transferred to the PVDF membrane, and after being fully blocked, PVDF membrane was incubated with the diluted antibodies against AMPK (1:2000), p-AMPK (1:1000), PPARγ (1:2000), and GAPDH (1:10000) at 4°C overnight. After the PVDF membrane was washed, it was incubated with the HRC-conjugated Goat Anti-Rabbit IgG for 2 h. Then a gel imaging system and a chemiluminescence kit were used to obtain the western blot photos.
Nuclear translocation of NF-κB p65
BV-2 cells were seeded into a 6-well plate with glass slides for cell climbing. After the cells were treated with or without salidroside for 12 h followed by LPS for 24 h, all cells were fixed with paraformaldehyde. After washing, the cells were covered with diluted anti-NF-κB p65 (1:100) antibody and incubated overnight. After washing the cells with PBS, the cells were covered with diluted Fluorescein (FITC)-conjugated Goat Anti-Rabbit IgG (1:100) and incubated for 2 h. After multiple washing, the cells were stained with DAPI and incubated for 5 min. After washing the BV-2 cells for at least 3 times, the cell slide was taken out, and the cell was covered on the glass slide using glycerin gelatin mounting solution. A fluorescence microscope was used to take pictures of cells.
Image pro plus6.0 was used to analyze immunofluorescence and immunohistochemistry pictures and export measurement data. Quantity One was used to analyze the blot data and perform relative quantitative analysis. After all the data were derived from the measurement data, the SPSS 23.0 statistics were used to perform a one-way analysis of variance on the measurement data first logarithm, and the measurement data are expressed as the mean ± standard deviation. P<0.05 indicated that the difference was statistically significant. Bonferroni’s posttest conducted the post hoc test. GraphPad Prism 7.0 (San Diego, CA, USA) was used to graph the measurement data.