In vivo
Animals
Thirty healthy adult Sprague-Dawley (SD) rats (250~280 g) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (SCXK (Hu) 2017-0005, Shanghai, China). All the rats were allowed to access food and water free and living in standard condition (humidity: 55~70%; room temperature: 23±2 ºC; light cycle: 12-h light/dark). All animal assays were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal experiments were in accordance with the guidelines of laboratory animal care and were approved by the Hangzhou Eyong Biotechnological Co., Ltd. Animal Experiment Center (Hangzhou, China).
Established KOA model and experimental group
The rats were divided into 3 groups randomly (n=10/group): control, KOA and KOA + polydatin. The KOA was induced according to previously study [12]. In detail, after anesthetized with pentobarbital (40 mg/kg), the KOA and KOA + polydatin rats were fixed in supine position. And the right medial knee joint was made a longitudinal incision (1.5 cm), the medial collateral ligament and joint capsule was exposed and broken. Then dislocated the patella to expose and cut off the anterior cruciate ligament. Later, the medial meniscus was extirpated and cut off the posterior crucial ligament. Finally, established KOA rat models were verified by drawer test. After fixed the patella, sutured the joint capsule and skin. In order to avoid infection, all the rats were treated with penicillin (40,000 U/mL, 1 mL) by intramuscularly injected after the surgery for consecutive 3 days. The normal group rats not received any treatment. All the rats were fed and drank freely and normally. After modeling 4 weeks [13], the KOA + polydatin rats were intraperitoneally injected with polydatin (4 mg/kg, dissolved in saline), while normal rats and model rats intraperitoneally injected with same volume normal saline and continued for 30 d.
Hematoxylin-eosin (HE) staining
The cartilage tissues of each group rats were fixed in 4% paraformaldehyde for 24 h at room temperature. Then, the cartilage tissues were routinely dehydrated by gradient ethanol, embedded, and cut into 4 μm sections. Then the sections were stained with HE. After washed, the slices were observed under an optical microscope (Olympus, Japan) to detected the histopathological changes. The cartilage tissues were scored as following: 0 point, normal; 1 point, mild cell infiltration and synovial invasion; 2 points, bone invasion; 3 points, more serious cell infiltration and synovial invasion accompanied by bone invasion; 4 points, severe cell infiltration and synovial invasion accompanied by bone invasion.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The apoptosis rate of chondrocytes was assessed by TUNEL assay. After fixed with 4% paraformaldehyde and dehydrated, the brain tissue was embedded and sectioned into 4-µm. When the slices were dewaxed and transparent, 100 µL TdT enzyme buffer was added and incubated at 37 °C for 1 h in a dark and humectation environment. Then the slices were incubated with 100 µL Streptavadin-HRP at 37 °C for 30 min. After washed, the sections were stained with diaminobenzidine (DAB) (MD912068, MDL, China), and re-stained with hematoxylin. Six fields were randomly selected for each group and quantitative analysis was detected by Image J. Apoptosis index (%) = TUNEL-positive neurons/total neurons×100%.
Western blotting
Radioimmunoprecipitation assay (RIPA) was used to extracted protein from the ischemic cartilage tissues. After crushed in liquid nitrogen, the tissue lysates were collected and centrifugated at 12000 g for 20 min to obtained the supernatant. After quantified the concentration of total cellular proteins by bicinchoninic acid (BCA, pc0020, Solarbio, China) kit. The proteins were loaded onto SDS-PAGE gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life, USA). After blocked by 5% dried skim milk for 30 min, primary antibodies were added and incubated with membranes overnight at 4 ºC, then incubated with secondary antibody at room temperature for 1 h. Enhanced chemiluminescence (ECL) (Solarbio, Beijing, China) method was used to detected the expression of protein. Image J software (National Institutes of Health) was used to detected the band intensities. The relative protein level was normalized to that of GAPDH. The primary antibodies including anti-LC3A/B antibody (1:500, AF5402, Affinity, USA), anti-Beclin 1 antibody (1:500, AF5128, Affinity, USA), anti-GAPDH antibody (1:3000, AF7021, Affinity, USA).
In vitro
Cell culture and grouping
C28/I2 cell lines were cultured in DMEM/F12 medium (SH30243.01, Hyclone, USA) at 37 °C and under 5% CO2 atmosphere. The medium was changed every 48 h. When the cells reached about 80-90% confluence, cells were used for the further experiments. The C28/I2 cell lines were seeded into six plates or flasks and divided into 3 groups: control (only DMEM/F-12), LPS (DMEM/F-12 contain 5 μg/mL LPS, cultured for 24 h), LPS+polydatin (DMEM/F-12 contain 5 μg/mL LPS and 1 μM polydatin, cultured for 24 h) [14].
MTT
In order to detected the effect of LPS and polydatin on C28/I2 cells, MTT assay was performed. Briefly, C28/I2 cells (1×105 cell/L, 100 µl/well) which was logarithmic growth phase were seeded into 96-well plates and incubated in a incubator (37°C, 5% CO2) for 24 h. After treated with LPS and polydatin for 24 h, 10 μL MTT was added and cultured for another 24 h. Finally, the plates were detected at 450 nm.
Flow Cytometry
Logarithmic growth phase C28/I2 cells (1.2×106/well) were seeded into 96-well plates. After induced by LPS and treated with polydatin for 24 h, the concentration of C28/I2 cells were diluted into 1×106 cells/mL by cool PBS. Then, annexin V/PI staining was processed to assess the anti-apoptotic effects of polydatin on C28/I2 cells induced by LPS. C28/I2 cells were re-suspend in binding buffer (100 μL), and co-incubated with Annexin V-FITC (5 μL) and PI solution (10 μL) at room temperature in the dark for 15 min. After added 400 μL binding buffer, the solution was analyzed by flow cytometry within 1 h.
Western blotting
After treated with LPS and polydatin, the C28/I2 cells were lysed with RIPA buffer to obtained protein. After measured the concentrations by BCA Kit, proteins were also separated by SDS-PAGE and transferred onto PVDF membranes. After blocked with 5% dried skim milk for 2 h, the membranes were incubated with dilute solution of antibodies at 4 °C overnight and secondary antibody for 2 h at room temperature. ECL method was used to detected the expression of protein. Image J software was used to detected the band intensities. The relative protein level was normalized to that of GAPDH. The primary antibodies including anti-LC3A/B antibody (1:500, AF5402, Affinity, USA), anti-Beclin 1 antibody (1:500, AF5128, Affinity, USA), phospho-AMPK alpha (Thr172) antibody (1:500, AF3423, Affinity, USA), anti-AMPK alpha antibody (1:500, AF6423, Affinity, USA), phospho-mTOR (Ser2448) antibody (1:500, AF3308, Affinity, USA) and anti-mTOR antibody (1:500, AF6308, Affinity, USA), anti-GAPDH antibody (1:3000, AF7021, Affinity, USA).
Effect of Dorsomorphin 2HCl on C28/I2 cells
The inhibitor of AMPK was used to assess the mechanism of polydatin on the C28/I2 cells. The C28/I2 cells were divided into four groups: control (only DMEM/F-12), LPS (DMEM/F-12 contain 5 μg/mL LPS, incubate for 24 h), LPS+polydatin (DMEM/F-12 contain 5 μg/mL LPS and 1 μM polydatin, incubate for 24 h), and LPS+polydatin+Dorsomorphin 2HCl (After treated with 10 μM Dorsomorphin 2HCl for 1 h, C28/I2 cells were cultured in DMEM/F-12 with 5 μg/mL LPS and 1 μM polydatin for 24 h) [15]. Then, the role of Dorsomorphin 2HCl on the proliferation and apoptosis of C28/I2 cells and the mechanism were also detected according to the above description.
Statistical analysis
All the assay data were analyzed by SPSS 19.0 statistical software. The data were shown as `χ ± s. Multi-group comparison were analysed by One-way-ANOAY. Inter-group comparisons were analysed by SNK. Variance data were analysed by Kruskal-Wallis H test. P < 0.05 was seen as statistically significant.