2.1. Induction and treatment of collagen-induced arthritis in mice
Male DBA/1 mice (8-10 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd, and were housed in specific pathogen free laboratory of Institute of Clinical Pharmacology, Anhui Medical University with a 12 light/12 dark cycle. All protocols of these experiments have been approved by the Ethics Committee of Institute of Clinical Pharmacology, Anhui Medical University (Approval ID: PZ-2020-045). After a week of adaptive feeding, the mice were injected subcutaneously with 0.1 ml of chick type II collagen (CCII) emulsion (2 mg/ml immunization grade CCII (Catalog #:20011, Chondrex, Inc. Woodinville, WA, USA) dissolved in 0.05M acetic acid was emulsified with equal volume of Complete Freund’s Adjuvant (CFA) containing 2 mg/ml heat-killed M. Tuberculosis H37 RA (Catalog #:7009, Chondrex, Inc. Woodinville, WA, USA) at the base of tail on day 0. Then on day 21, the same dose of CCII emulsion was subcutaneously injected into the tail or back of the mice to enhance immunity .
95% of mice injected with CCII emulsion successfully developed arthritis, then CIA mice were randomly divided into vehicle treatment; 5 mg/kg, 10 mg/kg, or 20 mg/kg of Ziyu I (Catalog #: B20294, HPLC≥98%, Shanghai yuanye Bio-Technology Co., Ltd, Shanghai, China) treatment; or 2 mg/kg of MTX treatment groups. Treatment was started on D28 with Ziyu I once daily and MTX once every 3 days for 4 weeks, with global assessments measured every 7 days before booster immunization and every 3 days after the onset of joint inflammation. The body weight gain (g) of each mouse was calculated by subtracting the body weight of each mouse on day 0. The arthritis severity of each paw was evaluated by using a macroscopic scoring system ranging from 0 to 4 (0, paws with no swelling or focal redness; 1, mild but definite redness and swelling of the ankle or wrist or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2, moderate redness and swelling of the ankle or wrist; 3, severe redness and swelling of the entire paw, including digits; and 4, maximally inflamed limbs with the involvement of multiple joints). The cumulative score for all four paws of each mouse was used as the polyarthritis index and had a maximum value of 16 .
2.2. Histopathological examination of the spleen and joints
The left rear ankle joint and spleen were collected after treatment. Thymus was weighted to calculate the thymus index by comparing the thymus weight (g) to body weight (g). All mice were sacrificed on day 55. The fur and muscle were removed from ankle joint before fixation. The knee joints were collected from indicated mice and fixed in formalin for 24 h, then were subjected to decalcification in 10% EDTA. The joints and spleens were embedded into paraffin blocks and 4 μm sections were prepared for H&E staining. The slides were scanned in a 3D HISTECH panoramic scanner (3DHISTECH Ltd. Budapest, Hungary) and analyzed using included software caseviewer. The histopathological evaluation indexes and grades of joints and spleen of mice were based on previous reports. The severity of joint destruction was classifed from grade 0 to grade 4 for intensity of the lining layer hyperplasia, mononuclear cell infltration, and pannus formation. Five parameters were applied for the pathological evaluation of the spleen: the amount of red pulp, the total number of germinal centres (GCs), cellularity of the periarteriolar lymphoid sheath (PALS), lymphoid follicles, and marginal zone. The infammation was ranked on a numerical scale from 0–4 (severe change) .
2.3. Serum cytokine detection with ELISA
After the mice were anesthetized, peripheral blood was collected for coagulation at 37℃ for 2 hours and centrifuged at 2500 RPM for 30 minutes. The supernatant was retained, and the cytokines (including IL-17 and TGF-β) were detected using Elisa kits . Mouse Interleukin-17 (IL-17) ELISA Kit (Catalog #: ml037866-J) and Mouse TGF-β ELISA Kit (Catalog #: ml057830-J) were products from Shanghai Enzyme-linked Biotechnology Co., Ltd. Standard curve was used to determine the amount of TGF-β or IL-17 in unknown samples and the absorbance were recorded at 450 nm using a BioTek Elx×808 microplate reader (Lonza Group, Ltd. Basel, Switzerland).
2.4. Viability of T lymphocytes
The vitality of T lymphocytes was determined by Cell Counting Kit-8 (CCK-8) assay as previously described . After treatment, T cells of thymus were prepared from the thymus of mice. 1 × 106 splenic cells were seeded evenly into each well of a 96-well plate and stimulated with Concanavalin A (ConA) (Catalog #: FMS-FZ203, FcMACS, Nanjing, China) at final concentration of 5mg/L in a 5% CO2 cell incubator for 16 hours. Two hours before the termination, 10 μl of CCK-8 solution (Catalog #: E-CK-A361, Elabscience Biotechnology, Wuhan, China) was added into each well and the cells were continued with the culture. The absorbance at 450 nm was measured on a BioTek Elx×808 microplate reader (Lonza Group, Ltd. Basel, Switzerland).
2.5. Flow cytometry
Flow cytometry was applied to detect the percentage of T lymphocyte subsets in the spleen of treated mice. After individual treatment, the mice were sacrificed through anesthetization. The spleens were ground to separate lymphocyte cells using mouse lymphocyte separation medium (Catalog #: DKW33-R0100, Dakewe Biotech Co., Ltd. Shenzhen, China). 1×106 cells were incubated with FITC Rat Anti-Mouse CD4 (Clone RM4-5, Catalog #: 553046, BD Biosciences, Franklin Lakes, NJ, USA) / APC Rat Anti-Mouse CD25 (Clone PC61, Catalog #: 561048, BD Biosciences, Franklin Lakes, NJ, USA) / PE Anti-Mo/Rt Foxp3 (Clone FJK-16a, Catalog #: 2344844, Elabscience Biotechnology, Wuhan, China)/ APC Rat Anti-Mouse CD3 (Catalog #: 554832, BD Biosciences, Franklin Lakes, NJ, USA) /PE Rat Anti-Mouse IL-17A (Catalog #: 553046, BD Biosciences, Franklin Lakes, NJ, USA). The samples were sufficiently mixed and incubated at 4 °C for 30 min in dark, and they were detected on Cytoflex Platform (Cytoflex S, Beckman Coulter Life Sciences, Indianapolis, IN, USA). The percentage of CD4+IL-17A+ Th17 cell and CD4+CD25+Foxp3+ Treg cell in total T cell were analyzed by using CellQuest™ software (BD, San Diego, CA, USA).
2.6. Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from spleen cells using Trizol reagent following the manufacturer’s protocol and then total RNA was extracted and was reversely transcribed into complementary DNA (cDNA) using cDNA synthesis kit (Catalog #: 634926, Takara Bio Inc. Otsu, Shiga, Japan) referring to the instructions. The specific primers for RORγt, Foxp3 and GAPDH are listed in Table 1. Subsequently, the cDNA template was amplified on a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) by applying a Fast SYBR Green Master Mix (Catalog #: 4385612, Thermo Fisher Scientific, Waltham, MA, USA). Expression changes were calculated with normalization of GAPDH values with the 2−ΔΔCt method. The primers sequences for the RT-qPCR are listed below as Table 1.
2.7. Molecular docking analysis
There is no complete protein three-dimensional structure of Akt1 in the PDB protein database. Obtain the protein primary structure of Akt1, that is, the amino acid sequence, from the NCBI-Protein database (https://www.ncbi.nlm.nih.gov/protein/). Use the homology modeling function of Discovery Studio 2020 software to predict the three-dimensional structure of Akt1. In homology modeling, use PDB _nr95 (PDB non-redundant structure database) to find the template, select the sequence with higher sequence identity, and perform the Build Homology Models operation in the DS-Create Homology Models section to obtain the three-dimensional Akt1 structure. Docking simulation of Ziyu I with Akt1 protein was carried out with the program Discovery Studio 2020 (DS 2020, Accelrys Software Inc.). The active sites were defined and sphere of 13.7 Å according to the important amino acid residues generated around the Akt1 active site pocket by the From Receptor Cavities program in the software Define Site settings, with the active site pocket of model using LipDock, molecular dynamics (MD) simulated-annealing based algorithm module from DS 2020. The structure of protein, substrate was subjected to energy minimization using CHARM force field as implemented in DS 2020. A full potential final minimization was then used to refine the substrate poses. Based on LipDock, energy docked conformation of the substrate was retrieved for post-docking analysis .
2.8. Western blotting
The expression of Akt、mTOR and phospho-Akt (p-Akt)、phospho- mTOR (p- mTOR) in splenic T cells were determined by Western blotting. Cell samples separated from individual group of mice were lysed with lysis buffer containing 10 mM of PMSF and 10 mM of phosphatase inhibitor on ice by an ultrasonic homogenizer. The soluble protein was collected by centrifugation at 12000 rpm for 30 min at 4°C and was quantified using a Pierce BCA Protein Assay Kit (Catalog #: 23227, Thermo Fisher Scientific, Waltham, MA, USA). The protein was denatured by mixing with 5× loading buffer, and then was separated by 10% SDS polyacrylamide gel electrophoresis. The PVDF membrane with transferred protein was blocked with 5% no-fat milk in 0.05% Tween 20-PBS at 37 °C for 2 hours followed by the incubation of primary antibodies against mTOR (1:200, Catalog #: 55306F, ABMART, Shanghai China), p-mTOR (1:500, Catalog #: 293133, Santa Cruz Biotechnology, CA), Akt1/2/3 mAb (1:200, Catalog #: 55561F, ABMART, Shanghai China), Phospho-Akt (Ser473) mAb (1:200, Catalog #: T56569F, ABMART, Shanghai, China), GAPDH (1:5000, Catalog #: AF0911, Affinity Biosciences, Changzhou, China) overnight at 4°C. After rinsing with 0.05% Tween 20-PBS 3 times, the membrane was incubated with corresponding secondary antibody (1:10000, Goat Anti-Rabbit IgG (H+L) HRP, Catalog #: S0001, Goat Anti-Mouse IgG (H+L) HRP, Catalog #: S0002, Affinity Biosciences, Changzhou, China) at 37°C for 2 hours. By applying the ECL Western Blotting Substrate (Catalog #: 32106, Thermo Fisher Scientific, Waltham, MA, USA), the membrane was imaged on the Image Quant LAS 500 imager (GE Healthcare Systems, Chicago, IL, USA) and the bands of the target protein were semi-quantified by GAPDH using ImageJ software (version 1.42q, NIH) .
In situ expression of mTOR/p-mTOR in T cells of treated mice was detected by immunofluorescence. 4 μm thick paraffin sections of mouse spleen tissues from each group were deparaffinized, Subsequently, the tissue was infiltrated with 0.1% Triton X-100 for 30 min and sealed in PBS containing 1% BSA for one hour in the dark. Each tissue section was incubated with a mixture of rabbit mAb anti-mTOR (1:100, Catalog #: 55306F, ABMART, Shanghai China) and mouse mAb anti-p-mTOR (1:100, Catalog #: 293133, Santa Cruz Biotechnology, CA) and PE Anti-Mouse IL-17A antiboy (1:00, Catalog #: 1995433, BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C. After rinsing with PBS for 3 times, the samples were incubated with the corresponding fluorescent secondary antibody Alexa Fluor 647 AffiniPure Goat Anti-Mouse IgG (Catalog #: 115-605-205, Jackson ImmunoResearch Inc., West Grove, PA, USA) or Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (Catalog #: 115-585-003, Jackson ImmunoResearch Inc., West Grove, PA, USA) for 2 hours. The nuclei were stained with DAPI for 10 min after the sections were washed 3 times with PBS, and the slides were dried and sealed with a drop of anti-fluorescence quenching mounting solution. Digital images were acquired on a Leica TCS SP8 laser scanning confocal microscope, ImageJ software (version 1.42q, NIH) was used to quantify the mean fluorescence intensity of mTOR and p-mTOR.
2.10. Statistical analysis
Data were expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine significant differences between groups. Values of P less than 0.05 were considered to be significant.