Human subjects and ethical considerations
Eight patients with TAK who had undergone surgical treatment were recruited from Zhongshan Hospital, Fudan University from January 1, 2010 to July 31, 2015. All patients were diagnosed according to the American College of Rheumatology 1990 (ACR 1990) criteria. The clinical characteristics were collected prior to surgery and presented in Supplementary Table I. In addition, normal aortic tissues were obtained from six donors for heart or kidney transplantation with matching ages.
A total of 16 patients diagnosed with TAK were enrolled in the curcumin treatment study, whose characteristics clinical were shown in Supplementary Table II. The inclusion criteria were as follows: i) over 14 years old; ii) patients with abnormal erythrocyte sedimentation rate (ESR), symptoms, or imaging progression; iii) no adjustment of immunosuppressants in the preceding one month; and iv) signing of an informed consent form. Exclusion criteria consisted of: i) presence of a recent active infection (e.g., tuberculosis); ii) patients with organ failure; iii) patients allergic to curcumin; (iv) subjects who had used other traditional Chinese medicine in the past month; (v) women pregnant, lactating, or preparing for pregnancy.
The protocol used in this study was approved by the Ethics Committees of Zhongshan Hospital and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All patients provided written informed consent for inclusion in this study.
Cell culture and reagents
AAFs were purchased from ScienCell Research Laboratories (Cat No.6120) and grown in Fibroblast Medium-2 (FM-2, ScienCell, Cat No. 2331) supplemented with 5% fetal bovine serum (FBS, ScienCell, Cat No.0025), 1% Fibroblast Growth Supplement-2 (FGS-sf, ScienCell, Cat No.2382), and a 1% Penicillin/Streptomycin Solution (P/S Solution, ScienCell, Cat No.0503). HSP65 was cloned, expressed and purified from Mycobacterium tuberculosis virulent strain H37Rv. EtEraser Endotoxin Removal Kit (Xiamen Bioendo Technology Co.,Ltd.) was used to remove the endotoxin in HSP65. In this study, 1 μg/mL HSP65 was used to stimulate AAFs, and the level of residual Lipopolysaccharide (LPS) was determined by Endpoint Chromogenic LAL Assays (Xiamen Bioendo Technology Co.,Ltd.), which was <0.0625 EU/mL. A group named BC was set to avoid the potential effect caused by the residual LPS in HSP65 by adding 0.0625 EU/mL extra LPS. The total RNA, cell culture supernatants, and non-phosphorylated total proteins were collected after 12 h, whereas the expression of phosphorylated signaling pathway proteins were detected within 2 h after stimulation (0, 15, 30, 60, 90, and 120 min).
mRNA-seq analysis
Total RNA of AAFs stimulated by purified HSP65 (1 μg/mL) with or without pretreatment with curcumin (10 μM), as well as AAFs without any treatment was extracted for mRNA transcriptome sequencing, each group was performed in triplicate. For all samples, the original gene sequence was counted with StringTie software (Johns Hopkins University; UT Southwestern Medical Centre, USA), and the gene expression was calculated via fragments per kilobase of transcript per million fragments mapped (FPKM). FPKM ≥ 0.1 indicates that the transcript is expressed. DESeq2 software was used to screen the differentially expressed genes (DEGs) among different sample groups. The genes that met the screening criteria of | log2FoldChange |≥1 and FDR ≤ 0.05 were the DEGs between the two groups. log2FoldChange was used to range the screened DEGs.
Immunohistochemical staining
After deparaffinizing and hydrating the samples, the arterial tissue sections were repaired with a citric acid buffer solution, treated with 3% H2O2 for 25 min at room temperature (RT) protected from light to block the endogenous peroxidase activity. The sections were then incubated in 5% BSA at RT for another 30 min. The slices were subsequently incubated with the following diluted primary antibodies overnight at 4℃: HSP65 (Cell Signal Technology), CCL2 (Abcam), IL-6 (Abcam) and IL-1β (Servicebio, China), then reacted with a secondary antibody conjugated with HRP (Yesen) for 1 h at RT. The slices were developed with DAB reagent and counterstained with hematoxylin. Photographs of random sites were captured under high-power 400× magnification with Leica QWin Plus v3 software with identical setting parameters. Positive staining was measured using Image J 1.8u software (National Institutes of Health, USA). Integrated optical density of the positive stains in each photograph was measured, and the area fraction (%) of the positive stains was calculated.
Double-labelled immunofluorescence
The first few steps of performing immunofluorescence on paraffin sections were the same as those of Immunohistochemical staining, except that each section was simultaneously incubated with two of the following primary antibodies: HSP65 (Cell Signal Technology) and alpha-smooth muscle actin (α-SMA, Abcam), HSP65 and CCL2 (Abcam), HSP65 and IL-6 (Abcam), HSP65 and IL-1β (Abclonal), or α-SMA and TLR4 (Servicebio), then reacted with species-specific Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L) and Alexa Fluor 594 AffiniPure Goat Anti-Mouse IgG (H+L), or Alexa Fluor® 488 AffiniPure Goat Anti-Mouse IgG (H + L), and Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H + L) (Yesen) for 1 h at RT in the dark. Finally, the sections were sealed with an antifade Mounting Medium with DAPI (Beyotime). Photographs of random sites were captured under high-power magnification by an Olympus FV3000 laser confocal microscope. The area fraction of positive staining was measured using Image J 1.8u software.
Drugs and small molecule inhibitors
Curcumin powder (Sigma-Aldrich, Cat No.C7727) was first dissolved in 13.57 mL DMSO to prepare a 100 mM storage solution. Prior to use, the storage solution was diluted with DMSO into a 2, 10, 20, 40, 60, 80 mM working solution. AG490 (Selleck, S1143), a selective JAK2 inhibitor, was dissolved in DMSO into a 100 mM working solution. Tofacitinib (Selleck, S2789) was dissolved in DMSO into a 500 μM working solution before use. MTX (Selleck, S1210) was dissolved in DMSO into a 100 μM working solution. The final concentrations of AG490, tofacitinib, and MTX were 50 μM, 250 nM, and 50 nM, respectively. All drugs had no significant cytotoxicity at the concentration used (cell viability>95%). DMSO was added in non-intervention groups to avoid the potential deviation due to its cytotoxicity.
Cell viability and proliferation assay
AAFs were plated at a density of 104 cells/well with 100 μL of the whole culture medium in 96-well culture plates and cultured in an incubator (37℃, 5%CO2). After adhering to the well, the cells were treated with different concentrations of curcumin (1, 10, 20, 30, 40, 50, and 60 μM) for 12 h. We washed the cells with D-PBS (Gibco), and added 10 μL of Cell Counting Kit-8 (CCK8) solution (Dojindo, JAPAN) to each well and incubated the plates in the incubator for 2 h. Finally, the absorbance at 450 nm was measured using a FlexStation3 Multi-Mode Microplate Reader (Molecular Devices, USA). Six replicates were performed in each experiment.
RT-qPCR
Total RNA was extracted from AAFs using TRIzol reagent (Sigma). The PrimeScript RT reagent kit (Takara, Cat No. RR036A) was used for reverse-transcription. The cDNA template was further amplified with Hieff® qPCR SYBR Green Master Mix (Yesen, China, Cat No.11202ES03) and gene-specific primers (Table I). Three replicates were set for each primer in each group. The relative mRNA expression was normalized to β-actin and reported as 2-△△Ct.
Western blot and antibodies
The total proteins were extracted from AAFs using a lysis buffer containing RIPA buffer (Beyotime, China), 10% PhosSTOP (Thermo), and 1 mM phenylmethanesulfonylfluoride (Beyotime). Next, 10% and 12% SDS-PAGE were used to separate the proteins, and then the proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked in TBS/Tween with 5% BSA and subsequently incubated with the following primary antibodies: p-JAK1, JAK1, p-JAK2, JAK2, p-JAK3, JAK3, p-AKT (Thr308), AKT, p-STAT3, STAT3 (Cell Signal Technology), IL-1β (Abclonal, China, Cat No.A11369), TLR4 (Santacruz), and β-actin (Abcam). Next, we incubated the membrane with an HRP-conjugated secondary antibody (Yesen). Ultimately, a super ECL Detection Reagent Kit (Yesen, Cat No.36208ES60) was used to visualize the target protein band.
Treatment
TAK patients were treated with curcumin granules (Yifang Pharmaceutical Co., Ltd., Guangdong, China) at a dose of 15 g/day, p.o. (equivalent to approximately 45 mg/kg/day curcumin) while maintaining the original treatment for three months. The patient serum was collected at the beginning of the study and after three months and stored at -80℃ after separate packaging.
Enzyme linked immunosorbent assay (ELISA)
We determined the concentrations of IL-1β, IL-6, and CCL2 in the cell culture supernatants and serum of patients with a Human IL-1β Quantikine ELISA Kit (R&D Systems, Cat No. DLB50), Human IL-6 Quantikine ELISA Kit (R&D Systems Cat No.D6050), and Human MCP-1/CCL2 ELISA Kit (Boster, China, Cat No.EK0441) in accordance with the manufacturer’s instructions.
Statistical analysis
PCR, western blot and Immunohistochemical staining were expressed by mean ± SD values and analyzed by the student t-test. Data regarding patient’ characteristics was expressed as the mean ± SD values. Paired t test (For data that conform to normal distribution) or Wilcoxon test (For data that did not conform to normal distribution) were used to analyze the differences of indicators of patients before and after curcumin treatment. Pearson and Spearman correlation analysis was also performed. All statistical analyses were performed using GraphPad prism 8.2.1 (GraphPad Software Inc., USA). All experiments have been performed in triplicate. p < 0.05 indicating statistical significance.