Abstract
Objectives
The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.
Methods
In this study, TGF-β1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-β1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours.
Results
A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-β1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats.
Conclusion
Mechanical stress-induced overexpression of TGF-β1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-β1 inhibitor can inhibit articular cartilage degradation.
Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1). Bone Joint Res 2018;7:587–594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1.
Article focus
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Transforming growth factor beta 1 (TGF-β1) and mechanical stress in progession of osteoarthritis (OA).
Key messages
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TGF-β1 overexpression from osteoclasts under mechanical stress has detrimental effects on chondrocytes and articular cartilage in OA progression.
Strengths and limitations
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The effects of TGF-β1, chondrocyte apoptosis, and articular degeneration were studied comprehensively.
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However, how TGF-β1 is secreted from osteoclasts, and how it affects cartilage degeneration in vivo, should be studied further.
Introduction
Osteoarthritis (OA) is characterized by degradation of articular cartilage, thickening of subchondral bone, and formation of osteophytes.1 In the early stages of OA, the possibility of accelerated subchondral bone remodelling has been confirmed.2 In general, ageing, vitamin and mineral deficiencies, and drug abuse lead to an increased risk of OA development.3 However, the pathogenic mechanisms of OA have not been clearly identified. There is evidence that mechanical overload leads to the activation of anabolic processes in chondrocytes, including matrix proteins and matrix-degrading enzymes such as matrix metallopeptidases (MMPs).4 Several in vitro and in vivo experiments suggest that mechanical stimulation contributes to OA development by altering specific signalling pathways in chondrocytes or joints.5-7 In addition, mechanical stimulation-induced chondrocyte apoptosis has been identified as being involved in OA progression.6 In ageing mice, disruption of Sirt1 in chondrocytes causes the accelerated progression of OA under mechanical stress.8 Mechanical stress can also affect bone homeostasis. Load can lead to bone apposition,9 whereas load removal can cause bone loss.10 The direct effect of mechanical stress on osteoclasts has been studied and demonstrates that mechanical load can suppress osteoclast differentiation and fusion by the increase of nitric oxide (NO) via inducible nitrix oxide synthase (iNOS).11 However, osteoclasts under mechanical stress and their contribution to chondrocyte metabolism remain unclear.
Transforming growth factor beta 1 (TGF-β1) is a cytokine and plays an important role in the induction of chondrogenesis.12 The expression of TGF-β1 in healthy cartilage is significantly higher than in OA cartilage.13 In the progression of chondrocytic phenotypic degeneration, including senescence and dedifferentiation, downregulation of TGF-β1 indirectly induces metabolic disorder in chondrocytes.14 TGF-β1 promotes proliferation of chondrocytes through β-catenin signalling and maintains chondrocytic phenotype by enhancing collagen II synthesis.14,15 In native mouse knee joints, injection of low-dose TGF-β1 increases the proteoglycan content of articular cartilage;16 however, overexpression of TGF-β1 in the knee joint results in OA-like changes, including hyperplasia of the synovium and osteophyte formation in the knee joints of C57Bl/6 mice.17 In co-cultured osteoclast precursors and OA synovial fibroblasts, TGF-β-induced osteoprotegerin (OPG) production in synovial fibroblasts inhibits osteoclast formation. Moreover, blocking TGF-β activity in OA joints promotes osteoclastogenesis and inhibits osteophyte formation.13 In the early phases of OA, upregulation of TGF-β stimulates chondrocyte proliferation and attempts to repair injured cartilage.18 In transgenic mice, overexpressed TGF-β1 leads to abnormal subchondral bone structure and articular cartilage degeneration.19 More and more evidence shows that changes in subchondral bone in OA occurs rapidly. These changes occur at the same time as, if not earlier than, articular cartilage changes.20 Furthermore, increased osteoclast activity is a main pathogenic factor in subchondral bone injury in OA, and dysregulation of TGF-β1 signalling is detected in subchondral bone by accelerating osteoclast bone resorption in OA.18 To study the mechanical stress and TGF-β signalling in OA, an in vitro 3D collagen scaffold culture model was used. It is difficult to determine whether cells in the culture experience the same mechanical stimulus as in the skeleton. The mechanical environment that osteoclasts or chondrocytes experience in vivo is still under debate; however, cellular deformation caused by mechanical stress is believed to be essential in subsequent mechanotransduction. Different in vitro mechanical loading models were reviewed by Yang et al.21 In our study, we investigated the role of mechanical overload in the development of OA, and studied the TGF-β1 signalling pathways in response to mechanical overload in the co-cultured cells of osteoclasts and chondrocytes, which was a valuable cell model for studying the pathogenesis of OA.
Patients and Methods
Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited from the Department of Orthopaedics, the Fifth Affiliated Hospital of Sun Yat-sen University (Zhuhai, China), between January 2015 and December 2015, and written informed consent was obtained. The study was approved by the Ethics Committee of the Department of Orthopaedics, the Fifth Affiliated Hospital of Sun Yat-sen University (Zhuhai, China). Femoral head was used to develop a cell culture of osteoclasts and chondrocyte.6,22 The subchondral bone from the femoral head was ground into powder before being washed and incubated in Medium 199 (Sigma-Aldrich, St Louis, Missouri). The supernatant was incubated in the culture medium at 37°C, 5% CO2 condition before use. Cartilage samples were cut from the femoral head and minced into small fragments, followed by digestion with 0.25% trypsin (Gibco/Invitrogen, Carlsbad, California) for 30 minutes at 37°C and then with 0.2 collagenase (Sigma-Aldrich) for five hours at 37°C. The cell suspension was then filtered through a 40 μm cell strainer (BD Falcon, San Jose, California), and cells were collected by centrifugation at 800 × g for ten minutes. The chondrocytes were then cultured in Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F-12; Gibco/Invitrogen), supplemented with 10% foetal bovine serum (FBS; Gibco/Invitrogen)). The adherent cells were further incubated in a fresh medium after culturing overnight.
Ten-week-old male Sprague-Dawley (SD) rats (Guangzhou University of Traditional Chinese Medicine, Guangzhou, China) were allowed to acclimatize to the environment for one week. All experimental procedures were carried out in accordance with the guidelines of the Fifth Affiliated Hospital of Sun Yat-sen University on animal care. The anterior cruciate ligament (ACL) was transected to induce abnormal mechanical loading-associated OA of the left knee. Sham surgery was done by opening the joint capsule without ACL transection in the left knee in control rats. The animals were sacrificed at six weeks post-surgery, and samples of the knee joints were collected for further molecular and histological analyses. The rats were randomly divided into three groups: 1) a control group, in which rats underwent sham surgery (n = 6); 2) a group, in which rats underwent ACL transection (ACLT) (n = 6); and 3) a SB-505124 group, in which rats underwent ACLT and received SB-505124, administrated abdominally, once after surgery (n = 6).
Cell culture, cyclic loading, and co-cultured system
Osteoclasts and chondrocytes were maintained in α-minimal essential medium (α-MEM; Thermo Fisher Scientific, Waltham, Massachusetts), supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific) at 37°C in a humidified 5% CO2 incubator (Thermo Fisher Scientific).23 Osteoclasts were seeded into 3D collagen sponges.24 The cyclic mechanical loading was induced by a computer-controlled biostretch device (Bio-Stretch; ICCT Technologies, Markham, Canada).5 After 24 hours, cyclic loading-processed osteoclasts were co-cultured with chondrocytes.
Cell viability and TUNEL staining analysis
Chondrocyte proliferation was monitored by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation/viability assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the guidelines.
Quantitative assessment of apoptotic cells was assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method, which examines Deoxyribose Nucleic Acid (DNA) strand breaks during apoptosis by using a BD ApoAlert DNA Fragmentation Assay Kit (Takara Bio, Inc., Mountain View, California). Cartilage were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. After being washed, the cells were incubated with the reaction mixture for 60 minutes at 37°C. Cells were immediately analyzed using FACScan and the Cellquest program (both Becton Dickinson, Franklin Lakes, New Jersey).
After fixation in 75% alcohol for seven days, the tibia (n = 6/group) specimens were decalcified in 0.5 M EDTA (pH = 8.0) and then embedded in paraffin as per standard histological procedures. Sections measuring 5 μm were cut and stained with safranin O (Nanjing SenBeiJia Biological Technology Co., Ltd, Nanjing, China), and examined under a microscope (DM 2500; Leica, Wetzlar, Germany).
Enzyme-linked immunosorbent assay analyses (ELISA) were used to detect the expression levels of TGF-β1 in the in vitro culture cells and tissue homogenates of the rat knee joints. All procedures followed the ELISA kit instruction manual (Bio-Rad Laboratories, Marnes-La-Coquette, France). In brief, cell lysates and tissue homogenates were prepared using a Heidolph Diax 900 homogenizer (Elektro GmbH, Kelheim, Germany) in an ice-cold buffer containing 50 mM hydroxymethyl and 150 mM sodium chloride (NaCl) (pH 8.0), supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 1 μg/ml aprotinin, leupeptin, and pepstatin. The solution was centrifuged for 15 minutes at 16 000 × g, 4°C, and a total of 50 μl of supernatant was added into the wells coated with specific TGF-β1 detection antibodies. After the addition of 50 μl of biotin-conjugated secondary antibody, horseradish peroxidase (HRP)-conjugated avidin, and substrate, sample absorbance values were recorded using a Wallac Victor2 microplate reader (Perkin Elmer, Gaithersburg, Maryland) at a wavelength of 650 nm. Standard curves were drawn based on the sample concentrations and absorbance values in order to calculate the concentration of TGF-β1 in the specimens.
Ribonucleic Acid (RNA) was extracted from chondrocytes using TRIzol (Invitrogen; Thermo Fisher Scientific) according to the manufacturer’s instructions. Synthesis of cDNA was performed on 2 μg total RNA using Moloney Murine Leukemia Virus reverse transcriptase (Promega Corporation, Madison, Wisconsin) and oligo (dT) 15 primers (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative Polymerase Chain Reaction (qPCR) was performed using the Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). SDS software version 2.1 (Applied Biosystems; Thermo Fisher Scientific) was used to calculate the quantification cycle fluorescence value (Cq), and the 2-ΔΔCq method for the relative expression levels of RANK19 normalizing to the internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences that were used are given in Table I.
Table I.
Gene | Forward | Reverse |
---|---|---|
TGF-β1 | 5’-GGTACCTGAACCCGTGTTGCT-3’ | 5’-TGTTGCTGTATTTCTGGTACAGCTC-3’ |
MMP-9 | 5’-GCATAAGGACGACGTGAATGGC-3’ | 5’-CGGTGTGGTGGTGGTTGGAG-3’ |
MMP-13 | 5’-CTTGATGCCATTACCAGTC-3’ | 5’-GGTTGGGAAGTTCTGGCCA-3’ |
Col-X | 5’-ACAGGGGAGGTGATAGCATT-3’ | 5’-GACCAAAAGCCTTCATACATCTC-3’ |
Ctsk | 5’-CTGAAGATGCTTTCCCATATGTGGG-3’ | 5’-GCAGGCGTTGTTCTTATTCCGAGC-3’ |
GAPDH | 5’-ACAGGGGAGGTGATAGCATT-3’ | 5’-GACCAAAAGCCTTCATACATCTC-3’ |
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TGF-β1, transforming growth factor beta 1; MMP, matrix metallopeptidase; Col-X, type X collagen; Ctsk, cathepsin K; GAPDH, glyceraldehyde 3-phosphate dehydrogenase
Cells and tibia were homogenized and lysed in NP-40 buffer (Beyotime Institute of Biotechnology). Following five to ten minute at boiling temperature, cells were centrifuged at 10 000 × g, 4°C, for ten minutes to obtain the supernatant. Protein samples (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, Massachusetts). Membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline and 0.1% (w/v) Tween 20 (TBST), and incubated at 4°C overnight with the following primary antibodies: TGF-β1; B-cell lymphoma 2 (Bcl-2); caspase-3; MMP-9; cathepsin K (Ctsk); and GAPDH (all from Santa Cruz Biotechnology, Inc., Dallas, Texas). After being washed, the membranes were incubated with HRP-conjugated anti-immunoglobulin G (IgG) at room temperature for two hours. Signal detection was carried out with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech Inc., Piscataway, New Jersey).
Statistical analysis
Mean and standard error of the mean (sem) were used for data description. All statistical analyses were performed using GraphPad Prism software, version 5.0 (GraphPad Software Inc., La Jolla, California). Groups were compared using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test as a post hoc test to compare the mean values of each group. A statistically significant difference was considered to be p < 0.05.
Results
Upregulated TGF-β1 expression in osteoclasts under mechanical stress
Human osteoclasts were isolated from the femoral head of patients with a fracture of the femoral neck and seeded into collagen sponges. After being subjected to a cyclic mechanical loading at 1 Hz for 12 hours, the expression of TGF-β1 from the in vitro cultured osteoclasts was significantly upregulated with the increase of the elongation (0% to 15%) (Fig. 1a). Moreover, at 10% elongation and 1 Hz cyclic loading, the expressions of TGF-β1 were increased in a time-dependent manner (0 to 24 hours) (Fig. 1b).
Fig.
Cyclic loading-processed osteoclasts inhibit chondrocyte proliferation and induce apoptosis in osteoclast/chondrocyte co-cultured system
Osteoclasts under cyclic loading, 10% elongation, and 1 Hz for 12 hours were co-cultured with chondrocytes. The chondrocyte viability was determined at 24 hours after the co-cultured system by MTT assay. Results showed that chondrocytes in this co-cultured system demonstrated cell growth inhibition as compared with unloaded co-cultured cells (Fig. 2a). Next, we examined apoptosis in co-cultured cells by flow cytometry. TUNEL staining results demonstrated that cyclic loading-processed osteoclasts co-cultured with chondrocytes induced cell apoptosis compared with the control group (Fig. 2b). However, SB-505124 treatment could significantly reverse the inhibition of chondrocyte proliferation caused by cyclic loading-processed osteoclasts, and induced apoptosis in the osteoclast/chondrocyte co-cultured system (Figs 2a and 2b). Furthermore, we found that mechanical pressure injury increased the caspase-3 protein levels and inhibited the Bcl-2 protein levels in loaded co-cultured cells as compared with unloaded co-cultured cells. However, administration of SB-505124 reversed the upregulation of caspase-3 and the downregulation of Bcl-2 in loaded co-cultured cells (Figs 2c and 2d).
Fig.
Cyclic loading-processed osteoclasts regulate cartilage degeneration-related markers in osteoclast/chondrocyte co-cultured system
We examined the mRNA levels of MMP-9 and MMP-13 in an osteoclast/chondrocyte co-cultured system. Indeed, mechanical stress significantly upregulated the mRNA levels of MMP-9 and MMP-13 in osteoclast/chondrocyte co-cultured cells compared with the control group. The addition of SB-505124 to the co-cultured cells with mechanical stress inhibited MMP-9 and MMP-13 expression significantly when compared with the non-treatment group (Figs 3a and 3b). Moreover, the mRNA levels of type X collagen (Col-X) were significantly higher in the co-cultured cells with mechanical stress than in the unloaded group. However, in the presence of SB-505124, upregulation of Col-X caused by mechanical stress was reversed (Fig. 3c). Furthermore, we found that administration of SB-505124 reversed the upregulation of Ctsk in loaded co-cultured cells (Fig. 3d), which is a lysosomal cysteine protease and is involved in matrix remodelling.
Fig.
TGF-β1 is involved in ACL-induced OA in a rat model
First, Safranin O staining demonstrated that the articular cartilage in ACLT rats exhibited accelerated proteoglycan loss as compared with that of the rats in the control group. However, SB-505124 treatment markedly attenuated articular cartilage degradation in the surgical rats (Fig. 4a). The mRNA and protein levels of TGF-β1 were significantly upregulated in the articular cartilage of ACLT rats as compared with the normal group. The upregulation of TGF-β1 mRNA and protein expression in the proximal tibia from ACLT rats was reversed by the administration of SB-505124 (Figs 4b, 4f, and 4g). The expression of matrix-degrading enzyme MMP-9 was significantly increased in surgically induced OA cartilage compared with the control group as shown by real-time PCR (Fig. 4c) and Western blotting (Figs 4f and 4h). Moreover, Col-X expression is affected following ACLT operation. This upregulation of Col-X could be completely blocked by SB-505124 in ACLT rats (Fig. 4d). We found that the mRNA and protein expression of Ctsk were significantly upregulated in the articular cartilage of ACLT rats as compared with the normal group. The upregulation of Ctsk mRNA and protein expression was reversed by administration of SB-505124 in ACLT rats (Figs 4e, 4f and 4i).
Fig.
Discussion
Cartilage degeneration is a prominent feature of OA. In this process, mechanical stress plays a crucial role in promoting metabolic disorder in chondrocytes.25 Evidence suggests that mechanical stress can directly contribute to matrix remodelling by inducing MMP expression in a variety of tissues.26 Previous studies indicate that chondrocytes are highly responsive to mechanical stress, which can induce TGFs, matrix-degrading enzymes, and collagenase to mediate biochemical signals in articular cartilage remodelling.3,18,25 In SW1353 chondrocyte-like cells, mechanical stress induces MMP-13 expression by runt-related transcription factor 2 (RUNX2).25
Our study demonstrates that mechanical stress can be a trigger for the upregulation of TGF-β1 in osteoclasts. Mechanical stress-processed osteoclasts inhibit chondrocyte proliferation and induce apoptosis in an osteoclast/chondrocyte co-cultured system. However, interruptive TGF-β1 signalling by SB-505124 can inhibit chondrocyte dysfunction and OA-like changes in vitro. In an ACLT rat model, we also show that overexpressed TGF-β1 is involved in articular cartilage degeneration and OA-like changes. Our results are in line with those of Yang et al5 who showed that the expression of MMP-13 and Col-X were significantly increased in ACLT-induced OA rats.
TGF β1 is considered a central regulator of articular cartilage remodelling under mechanical stress, which is a crucial indirect mediator of OA via induction of TGF-β1 production.3 Blockage of TGF-β1 signalling attenuates articular cartilage degeneration in the ACLT rodent model of OA.27 It has been previously reported that excessive TGF-β 1 action in subchondral bone can induce temporo-mandibular joint OA.28 In the progression of osteoclast bone resorption, dysregulation of TGF-β 1 alters the fate of bone marrow mesenchymal stem cells (MSCs), uncoupling bone remodelling, and causing skeletal disorders.12 A previous study indicates that TGF-β1 is essential in the mediation of osteoclast differentiation in a co-cultured system with rheumatoid arthritis (RA) synovial fibroblasts.13 However, a complete understanding of the TGF-β1 signalling in an osteoclast/chondrocyte co-cultured system remains elusive. In the present study, we demonstrated that not only magnitudes of elongation to osteoclasts but also the duration of time increased the mRNA and protein expression of TGF-β1. Our study has shown osteoclasts co-cultured with chondrocytes as a valuable cell model for studying the pathogenesis of OA under mechanical stress. It also showed that chondrocyte growth was repressed by cyclic loading-processed osteoclasts. Moreover, chondrocyte apoptosis was induced in a co-cultured system. However, the TGF-β1 receptor inhibitor, SB-505124, could significantly reverse the suppression of chondrocyte growth and the induction of apoptosis. Interestingly, transgenic expression of active TGF-β1 in osteoblastic cells is sufficient to induce osteoarthritis, whereas direct inhibition of TGF-β1 activity in subchondral bone attenuated the degeneration of articular cartilage.12 These observations suggest that high levels of active TGF-β in bone remodelling are associated with the progression of OA.
Various proteins are involved in the phenotypic change of chondrocytes by regulating the expression of chondrocyte-specific proteins such as collagen type II (Col-II), glycoproteins, and proteoglycans (PGs).14,29 In senescence stages, chondrocytes exhibit high expression of Col-II and low expression of Col-X. Moreover, the cartilage-degrading enzyme, MMP-13, is actively produced in senescent chondrocytes.14 In our study, we found that cyclic loading-processed osteoclasts regulated cartilage degeneration-related markers in an osteoclast/chondrocyte co-cultured system, including the upregulation of Col-X, MMP-9, and MMP-13 in co-cultured cells. Importantly, the TGF-β1 receptor inhibitor, SB-505124, could inhibit levels of Col-X, MMP-9, and MMP-13 in co-cultured cells. Based on these observations, we suggest that the direct suppression of TGF-β1 signalling in chondrocytes could alleviate mechanical stress-induced chondrocyte dysfunction. Intriguingly, TGF-β1 has varying roles in chondrogenesis in vivo; introducing TGF-β1 into the periosteum of a femur causes chondrocyte differentiation and cartilage formation,30 whereas inhibition of the TGF-β1 signalling pathway delays the development of osteoarthritis in both mouse and rat models.12 In our work, elevation of TGF-β1 levels had been observed in ACLT-induced OA rats, and articular cartilage degradation was accompanied by the progression of OA in ACLT surgical rats. However, SB-505124 treatment markedly reduced articular cartilage degradation and inhibited the expression of TGF-β1 in the surgical rats.
In conclusion, TGF-β1 is one of the key cytokines responsible for chondrocyte dysfunction in a cyclic loading-processed osteoclast/chondrocyte co-cultured system. We also found that overexpressed TGF-β1 was proven to have a detrimental effect in response to mechanical stress-induced articular cartilage degradation in a rat model. These results suggest that TGF-β1 can be used as a potential pathway, worthy of further study, in the regulation of mechanical stress-induced OA.
Open access
This is an open-access article distributed under the terms of the Creative Commons Attributions licence (CC-BY-NC), which permits unrestricted use, distribution, and reproduction in any medium, but not for commercial gain, provided the original author and source are credited.
This work was supported by grants obtained from the China Scholarship Foundation (Grant No. 201506385051), the Natural Science Foundation of Guangdong Province, China (Grant No. 2015A030310240), the Medical Research Foundation of Guangdong Province, China (Grant No. A2014261), the Scientific and Technological Projects of Zhuhai City, Guangdong Province, China (Grant No. 2013D0401990009), the Science and Technology Planning Project of Tianhe District, Guangdong, China (201404KW005), the Medical Scientific Research Foundation of Guangdong Province, China (A2015422), the National Natural Science Foundation of China (81371990, 81601945), and a scientific research grant for young investigators from Southern Medical University to H. Fang.
None declared
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