Wenxian Zhou1,2,3, Yifeng Shi1,2,3, Hui Wang1,2,3, Caiyu Yu4, Huanqing Zhu3, Aimin Wu1,2,3. 1. Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, China. 2. Zhejiang Provincial Key Laboratory of Orthopedics, Wenzhou, China. 3. The Second School of Medicine, Wenzhou Medical University, Wenzhou, China. 4. The School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, China.
Abstract
As a common degenerative disease, osteoarthritis (OA) usually causes disability in the elderly and socioeconomic burden. Previous studies have shown that proper autophagy has a protective effect on OA. Sinensetin (Sin) is a methylated flavonoid derived from citrus fruits. Studies have shown that Sin is a good autophagy inducer and has shown excellent therapeutic effects in a variety of diseases; however, its role in the treatment of OA is not fully understood. This study proved the protective effect of Sin on OA through a series of in vivo and in vitro experiments. In vitro experiments have shown that Sin may inhibit chondrocyte apoptosis induced by tert-butyl hydroperoxide (TBHP); at the same time, it might also inhibit the production of MMP13 and promote the production of aggrecan and collagen II. Mechanism studies have shown that Sin promotes chondrocyte autophagy by activating AMPK/mTOR signaling pathway. On the contrary, inhibition of autophagy can partially abolish the protective effect of Sin on TBHP-treated chondrocytes. In vivo experiments show that Sin may protect against DMM-induced OA pathogenesis. These results provide evidence that Sin serves as a potential candidate for the treatment of OA.
As a common degenerative disease, osteoarthritis (OA) usually causes disability in the elderly and socioeconomic burden. Previous studies have shown that proper autophagy has a protective effect on OA. Sinensetin (Sin) is a methylated flavonoid derived from citrus fruits. Studies have shown that Sin is a good autophagy inducer and has shown excellent therapeutic effects in a variety of diseases; however, its role in the treatment of OA is not fully understood. This study proved the protective effect of Sin on OA through a series of in vivo and in vitro experiments. In vitro experiments have shown that Sin may inhibit chondrocyte apoptosis induced by tert-butyl hydroperoxide (TBHP); at the same time, it might also inhibit the production of MMP13 and promote the production of aggrecan and collagen II. Mechanism studies have shown that Sin promotes chondrocyte autophagy by activating AMPK/mTOR signaling pathway. On the contrary, inhibition of autophagy can partially abolish the protective effect of Sin on TBHP-treated chondrocytes. In vivo experiments show that Sin may protect against DMM-induced OA pathogenesis. These results provide evidence that Sin serves as a potential candidate for the treatment of OA.
Osteoarthritis (OA) is the most common degenerative disease, involving the entire joint, causing pain, and eventually leading to disability, which causes huge socioeconomic burden (Vina and Kwoh, 2018; Safiri et al., 2020). The pathological features of OA are progressive destruction of articular cartilage, chronic inflammation of the synovium, and subchondral bone sclerosis (Vina and Kwoh, 2018). Chondrocytes are the only cell type in articular cartilage, and pathological factors such as inflammation and oxidative stress lead to excessive apoptosis of chondrocytes, reduced cell density, and degradation of extracellular matrix (ECM), thereby aggravating the development of OA (van den Bosch, 2021; Goutas et al., 2018). In addition, ECM is an essential components for maintaining function and structure of cartilage (Kronenberg, 2003). Therefore, preventing excessive apoptosis of chondrocytes may be an effective treatment method to prevent the progression of OA.Autophagy is a protective mechanism through engulfing and degrading misfolded proteins and senescent or damaged organelles to realize the metabolic needs of the cell itself and the renewal of some organelles (Klionsky et al., 2016; Mizushima, 2007). Autophagy occurs in both physiological and pathological conditions. A large number of researches showed that autophagy plays an important role in the homeostasis of articular cartilage (Caramés et al., 2015; Li et al., 2016). Autophagy enhanced by specific deletion of mTOR could significantly protect OA induced by destabilisation of medial meniscus (DMM) (Zhang et al., 2015), indicating that autophagy plays a protective role in OA.Sinensetin (Sin) is a polymethoxylated flavonoid, which is found in citrus fruits and possess potent anti-inflammatory, anti-angiogenesis and anticancer activities (Han Jie et al., 2021). Sin is also involved in the regulation of a variety of signaling pathways, such as NF-kappaB, AKT/mTOR and MAPKs signaling (Tan et al., 2019; Li et al., 2020). A recent study showed that Sin promotes autophagy through AMPK/mTOR signaling pathway to induce hepatocellular carcinoma cell apoptosis (Kim et al., 2020). Although studies have conducted extensive studies on the function of Sin, its effect on chondrocyte autophagy and its potential therapeutic effect on the progression of OA remain unclear.In this study, we investigated the effect of sin on chondrocyte autophagy under tert-butyl hydroperoxide (TBHP) treatment and explored its potential molecular mechanism. We also used the DMMmouse model to study the in vivo effects of Sin on OA.
Materials and Methods
Ethics Statement
All experimental procedures in this study, such as surgery, treatment, and post-operative care of animals, followed the “Guidelines for the Care and Use of Laboratory Animals” issued by the National Institutes of Health. All animal experiments were approved by the Animal Care and Use Committee of Wenzhou Medical University. No clinical trial was involved in this study.
Reagents and Chemicals
Sinensetin (purity >99%), 3-methyladenine (3-MA), chloroquine (CQ), and bafilomycin A1 were purchased from MCE (Monmouth Junction, NJ, United States). tert-butyl hydroperoxide (TBHP) solution was purchased from Sigma-Aldrich (St. Louis, MO, United States). Safranin-O, dimethyl sulfoxide (DMSO), and collagenase II were purchased from Solarbio (Beijing, China). The primary antibodies against aggrecan, collagen II, MMP13, p62, Bcl-2, Bax were obtained from Abcam (Cambridge, MA, United States); antibodies against AMPK, p-AMPK, mTOR, p-mTOR, cleaved caspase 3, LC3 antibodies against Cell Signaling Technology (Danvers, MA, United States); antibodies against Beclin1 and GADPH were acquired from Proteintech (Chicago, IL, United States). Cell counting kit-8 (CCK8) was obtained from m Dojindo (Kumamoto, Japan). Alexa Fluor 488-labeled and Alexa Fluor 594-labeled goat anti-rabbit/mouse IgG H&L secondary antibodies were acquired from Abcam (Cambridge, MA, United States). 4,6-Diamidino-2-phenylindole (DAPI) was purchased from Yeasen Biochemical (Shanghai, China). The reagents for culturing cartilage were obtained from Gibco (Grand Island, NY, United States).
Primary Mice Chondrocytes Extraction and Culture
Under aseptic conditions, the knee joint cartilages of immature C57BL/6 mice were collected, completely cut into small pieces, washed three times with phosphate-buffered saline (PBS), and digested with 2 mg/ml collagenase II at 37°C for 4 h. Subsequently, the digested cartilage tissue was centrifuged, washed three times with PBS, and resuspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin/penicillin) in 5% CO2 at 37°C. Replace with fresh medium every other day. All experiments used second-passage cells to ensure consistent cell phenotypes.
Cell Viability Assay
The CCK8 kit was used to detect the effect of Sin on the viability of chondrocytes. According to the manufacturer’s protocol, the chondrocytes of the same generation were seeded in 96-well plates (5*104 cells per well) and cultured for 24 h, and then treated with different concentrations of Sin (0, 10, 20, 30, 40, and 50 μM) for 24 h. Then, the cells were washed with PBS and incubated with 100 μl of serum-free DMEM/F12 medium containing 10% CCK8 solution for 2 h at 37°C. The absorbance was measured at 450 nm using an ultraviolet spectrophotometer (Thermo Fisher). In addition, we also detected the effect of 40 μM Sin on chondrocyte viability in different time points (0, 3, 6, 12, 24, and 48 h), and the required steps are the same as above.
Western Blotting
The chondrocytes were lysed with RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) for 15 min, and then centrifuged at 12,000 rpm and 4°C for 30 min to extract total cell protein. Use the BCA reagent (Beyotime, Shanghai, China) to detect protein concentration. Then, 40 ng protein was separated using 8–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, California, United States). The membranes were blocked with 5% skimmed milk for 2 h at room temperature, and washed three times with Tris-buffered saline with 0.1% Tween-20 (TBST). Then the membranes were cut, placed in the corresponding primary antibodies, and incubated overnight at 4°C. Next, the membranes were washed three times with TBST and incubated with the corresponding secondary antibody at room temperature for 90 min. Finally, the membranes were washed three times with TBST, and the membrane blots were developed using super-sensitive ECL chemiluminescence kit, and visualized by the ChemiDoc XRS + Imaging System (Bio-Rad). Quantitative analysis was performed by ImageJ software.
Immunofluorescence
The chondrocytes were seeded in a 6-well plate for 24 h, and then treated with TBHP (30 μM) or treated with TBHP and Sin (40 μM) for 24 h. Subsequently, chondrocytes were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and then infiltrated with 0.1% Triton X-100 in PBS for 15 min. After blocking the cells with 5% bovine serum albumin at 37°C for 1 h, they were incubated with the corresponding primary antibody [MMP13 (1:200), cleaved caspase 3 (1:100), and LC3 (1:200)] overnight at 4°C. Finally, the cells were incubated with Alexa Fluor 488 or Alexa Fluor 594 conjugated secondary antibodies for 1 h at room temperature, and stained with nuclear staining dye DAPI for 5 min. A Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan) was used to observe the stained cells under five random different fields for each slide. Fluorescence intensity was measured with ImageJ.
TUNEL Staining
TUNEL staining was used to detect the level of DNA damage of chondrocytes. According to the manufacturer’s protocol, the chondrocytes were fixed, stained with in situ cell death detection kit (Promega Corporation, Wisconsin, United States) at 37°C for 30 min, and the nuclei were stained with DAPI. Twenty-five fields of view were randomly selected from each slide, and TUNEL positive cells were counted under a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).
Animal Models
Thirty ten-week-old C57BL/6 male mice were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences and randomly divided into sham, DMM and DMM + Sin groups (n = 10 per group). The mouseosteoarthritis model was caused by the destabilization of the medial meniscus (DMM) through surgery (Glasson et al., 2007). In brief, mice were anesthetized by intraperitoneal injection of 2% (w/v) pentobarbital (40 mg/kg), the right knee joint capsule was cut inside the patellar tendon, and the tibial ligament of the medial meniscus was cut with microsurgery scissors. Meanwhile, the same operation was performed on the left knee. The joint incision without resection of the medial meniscus ligament was used as the sham group. After that, mice in the sham group and DMM group were given saline by gavage every day, while mice in the DMM + Sin group received 50 mg/kg/day Sin dissolved in saline by gavage. Eight weeks after the operation, the mice were sacrificed, and the knee joints were collected for imaging and histological evaluation.
X-Ray Imaging Analysis
X-ray examinations were performed on mice in all groups 8 weeks after operation. A digital X-ray machine (Kubtec Model XPERT.8; KUB Technologies Inc.) was used to perform X-ray imaging of mice at 50 Kv and 160 μA to evaluate the joint space and cartilage surface calcification changes.
Immunohistochemical Assay
The knee joint was embedded in paraffin, sectioned, deparaffinized and hydrated, and the endogenous peroxidase was blocked by 3% hydrogen peroxide for 15 min. Then, the sections were incubated with 0.4% pepsin (Biotech, Shanghai, China) in 5 mM HCl at 37°C for 20 min for antigen retrieval. Next, the sections were incubated with 5% bovine serum albumin at room temperature for 30 min, then with the corresponding primary antibody [MMP13 (1:100), and LC3 (1:100] overnight at 4°C, and finally incubated with the HRP-conjugated secondary antibody for 1 h. The rate of positive cells was quantitated by researchers who were blinded to the experimental group. Five mice in each group were quantitatively analyzed.
Histopathological Analysis
The three groups of mouse joint specimens were sliced and stained with Safranin O (S-O) to evaluate the destruction of articular cartilage. Another group of experienced histology researchers examined the cellularity and morphology of cartilage and subchondral bone with a microscope in a blinded manner, and used the Osteoarthritis Research Society International (OARSI) to evaluate the degree of cartilage degeneration as described previously (Glasson et al., 2010).
Statistical Analysis
The results are presented as mean ± S.D. and are from three independent experiments. SPSS 20.0 (IBM, Armonk, NY, United States) was used for statistical analysis. Parametric data were analyzed by one-way analysis of variance (ANOVA), and then the Tukey’s test was used to compare the groups. Nonparametric data (OARSI scores) were compared by the Kruskal–Wallis H test. p < 0.05 was considered significant.
Results
Effect of Sin on Chondrocyte Viability
The chemical structure of Sin is shown in Figure 1A. To evaluate the toxicity of Sin to chondrocytes, we used the CCK-8 assay to determine the viability of chondrocytes treated with different concentrations of Sin (0, 10, 20, 30, 40, and 50 μM) for 24 h. The results showed that Sin concentrations below 40 μM had no obvious cytotoxicity to chondrocytes, but 50 μM Sin reduced the viability of chondrocytes (Figure 1B). Meanwhile, the cytotoxicity of 40 μM Sin at different time points (0, 3, 6, 12, 24, and 48 h) was also determined, and it was found that 40 μM Sin had no significant effect on the viability of chondrocytes within 48 h (Figure 1C).
FIGURE 1
Sin’s effect on chondrocyte viability. (A) Chemical structure of Sin. (B) The results of CCK-8 assay show the viability of chondrocytes after treatment with 0, 10, 20, 30, 40, and 50 μM Sin for 24 h. (C) The results of CCK-8 assay show the viability of chondrocytes after treatment with 40 μM Sin for 0, 3, 6, 12, 24, and 48 h. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin’s effect on chondrocyte viability. (A) Chemical structure of Sin. (B) The results of CCK-8 assay show the viability of chondrocytes after treatment with 0, 10, 20, 30, 40, and 50 μM Sin for 24 h. (C) The results of CCK-8 assay show the viability of chondrocytes after treatment with 40 μM Sin for 0, 3, 6, 12, 24, and 48 h. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Effect of Sin on Apoptosis in TBHP-Treated Chondrocytes
TBHP significantly reduces the viability of chondrocytes (Supplementary Figure 1). We analyzed whether Sin protects chondrocytes from apoptosis through the TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, immunofluorescence and western blotting analysis of the expression of apoptosis-related proteins, cleaved caspase 3 (C-caspase3), Bcl-2 and Bax. As shown in Figures 2A,B, the number of TUNEL-positive chondrocytes increased after TBHP treatment, but significantly decreased in the Sin treatment group. Meanwhile, TBHP treatment upregulated the expression of cleaved caspase 3 and Bax, and downregulated the expression of Bcl-2, while Sin treatment could reverse these conditions (Figures 2C,D). Similarly, the results of cleaved caspase 3-labeled immunofluorescence further confirmed that Sin treatment reduces the intensity of cleaved caspase 3 (Figures 2E,F). These results indicate that Sin protects chondrocytes from TBHP-induced apoptosis.
FIGURE 2
Sin protects TBHP-treated chondrocytes from apoptosis. (A, B) The results of TUNEL assay show the number of TUNEL-positive chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h (Scale bar: 50 μm). (C, D) The results of western blotting show the levels of cleaved caspase 3, Bcl-2 and Bax proteins in the chondrocytes as treated above. (E, F) The results of immunofluorescence show the intensity of cleaved caspase 3 in the chondrocytes as treated above (Scale bar: 20 μm). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin protects TBHP-treated chondrocytes from apoptosis. (A, B) The results of TUNEL assay show the number of TUNEL-positive chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h (Scale bar: 50 μm). (C, D) The results of western blotting show the levels of cleaved caspase 3, Bcl-2 and Bax proteins in the chondrocytes as treated above. (E, F) The results of immunofluorescence show the intensity of cleaved caspase 3 in the chondrocytes as treated above (Scale bar: 20 μm). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Effect of Sin on ECM Degradation in TBHP-Treated Chondrocytes
Next, we evaluated the effect of Sin on TBHP-induced ECM degradation by detecting the expression of collagen II, aggrecan and matrix metallopeptidase 13 (MMP13). Western blotting showed that TBHP treatment significantly reduced the expression of aggrecan and collagen II, and increased the expression of MMP13, while Sin could reverse these effects induced by TBHP (Figures 3A,B). Additionly, the results of immunofluorescence labeled with MMP13 are consistent with the results of western blotting (Figures 3C,D). In summary, these results indicate that Sin has the effect of protecting chondrocytes from TBHP-induced ECM degradation.
FIGURE 3
Sin protects TBHP-treated chondrocytes from ECM degradation. (A, B) The results of western blotting show the levels of aggrecan, collagen II and MMP13 proteins in the chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h. (C, D) The results of immunofluorescence show the intensity of MMP13 in the chondrocytes as treated above (Scale bar: 20 μm). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin protects TBHP-treated chondrocytes from ECM degradation. (A, B) The results of western blotting show the levels of aggrecan, collagen II and MMP13 proteins in the chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h. (C, D) The results of immunofluorescence show the intensity of MMP13 in the chondrocytes as treated above (Scale bar: 20 μm). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Effect of Sin on Autophagy in Chondrocytes
Autophagy is an intracellular degradation mechanism, which transports cytoplasmic components to the lysosome for degradation, and plays an important role in the progression of OA (Mizushima, 2007; Li et al., 2016). We analyzed whether Sin activates autophagy in chondrocytes by detecting autophagy markers, such as p62, Beclin1, and LC3II/LC3I ratio, using a dose- and time-dependent method. Dose-dependent western blotting showed that Sin treatment increases the expression of Beclin1 and the ratio of LC3II/LC3I, while p62 expression decreased (Figures 4A,B). And the immunofluorescence results of labeled LC3 showed the same result (Figures 4E,F). And time-dependent western blotting showed that Sin treatment increased the expression of Beclin1 and the ratio of LC3II/LC3I, and reached a peak at 24 h. On the contrary, the expression of p62 after Sin treatment gradually decreased (Figures 4C,D).
FIGURE 4
Sin activates autophagy in chondrocytes. (A, B) The results of dose-dependent western blotting show that the levels of p62, Beclin1, and LC3II/LC3I ratio in chondrocytes treated with different concentrations of Sin (0, 10, 20, 30, and 40 μM) for 24 h. (C, D) The results of time-dependent western blotting show that the levels of p62, Beclin1, and LC3II/LC3I ratio in chondrocytes treated with 40 μM Sin for different times (0, 6, 12, 24,and 48 h). (E, F) The results of immunofluorescence show the intensity of LC3 in the chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h (Scale bar: 20 μm). (G, H) The western blotting results show that the levels of LC3II/LC3I ratio in chondrocytes that were not treated, or treated with 100 nM bafilomycin A1 for 1 h, or treated with 40 μM Sin for 24 h, or treated with bafilomycin A1 and Sin. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin activates autophagy in chondrocytes. (A, B) The results of dose-dependent western blotting show that the levels of p62, Beclin1, and LC3II/LC3I ratio in chondrocytes treated with different concentrations of Sin (0, 10, 20, 30, and 40 μM) for 24 h. (C, D) The results of time-dependent western blotting show that the levels of p62, Beclin1, and LC3II/LC3I ratio in chondrocytes treated with 40 μM Sin for different times (0, 6, 12, 24,and 48 h). (E, F) The results of immunofluorescence show the intensity of LC3 in the chondrocytes treated with or without 40 μM Sin for 24 h and 30 μM TBHP for 24 h (Scale bar: 20 μm). (G, H) The western blotting results show that the levels of LC3II/LC3I ratio in chondrocytes that were not treated, or treated with 100 nM bafilomycin A1 for 1 h, or treated with 40 μM Sin for 24 h, or treated with bafilomycin A1 and Sin. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.In addition, autophagy flux is also a reliable indicator for detecting autophagy (Zheng et al., 2018). By treating chondrocytes with Sin and the lysosomal inhibitor bafilomycin A1, we analyzed whether Sin also enhances autophagy flux. As shown in Figures 4G,H, the autophagy flux of chondrocytes increased after Sin treatment.
Sin Activates the AMPK/mTOR Pathway in Chondrocytes
AMP-activated protein kinase (AMPK) is a key regulator of autophagy by inhibiting mammalian target of rapamycin C1 (mTORC1) (Lahiri et al., 2019). In order to study whether the mechanism of Sin’s activation of autophagy is related to the AMPK/mTOR signaling pathway, we used western blot to detect the expression levels of p-AMPK and p-mTOR. The dose-dependent western blotting showed that after Sin treatment, the expression of p-AMPK in chondrocytes is significantly increased, but the expression of total AMPK is not affected, and the expression of p-mTOR is decreased (Figures 5A,B). Additionally, time-dependent western blotting showed that Sin treatment increases the expression of p-AMPK and decreases the expression of p-mTOR (Figures 5C,D). These results indicate that Sin activates the AMPK/mTOR pathway, and the autophagy activated by Sin may be related to the AMPK/mTOR pathway.
FIGURE 5
Sin activates the AMPK/mTOR pathway in chondrocytes. (A, B) The results of dose-dependent western blotting show that the levels of p-mTOR and p-AMPK in chondrocytes treated with different concentrations of Sin (0, 10, 20, 30, and 40 μM) for 24 h. (C, D) The results of time-dependent western blotting show that the levels of p-mTOR and p-AMPK in chondrocytes treated with 40 μM Sin for different times (0, 6, 12, 24,and 48 h). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin activates the AMPK/mTOR pathway in chondrocytes. (A, B) The results of dose-dependent western blotting show that the levels of p-mTOR and p-AMPK in chondrocytes treated with different concentrations of Sin (0, 10, 20, 30, and 40 μM) for 24 h. (C, D) The results of time-dependent western blotting show that the levels of p-mTOR and p-AMPK in chondrocytes treated with 40 μM Sin for different times (0, 6, 12, 24,and 48 h). The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Inhibition of Autophagy Attenuates Sin-Induced Anti-apoptosis and Anti-ECM Degradation
To further prove that Sin protects chondrocytes from apoptosis and ECM degradation by activating autophagy, we used two autophagy inhibitors Chloroquine (CQ) and 3-Methyladenine (3-MA) to block autophagy. The results of western blotting showed that CQ or 3-MA significantly increased the expression of cleaved caspase 3 and Bax, and decreased the expression of Bcl-2 in Sin-treated chondrocytes (Figures 6A,B). As shown in Figures 6C,D, in Sin-treated chondrocytes, CQ or 3-MA also significantly increased the expression of MMP13 and decreased the expression of Aggrecan and Collagen II. The above results indicate that Sin inhibits chondrocyte apoptosis and ECM degradation by enhancing chondrocytes autophagy.
FIGURE 6
Inhibition of autophagy offsets the beneficial effect of Sin. Chondrocytes were untreated, or treated with TBHP (30 μM for 24 h) alone, or treated with Sin (40 μM for 24 h) and TBHP, or treated with TBHP and Sin combined with CQ (50 μM for 2 h) or 3-MA (10 mM for 2 h). (A, B) The results of western blotting show that the levels of Bcl-2, Bax and cleaved caspase 3 in chondrocytes treated above. (C, D) The results of western blotting show that the levels of Aggrecan, collagen II and MMP13 in chondrocytes treated above. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Inhibition of autophagy offsets the beneficial effect of Sin. Chondrocytes were untreated, or treated with TBHP (30 μM for 24 h) alone, or treated with Sin (40 μM for 24 h) and TBHP, or treated with TBHP and Sin combined with CQ (50 μM for 2 h) or 3-MA (10 mM for 2 h). (A, B) The results of western blotting show that the levels of Bcl-2, Bax and cleaved caspase 3 in chondrocytes treated above. (C, D) The results of western blotting show that the levels of Aggrecan, collagen II and MMP13 in chondrocytes treated above. The data are presented as the means ± SD (n = 3); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin Ameliorates in vivo OA Progression in DMM Model Mice
In order to investigate the therapeutic effect of Sin on OA in vivo, we administered Sin to DMMmice by gavage. We established the OA model in mice by DMM, and for the following 8 weeks, the DMMmice were given Sin (Sin group) or saline (DMM group) by gavage once a day, and the joints were analyzed by X-ray, safranin O staining and immunofluorescence. The results of X-ray showed that the joint space was severely narrowed and cartilage was obviously ossified in DMMmice, while the joint space narrowing and cartilage solidification were improved after Sin treatment (Figure 7A). Eight weeks after surgery, safranin O staining showed that the joint surface was rough and chondrocytes were reduced in the DMM group, but Sin treatment partially rescued these conditions (Figure 7B). Consistent with the staining results, Sin treatment reduced the Osteoarthritis Research Society International (OARSI) score (Figure 7D).
FIGURE 7
Sin protects against DMM-induced OA pathogenesis. (A) Representative X-ray images of the knee joints of mice in different experimental groups. White arrows indicate narrowing of the joint space, and black arrows indicate calcification of the cartilage surface. (B) Representative images of cartilage S-O staining in the three groups at 8 weeks postoperatively. (C) Immunohistochemical staining assay of LC3 II and MMP13 in the mouse cartilage (Scale bar: 50 μm). (D) OARIS scores of articular cartilage in three groups. (E) Quantitative analysis of LC3 II and MMP13 positive expression in sections. The data are presented as the means ± SD (n = 10); *p < 0.05, **p < 0.01, and ***p < 0.001.
Sin protects against DMM-induced OA pathogenesis. (A) Representative X-ray images of the knee joints of mice in different experimental groups. White arrows indicate narrowing of the joint space, and black arrows indicate calcification of the cartilage surface. (B) Representative images of cartilage S-O staining in the three groups at 8 weeks postoperatively. (C) Immunohistochemical staining assay of LC3 II and MMP13 in the mouse cartilage (Scale bar: 50 μm). (D) OARIS scores of articular cartilage in three groups. (E) Quantitative analysis of LC3 II and MMP13 positive expression in sections. The data are presented as the means ± SD (n = 10); *p < 0.05, **p < 0.01, and ***p < 0.001.The results of immunohistochemistry showed that the Sin treatment group could effectively reduce the expression of MMP13 and increase the expression of LC3. These results indicate that Sin improves the OA situation and reduces the degradation of ECM by enhancing the autophagy of chondrocytes in the OA mice (Figures 7C,E).
Discussion
Osteoarthritis (OA) is a common degenerative chronic disease that lacks effective treatment methods (Nelson, 2018). In this study, we investigated the role of Sinensetin (Sin) in alleviating the progression of OA and its underlying mechanism. We found that Sin not only inhibited apoptosis and extracellular matrix (ECM) degradation in TBHP-induced chondrocytes, but also promoted the autophagy function of chondrocytes. In addition, we further proved that Sin enhances autophagy through AMPK/mTOR signaling pathway, thereby ameliorating OA.Previous studies have shown that Sin is a plant extract with good antioxidant (Yao et al., 2009), anti-inflammatory (Chae et al., 2017) and antimicrobial properties (Vikram et al., 2013). Pathological factors such as inflammation and oxidative stress are involved in the occurrence and development of OA (Goutas et al., 2018; Wang and He, 2018), and reactive oxygen species (ROS) play an important role in the pathology of OA (Lepetsos and Papavassiliou, 2016). Therefore, we speculate that Sin may weaken ROS-induced apoptosis and ECM degradation in chondrocytes, and ameliorate the progression of OA. First, we found that Sin at a concentration of less than 40 M has no significant cytotoxicity to chondrocytes. Subsequently, we demonstrated that Sin protects TBHP (as ROS donor)-induced chondrocytes from apoptosis and ECM degradation, and activates chondrocyte autophagy.Apoptosis of chondrocytes causes OA cartilage destruction (Charlier et al., 2016), so inhibiting apoptosis of chondrocytes protects against OA pathogenesis (Son et al., 2019). We thus investigated whether Sin has a protective effect on chondrocyte apoptosis. Our experimental results show that Sin reduces the apoptosis of chondrocytes treated by TBHP, indicating Sin’s therapeutic potential for OA.The up-regulation of matrix degrading enzymes and/or the degradation of cartilage extracellular matrix (ECM) is an important cause of cartilage destruction (Troeberg and Nagase, 2012). Our research proves that Sin reduces the level of matrix metalloproteinase 13 (MMP13) protein and increases the level of collagen II and aggrecan in TBHP-treated chondrocytes, indicating that Sin can maintain the homeostasis of ECM.Autophagy is the degradation process of cells, which helps maintain cell homeostasis and improve cell survival and function (Mizushima, 2007). Autophagy has been proved to be a potential therapeutic target for OA, because it helps to alleviate the apoptosis and senescence of chondrocytes (Appleton, 2018). Previous studies have shown that Sin is an autophagy inducer used to treat colitis and hepatocellular carcinoma (Xiongjian et al., 2019; Kim et al., 2020). In our study, Sin also significantly improved the autophagy function of chondrocytes, and its protective effect on TBHP-treated chondrocytes was abrogated when autophagy was inhibited, indicating that Sin plays a protective role by enhancing autophagy.A variety of upstream signaling pathways are involved in the activation of autophagy. Among them, AMP activated protein kinase (AMPK), as a metabolic sensor, plays an important role in regulating autophagy (Mihaylova and Shaw, 2011). In addition, mTOR is a negative regulator of autophagy, and its activity is involved in AMPK-mediated autophagy (Klionsky et al., 2016). Our results showed that Sin activates AMPK activity and reduces mTOR activity in chondrocytes in a time- and dose-dependent manner, indicating that Sin may activate autophagy through AMPK/mTOR signaling pathway.Our research has several limitations. We cannot establish a direct interaction between Sin and AMPK, and we should verify the in vivo effects of Sin in joint-specific knockdown AMPKmice. Another shortcoming of our study is that there are a few indicators to evaluate the protective effect of Sin on OA in vivo. Further in vivo characterization and functional studies are needed to prove the therapeutic effect of Sin on OA. In conclusion, our research shows that Sin activates autophagy via AMPK/mTOR signaling pathway, thereby effectively inhibiting the apoptosis and ECM degradation of TBHP-treated chondrocytes and the pathogenesis of OA in DMM model mice (Figure 8).
FIGURE 8
Schematic diagram shows the potential protective effect of Sin on OA.
Schematic diagram shows the potential protective effect of Sin on OA.
Authors: Saeid Safiri; Ali-Asghar Kolahi; Emma Smith; Catherine Hill; Deepti Bettampadi; Mohammad Ali Mansournia; Damian Hoy; Ahad Ashrafi-Asgarabad; Mahdi Sepidarkish; Amir Almasi-Hashiani; Gary Collins; Jay Kaufman; Mostafa Qorbani; Maziar Moradi-Lakeh; Anthony D Woolf; Francis Guillemin; Lyn March; Marita Cross Journal: Ann Rheum Dis Date: 2020-05-12 Impact factor: 19.103
Authors: Daniel J Klionsky; Kotb Abdelmohsen; Akihisa Abe; Md Joynal Abedin; Hagai Abeliovich; Abraham Acevedo Arozena; Hiroaki Adachi; Christopher M Adams; Peter D Adams; Khosrow Adeli; Peter J Adhihetty; Sharon G Adler; Galila Agam; Rajesh Agarwal; Manish K Aghi; Maria Agnello; Patrizia Agostinis; Patricia V Aguilar; Julio Aguirre-Ghiso; Edoardo M Airoldi; Slimane Ait-Si-Ali; Takahiko Akematsu; Emmanuel T Akporiaye; Mohamed Al-Rubeai; Guillermo M Albaiceta; Chris Albanese; Diego Albani; Matthew L Albert; Jesus Aldudo; Hana Algül; Mehrdad Alirezaei; Iraide Alloza; Alexandru Almasan; Maylin Almonte-Beceril; Emad S Alnemri; Covadonga Alonso; Nihal Altan-Bonnet; Dario C Altieri; Silvia Alvarez; Lydia Alvarez-Erviti; Sandro Alves; Giuseppina Amadoro; Atsuo Amano; Consuelo Amantini; Santiago Ambrosio; Ivano Amelio; Amal O Amer; Mohamed Amessou; Angelika Amon; Zhenyi An; Frank A Anania; Stig U Andersen; Usha P Andley; Catherine K Andreadi; Nathalie Andrieu-Abadie; Alberto Anel; David K Ann; Shailendra Anoopkumar-Dukie; Manuela Antonioli; Hiroshi Aoki; Nadezda Apostolova; Saveria Aquila; Katia Aquilano; Koichi Araki; Eli Arama; Agustin Aranda; Jun Araya; Alexandre Arcaro; Esperanza Arias; Hirokazu Arimoto; Aileen R Ariosa; Jane L Armstrong; Thierry Arnould; Ivica Arsov; Katsuhiko Asanuma; Valerie Askanas; Eric Asselin; Ryuichiro Atarashi; Sally S Atherton; Julie D Atkin; Laura D Attardi; Patrick Auberger; Georg Auburger; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Maria Laura Avantaggiati; Limor Avrahami; Suresh Awale; Neelam Azad; Tiziana Bachetti; Jonathan M Backer; Dong-Hun Bae; Jae-Sung Bae; Ok-Nam Bae; Soo Han Bae; Eric H Baehrecke; Seung-Hoon Baek; Stephen Baghdiguian; Agnieszka Bagniewska-Zadworna; Hua Bai; Jie Bai; Xue-Yuan Bai; Yannick Bailly; Kithiganahalli Narayanaswamy Balaji; Walter Balduini; Andrea Ballabio; Rena Balzan; Rajkumar Banerjee; Gábor Bánhegyi; Haijun Bao; Benoit Barbeau; Maria D Barrachina; Esther Barreiro; Bonnie Bartel; Alberto Bartolomé; Diane C Bassham; Maria Teresa Bassi; Robert C Bast; Alakananda Basu; Maria Teresa Batista; Henri Batoko; Maurizio Battino; Kyle Bauckman; Bradley L Baumgarner; K Ulrich Bayer; Rupert Beale; Jean-François Beaulieu; George R Beck; Christoph Becker; J David Beckham; Pierre-André Bédard; Patrick J Bednarski; Thomas J Begley; Christian Behl; Christian Behrends; Georg Mn Behrens; Kevin E Behrns; Eloy Bejarano; Amine Belaid; Francesca Belleudi; Giovanni Bénard; Guy Berchem; Daniele Bergamaschi; Matteo Bergami; Ben Berkhout; Laura Berliocchi; Amélie Bernard; Monique Bernard; Francesca Bernassola; Anne Bertolotti; Amanda S Bess; Sébastien Besteiro; Saverio Bettuzzi; Savita Bhalla; Shalmoli Bhattacharyya; Sujit K Bhutia; Caroline Biagosch; Michele Wolfe Bianchi; Martine Biard-Piechaczyk; Viktor Billes; Claudia Bincoletto; Baris Bingol; Sara W Bird; Marc Bitoun; Ivana Bjedov; Craig Blackstone; Lionel Blanc; Guillermo A Blanco; Heidi Kiil Blomhoff; Emilio Boada-Romero; Stefan Böckler; Marianne Boes; Kathleen Boesze-Battaglia; Lawrence H Boise; Alessandra Bolino; Andrea Boman; Paolo Bonaldo; Matteo Bordi; Jürgen Bosch; Luis M Botana; Joelle Botti; German Bou; Marina Bouché; Marion Bouchecareilh; Marie-Josée Boucher; Michael E Boulton; Sebastien G Bouret; Patricia Boya; Michaël Boyer-Guittaut; Peter V Bozhkov; Nathan Brady; Vania Mm Braga; Claudio Brancolini; Gerhard H Braus; José M Bravo-San Pedro; Lisa A Brennan; Emery H Bresnick; Patrick Brest; Dave Bridges; Marie-Agnès Bringer; Marisa Brini; Glauber C Brito; Bertha Brodin; Paul S Brookes; Eric J Brown; Karen Brown; Hal E Broxmeyer; Alain Bruhat; Patricia Chakur Brum; John H Brumell; Nicola Brunetti-Pierri; Robert J Bryson-Richardson; Shilpa Buch; Alastair M Buchan; Hikmet Budak; Dmitry V Bulavin; Scott J Bultman; Geert Bultynck; Vladimir Bumbasirevic; Yan Burelle; Robert E Burke; Margit Burmeister; Peter Bütikofer; Laura Caberlotto; Ken Cadwell; Monika Cahova; Dongsheng Cai; Jingjing Cai; Qian Cai; Sara Calatayud; Nadine Camougrand; Michelangelo Campanella; Grant R Campbell; Matthew Campbell; Silvia Campello; Robin Candau; Isabella Caniggia; Lavinia Cantoni; Lizhi Cao; Allan B Caplan; Michele Caraglia; Claudio Cardinali; Sandra Morais Cardoso; Jennifer S Carew; Laura A Carleton; Cathleen R Carlin; Silvia Carloni; Sven R Carlsson; Didac Carmona-Gutierrez; Leticia Am Carneiro; Oliana Carnevali; Serena Carra; Alice Carrier; Bernadette Carroll; Caty Casas; Josefina Casas; Giuliana Cassinelli; Perrine Castets; Susana Castro-Obregon; Gabriella Cavallini; Isabella Ceccherini; Francesco Cecconi; Arthur I Cederbaum; Valentín Ceña; Simone Cenci; Claudia Cerella; Davide Cervia; Silvia Cetrullo; Hassan Chaachouay; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Georgios Chamilos; Edmond Yw Chan; Matthew Tv Chan; Dhyan Chandra; Pallavi Chandra; Chih-Peng Chang; Raymond Chuen-Chung Chang; Ta Yuan Chang; John C Chatham; Saurabh Chatterjee; Santosh Chauhan; Yongsheng Che; Michael E Cheetham; Rajkumar Cheluvappa; Chun-Jung Chen; Gang Chen; Guang-Chao Chen; Guoqiang Chen; Hongzhuan Chen; Jeff W Chen; Jian-Kang Chen; Min Chen; Mingzhou Chen; Peiwen Chen; Qi Chen; Quan Chen; Shang-Der Chen; Si Chen; Steve S-L Chen; Wei Chen; Wei-Jung Chen; Wen Qiang Chen; Wenli Chen; Xiangmei Chen; Yau-Hung Chen; Ye-Guang Chen; Yin Chen; Yingyu Chen; Yongshun Chen; Yu-Jen Chen; Yue-Qin Chen; Yujie Chen; Zhen Chen; Zhong Chen; Alan Cheng; Christopher Hk Cheng; Hua Cheng; Heesun Cheong; Sara Cherry; Jason Chesney; Chun Hei Antonio Cheung; Eric Chevet; Hsiang Cheng Chi; Sung-Gil Chi; Fulvio Chiacchiera; Hui-Ling Chiang; Roberto Chiarelli; Mario Chiariello; Marcello Chieppa; Lih-Shen Chin; Mario Chiong; Gigi Nc Chiu; Dong-Hyung Cho; Ssang-Goo Cho; William C Cho; Yong-Yeon Cho; Young-Seok Cho; Augustine Mk Choi; Eui-Ju Choi; Eun-Kyoung Choi; Jayoung Choi; Mary E Choi; Seung-Il Choi; Tsui-Fen Chou; Salem Chouaib; Divaker Choubey; Vinay Choubey; Kuan-Chih Chow; Kamal Chowdhury; Charleen T Chu; Tsung-Hsien Chuang; Taehoon Chun; Hyewon Chung; Taijoon Chung; Yuen-Li Chung; Yong-Joon Chwae; Valentina Cianfanelli; Roberto Ciarcia; Iwona A Ciechomska; Maria Rosa Ciriolo; Mara Cirone; Sofie Claerhout; Michael J Clague; Joan Clària; Peter Gh Clarke; Robert Clarke; Emilio Clementi; Cédric Cleyrat; Miriam Cnop; Eliana M Coccia; Tiziana Cocco; Patrice Codogno; Jörn Coers; Ezra Ew Cohen; David Colecchia; Luisa Coletto; Núria S Coll; Emma Colucci-Guyon; Sergio Comincini; Maria Condello; Katherine L Cook; Graham H Coombs; Cynthia D Cooper; J Mark Cooper; Isabelle Coppens; Maria Tiziana Corasaniti; Marco Corazzari; Ramon Corbalan; Elisabeth Corcelle-Termeau; Mario D Cordero; Cristina Corral-Ramos; Olga Corti; Andrea Cossarizza; Paola Costelli; Safia Costes; Susan L Cotman; Ana Coto-Montes; Sandra Cottet; Eduardo Couve; Lori R Covey; L Ashley Cowart; Jeffery S Cox; Fraser P Coxon; Carolyn B Coyne; Mark S Cragg; Rolf J Craven; Tiziana Crepaldi; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Maria Teresa Cruz; Ana Maria Cuervo; Jose M Cuezva; Taixing Cui; Pedro R Cutillas; Mark J Czaja; Maria F Czyzyk-Krzeska; Ruben K Dagda; Uta Dahmen; Chunsun Dai; Wenjie Dai; Yun Dai; Kevin N Dalby; Luisa Dalla Valle; Guillaume Dalmasso; Marcello D'Amelio; Markus Damme; Arlette Darfeuille-Michaud; Catherine Dargemont; Victor M Darley-Usmar; Srinivasan Dasarathy; Biplab Dasgupta; Srikanta Dash; Crispin R Dass; Hazel Marie Davey; Lester M Davids; David Dávila; Roger J Davis; Ted M Dawson; Valina L Dawson; Paula Daza; Jackie de Belleroche; Paul de Figueiredo; Regina Celia Bressan Queiroz de Figueiredo; José de la Fuente; Luisa De Martino; Antonella De Matteis; Guido Ry De Meyer; Angelo De Milito; Mauro De Santi; Wanderley de Souza; Vincenzo De Tata; Daniela De Zio; Jayanta Debnath; Reinhard Dechant; Jean-Paul Decuypere; Shane Deegan; Benjamin Dehay; Barbara Del Bello; Dominic P Del Re; Régis Delage-Mourroux; Lea Md Delbridge; Louise Deldicque; Elizabeth Delorme-Axford; Yizhen Deng; Joern Dengjel; Melanie Denizot; Paul Dent; Channing J Der; Vojo Deretic; Benoît Derrien; Eric Deutsch; Timothy P Devarenne; Rodney J Devenish; Sabrina Di Bartolomeo; Nicola Di Daniele; Fabio Di Domenico; Alessia Di Nardo; Simone Di Paola; Antonio Di Pietro; Livia Di Renzo; Aaron DiAntonio; Guillermo Díaz-Araya; Ines Díaz-Laviada; Maria T Diaz-Meco; Javier Diaz-Nido; Chad A Dickey; Robert C Dickson; Marc Diederich; Paul Digard; Ivan Dikic; Savithrama P Dinesh-Kumar; Chan Ding; Wen-Xing Ding; Zufeng Ding; Luciana Dini; Jörg Hw Distler; Abhinav Diwan; Mojgan Djavaheri-Mergny; Kostyantyn Dmytruk; Renwick Cj Dobson; Volker Doetsch; Karol Dokladny; Svetlana Dokudovskaya; Massimo Donadelli; X Charlie Dong; Xiaonan Dong; Zheng Dong; Terrence M Donohue; Kelly S Doran; Gabriella D'Orazi; Gerald W Dorn; Victor Dosenko; Sami Dridi; Liat Drucker; Jie Du; Li-Lin Du; Lihuan Du; André du Toit; Priyamvada Dua; Lei Duan; Pu Duann; Vikash Kumar Dubey; Michael R Duchen; Michel A Duchosal; Helene Duez; Isabelle Dugail; Verónica I Dumit; Mara C Duncan; Elaine A Dunlop; William A Dunn; Nicolas Dupont; Luc Dupuis; Raúl V Durán; Thomas M Durcan; Stéphane Duvezin-Caubet; Umamaheswar Duvvuri; Vinay Eapen; Darius Ebrahimi-Fakhari; Arnaud Echard; Leopold Eckhart; Charles L Edelstein; Aimee L Edinger; Ludwig Eichinger; Tobias Eisenberg; Avital Eisenberg-Lerner; N Tony Eissa; Wafik S El-Deiry; Victoria El-Khoury; Zvulun Elazar; Hagit Eldar-Finkelman; Chris Jh Elliott; Enzo Emanuele; Urban Emmenegger; Nikolai Engedal; Anna-Mart Engelbrecht; Simone Engelender; Jorrit M Enserink; Ralf Erdmann; Jekaterina Erenpreisa; Rajaraman Eri; Jason L Eriksen; Andreja Erman; Ricardo Escalante; Eeva-Liisa Eskelinen; Lucile Espert; Lorena Esteban-Martínez; Thomas J Evans; Mario Fabri; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Nils J Færgeman; Alberto Faggioni; W Douglas Fairlie; Chunhai Fan; Daping Fan; Jie Fan; Shengyun Fang; Manolis Fanto; Alessandro Fanzani; Thomas Farkas; Mathias Faure; Francois B Favier; Howard Fearnhead; Massimo Federici; Erkang Fei; Tania C Felizardo; Hua Feng; Yibin Feng; Yuchen Feng; Thomas A Ferguson; Álvaro F Fernández; Maite G Fernandez-Barrena; Jose C Fernandez-Checa; Arsenio Fernández-López; Martin E Fernandez-Zapico; Olivier Feron; Elisabetta Ferraro; Carmen Veríssima Ferreira-Halder; Laszlo Fesus; Ralph Feuer; Fabienne C Fiesel; Eduardo C Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; John H Fingert; Steven Finkbeiner; Toren Finkel; Filomena Fiorito; Paul B Fisher; Marc Flajolet; Flavio Flamigni; Oliver Florey; Salvatore Florio; R Andres Floto; Marco Folini; Carlo Follo; Edward A Fon; Francesco Fornai; Franco Fortunato; Alessandro Fraldi; Rodrigo Franco; Arnaud Francois; Aurélie François; Lisa B Frankel; Iain Dc Fraser; Norbert Frey; Damien G Freyssenet; Christian Frezza; Scott L Friedman; Daniel E Frigo; Dongxu Fu; José M Fuentes; Juan Fueyo; Yoshio Fujitani; Yuuki Fujiwara; Mikihiro Fujiya; Mitsunori Fukuda; Simone Fulda; Carmela Fusco; Bozena Gabryel; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Sehamuddin Galadari; Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; Nelly Godefroy; Robert M Gogal; Kuppan Gokulan; Gustavo H Goldman; Delia Goletti; Michael S Goligorsky; Aldrin V Gomes; Ligia C Gomes; Hernando Gomez; Candelaria Gomez-Manzano; Rubén Gómez-Sánchez; Dawit Ap Gonçalves; Ebru Goncu; Qingqiu Gong; Céline Gongora; Carlos B Gonzalez; Pedro Gonzalez-Alegre; Pilar Gonzalez-Cabo; Rosa Ana González-Polo; Ing Swie Goping; Carlos Gorbea; Nikolai V Gorbunov; Daphne R Goring; Adrienne M Gorman; Sharon M Gorski; Sandro Goruppi; Shino Goto-Yamada; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Yacine Graba; Martin Graef; Giovanna E Granato; Gary Dean Grant; Steven Grant; Giovanni Luca Gravina; Douglas R Green; Alexander Greenhough; Michael T Greenwood; Benedetto Grimaldi; Frédéric Gros; Charles Grose; Jean-Francois Groulx; Florian Gruber; Paolo Grumati; Tilman Grune; Jun-Lin Guan; Kun-Liang Guan; Barbara Guerra; Carlos Guillen; Kailash Gulshan; Jan Gunst; Chuanyong Guo; Lei Guo; Ming Guo; Wenjie Guo; Xu-Guang Guo; Andrea A Gust; Åsa B Gustafsson; Elaine Gutierrez; Maximiliano G Gutierrez; Ho-Shin Gwak; Albert Haas; James E Haber; Shinji Hadano; Monica Hagedorn; David R Hahn; Andrew J Halayko; Anne Hamacher-Brady; Kozo Hamada; Ahmed Hamai; Andrea Hamann; Maho Hamasaki; Isabelle Hamer; Qutayba Hamid; Ester M Hammond; Feng Han; Weidong Han; James T Handa; John A Hanover; Malene Hansen; Masaru Harada; Ljubica Harhaji-Trajkovic; J Wade Harper; Abdel Halim Harrath; Adrian L Harris; James Harris; Udo Hasler; Peter Hasselblatt; Kazuhisa Hasui; Robert G Hawley; Teresa S Hawley; Congcong He; Cynthia Y He; Fengtian He; Gu He; Rong-Rong He; Xian-Hui He; You-Wen He; Yu-Ying He; Joan K Heath; Marie-Josée Hébert; Robert A Heinzen; Gudmundur Vignir Helgason; Michael Hensel; Elizabeth P Henske; Chengtao Her; Paul K Herman; Agustín Hernández; Carlos Hernandez; Sonia Hernández-Tiedra; Claudio Hetz; P Robin Hiesinger; Katsumi Higaki; Sabine Hilfiker; Bradford G Hill; Joseph A Hill; William D Hill; Keisuke Hino; Daniel Hofius; Paul Hofman; Günter U Höglinger; Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; 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Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; 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Kuen-Jer Tsai; Mario P Tschan; Yi-Hsin Tseng; Takayuki Tsukuba; Allan Tsung; Andrey S Tsvetkov; Shuiping Tu; Hsing-Yu Tuan; Marco Tucci; David A Tumbarello; Boris Turk; Vito Turk; Robin Fb Turner; Anders A Tveita; Suresh C Tyagi; Makoto Ubukata; Yasuo Uchiyama; Andrej Udelnow; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Benjamin R Underwood; Christian Ungermann; Rodrigo P Ureshino; Ryo Ushioda; Vladimir N Uversky; Néstor L Uzcátegui; Thomas Vaccari; Maria I Vaccaro; Libuše Váchová; Helin Vakifahmetoglu-Norberg; Rut Valdor; Enza Maria Valente; Francois Vallette; Angela M Valverde; Greet Van den Berghe; Ludo Van Den Bosch; Gijs R van den Brink; F Gisou van der Goot; Ida J van der Klei; Luc Jw van der Laan; Wouter G van Doorn; Marjolein van Egmond; Kenneth L van Golen; Luc Van Kaer; Menno van Lookeren Campagne; Peter Vandenabeele; Wim Vandenberghe; Ilse Vanhorebeek; Isabel Varela-Nieto; M Helena Vasconcelos; Radovan Vasko; Demetrios G Vavvas; Ignacio Vega-Naredo; Guillermo Velasco; 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