Autophagy and mitochondrial dysfunction in adjuvant-arthritis rats treatment with resveratrol

Resveratrol is a polyphenol derivatives which exhibits a pro-apoptotic effect in a variety of human cancers by triggering mitochondria apoptosis pathway and autophagy. However, there are scarcely reports on its apoptosis-promoting effect in abnormal proliferation fibroblast-like synoviocytes (FLSs). In this study, we investigated the underlying mechanism and apoptosis-inducing effects of resveratrol on the abnormal proliferation of FLSs in adjuvant-arthritis (AA) rats. Since using resveratrol for 12 days resulted in a significant decreasing the swelling degree of the paw, reducing malondialdehyde (MDA) content and enhancing superoxide dismutase (SOD) activity, antioxidant capacity, glutathione peroxidase and glutathione reductase ratio in AA rats. Moreover, we found that 5 μMH2O2 could increase cells viability, Beclin1, LC3A/B, MnSOD, SIRT3 protein expression in FLSs. But, resveratrol could reverse these effects by changing mitochondrial membrane potential (Δψm) to promote mitochondrial reactive oxygen species (mtROS) generation in 5 μMH2O2-treatment FLSs. These results suggest that oxidative stress existed in AA rats. Resveratrol could suppress oxidative stress in AA rats and increase mtROS production by reducing autophagy protein Beclin1, LC3A/B and oxidative stress protein MnSOD to promoted the apoptosis of FLSs. Thus, targeting of mtROS may be a crucial mechanism of resveratrol confers patients with rheumatoid arthritis.

the glutathione disulfide generates glutathione with H + which was provided by NADPH 12 . Furthermore, some studies reported that alterations in extracellular GSH/GSSG could affect proliferation of colorectal carcinoma and lung fibroblast cells 14,15 . So the activity of GSH and GR are very important. Due to the imbalance between oxidation system and antioxidation system increased chemical reaction or insufficient antioxidant defence system results in oxidative stress 16 . Some studies suggested mitochondrial ROS (mtROS) directly regulated the composition of autophagosomes 17 . Although the regulation of autophagy is unknown, it is highly likely that it is affected by oxidative modification of transcription factors 17 . Excessive generation of ROS cause mitochondrial damage resulting in the loss of its function and then cause mitochondrial autophagy. Mitochondrial autophagy remove damaged mitochondria by preventing mtROS accumulating which is a scavenger to maintain normal mitochondrial function 18 .
Resveratrol (3,5,4′ -trihydroxy-trans-stilbene, Res) is a natural polyphenol abundantly found in grape skins and polygonum cuspidatum which possesses a variety of biochemical and physiological effects including anti-inflammatory, anti-oxidation, anti-proliferation and chemopreventive 18 . Low concentration of resveratrol is an excellent scavenger of hydroxyl, superoxide, and other radicals. Resveratrol is also able to avoid excessive ROS induced lipid peroxidation and DNA damage 19 . It is well known that a high dose of resveratrol can induce ovarian cancer cells apoptosis 20,21 . Silent mating-type information regulation 2 homolog 3 (Sirt3) is the primary mitochondrial deacetylase 22 and directly regulates biological functions involved in mitochondrial energy production 23 which also plays a key role in regulating mtROS homeostasis. Some seminal papers have been reported that Sirt3 regulates manganese superoxide dismutase (MnSOD) deacetylation and identified the target lysines [24][25][26] . At the same time, lysine acetylation is also involved in the regulation of p53 which also plays a role in mitochondrial redox regulation 27 . The study also showed that the forms of oxidative stress induce Sirt3 deacetylation activity and MnSOD activity 28 . It is well established that over expression of Sirt3 decreases both intracellular mitochondrial O 2 • − and total ROS levels, suggesting that Sirt3 regulates both energy production and mtROS scavenging pathways 29 . Some research pointed that mitochondrial oxidative stress induced compensatory upregulation of SIRT3, which might in turn activate phosphorylation of AMPK and subsequently triggered autophagy by upregulating Beclin1 expression and LC3 II/I conversion 30 . Furthermore, Res induced apoptosis via ROS-triggered autophagy in human colon cancer cells 31 , while the study indicated Res enhanced temozolomide-induced apoptotic cell death in malignant glioma by inhibiting autophagy 32 . Similarly, It is unknown whether resveratrol can regulates mtROS level to promote FLSs apoptosis by Sirt3-MnSOD axis or autophagy pathway in H 2 O 2 -treatment FLSs.

Results
Arthritis induction in SD rats. The Fig. 1 displays that after the injection FCA in SD rats 20 days, compared with the secondary began to appear obvious swollen (Fig. 1A) and the nose and double fore legs appear different degree of inflammation injury (Fig. 1B). Besides, after the injection for 8, 12, 18, 20 days, compared with the control group, arthritis index was gradually increased (Fig. 1C) and the swelling degree of the paw was gradually increased, too (Fig. 1D). But, the change of 15 mg/kg resveratrol group was significantly decreased (P < 0.05), and 45 mg/kg resveratrol and 200 mg/kg NAC group changes were most obvious (p < 0.01), after 12 days can reach 0.80 ml, 0.65 ml, 0.41 ml ( Fig. 2A). And the content of MDA in the model group was about 2 folds more than control group. After administration 5 mg/kg, 15 mg/kg, 45 mg/kg resveratrol and 200 mg/kg NAC the contents of MDA were gradually decreased in rats serum (Fig. 2B). And the control of total antioxidant capacity (T-AOC) (Fig. 2C), total SOD activity (T-SOD) (Fig. 2D), the ratio of glutathione peroxidase and glutathione reductase (GSH-PX/GR) (Fig. 2E) was significantly higher than the model group. Compared with the model group, 15 mg/kg, 45 mg/kg resveratrol and 200 mg/kg NAC group was significantly increased. In model group, the synovial tissue obviously thickened, and the cartilage tissue was eroded. A large number of fragments appeared in the synovial cavity. However, after treatment with resveratrol, the above symptoms relieved or even disappeared (Fig. 2F).  (Fig. 3A). Before adding 5 μ MH 2 O 2 treatment for 12 h, different concentrations of resveratrol pretreatment for 24 h, 50-400 μ MRes can inhibite the proliferation of FLSs. 5 μ MH 2 O 2 cell activity enhanced to 105.1%, after adding resveratrol to cells, the viability reduced to 93.2%, 80.6%, 45.2%, 32.3%, in a dose-dependent (Fig. 3B). The same, 0-10 μ MH 2 O 2 early apoptotic cells and late apoptotic/necrotic cells with few changes. Compared with the control group, 20-80 μ M H 2 O 2 markedly increase early apoptotic cells and late apoptotic/necrotic cells (Fig. 3C-E). Compared with the 5 μ MH 2 O 2 treatment group, Res also can increase 5 μ MH 2 O 2 treatment-FLSs early apoptotic cells and late apoptotic/necrotic cells (Fig. 3C,F,G).

Resveratrol inhibited H
Resveratrol enhanced mitochondrial superoxide generation and the loss of mitochondrial membrane potential in FLSs. The effects of resveratrol on regulation of mitochondrial redox status and mitochondrial membrane potential (Δ ψ m) were evaluated using MitoSOX Red (Invitrogen, USA) and assay kit with JC-1 (Life technologies, USA) in FLSs. Once MitoSOX Red reagent is selectively targeted to mitochondriain, MitoSOX Red is oxidized by O 2 • − and exhibits red fluorescence. The 5 μ MH 2 O 2 group significantly decreased mitochondrial O 2 • − generation compared with the control group, which was notably increased by resveratrol pretreatment (Fig. 4A,C). JC-1 can selectively enter mitochondria and reversibly changes color as the Δ ψ m changes. There was a significantly increased red fluorescence in 5 μ MH 2 O 2 group. However, Res pretreatment promoted green fluorescence generation, suggesting that the mitochondrial membrane was depolarized (Fig. 4B,D).These results suggested that resveratrol remarkably enhanced collapse of mitochondrial membrane potential in FLSs (P < 0.05).

Resveratrol declined autophagy related Beclin1 and LC3A/B proteins expression.
Immunofluorescence images indicated that the control group Beclin1 protein was located in the cytoplasm and abundantly existed in the nucleus surrounding. LC3A/B protein mainly enriched in nucleus and few part   Resveratrol enhanced mtROS generation through declining mitochondrial oxdative stress related proteins SIRT3 and MnSOD expression. Immunofluorescence images indicated that the SIRT3 protein was located in the cytoplasm and nucleus. MnSOD protein merely enriched in the cytoplasm. However, the 5 μ M H 2 O 2 group SIRT3 protein mainly enriched in nucleus surrounding, especially the nucleus surrounding fluorescence is very strong and with increasing concentration of Res, SIRT3 and MnSOD fluorescence intensity gradually reduced (Fig. 6A,B). Western blotting analysis, compared with control group 5 μ MH 2 O 2 can increase the expression of SIRT3 and MnSOD protein. With increasing concentration of Res, the expression gradually reduced and high doses of Res protein expression level reducing is most obvious (Fig. 6C-E).

Discussion
The main features of RA patients rheumatoid arthritis showed the formation of pannus invasion and destruction of articular structure in local thickening of the synovium. Pannus is mainly composed of permeate combination of synovial fibroblasts and a large number of lymphocytes and macrophages 33,34 . Human FLSs plays an important role in the pathogenesis of RA. TNF-α and IL-1β are extremely important in the induction of arthritis, can trigger the production of matrix metalloproteinases (MMPs), and ultimately damage the synovial and articular cartilage 35 , In addition, macrophages and FLSs can produce proinflammatory cytokines such as TNF-α , IL-1β , IL-6, IL-8. Excessive production of pro-inflammatory cytokines stimulates neutrophils and activated macrophages the secreting ROS which making them as the medium of joint damage 36,37 . So the accumulation of ROS leads to the arthritis which is the main reason. Under the condition of oxidative stress, ROS was produced in excess leading to lipid peroxidation, protein oxidation and DNA fragmentation 38 . MDA, a marker for lipid peroxidation, is frequently used as an indicator for measurement of cellular membrane damage 39 . Superoxide anion is regulated by enzymes such as superoxide dismutase (SOD) and peroxidases, as well as endogenous supplies of antioxidants such as glutathione (GSH) 40 .
Resveratrol is a phenolic compound, which is widely found in various fruits and vegetables, possess antioxidant, anti-aging, regulation of lipid metabolism, anti-cancer properties 18 . Similarly, in vivo experiments, it was found that the content of MDA in the model group was significantly higher than the normal group, and the 5mg/kg, 15 mg/kg, 45 mg/kg resveratrol group and 200 mg/kg NAC group were significantly decreased in AA rats serum. The total SOD activity, total antioxidant capacity and the ratio of GSH-PX and GSH reductase in the 15 mg/kg resveratrol group were significantly lower than the normal control group, after successively intragastric administration of 5 mg/kg, 15 mg/kg, 45 mg/kg resveratrol and 200 mg/kg NAC, those were significantly higher. HE staining showed that 15 mg/kg, 45 mg/kg resveratrol and 200 mg/kg NAC can significantly suppressed the AA rat knee joint synovium hyperplasia, relieved the pannus formation and synovial endothelial swelling, also can effectively reduce the subsynovial layer of a large number of lymphocytes and plasma cells infiltration, inhibited synovial invasion of cartilage and subchondral substrate. Especially 45 mg/kg resveratrol improved more obviously. Thus, we further confirmed that AA rats had oxidative stress. This oxidative stress can promote the proliferation of synovial cells and the thickening of the synovial layer. However, the resveratrol could inhibit proliferation of the synovial layer in AA rats.
In recent years, more and more evidences show that resveratrol can promote the apoptosis of cancer cells 41 . High concentrations of resveratrol can induce many kinds of tumor cells apoptosis. FLSs has the characteristics of tumor like properties. Therefore, we also show that the apoptotic effect of 50-200 μ M resveratrol on FLSs were not obviously, but the 200-400 μ M resveratrol significantly promoted the apoptosis of FLSs, and even 400 μ M of resveratrol obviously increased the apoptosis rate of FLSs. In addition, mitochondria can also regulate the apoptosis of tumor cells. In the physiological state, more than 90% of ROS are produced by the mitochondria, and in the process of respiratory chain transfer, the electron from the mitochondrial respiratory chain of the mitochondrial respiratory chain is formed by the oxidation of the complex of the oxidative phosphorylation of the complex I and III is the formation of O 2 ▪ − 42 . O 2 ▪ − mainly through manganese superoxide dismutase (MnSOD) to form H 2 O 2 ; H 2 O 2 was decomposed into H 2 O by the body's hydrogen peroxide enzymes. There is a good balance between them, once the balance is broken, it will lead to the occurrence of oxidative stress, resulting in a large number of active ingredients. In our experiment, we found excessive H 2 O 2 to cause early apoptotic FLSs and late apoptotic/ necrotic FLSs increasing and we also found that resveratrol can increase mtROS generation and early apoptotic cells and late apoptotic/necrotic cells in H 2 O 2 -treatmnet FLSs. So ROS plays a very important role in cell proliferation and apoptosis. At low concentrations, ROS can be used as a signal molecule to regulate cell proliferation and other functions of which can lead to cell senescence and death at high concentrations of ROS 43 .
Resveratrol interferes into the signal pathway in tumor cells to regulate cell survival and apoptosis, but also regulates the mitochondrial permeability, damage the mitochondrial membrane potential 44,45 , the destruction of electronic respiratory chain 46 , and increase the production of mitochondrial superoxide. In our experiment, the mtROS and mitochondrial membrane potential (Δ ψ m) were significantly increased comparing with normal control group. With the increase of the concentration of resveratrol, and the apoptosis rate was higher. Recent studies have found that resveratrol can cause the change of SIRT3 in the SIRTs family, SIRT3 plays a key role in regulating the balance of MnSOD in cells. It is used to change the content of mitochondrial ROS by MnSOD 47 . In our experiments, 5 μ MH 2 O 2 was found to promote the expression of SIRT3 and MnSOD protein, and MnSOD protein expression decreased with the increasing resveratrol concentration, which indicated that the antioxidant capacity of SIRT3 was decreased. This also further suggests that mitochondrial ROS content is increased. This means that MtROS could trigger early apoptotic cells and late apoptotic/necrotic cells in H 2 O 2 -treatmnet FLSs. In addition, ROS plays a very important role in the induction of autophagy, and it is likely that transcription factors are regulated by oxidative regulation of autophagy. Recently, it was found that resveratrol can induce FLSs apoptosis in RA patients with caspase-8 and caspase-9 in the caspase pathway, which activates the caspase-3 pathway. And then, Bcl-2, Bcl-xL or Bax activity can also be changed in the mitochondrial membrane and cytoplasm, and then promote the apoptosis of cells 48 . In addition, studies have found that, under normal conditions, autophagy related proteins Beclin1, AMBRA1 and Bcl-2 or Bcl-XL combine together. When BH3-only was combined with Bcl-2 or Bcl-XL, Beclin1 and AMBRA1, VPS34 and could be formed by the combination of and VPS15, extend the wrapping of mitochondria in LC3-II under the action of mature and lysosomal binding, mitochondrial autophagy triggered 49 . In order to further understand the signal pathway of resveratrol induced apoptosis in FLSs, we studied the expression levels of LC3A/B protein and Beclin1 protein associated with autophagy maker. The results showed that after treatment with 5 μ MH 2 O 2 , the expression level of LC3A/B had no significant change and the expression level of Beclin1 increased in FLSs. With different resveratrol concentration, the expression level of LC3A/B and Beclin1 decreased. Therefore, the signaling pathway of FLSs is likely to be caused by the accumulation of MtROS by autophagy pathway and oxidative stress combined action, and excessive MtROS induced apoptosis in FLSs. To sum up, resveratrol plays a very important role in the inhibition of abnormal proliferation of FLSs in AA rats, but the specific detailed mechanism of its induction of apoptosis is not clear, so the results of this study can provide a theoretical basis for the further study on the mechanism of apoptosis, and it is very important to the pathogenesis and treatment of RA. Experimental animals and design. Male SD rats weighed 180 ± 20 g were provided from laboratory animal Center of Anhui Medical University in this experiment. Rats were acclimatized in temperature and humidity controlled rooms for one week. Rats can be freely available standard food and tap water. The left hind toe of male SD rats were injected with 150 μ l Freund's complete adjuvant (FCA, Sigma, USA) for 20 days. Control rats were injected with 150 μ l physiological saline. Then adjuvant-arthritis rats and control rats were randomly assigned to six groups treated with 5 mg/kg, 15 mg/kg, 45 mg/kg resveratrol and 200 mg/kg N-acetyl-L-cysteine (NAC, Sigma, USA) for 12 days by continuous intragastric administration. These doses have been selected based on previously described 50,51 . All animals were sacrificed after above treatment 12 days. Pairs of legs was excised, fixed in 4% paraformaldehyde or stored in − 80 °C for later analysis. Blood samples were collected, and then 4 °C for the night. Next day, those samples were centrifuged at 3500 rpm/min for 20 min to obtain supernatant fluid which was stored at − 80 °C for further analyses.

Chemicals and reagents.
Isolation and culture FLSs. FLSs were isolated from model group rats as described previously 52 . In brief, sterile synovial tissue were cut into 1 mm 3 size, with twice the volume of 0.2%-II type collagenase (Sigma, USA) which contained 10% fetal bovine serum (FBS) digesting 2 to 2.5 h (percussion one time every 30 min). And then using 0.25% trypsin digest 30 min before ending to digestion. Resuspend the cells with DMEM (Gibco, USA) containing 15% fetal bovine serum (Gibco, USA) in the 25 cm 2 flask, then incubated in a humidified atmosphere containing 5% CO 2 at 37 °C. All cells used in experiments for the 3-5 passengers.
The swelling degree of the paw and arthritis index score. The foot volume were measured before each rat left hind toe volume injected FCA. After injection occurs, every 3 or 4 days right hind foot volume measurement were executed by Toe swelling measuring instrument (ZH-YLS-7C, China). The swelling degree of the paw to calculate: swelling of the feet (ml) = model group of right hind foot volume (ml)-control group of right hind foot volume (ml) and medication after foot swelling (ml) = after administration of right hind foot volume (ml)-before administration of right hind foot volume(ml). The degree of arthritis index was determined using a scoring protocol 53 , where by severity was scored on a scale of 0-4, where 0 = absent, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe.
Oxidative injury parameters. Lipid peroxidation was evaluated by means of the TBARS (thiobarbituric acid-reactive substances) assay. Malondialdehyde (MDA) comes form Lipid peroxide degradation products which can be combined with thiobarbituric acid (TBA) to form pink products. MDA content was determined by using the MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. briefly, Serum of rats with various reagents blending were incubated in 95 °C water bath for 40 min and subsequently centrifuged at 3000 rpm for 10 min to obtain supernate which was estimated absorbance by spectrophotometry at 532 nm and the values expressed as nmol (mg protein) −1 . SOD activity, Antioxidant capacity, glutathione peroxidase and glutathione reductase ratio were determined by SOD activity assay kit, antioxidant capacity assay kit, glutathione peroxidase assay kit, glutathione reductase assay kit (Nanjing Jiancheng Bioengineering Institute, Nangjing, China). Simply, serum of rats blend with reagents for several minutes. Respectively, obtaining supernate was detected absorbance by spectrophotometry at 550 nm, 520 nm, 412 nm, 340 nm and the values expressed as percentage.
Hematoxylin and eosin staining. After 12 days of continuous intragastric administration, AA rats were sacrificed. Knee-joint was harvested and fixed in 4% paraformaldehyde. Then fixed tissue was dehydrated and embedded in paraffin after 6 months of decalcification of knee joint. Knee-joint was cut into 4 micron size and mounted on glass slides. The section was stained with hematoxylin and eosin (Beyotime, Shanghai, China). Images of the stained tissue were obtained using light microscopy.
Cell growth inhibition assay. Cell viability was performed using cck-8 assays kit (Biosharp, Hefei, China) according to the manufacturer's protocols. Briefly, the cells was plated in 96-well plates and grown 24 h. Next day, the cells were treated with various concentration of hydrogen peroxide (H 2 O 2 ) for 12 h, or after various doses of resveratrol pretreatment in FLSs for 24 h, selected dosage of H 2 O 2 incubation 12 h. After above treatment, A volume of 10 μ l cck-8 was added to those wells. Cells were cultured for 3 h at 37 °C. Then absorbance of the 96-well plates was determined on enzymelinked immunosorbent assay plate reader at 450 nm.
Cell apoptosis detection by flow cytometry. The apoptotic rate of FLSs was detected by flow cytometry using Annexin V-FITC/PI staining kit (BestBio, Shanghai, China) according to the manufacturer's protocol. Briefly, FLSs were seeded and incubated in 6-well plates and treated with above dosage and time.after that cells were digested with pancreatic enzymes, resuspended in 400 μ l Annexin V binding buffer at a density of 1 × 10 6 cells/ml and incubated with 5 μ l Annexin V-FITC and 10 μ l propidium iodide (PI) for 15 min at 4 °C in dark. Finally, cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences).

MtROS assessment. The levels of mtROS were detected by MitoSOX Red Mitochondrial Superoxide
Indicator (life technologies, CA, USA). FLSs were grown on glass coverslips and then harvested in 6-well plates. After treated with above dosage and time, the cells were cultured with MitoSOX Red Mitochondrial Superoxide Indicator at 37 °C for 10 min. After staining, wash the cells in fresh growth medium 3 times. Images were collected by confocal laser scanning microscope (model LEICA.SP5-DMI6000-DIC; Leica Microsystems GmbH). The quantitative analysis of the red fluorescence signal was detected by using the built-in evaluation software (Leica LAS AF Lite, Mannheim, Germany).
Mitochondrial membrane potential (Δψm) determination. Mitochondrial membrane potential (Δ ψ m) was detected using Mitochondrial membrane potential assay kit with JC-1 (life of technology, CA, USA) according to the manufacturer's protocol. JC-1 probe was accumulated in the mitochondrial matrix polymer which gave off a strong red fluorescence in normal mitochondrion. However, JC-1 probe exists as a monomer at unhealthy mitochondrion which gave off a strong green fluorescence. The cells were grown on glass coverslips. After treated with above dosage and time, the cells were cultured with 5 μ g/ml JC-1 at 37 °C for 20 min. After staining, wash the cells 3 times with PBS. The fluorescence intensity was determined by confocal laser scanning microscope (model LEICA.SP5-DMI6000-DIC; Leica Microsystems GmbH). The quantitative analysis of the red/ green fluorescence signal was detected by using the built-in evaluation software (Leica LAS AF Lite, Mannheim, Germany). The Δ ψ m was represented as the ratio of red to green fluorescence intensity.
Western blotting. Cells were seeded and incubated in 6-well plates and treated with above dosage and time and washed with ice-cold PBS and then suspended in 150 μ l of RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was determined using the BCA assay kit (Beyotime, Shanghai, China). An equal amount of proteins was added into each lane. Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After the membranes were blocked with 5% skim milk for 2 h, the membranes were incubated with the primary antibody overnight at 4 °C, washed with Tris-buffered saline-Tween solution (TBST) 3 times and incubated with a 1:1000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Beyotime, Shanghai, China) for 1 h. Finally, bands were detected using enhanced chemiluminescence reagents (BOSTER, Wuhan, China). LEICA.SP5-DMI6000-DIC; Leica Microsystems GmbH) and the fluorescence intensity was expressed as the percentage relative to the control group (set as 100%), respectively. Statistical analysis. Using SPSS19.0 statistical software, data were expressed by mean ± SD. Three or more groups was compared with single factor analysis of variance (one-way ANOVA) and t test was used in the two groups. Values of p < 0.05 and p < 0.01 were considered statistically significant.

Ethics statement. This study was approved by the Association of Laboratory Animal Sciences and the
Center for Laboratory Animal Sciences at Anhui Medical University (Permit Number: 15-0026). All experiments on animals complied with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health, including all use, care and operative procedures. And all experimental procedures were followed the guidelines for humane treatment set by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at Anhui Medical University.