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Folate deficiency provokes apoptotic lethality in synoviocytes. HIG-82 synoviocytes (1.5×105) were plated in 60-mm cultured dishes for 24 h. The culture medium was replaced with FC, MFD, and FD media and then continued cultivating for additional 48 h. (A) Cells were then collected, washed with PBS, fixed in PBS-methanol (1:2 v/v) solution and maintained at 4°C for at least 18 h. After one washed with PBS, the cell pellets were then stained with a PI solution containing PBS, PI (40μg/mL), and DNase-free RNase A (40μg/mL) for 30 min at RT in the dark. The cell pellets were then analyzed using a Becton-Dickinson FACSan flowcytometer. The epirubicin (500 nM) treatment (Epi) is a positive control assay of apoptosis. The blank bar, gray bar, right slash bar and left slash bar represent FC, MFD, FD and Epi treatment, respectively. The percentages of subG1 population determined by the PI fluorescent intensity in apoptosis cells which was weaker than that of cells in the G1 phase. The percentages of apoptosis cells were characterized as the percentages of cells in the SubG1 region of the DNA distribution histograms. The FD subG1 bar graph is compared with FC or MFD. A p<0.05 (*) was considered statistically significant. (B) Cells were fixed in 1% paraformaldehyde in PBS for 30 min, then washed with PBS, and stored in 70% methanol at 4°C. After rehydration in PBS, cells were evaluated with TUNEL assay. The values shown are mean ± SD (n = 5–8 samples per experiment). Significant differences from the FC or MFD groups are p<0.05 (*), p<0.01 (**), p<0.001 (***).
Source publication
Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight i...
Citations
... superoxide, thus modulating the oxidative stress-mediated pathway that has been shown to play a pivotal role in chronic pain. [43][44][45] In addition, experimental data have shown AP to inhibit NF-κB activation and upregulate Nrf2 gene expression with consequent downstream effects. 46 As for PA, it exerts its effect via inhibiting NF-κB activation with modulation of the inflammatory responses, through inhibition of proinflammatory cytokines TNF-α, IL-1β, IL-6, metalloproteinases and of MCP-1 expression. ...
... Experimental data in rodents indicate that AP may inhibit the pathway involved in transmitting neuropathic pain, 54 and several reports indicating a beneficial effect of these two compounds evaluated individually in animal models of neuropathic pain have been published. 43,44 In a streptozotocin-induced diabetic neuropathy rat model, AP was found to dose-dependently increase the pain threshold of the animals, and appeared to do so by decelerating the oxidative-stress-mediated pathology in the sciatic nerve under study. 55 Similarly, in an identical model, AP was found to partially reverse allodynia, along with reversal of oxidative-stress markers in the spinal cord. ...
... Specifically, recent studies have demonstrated that synoviocytes cultivated under FD conditions exhibit elevated reactive oxygen species (ROS) production and increased cytosolic Ca 2+ release, leading to apoptotic lethality [20]. This mechanistic insight is consistent with our results, suggesting that folate deficiency contributes to OA pathogenesis not only through impaired immune response but also by directly exacerbating cellular stress and apoptosis in joint tissues. ...
... This convergence of evidence highlights folate's potential as a modifiable factor in OA management and prevention. The protective effect of folate against OA progression, as indicated in both studies, suggests that folate supplementation could be a beneficial intervention in OA in reducing the risk or severity of OA in critical joints, underlining its role in preserving the integrity of chondrocytes and synoviocytes [20,21]. On the other hand, the identification of succinate dehydrogenase (SDH)/complex II as a major site for ROS production under FD conditions offers a potential therapeutic target to mitigate these effects [20]. ...
... The protective effect of folate against OA progression, as indicated in both studies, suggests that folate supplementation could be a beneficial intervention in OA in reducing the risk or severity of OA in critical joints, underlining its role in preserving the integrity of chondrocytes and synoviocytes [20,21]. On the other hand, the identification of succinate dehydrogenase (SDH)/complex II as a major site for ROS production under FD conditions offers a potential therapeutic target to mitigate these effects [20]. Considering our results, checking folate levels in OA patients could be recommended as part of the evaluation process. ...
Context
Folate deficiency is often observed in patients with inflammatory diseases, raising questions about its role in knee osteoarthritis (OA) progression.
Objectives
This study aimed to assess the association of folate deficiency with the clinical and radiological severity of knee OA.
Methods
A prospective cross-sectional study was conducted from January 1, 2019 to January 1, 2020. Primary knee OA patients referred to orthopedic clinics in Zabol, Iran were included. Radiographic severity was gauged utilizing the Kellgren–Lawrence (KL) classification. For clinical severity, patients completed the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire. IBM SPSS v.27 facilitated the statistical analysis.
Results
Forty-nine knee OA patients, averaging 67.45±13.44 years in age, were analyzed. Spearman correlation analysis revealed a negative correlation between folate levels and both WOMAC and KL scores. The correlation was stronger between folate and KL score (Spearman correlation coefficient: −0.75) than between folate and WOMAC total score (Spearman correlation coefficient: −0.46). Additionally, a significantly higher KL score was observed in patients with folate deficiency (p=0.004).
Conclusions
Our study highlights a significant correlation between folate deficiency and increased severity of OA, which is evident in radiological and clinical assessments. These findings suggest that folate plays a key role in OA pathogenesis and could be a modifiable factor in its management.
... It has been highlighted that the interaction between highest and lowest levels of serum folate and vitamin B12 respectively, being a common finding in these subjects, was associated to an increased risk of gestational diabetes [3]. For instance, the evidence by Saravanan et al. is challenging and essential to definitely confirm and translate into this clinical framework the association between the risk of diabetes and the vitamins' status observed using in vitro/in vivo models [3][4][5][6]. Indeed, as coenzymes, folate and B12 are involved in various biological pathways (i.e., aforementioned for the folate, whereas B12 mainly promotes fatty acids/amino acids degradation) whose balance is warranted by the maintenance of adequate levels of both vitamins in order to assure the normal physiological functions [6]. ...
... Indeed, as coenzymes, folate and B12 are involved in various biological pathways (i.e., aforementioned for the folate, whereas B12 mainly promotes fatty acids/amino acids degradation) whose balance is warranted by the maintenance of adequate levels of both vitamins in order to assure the normal physiological functions [6]. Undoubtedly, folate and particularly for its depletion, is the most investigated vitamin in the framework of gestational risk and diabetes [4][5][6][7]. In vitro models of the pathophysiology of diabetes have shown that the folate deficiency is associated with the apoptosis of islet β cells [4]. ...
... Undoubtedly, folate and particularly for its depletion, is the most investigated vitamin in the framework of gestational risk and diabetes [4][5][6][7]. In vitro models of the pathophysiology of diabetes have shown that the folate deficiency is associated with the apoptosis of islet β cells [4]. Noteworthy, the excessive exposure to FA may promote DNA methylation patterns thus contributing to trigger the inflammatory response, well characterized, although differentiated, in diabetes type I and II [5]. ...
... Previous studies have indicated that the folate receptor is overexpressed on activated macrophages, which are closely related to osteoarthritis (Tsuneyoshi et al., 2012). Some literature has also showed that folic acid deficiency (FD) can mediate synovial cell apoptosis by affecting the excessive production of reactive oxygen species (ROS) induced by mitochondrial complex II and NOX and the sharp release of intracellular calcium (Ca2 +) concentration (Hsu et al., 2016). Besides, Duan W et al. proved that a folate receptor-targeted nanocarrier system can effectively block the NF-κB signaling pathway and reduce the expression of proinflammatory cytokines, so as to significantly inhibit the progression of arthritis in a mouse model (Duan and Li, 2018). ...
Background: Osteoarthritis (OA) is a degenerative disease which serious affects patients. Ligusticum chuanxiong (CX) has been shown to have a certain curative effect on osteoarthritis in traditional Chinese medicine therapy. This study is based on network pharmacology and molecular docking technology to explore the potential mechanism of CX.
Methods: Components of CX to treat osteoarthritis were screened in the TCMSP database and targets were predicted by the PharmMapper database, the osteoarthritis targets were collected from the GeneCards database, and intersection genes were found to be the possible targets of CX anti-OA. The STRING database and Cytoscape software were utilized for protein-protein interaction analysis and further screening of core targets. The Metascape database was used for KEGG and GO enrichment analyses. Then, the top 10 pathways were selected to construct “drug-compound-target-pathway-disease” network analysis. Finally, molecular docking was used to analyze the binding affinity of seven compounds with core targets and TNF-α.
Results: Seven compounds with 253 non-repetitive targets of CX were screened from the TCMSP database and 60 potential intersection targets of CX anti-OA were found. PPI network analysis showed that the core targets were ALB, AKT1, IGF1, CASP3, MAPK1, ANXA5, and MAPK14, while GO and KEGG pathway enrichment analyses showed that the relevant biological processes involved in the treatment of osteoarthritis by CX might include the MAPK cascade and reactive oxygen species metabolic process. The KEGG pathway analysis result was mainly associated with the MAPK signaling pathway and PI3K-AKT signaling pathway. We further docked seven ingredients with MAPK1 and MAPK14 enriched in the MAPK pathway, and TNF-α as the typical inflammatory cytokine. The results also showed good binding affinity, especially FA, which may be the most important component of CX anti-OA.
Conclusion: Our research revealed the potential mechanism of CX in the treatment of OA, and our findings can also pave the way for subsequent basic experimental verification and a new research direction.
... As mentioned in the introduction section, Hcy is considered a pro-oxidant agent that compromises the oxidative balance by different mechanisms [46,49,50]. Among its proposed mechanisms is the increase of multicomponent NOX enzyme complexes, which are one of the major producers of endogenous ROS in different tissues [11][12][13]45]. In this context, NOX plays a central role in liver fibrogenesis. ...
... Moreover, the ratio of cleaved caspase-3/caspase-3 is also reduced, indicating that apoptosis is decreased when FA hepatic levels are balanced, and that this vitamin plays an important role during BD liver damage. FA is known for its anti-apoptotic properties in different types of cells [13,[68][69][70][71]. These authors attribute this action to different mechanisms: through decreasing NOX activity-ROS generation-mitochondrial complex II; decreasing OS-, preventing telomeric DNA oxidation and attrition; regulating the expression of apoptosis-related genes (decreased caspase-3 and upregulated BCL2/BAX ratio); modulating microRNA-34a, associated with Bcl-2 signaling; or decreasing caspase-9 activity. ...
Chronic ethanol consumption and liver disease are intimately related to folic acid (FA) homeostasis. Despite the fact that FA decreases lipid oxidation, its mechanisms are not yet well elucidated. Lately, adolescents have been practising binge drinking (BD), consisting of the intake of a high amount of alcohol in a short time; this is a particularly pro-oxidant form of consumption. The aim of this study is to examine, for the first time, FA homeostasis in BD adolescent rats and its antioxidant properties in the liver. We used adolescent rats, including control rats and rats exposed to an intermittent intraperitoneal BD model, supplemented with or without FA. Renal FA reabsorption and renal FA deposits were increased in BD rats; hepatic deposits were decreased, and heart and serum levels remained unaffected. This depletion in the liver was accompanied by higher transaminase levels; an imbalance in the antioxidant endogenous enzymatic system; lipid and protein oxidation; a decrease in glutathione (GSH) levels; hyper-homocysteinemia (HHcy); an increase in NADPH oxidase (NOX) 1 and NOX4 enzymes; an increase in caspase 9 and 3; and a decrease in the anti-apoptotic metallopeptidase inhibitor 1. Furthermore, BD exposure increased the expression of uncoupled endothelial nitric oxide synthase (eNOS) by increasing reactive nitrogen species generation and the nitration of tyrosine proteins. When FA was administered, hepatic FA levels returned to normal levels; transaminase and lipid and protein oxidation also decreased. Its antioxidant activity was due, in part, to the modulation of superoxide dismutase activity, GSH synthesis and NOX1, NOX4 and caspase expression. FA reduced HHcy and increased the expression of coupled eNOS by increasing tetrahydrobiopterin expression, avoiding nitrosative stress. In conclusion, FA homeostasis and its antioxidant properties are affected in BD adolescent rats, making it clear that this vitamin plays an important role in the oxidative, nitrosative and apoptotic hepatic damage generated by acute ethanol exposure. For this, FA supplementation becomes a potential BD therapy for adolescents, preventing future acute alcohol-related harms.
... Similarly, disrupted cellular redox homeostasis by the accumulation of unmetabolized homocysteine has also been reported in vitamin B 12 -deficient worms that showed significant reduction in the activity of cobalamin-dependent methionine synthase [49]. Moreover, the results reported above were similar phenomena to those observed in mammals during folate deficiency [50,51], which reportedly induces severe oxidative stress, leading to DNA damage [52,53] and increased blood pressure and insulin resistance [54] in mammals. ...
Using Caenorhabditis elegans as a model animal, we evaluated the effects of chronical supplementation with high-dose folic acid on physiological events such as life cycle and egg-laying capacity and folate metabolism. Supplementation of high-dose folic acid significantly reduced egg-laying capacity. The treated worms contained a substantial amount of unmetabolized folic acid and exhibited a significant downregulation of the mRNAs of cobalamin-dependent methionine synthase reductase and 5,10-methylenetetrahydrofolate reductase. In vitro experiments showed that folic acid significantly inhibited the activity of cobalamin-dependent methionine synthase involved in the metabolism of both folate and methionine. In turn, these metabolic disorders induced the accumulation of unmetabolized homocysteine, leading to severe oxidative stress in worms. These results were similar to the phenomena observed in mammals during folate deficiency.
... Previous studies investigating synovial-caused OA pathologies have several limitations (59)(60)(61)(62)(63): i) Cells are cultured only under normal oxygen partial pressure, thus physiological or pathological oxygen fluctuations are not considered; and ii) the effect on hypoxic tolerance of chondrocytes by fluctuating oxygen have been studied, while mitochondrial damage and respiratory dysfunction in oxygen dependent FLS have not been investigated in depth. Therefore, due to physiological and pathological H/R processes in joint organs, previous studies may have a certain level of bias (15). ...
Hypoxia/reoxygenation (H/R) may play an important role via senescence in the mechanism of osteoarthritis (OA) development. The synovial membrane is highly sensitive to H/R due to its oxygen consumption feature. Excessive mechanical loads and oxidative stress caused by H/R induce a senescence‑associated secretory phenotype (SASP), which is related to the development of OA. The aim of the present study was to investigate the differences of SASP manifestation in synovial tissue masses between tissues from healthy controls and patients with OA. The present study used tumor necrosis factor‑α (TNF‑α) to pre‑treat synovial tissue and fibroblast‑like synoviocytes (FLS) to observe the effect of inflammatory cytokines on the synovial membrane before H/R. It was determined that H/R increased interleukin (IL)‑1β and IL‑6 expression levels in TNF‑α‑induced cell culture supernatants, increased the proportion of SA‑β‑gal staining, and increased the expression levels of high mobility group box 1, caspase‑8, p16, p21, matrix metalloproteinase (MMP)‑3 and MMP‑13 in the synovium. Furthermore, H/R opened the mitochondrial permeability transition pore, caused the loss of mitochondrial membrane potential (ΔΨm) and increased the release of reactive oxygen species (ROS). Moreover, H/R caused the expansion of the mitochondrial matrix and rupture of the mitochondrial extracorporeal membrane, with a decrease in the number of cristae. In addition, H/R induced activation of the JNK signaling pathway in FLS to induce cell senescence. Thus, the present results indicated that H/R may cause inflammation and escalate synovial inflammation induced by TNF‑α, which may lead to the pathogenesis of OA by increasing changes in synovial SASP and activating the JNK signaling pathway. Therefore, further studies expanding on the understanding of the pathogenesis of H/R etiology in OA are required.
... The underlying inflammation in the DRM + I group may have caused mitochondrial dysfunction, which would have been less pronounced in the DRM-I group. Several studies have related malnutrition with a rise of ROS in different types of diseases [11,[26][27][28]. In this sense, Hsu et al. reported high levels of ROS and altered mitochondrial function in subjects with folate deficiency, which suggests that mitochondrial dysfunction is a result of malnutrition [29]. ...
Background:
The aim of this study was to evaluate markers of inflammation, oxidative stress and endothelial function in a disease-related malnutrition (DRM) outpatient population.
Methods:
For this cross-sectional study, a total of 83 subjects were included and clustered in 3 groups: 34 with normonutrition (NN), 21 with DRM without inflammation (DRM-I) and 28 with DRM and inflammation (DRM + I). Nutritional diagnosis was conducted for all subjects according to ASPEN. Biochemical parameters, proinflammatory cytokines, reactive oxygen species production, glutathione, mitochondrial membrane potential, oxygen consumption, adhesion molecules and leukocyte-endothelium interactions were evaluated.
Results:
DRM + I patients showed lower albumin, prealbumin, transferrin, and retinol-binding protein levels with respect to the NN group (p < 0.05), differences that were less noticeable in the DRM-I group. DRM + I was associated with a significant increase in hsCRP and IL6 vs the NN and DRM-I groups, and TNFα was increased in both DRM vs NN. DRM was characterised by increased oxidative stress, which was marked by a significant increase in ROS levels and a decrease in mitochondrial membrane potential in the DRM + I group. An evident reduction in mitochondrial oxygen consumption and glutathione concentration was observed in both DRM groups, and was accompanied by increased leukocyte adhesion and adhesion molecules and decreased rolling velocity in the DRM + I group. Furthermore, percentage of weight loss was negatively correlated with albumin, prealbumin, transferrin, O2 consumption, glutathione and leukocyte rolling velocity, and positively correlated with hsCRP, IL6, TNFα, ROS, leukocyte adhesion, and VCAM-1.
Conclusions:
Our results show that DRM is associated with oxidative stress and an inflammatory state, with a deterioration of endothelial dysfunction in the DRM + I population.
... Furthermore we investigated the intracellular ROS generation in isolated pancreatic islet cells as Hsu et al. (2016) reported that pancreatic islet cells express an extremely low amount of antioxidant enzyme gene which make these cells more prone to oxidative damage. Intracellular ROS generation was measured by the rate of oxidation of DCHF-DA to DCF by free radicals. ...
... Synovial cell lines were established and are valuable for investigating various synovial cell functions in vitro. Some synovial cell lines (27)(28)(29) have been utilized for investigating cellular and molecular mechanisms underlying the emergence of rheumatoid arthritis (RA) and OA (30)(31)(32). However, synovial cell lines derived from the TMJ are difficult to find; we found only one study stating that fibroblast-like cells derived from baboon TMJs were successfully immortalized by the ectopic expression of human telomerase reverse transcriptase (hTERT) (33). ...
Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast‑like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho‑associated coiled‑coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α‑smooth muscle actin (α‑SMA) and type I collagen; and ii) an inhibitor against the actin‑polymerizing agent ROCK, Y‑27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor‑regulated transcription, CCG‑100602, clearly suppressed the mRNA levels of α‑SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor‑1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ‑OA and may aid in identifying drug targets for treating this condition at the molecular level.
