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Introduction: Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a dietary flavonoid with good antioxidant and anti-inflammatory properties. The present study aims to determine these effects in peripheral blood mononuclear cells (PBMCs) evoked by lipopolysaccharides (LPS). Aims: The present study aims to determine these effects in peripheral blood mononuclear cells (PBMCs) evoked by lipopolysaccharides (LPS). Methods: The mRNA expression and protein secretion of inflammatory mediators were evaluated by enzyme- linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (PCR), respectively. Western blotting was utilized for assessing p65-NF-κB phosphorylation. Ransod kits evaluated the glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in the cell lysates. Ultimately, the molecular docking approach was performed to investigate the biological activity of Quercetin against NF-κB pathway proteins and antioxidant enzymes. Results: The findings revealed that quercetin significantly attenuated the expression and secretion of inflammatory mediators and p65-NF-κB phosphorylation in LPS-induced PBMCs. Additionally, quercetin dose-dependently improved the activities of SOD and GPx enzymes and decreased LPS-mediated oxidative stress in PBMCs. Moreover, quercetin has a considerable binding affinity to IκKb, the core element of the NF-κB pathway and the antioxidant enzyme SOD. Conclusion: The data show that quercetin plays a vital role in ameliorating inflammation and oxidative stress caused by LPS in PBMCs.

Background: Oxidative stress has been shown to be an important factor, which plays a significant role in the pathogenesis of neurodegenerative disorders. Heat Shock Protein-27 (HSP-27) has been implicated in antioxidant responses against oxidative stress. Trehalose is a natural disaccharide widely used in a variety of food products with demonstrated protective effects against several neurodegenerative diseases. This study investigated the effects of trehalose on antioxidant responses, and the gene expressions for HSP-27 and caspase-3 against hydrogen peroxide (H2O2) induced oxidative injury in PC-12 cell line. Methods: The PC-12 cells were treated with various concentrations of H2O2 and trehalose for 24hr. The cell viability was assessed, using MTT and Lactate Dehydrogenase (LDH) release assays. Moreover, the activity of Catalase (CAT) and Glutathione Peroxidase (GPx) enzymes, and the Malondialdehyde (MDA) levels were determined. In addition, the levels of HSP-27 and caspase-3 gene expressions were measured. Results: The results indicated that the pretreatment with trehalose increased cell survival against the H2O2-induced oxidative injury. Furthermore, trehalose elevated the CAT and GPx activities and reduced MDA levels compared to that of control group (P˂0.05). Moreover, trehalose upregulated the HSP-27 gene expression, while reducing the expression of caspase-3 gene compared to that of the untreated cells (P˂0.05). All of these biochemical changes were found to be dose-dependent for trehalose. Conclusion: Based on the study findings, trehalose had the capacity to attenuate the oxidative stress and cell injury. Therefore, trehalose may be suggested as a therapeutic agent to treat neurodegenerative disorders caused by oxidative stress damages.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.

Evidence has shown that inflammation and oxidative stress are implicated in the development of a great number of human diseases. Trehalose possesses various biological effects including antioxidant and anti-inflammatory activities. However, there is little data on the effects of trehalose on human cells including peripheral blood mononuclear cells (PBMCs). Here, we aimed to investigate whether trehalose could attenuate oxidative stress and inflammation induced by lipopolysaccharides (LPS) in PBMCs.The enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to assess the levels of inflammatory cytokines. To investigate the phosphorylation of c-Jun N-terminal kinase (JNK) and NF-κB, western blot analysis was utilized. Oxidant-antioxidant markers were assessed using ELISA and colorimetric procedures.The results revealed that trehalose significantly mitigated the effect of LPS on the phosphorylation of JNK and NF-κB-P65 (The results of this study indicated that trehalose prevented inflammation and oxidative stress in the LPS-stimulated PBMCs, providing evidence for the benefits of trehalose as a potential therapeutic agent in inflammatory conditions.LPS: Lipopolysaccharide; NAC: N-Acetyl cysteine; ROS: Reactive oxygen species; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; MDA: Malondialdehyde; MAPK: Mitogen-activated protein kinases; JNK: c-Jun N-terminal kinase; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells.

Aging is associated with an increase in oxidative stress, which damages organs such as the kidney. Trehalose has abundant beneficial activities including antioxidative effects. This study aimed to investigate the effects of trehalose on several antioxidant parameters of the aged kidney. Wistar rats were divided into three groups: young (4 months), aged (24 months), and aged-trehalose. The third group was treated with 2% trehalose for one month. The expression of target genes and enzyme activities in the kidney of the animals were evaluated by quantitative polymerase chain reaction (qPCR) and enzyme colorimetric procedures, respectively. Protein levels of NFE2L2 showed a 50% reduction in aged rats compared to young rats (P0.001), which was restored by trehalose intervention. In addition, the activity and mRNA levels of catalase (CAT) increased in aged rats while treatment with trehalose reversed this trend. On the other hand, superoxide dismutase (SOD) activity was reduced in the kidneys of aged rats but was not affected by trehalose intervention .It is concluded that trehalose supplementation alleviates the antioxidant system impairments in the kidneys of aged rats. However, further investigations are needed to thoroughly describe the antioxidative impacts of trehalose on the kidneys during aging.

Background Insulin resistance has a vital role in the pathophysiology of polycystic ovary syndrome (PCOS). Previous investigations have shown that some lipid ratios could be a simple clinical indicator of insulin resistance (IR) in some disorders and ethnicities. The present study was conducted to evaluate the correlation between triglyceride to HDL-cholesterol (TG/HDL-C), total cholesterol to HDL-cholesterol (TC/HDL-C), as well as fasting triglyceride-glucose (TyG) indices with IR (as measured by homeostasis model assessment of IR (HOMA-IR), quantitative insulin sensitivity check index (QUICKI) and fasting glucose to insulin ratio (FGIR)) among the Iranian women diagnosed with PCOS. Methods In the current study, a total of 305 women with PCOS were evaluated. TG/HDL-C, TC/HDL-C, and TyG indices were calculated. Fasting insulin level was measured using ELISA technique. IR was defined as a HOMA-IR value of ≥2.63, FG-IR value of Results The insulin-resistant (IR) and insulin-sensitive (IS) groups, established by the HOMA-IR, FG-IR, and QUICKI values were different in terms of TG/HDL-C, TC/HDL-C, and TyG indices. These indices were associated with IR even after adjusting for age and BMI. ROC curve analyses showed that TyG, TG/HDL-C, and TC/HDL-C strongly predicted HOMA-IR with area under the curve (AUC) of 0.639, 0.619, and 0.623, respectively (P P = 0.04). Conclusion TyG, TG/HDL-C, and TC/HDL-C indices might be good indicators of IR among Iranian women diagnosed with PCOS.

Diabetes mellitus is an associated risk factor for coronary artery disease (CAD). There is a growing interest in myocardial infarction (MI) to better understand the relationship between hyperglycemia and CVD. Glycated hemoglobin (HbA1c) is the standard measure for assessing glucose regulation in diabetes mellitus and an indicator of potential cardiovascular risk and exposure to glucose concentrations over a duration of approximately 3 months. Glycated albumin (GA) may be useful for evaluating hyperglycemia in settings in which HbA1c screening is problematic or inaccessible. Another indicator that has been considered recently is the GA/HbA1c ratio. The goal of this study was to investigate the relationship between GA/HbA1c and the presence of MI and identify the sugar fluctuations as a suitable indicator for early screening and prevention of MI. This was a case-control study of diabetic patients with MI and patients with type 2 diabetes with no history of heart problems. Each group consisted of 117 participants (117 cases and 117 controls), and the total number of participants was 234. The data collection tools included laboratory tests and a demographic questionnaire. Each patient was assigned to either the case or the control group in a simple and convenient way and the relationship between MI and the GA/HbA1c ratio was investigated. The findings of this study indicated that the ratio of glycated albumin to glycated hemoglobin was higher in the case group with MI than the control group (p = 0.001). To achieve the objectives of the study, descriptive statistics and the Mann-Whitney test were used to analyze the data. This study revealed the relationship between MI and the GA/HbA1c ratio. It can be said that prior to the occurrence of MI, blood glucose levels are a good indicator of recent glucose fluctuations, as well as the prediction and occurrence of MI. Therefore, the improved control of blood sugar and avoidance of high fluctuation may prevent MI from occurring.

Rosmarinic acid (RA) is a potential herbal medicine and has received considerable attention due to its strong antioxidant properties. The aim of this study is to investigate the impact of RA on inflammation and oxidative stress induced by lipopolysaccharide (LPS) in peripheral blood mononuclear cells (PBMCs). PBMCs were pre-treated with various contents of RA (20, 40, 80 µM) for 24 h, then, stimulated with LPS (10 ng/ml) for more 6 h. ELISA and Real-time PCR were done to detect the levels of IL-6, TNF-α, COX-2, IL-1β and IL-10. Western blot was done to investigate the phosphorylated amounts of P65-NF-κB and JNK. Inflammatory cytokines and oxidant-antioxidant parameters were determined by colorimetric and ELISA methods. The results indicated that LPS augmented the protein levels of IL-6, TNF-α, and IL-1β cytokines as well as the mRNA levels of IL-6, TNF-α, IL-1β, COX-2, and IL-10 cytokines in in PBMCs. However, pretreatment with RA could reduce the impact of LPS on inflammatory markers. In addition, RA inhibited P65-NF-κB and JNK phosphorylation. LPS also caused a decrease in antioxidant enzymes, total thiol, and total antioxidant capacity as well as an increment in malondialdehyde and nitric oxide metabolite contents that RA abrogated them. Collectively, our finding demonstrated that RA ameliorates LPS-induced inflammation in PBMCs. RA reduces oxidative stress by preventing lipid peroxidation and nitric oxide production as well as restarting the activity of the GPx and SOD enzymes. Furthermore, our findings indicated that RA was able to protect PBMCs from inflammation via inhibiting the NF-κB and JNK MAPK pathways. This evidence shows a promising therapeutic role for RA in inflammatory status.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.

Polycystic ovarian syndrome (PCOS) is a common poignant endocrine disorder affecting women, posing a close association with metabolic syndrome and obesity. Existing literature characterizes PCOS with deranged levels of several adipokines and myokines. CTRP15 is a paralogue of adiponectin, mainly expressed by skeletal muscles, and plays a key role in insulin, glucose, and lipid metabolism. In the current study, we aim to determine the circulating levels of CTRP15 and evaluate its association with cardiometabolic and inflammatory parameters in PCOS women. This case-control study included 120 PCOS patients (60 Recurrent pregnancy loss (RPL) and 60 infertile (inf) PCOS) and 60 healthy non-PCOS controls. Serum levels of hs-CRP were measured by commercial kits, while serum levels of adiponectin and CTRP15 were determined using the ELISA technique. Serum levels of CTRP15 were significantly elevated in PCOS-RPL and PCOS-inf subgroups when compared to controls (94.80 ± 27.08 and 87.77 ± 25.48 vs. 54.78 ± 15.45, both P < 0.001). Moreover, serum adiponectin was considerably lower in the PCOS group and subgroups (P < 0.001), while serum hs-CRP, fasting insulin, HOMA-IR, and free testosterone were significantly higher when compared to the non-PCOS group (P < 0.05). Furthermore, CTRP15 closely associated with FSH, HOMA-IR, hs-CRP, and BMI. These results highlight a possible involvement of CTRP15 in the pathogenesis of PCOS. The elevated levels of CTRP15 might be a compensatory mechanism for the metabolic dysregulations (excess adiposity, insulin resistance, metaflammation) associated with the syndrome. Nevertheless, future studies are necessary to unravel the underlying mechanism.

Background: Insulin resistance has a key role in the pathophysiology of polycystic ovary syndrome (PCOS). Previous investigations have informed that some lipid ratios could be a simple clinical indicator of insulin resistance (IR) in some disorders and ethnicities. We aimed to examine the correlation between triglyceride to HDL-cholesterol (TG/HDL-C), total cholesterol to HDL-cholesterol (TC/HDL-C) and fasting triglyceride-glucose (TyG) indices with IR (as measured by homeostasis model assessment of IR [HOMA-IR], quantitative insulin sensitivity check index [QUICKI] and fasting glucose to insulin ratio [FGIR]), and determine a good clinical predictor for IR in Iranian PCOS woman. Methods: We evaluated 305 PCOS women. After physical evaluations, biochemical parameters were measured using commercial kits and TG/HDL-C, TC/HDL-C and TyG indices were calculated using formula. Fasting insulin level measured using ELISA technique. IR was defined as a HOMA-IR value ≥2.63, FG-IRResults: The insulin-resistance and insulin-sensitive groups, which established by HOMA-IR, FG-IR and QUICKI values, were different in terms of TG/HDL-C, TC/HDL-C and TyG indices. These indices were associated with IR after adjusting for age and BMI. The under ROC curves (AUC) of TyG, TG/HDL-C and TC/HDL-C for predicting HOMA-IR index were 0.639, 0.619 and 0.623 respectively which were significant, with a p-value 0.012, 0.033 and 0.027, respectively. The AUC of TC/HDL-C (0.614) was significant (p-value 0.04) for predicting FG-IR.Conclusion: Our findings demonstrated that the elevated TyG, TG/HDL-C and TC/HDL-C were significantly associated with IR and could be utilized as indicators of IR among PCOS women in Iran.

Malvidin is an anthocyanin which is involved in inhibiting inflammatory-related mediators in inflammatory diseases; however, its mechanism of action in THP-1 cells is not yet known. THP-1 is a human monocytic cell line that is derived from patients with acute monocytic leukemia. The present study aimed to investigate the effect of malvidin on inflammatory responses and oxidative stress in lipopolysaccharide (LPS)-induced THP-1 cells. THP-1 cells were stimulated with LPS (50 ng/ml) to induce inflammation in the presence or absence of malvidin. The anti/proinflammatory cytokines were evaluated by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Total protein levels/phosphorylation of c-Jun N-terminal kinase (JNK), P65-NF-κB, and IKKα/IKKβ were evaluated by western blot analysis. Malondialdehyde (MDA) and nitric oxide (NO) metabolite levels, ferric reducing antioxidant power (FRAP), total thiol (T-SH) content, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were measured to evaluate the antioxidant activity of malvidin in THP-1 cells. Treatment of LPS-stimulated THP-1 cells with malvidin (100 and 200 μM) led to the significant inhibition of interleukin-6 (IL-6), tumor necrosis factor-α, and IL-1β messenger RNA (mRNA) expression and protein levels as well as a significant increase in the IL-10 mRNA expression and protein secretion. Moreover, 200 μM malvidin treatment reduced the phosphorylation of JNK, IKKα/IKKβ, and P65-NF-κB. These findings showed that malvidin not only decreased the MDA and NO metabolite levels but also increased the FRAP and T-SH content as well as SOD and GPx activities. The findings of the present study demonstrated the potential role of malvidin in blocking inflammation and oxidative stress induced by LPS in THP-1 cell line, suggesting that malvidin is likely to be a therapeutic agent for inflammatory diseases.

Resveratrol was reported to inhibit inflammatory responses; however, the role of this polyphenol in obesity-induced skeletal muscle inflammation remains unknown. Mice fed a high fat diet (HFD) were treated with resveratrol for 16 weeks. Resveratrol treatment decreased macrophage infiltration into skeletal muscle of HFD-fed mice. Resveratrol also led to the polarization of macrophages to the M2 direction, as well as decreasing the expression of a number of M1 pro-inflammatory cytokines [tumor necrosis factor α (TNF-α), interleukin 1 β (IL-1β) and interleukin 6 (IL-6)]. In addition, increased infiltration of regulatory T cells (Treg cells) was found following resveratrol treatment in skeletal muscle of mice. Decreased intramyocellular lipid deposition was associated with reduced expression levels of toll-like receptors 2 (TLR2) and TLR4 in resveratrol treated mice. We also found that diminished inflammation in skeletal muscle following resveratrol treatment was accompanied by increasing phosphorylation of 5’-adenosine monophosphate-activated protein kinase (AMPK) and decreasing phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Taken together, these findings suggest that resveratrol ameliorates inflammation in skeletal muscle of HFD-induced model of obesity. Therefore, resveratrol might represent a potential treatment for attenuation of inflammation in skeletal muscle tissue.

It is indicated that malvidin has anti-inflammatory and antioxidant effects on various cells, although the function of malvidin in preventing inflammatory reactions caused by lipopolysaccharide (LPS) in peripheral blood mononuclear cells (PBMCs) is still not known. The objective of this study was to examine the impact of malvidin on inflammatory responses and oxidative stress in PBMCs as caused by LPS. The present findings showed that LPS significantly increased the expression of IL-6, TNF-α, IL-1β, and COX-2 mRNA and protein release from PBMCs 22 hr after treatments. It was also revealed that increased levels in cytokine expression coincided with increased phosphorylation of JNK, P65-NF-κB, and IKKα/IKKβ. Also, the expression of IL-6, TNF-α, IL-1β, and COX-2 mRNA induced by LPS as well as secretion of protein in PBMC has been significantly decreased by pretreatment of malvidin. Importantly, pretreatment of the cells with malvidin completely abrogated the phosphorylation of P65-NF-κB, JNK, and IKKα/IKKβ in LPS treated cells. Malvidin protection against LPS-induced inflammation was coupled with a decline in the levels of nitric oxide metabolite and malondialdehyde, along with an increase in the ferric reducing antioxidant power, total thiol activity, and also superoxide dismutase and glutathione peroxidase activity. In accordance with this finding, malvidin may represent a promising therapeutic agent for the prevention of inflammation in PBMCs.

Autophagy was shown to modulate inflammation in immune cells. This study was designed to evaluate the association between autophagy and inflammation in peripheral blood mononuclear cells (PBMCs) of type 2 diabetic (T2D) and non-diabetic (ND) subjects. The autophagy markers were measured by real-time PCR and western blot. The gene expression of pro- and anti-inflammatory cytokines was assessed by real-time PCR. Reduced transcription of BECN1 and LAMP2 and unchanged expression of MAP1LC3B and ATG5 were observed in PBMCs of T2D patients. Decreased LC3B-II and increased p62/SQSTM1 levels were found in PBMCs of diabetic patients. The p-mTOR level was higher in PBMCs of diabetic patients. An increase in both IL-1β and TNF-α gene expression, along with a decrease in the expression of IL-10, was observed in PBMCs of T2D patients. TNF-α mRNA expression was inversely correlated with the mRNA expression of BECN1 and LAMP2. TNF-α and IL-1β expression were negatively correlated with the protein levels of LC3B-II. TNF-α and IL-1β expression had also a positive correlation with protein level of p62. IL-10 mRNA expression was positively correlated with the mRNA expression of BECN1 and LAMP2 and protein levels of LC3B-II and negatively correlated with protein level of p62. In addition, p-mTOR level was positively correlated with IL-1β and TNF-α mRNA expression. The results revealed a reduced autophagy in PBMCs of T2D patients that is liked with an enhanced inflammation. The suppression of autophagy in PBMCs of diabetic patients may be associated with the activation of the mTOR signaling.

Previous works have linked high concentrations of palmitate to cellular toxicity by autophagy modulation. However, the ways in which palmitate regulates inflammation and apoptosis in peripheral blood mononuclear cells (PBMCs), has not been well characterized. In the present study, we therefore aimed to investigate the role autophagy in inflammatory responses and apoptotic death of PBMCs treated with palmitate. 0.5mM palmitate increased the level of LC3-II at 6h, peaked at 12h and then decreased at 24h. The protein level of p62 was significantly increased at 6h and 12h, suggesting an impairment of autophagic flux in palmitate-treated PBMCs. Inhibiting autophagy with chloroquine (CQ) and 3-Methyladenine (3-MA) significantly augmented palmitate-induced PBMCs apoptotic death as demonstrated by increased cleaved PARP level and increased the percentage of apoptotic (YO-PRO-1 positive and PI negative) cells. Furthermore, CQ pretreatment exacerbated palmitate-induced TNF-α and IL-6 mRNA expression in PBMCs. Moreover, induction of autophagy by pretreatment of PBMCs with rapamycin resulted in a distinct increase of palmitate-induced apoptosis. The induction of autophagy also led to a further increase in palmitate-induced expression of TNF-α and IL-6. These results indicate that the excess palmitate could impair autophagy, hence contributing to palmitate-induced-inflammation and apoptosis in PBMCs. Therefore, dysregulated autophagy in PBMCs may provide a novel mechanism that connects diet-induced obesity to low grade inflammation in patients with type 2 diabetes.

Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders affecting females of reproductive age. It has been associated with cardiometabolic disorders including diabetes mellitus and cardiovascular disorders, and increases the risk of developing fecundity pathologies including recurrent pregnancy loss (RPL) and infertility. C1q/tumor necrosis factor-α-related protein-6 (CTRP6) is a novel adipokine involved in glucose and lipid metabolism, host inflammation, and organogenesis. In the present study, we aimed to determine the association of serum CTRP6 levels with some components of metabolic syndrome in PCOS patients (infertile PCOS [inf-PCOS] and PCOS-RPL). This case-control study included 120 PCOS patients (60 inf-PCOS and 60 PCOS-RPL) and 60 healthy controls. Serum high-sensitivity C-reactive protein (hs-CRP) and homocysteine were measured using commercial kits, while adiponectin and CTRP6 levels were assessed using ELISA technique. Inf-PCOS and PCOS-RPL individuals had higher levels of serum CTRP6 than controls (546.15 ± 125.02 ng/ml and 534.04 ± 144.19 ng/ml vs. 440.16 ± 159.24 ng/ml; both p < .001). Moreover, serum adiponectin levels were significantly reduced, while fasting insulin, homeostasis model assessment of insulin resistance, free testosterone, and hs-CRP levels were significantly elevated in PCOS group, when compared with controls. Furthermore, serum CTRP6 positively associated with body mass index in all subjects. It showed an inverse correlation with adiponectin in PCOS group and subgroups. However, it had a direct association with hs-CRP in PCOS group and inf-PCOS subgroup, but not PCOS-RPL subgroup. These findings unravel a probable role of CTRP6 in PCOS pathogenesis, which poses a possibility to be a good diagnostic target. However, further investigation is needed.

Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare, slow-growing mass, which is locally aggressive and classified as tumors of borderline malignancy. In this study, we report a case of subcutaneous ankle mass in 43-year-old female since 2 years ago with a pathological diagnosis of PHAT, characterized by clusters of ectatic, fibrin-lined, and thin-walled vessels which are surrounded by a spindled or pleomorphic neoplastic stroma.

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.

Aim Angiopoietin-like protein 3 (ANGPTL3) is recognized as a regulator of lipid metabolism. However, little is known about its association with insulin resistance in polycystic ovary syndrome (PCOS) setting. The present study aimed to investigate the serum levels of ANGPTL3 and adiponectin in PCOS women compared to healthy controls. Main method In this study, a total of 175 premenopausal women (117 PCOS and 58 non-PCOS) were enrolled. Serum concentrations of ANGPTL3, adiponectin, fasting insulin, and other hormonal variables were measured using ELISA technique. Key findings Results showed that adiponectin levels were significantly lower in PCOS group than those of non-PCOS group. However, serum levels of ANGPTL3, high-sensitivity C-reactive protein (hs-CRP), and homocysteine (Hcy) were found to be higher in PCOS patients, when compared to non-PCOS ones. Moreover, serum ANGPTL3 positively correlated with BMI and serum triglyceride, while it inversely correlated with serum HDL-C in PCOS patients. Significance Our results demonstrated that increased levels of ANGPTL3 correlated with insulin resistance and dyslipidemia in PCOS patients, highlighting the need for future studies targeting its role in the pathogenesis of this disease.

Autophagy is a cellular process activated in response to various stresses such as starvation, hypoxia, and oxidative stress. Autophagy was reported to modulate the inflammatory pathways. However, whether autophagy is involved in regulation of palmitate-induced inflammation of skeletal muscle C2C12 cells is still unknown. The present study aimed to investigate the autophagic pathway in C2C12 cells treated with 0.5 mM palmitate. The results showed that the protein levels of LC3BII and P62 were increased in C2C12 cells after 12 h palmitate treatment. Besides, inhibition of autophagy by chloroquine or 3-methyladenin and its activation by rapamycin were associated with elevated mRNA and protein levels of IL-6 and TNF-α inflammatory cytokines in C2C12 cells. To study the mechanism by which autophagy impairment leads to activation of inflammatory responses, reactive oxygen species (ROS) levels in palmitate-treated cells were measured. The results showed that while palmitate stimulates ROS production, pretreatment of the cells with N-acetyl cysteine (NAC), a ROS scavenger, reduced inflammatory responses and also improved LC3-BII and P62 protein in the C2C12 cells exposed to palmitate. These findings suggest that palmitate-induced defect of autophagic flux leads to elevated inflammatory cytokine expression in the skeletal muscle cells by regulating the oxidative stress process.

Curcumin, a natural polyphenol compound, has the beneficial effects on several diseases such as metabolic syndrome, cancer, and diabetes. The anti-inflammatory property of curcumin has been demonstrated in different cells; however, its role in prevention of palmitate-induced inflammation in skeletal muscle C2C12 cells is not known. In this study, we examined the effect of curcumin on the inflammatory responses stimulated by palmitate in C2C2 cells. The results showed that palmitate upregulated the mRNA expression and protein release of IL-6 and TNF-α cytokines in C2C12 cells, while pretreatment with curcumin was able to attenuate the effect of palmitate on inflammatory cytokines. The anti-inflammatory effect of curcumin was associated with the repression of phosphorylation of IKKα-IKKβ, and JNK. Palmitate also caused an increase in reactive oxygen species (ROS) level that curcumin abrogated it. Collectively, these findings suggest that curcumin may represent a promising therapy for prevention of inflammation in skeletal muscle cells.

Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor (PPAR) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPAR antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75mM palmitate reduced cell viability by 43 and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10M) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPAR-dependent and PTP1B-independent mechanisms. Copyright (c) 2014 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDYSaturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPAR-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc.

Background: The leptin (LEP G2548A) and leptin receptor (LEPR Q223R) gene polymorphisms have been variably associated with type 2 diabetes (T2D) in different populations. In this study we hypothesized that these variants might be associated with T2D and related metabolic traits in an Iranian population. Methods: The LEP G2548A and LEPR Q223R genotypes were determined by PCR-RFLP in 378 normoglycemic controls and 154 T2D patients. Bonferroni correction was applied for the correction of multiple testing. Results: The A allele of the LEP G2548A polymorphism was more prevalent in females of the T2D group than the controls (p = 0.009). In a recessive model (GG+GA vs. AA), the frequency of the AA genotype was higher in female patients compared to normoglycemic subjects 34.9% vs. 19.3%, OR 2.60 (1.27 - 5.31), p = 0.009. Multivariate logistic regression analysis also showed that the AA genotype of the LEP G2548A polymorphism is an independent risk factor for T2D in females. No significant association was found between the allele and genotype frequencies of the LEPR Q223R variant with T2D in female and male groups. In addition, no significant difference in an-thropometrical and biochemical parameters was observed between the genotypes of LEP and LEPR variants in gender-specific groups in both non-diabetic and diabetic subjects. Conclusions: Our results suggest that the LEP G2548A polymorphisms might associate with T2D among Iranian female subjects, whereas the LEPR Q223R variant is not associated with T2D and its related metabolic traits in this population.

The aim of the study was to investigate the effect of PTP1B modulation on palmitate-induced cytokine production in macrophages.Lentiviruses carrying PTP1B-shRNA or cDNA at different multiplicities of infection (MOIs) were used to decrease and increase PTP1B expression in Raw 264.7 cells, respectively. mRNA and protein levels of TNF-α and IL-6 were measured by real-time PCR and ELISA, respectively.0.5 mM palmitate reduced PTP1B mRNA and protein levels by 25 and 19 %, respectively, compared to untreated cells. Overexpression of PTP1B decreased mRNA and protein levels of TNF-α and IL-6 in macrophages stimulated with palmitate. We found that protein and mRNA levels of cytokines significantly increased in knockdown cells stimulated by palmitate in a dose-dependent manner with increased MOI. NF-kB, JNK, p38 and ERK specific inhibitors significantly reduced the production of TNF-α and IL-6 in macrophages stimulated with palmitate and also PTP1B knockdown cells. Furthermore, inhibition of PTP1B resulted in increased phosphorylation of JNK, p38, ERK and NF-kB p65 in macrophage cells.The data of this study demonstrate that PTP1B negatively regulates palmitate-induced cytokine secretion in macrophages by mechanisms involving the activation of MAPKs and NF-kB pathways.

Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor γ (PPARγ) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPARγ antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75 mM palmitate reduced cell viability by 43% and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10 μM) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPARγ-dependent and PTP1B-independent mechanisms.Saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPARγ-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling which is overexpressed in the liver of diabetic animals. The aims of this study were to generate liver-specific PTP1B knockout mice using a PTP1B-short hairpin RNA (shRNA) plasmid and to investi- gate the effect of PTP1B inhibition on plasma glucose levels in streptozotocin-induced diabetic mice. We first validated the hydrodynamic tail vein injection in mice using a vector carrying the luciferase gene. Expression of the PTP1B gene was quantified by real-time PCR. The level of phosphorylated Akt was examined by western blot analysis. The injection of the plasmid containing firefly luciferase revealed that the highest transfer of the vector into the liver was obtained 24 h after the injection of 20 µg plasmid. The injection of PTP1B-shRNA, but not the scrambled shRNA plasmid, resulted in a reduction in PTP1B expression levels by up to 84% in the liver of the diabetic mice. Plasma glucose levels following the injection of PTP1B-shRNA remained significantly lower in the diabetic mice for 5 days. In addition, mice receiving PTP1B-shRNA in the basal and insulin-stimulated states had higher levels of Akt phosphorylation in the liver cells compared with mice that were injected with the scrambled sequence (35 and 60%, respectively; p

To develop a convenient animal model of T2D by pretreatment with low-dose 10% w/v fructose (FRC) solution followed by the injection of low doses of streptozotocin (STZ) in Wistar rats. For this 8-week experimental study; rats were first fed a standard chow ad-libitum diet and either tap water (n=40) or 10% w/v FRC solution (n=40) for 4 weeks. Next, rats in each category were randomly allocated to 4 subgroups (n=10 each) of low-dose STZ (25,35, and 45 mg/kg). The final mean fasting blood sugar (FBG) of FRC+STZ45 (197±55.87 mg/dl) were significantly higher than that of the STZ45 (P=0.015) and FRC (P=0.019) groups. FRC+STZ45 showed the highest insulin resistance demonstrated by insulin tolerance test [area under the curve (AUC) of insulin tolerance test; P