Heart failure with preserved ejection fraction (HFpEF) is a public health epidemic that is projected to double over the next two decades. Despite the high prevalence of HFpEF, there are currently no FDA approved therapies for health-related outcomes in this clinical syndrome making it one the greatest unmet needs in cardiovascular medicine. Aging and obesity are hallmarks of HFpEF and therefore there is a high incidence of sarcopenic obesity (SO) associated with this syndrome. The presence of SO in HFpEF patients is noteworthy as it is associated with co-morbidities, worsened cardiovascular health, hospitalizations, quality of life, and mortality. Furthermore, SO plays a central role in exercise intolerance, the most commonly reported clinical symptom of this condition. The aim of this review is to provide insights into the current knowledge pertaining to the contributing pathophysiological mechanisms and clinical outcomes associated with HFpEF-related SO. Current and prospective therapies to address SO in HFpEF, including lifestyle and pharmaceutical approaches, are discussed. The urgent need for future research aimed at better understanding the multifaceted physiological contributions to SO in HFpEF and implementing interventional strategies to specifically target SO is highlighted. Heart failure (HF) is a rapidly growing public health epidemic affecting over 6.2 million Americans, with about half having HF with preserved ejection fraction (HFpEF) . As the The diagnosis of HFpEF remains a clinical challenge in cardiology, however, HFpEF is typically characterized by an ejection fraction >50% accompanied by diastolic dysfunction, high filling pressures and exercise intolerance . The comWithin this clinical syndrome, there are distinct phenotypes emerging that could potentially redefine how we design clinical trials and treat HFpEF patients , 7. The In regard to the identification of HFpEF phenotypes, obesity has gained attention as a potential one of HFpEF , 9, withToward developing effective therapies for these patients, patient reported QoL has emerged as a predominant outcome measure, with an understanding of the physiological contributors to QoL emerging as an important area of investigation. Exercise intolerance is the most commonly reported symptom of HFpEF. The downstream consequences of exercise intolerance include reduced physical activity levels and physical function that have detrimental implications on patient QoL, especially in those individuals with HFpEF who are younger and with concomitant obesity and diabetes mellitus . While tSarcopenic obesity (SO), a clinical and functional condition defined by the coexistence of excess adiposity  with a decline in muscle mass and related strength and functionality , is consistently reported in HFpEF patients and poses as a major limitation to exercise capacity . PatientThe aim of this review is to provide insights into the contributing physiological mechanisms and outcomes associated with HFpEF-related SO toward improving exercise intolerance and potentially QoL.SO is commonly defined as the coexistence of sarcopenia and obesity. A recent systematic review of the definitions and diagnosis of sarcopenic obesity reported large heterogeneity in the definition of SO and diagnostic approaches . This isA recently revised consensus from the European Working Group on Sarcopenia in Older People (EWGSOP) defined sarcopenia as a progressive skeletal muscle disorder that increases the risk of adverse physical outcomes such as falls, fractures, impaired physical function, disability, and mortality . While fObesity is defined as an excessive accumulation and storage of fat in the body that impairs health. Commonly used methodology to diagnose obesity in SO are body mass index (BMI), waist circumference (WC), and when BIA or DXA are available, with total body fat mass percentage . There hIn the recent years, the role of obesity on atrial function, in addition to ventricles and skeletal muscle has received much attention . Of noteBoth sarcopenia and obesity are well-documented as separate entities in HFpEF patients , 26. Fir2 and patients with a BMI <23.5 kg/m2 compared with BMI categories 23.5–26.4 kg/m2, 26.5–30.9 kg/m2, and 31–34.9 kg/m2 , the gold standard measurement of exercise capacity  class  class . The eff obesity . This suIn addition, patients with obesity and HFpEF present with greater plasma volume and increased filling pressures, which have not been consistently identified in patients without HFpEF . MoreoveAn overview of the multifaceted pathophysiological mechanisms that contribute to HFpEF related SO are highlighted in There is abundant evidence for inflammation in patients with HFpEF with circulating biomarkers of inflammation consistently reported to be substantially high in these patients , 50. SysCirculating markers of oxidative stress are increased in the setting of HFpEF and SO . The rolMultihormone hormone deficiency (MHDS) has been cited as a cause and consequence of HF , 68. AltChronic low grade inflammation in obesity that may contribute to muscle wasting is regulated in part by adiponectin, leptin, and insulin controlling various inflammatory and anti-inflammatory processes –74. In oSkeletal muscle mitochondrial dysfunction is sentinel to the pathogenesis of sarcopenia through its dysregulation of myocyte function and viability . ImpairmAdditionally, skeletal muscle mitochondrial dysfunction results in a disruption in metabolism due to a reduced capacity for lipid oxidation. In the presence of increased circulating free fatty acids and triglycerides, incomplete fatty acid oxidation, and insulin resistance, an impairment in mitochondrial oxidative capacity could result in substrate overload and the previously described accumulation of lipid intermediates in the skeletal muscle . As prevIron deficiency (ID) is highly prevalent in HFpEF and is more prominent in patients with more severe diastolic dysfunction. HFpEF related ID is thought to be a result of inflammation mediated reductions hepcidin, the principal regulator of iron absorption and tissue distribution –85. In aIndividuals with HFpEF are highly sedentary and therefore the resultant reduction in energy expenditure combined with disuse atrophy may contribute to SO in these patients , 89, 90.Certainly, the co-morbidities and symptoms associated with HFpEF may contribute to exertional fatigue and the reduction in physical activity, thus augmenting a vicious cycle of disuse atrophy and increased adiposity . AlthougDespite relatively few randomized controlled trials that investigated exercise training in HFpEF, exercise training has consistently been shown to be efficacious at improving exercise capacity and QoL in this patient population , 95–99. 2peak in HFpEF patients (k = −81 ± 99 s-1) and ventricular-arterial coupling (difference in Ees:Ea= −0.2 ± 0.3) when combined with sodium restriction in HFpEF patients , as well as a reduction in FM  and percent body FM   which resulted in an increase in VO CI−1−3) . However CI−1−3) . Notably CI−1−3) . The curIn addition to weight loss induced by caloric restriction, there is a growing interest in understanding the role of bariatric surgery in this population. In HF, observational studies suggest clinical benefits of this intervention , howeverConsidering the lack of available therapies in HFpEF and prevalence of SO in this population, there is an urgent need to identify whether dietary pattern, protein supplementation or caloric restriction as well as bariatric surgery are able to improve outcomes in this population.The discouraging lack of clinical outcomes pertaining to pharmacological therapies in HFpEF has provided a major challenge to cardiologists managing this syndrome. Despite a lack of beneficial outcomes pertaining to hospitalization and mortality in these patients, pharmaceutical therapies have resulted in clinical meaningful improvements in exercise capacity and QoL . In thisIn summary, SO is prevalent in patients with HFpEF and has noteworthy adverse consequences on end point outcomes, health related outcomes and patient reported QoL. Although the multifaceted pathophysiology of HFpEF related SO is not yet fully understood, inflammation, oxidative stress, hormonal imbalances, iron deficiency, and sedentary lifestyle appear to contribute to this condition. In addition, the plethora of comorbidities that accompany this syndrome further exacerbate the physiological milieu implicated in SO. Moreover, being obesity a major risk factors for several comorbidities, including coronary heart disease, future research should try to assess the diverse effects of obesity and HFpEF on clinical outcomes.Whilst exercise training as well as some dietary and pharmaceutical approaches appear to be efficacious at improving exercise capacity and QoL, there is a lack of interventional trials that specifically target SO in these patients. There is therefore an urgent need for lifestyle, pharmaceutical or nutraceutical approaches to address SO toward improving clinical and disease modifying outcomes in HFpEF.DK, NB, HB, and SC contributed to the content of the manuscript. DK, NB, HB, and SC edited manuscript drafts. DK, NB, HB, and SC approved the final version of the manuscript. All authors agree to be accountable for the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vascular inflammation plays a key role in the progression of hypertension. Progranulin (PGRN), an anti-inflammatory growth factor, mediated inhibition of tumor necrosis factor-α (TNF-α), a pleiotropic cytokine, activity has been well-established. Despite the role of chronic low-grade inflammation in hypertension, serum levels of PGRN and PGRN/TNF-α ratio and, their association with systolic and diastolic blood pressure has not been determined in hypertensive patients till now. This study aims to find and correlate the serum levels of pro-inflammatory cytokine (TNF-α), anti-inflammatory growth factor (PGRN), and PGRN/TNF-α ratio with the blood pressure in systolic-diastolic hypertension (SDH) and isolated systolic hypertension (ISH) patients.n = 25 each). Twenty-five age and body mass index (BMI)-matched healthy subjects  were considered as control. All patients and healthy subjects were overweight . Overnight fasting blood samples of subjects were taken and levels of PGRN and TNF-α were measured using ELISA diagnostic kits. PGRN and TNF-α levels were found significantly high, whereas PGRN/TNF ratio was found very low, in SDH and ISH patients as compared to healthy subjects. Reduced PGRN/TNF-α ratio and pulse pressure were found as independent predictors of SBP both in SDH and ISH patients.A cross-sectional study was conducted on SDH patients  number = 15/10) and ISH patients  (Findings of elevated PGRN levels in response to raised TNF-α levels depict the counter regulation by PGRN to neutralize TNF-α. Findings of reduced PGRN/TNF ratio, and it being an independent predictor of SBP, ascertain the key role of imbalance in pro- and anti-inflammatory environment in hypertension. Thus, it strengthens the cross-link between the concept of immunity–adiposity–inflammation–blood pressure¸ a vicious network. Further, this cross-link of SBP and progranulin must be explored in longitudinal studies. New researches should be focused not only on impact of pro-inflammatory environment rather to find on a balance between pro- and anti-inflammatory status, so that new target sites could be explored for therapeutic management of hypertension. Hypertension globally accounts for 9.4 million deaths worldwide every year WHO, 2015). Vascular inflammation plays a key role in the progression of essential hypertension 5. Vascul–5. PGRN-A prospective, cross-sectional study was conducted in hypertensive patients. The study protocol was approved  by human institutional ethics committee (IEC) of the academic institute and was performed in accordance with the code of Good Clinical Practice and followed STROBE guidelines for observational studies. All patients provided written informed consent to participate after a full explanation of the study. Hypertensive patients visiting the hospital for their regular checkup/new diagnosis/or their ongoing treatment for hypertension (systolic-diastolic hypertension or isolated systolic hypertension) during a period of 8 months (March 2015 to October 2015) were assessed for eligibility (inclusion/exclusion) criteria of the study. Simple random sampling design was selected for inclusion of the subjects to exclude bias in selection of patients. Overall 80 patients were screened for hypertension and isolated systolic hypertension for eligibility criteria. Finally, 25 patients (age ≥ 18 years) of isolated systolic hypertension (ISH), defined as SBP ≥ 140 mmHg and DBP ≤ 90 mm Hg; and 25 patients of systolic-diastolic hypertension (SDH) defined as SBP ≥ 120 mmHg and DBP ≥ 90 mmHg according to the Eighth Joint National Committee (JNC)-VIII guidelines for hypertension [Clinical evaluations included the demographic profile and serum biomarkers. Overnight fasting blood samples of the patients and healthy subjects were withdrawn, serum was separated out and stored at – 40 °C till further analysis. The blood pressure was measured using manual mercurial sphygmomanometer on the same day just before withdrawing blood sample. Body mass index of subjects was calculated as weight in kilogram/square meter height . Serum pp ≤ 0.05.Results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) test was used to compare the variables between the groups. The correlation between various clinical variables was examined by Pearson’s univariate correlation analysis. Multiple linear regression was used to reveal dependency of one variable on the other. All data were analyzed using Sigma Stat 3.5. Statistical significance was accepted at Samples size was determined by considering serum progranulin level as primary marker for predicting blood pressure. Serum progranulin levels have been reported as 40.1 ± 8.7 ng/ml in health subjects by Yamamoto et al. 2014 . Based oThe distribution of demographic, biochemical and hemodynamic characteristics are presented in Table p > 0.05). Since the sample size was very less, patients’ data was not subcategorized on the basis of age, sex, and BMI. All the patients and healthy subjects were overweight , but none was having dyslipidemia and on any weight reduction/hypolipidemic treatment. In this study, low TNF-α levels and high PGRN levels in healthy subjects revealed absence of low-grade inflammation and a balance between endogenous pro- and anti-inflammatory cytokines. Both progranulin and TNF-α levels were found high in SDH and ISH patients as compared to healthy subjects. High TNF-α levels depict the presence of low-grade inflammation in hypertension. Elevated PGRN levels in response to raised TNF-α levels depicts the counter regulation by PGRN to neutralize TNF-α. Moreover, PGRN/TNF-α ratio was found significantly decreased in ISH and SDH patients as compared to healthy subjects.On comparing the groups, age and BMI of healthy and patients were not significantly different  and PP ; SBP with PGRN  and PP ; PGRN/TNF-α ratio with PGRN , TNF-α , and PP .Among all variables in ISH patients, age was significantly associated with SBP , DBP , PGRN , and TNF-α ; PP with TNF-α  and PGRN . PGRN/TNF-α ratio  with PGRN  and TNF-α . No other significant association was found between remaining variables .Among all variables in SDH patients, both age and BMI were not significantly correlated with any parameter like SBP, DBP, PP, PGRN, and TNF-α. SBP was correlated with PP  and anti-inflammatory growth factor (PGRN) in hypertensive patients. Reduced PGRN/TNF-α ratio further supports the inflammatory basis of the disease. Moreover, PGRN/TNF ratio was also found as independent predictor of SBP that ascertain the significance of inflammatory component responsible for increasing SBP.ISH is the most common form of hypertension in those older than 65 years . Lloyd-JTNF-α is considered a potent pro-inflammatory cytokine which plays a crucial role in the initiation and continuation of inflammation and immunity. Various studies have shown raised levels of TNF-α in cardio-vascular diseases characterized by presence of low-grade inflammation including obesity , diabetePGRN acts as a strong anti-inflammatory mediator by antagonizing TNF-α signaling. The anti-inflammatory effects of PGRN are exerted through inhibition of TNF receptor (TNFR)-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling by competitively binding to TNFR, especially TNFR-2, which is found in bone marrow derived macrophages . Many clGiven that PGRN acts as a strong anti-inflammatory mediator by antagonizing TNF-α signaling, we further investigated the relationship between serum PGRN levels and TNF-α levels in patients with ISH. We found that serum PGRN levels increased with the rising of TNF-α levels. Based on these results, we speculated that PGRN, as a competitive molecule of TNF-α, is highly expressed in hypertension secondary to the increased inflammatory cytokines, particularly, TNF-α. We speculated that the ratio of PGRN/TNF-α in serum is an important factor to evaluate the inflammatory microenvironment of patient. The findings of low PGRN/TNF ratio in hypertensives as compared to healthy subjects depicted the feedback control/regulation of TNF-α by PGRN. To confirm this speculation, we studied the relationship between serum PGRN/TNF-α ratio and SBP and found that serum PGRN/TNF-α ratio was an independent predictor of SBP. These results create a link between the concept of immunity–inflammation–blood pressure¸ a sequential and vicious network.Present study had several limitations such as small sample size, a few numbers of variables, not measured serum soluble TNF receptor 1 (TNFR1) and TNFR2 level, no differentiation of results based on age, gender, smoking, disease severity, and other risk factors/or concomitantly present diseases. The results are based on evaluation of few patients and healthy subjects which may not justify the findings based on low statistical power of the study. In many published studies, blood pressure is taken as one of the co-variables with other main variables for observing association of serum PGRN with the disease profile. This is the first study reporting the correlation of serum PGRN/TNF ratio in systolic hypertensives in comparison to systolic-diastolic hypertensive subjects. Although results may not be robust enough but provide a preliminary data for futuristic clinical studies in hypertensive patients.The findings of reduced PGRN/TNF ratio as an independent predictor of SBP, ascertain the key role of imbalance in pro- and anti-inflammatory environment in hypertension. Thus, it strengthens the link between the concept of immunity–adiposity–inflammation–blood pressure¸ a sequential and vicious network. As PGRN is widely explored anti-inflammatory marker in various diseases, its serum levels and correlation status must be further explored with regard to age, sex, BMI, duration of hypertension, various stages, severity, and types of hypertension, as well as presence of other pro-/anti-inflammatory markers and concomitant cardiovascular disease risk variables. Further, this cross-link of SBP and progranulin must be explored in longitudinal studies to generalize the findings. New Researches should be focused not only on impact of pro-inflammatory environment rather to find on a balance between pro- and anti-inflammatory status, so that new target sites could be explored for therapeutic management of hypertension.


To better understand heart failure with preserved ejection fraction (HFpEF), we need to better characterize the transition from asymptomatic pre-HFpEF to symptomatic HFpEF. The current emphasis on left ventricular diastolic dysfunction must be redirected to microvascular inflammation and endothelial dysfunction that leads to cardiomyocyte remodeling and enhanced interstitial collagen deposition. A pre-HFpEF patient lacks signs or symptoms of heart failure (HF), has preserved left ventricular ejection fraction (LVEF) with incipient structural changes similar to HFpEF, and possesses elevated biomarkers of cardiac dysfunction. The transition from pre-HFpEF to symptomatic HFpEF also involves left atrial failure, pulmonary hypertension and right ventricular dysfunction, and renal failure. This review focuses on the non-left ventricular mechanisms in this transition, involving the atria, right heart cavities, kidneys, and ultimately the currently accepted driver—systemic inflammation. Impaired atrial function may decrease ventricular hemodynamics and significantly increase left atrial and pulmonary pressure, leading to HF symptoms, irrespective of left ventricle (LV) systolic function. Pulmonary hypertension and low right-ventricular function are associated with the incidence of HF. Interstitial fibrosis in the heart, large arteries, and kidneys is key to the pathophysiology of the cardiorenal syndrome continuum. By understanding each of these processes, we may be able to halt disease progression and eventually extend the time a patient remains in the asymptomatic pre-HFpEF stage. The incidence of heart failure with preserved ejection fraction (HFpEF) is growing worldwide, mainly due to the overall aging of the population and the pandemic of cardiovascular risk conditions, such as hypertension, diabetes, dyslipidemia, overweight, and physical inactivity. Over the past three decades, the syndrome of HFpEF has moved from a hidden corner of HF research to the center stage ,2,3.HFpEF currently accounts for >50% of all HF cases, and its prevalence, relative to heart failure with reduced ejection fraction (HFrEF), continues to increase ,5. This The diagnosis of HFpEF rests on the presence of the signs and symptoms of HF and a normal left ventricular ejection fraction (LVEF) together with cardiac functional and structural alterations and elevated levels of natriuretic peptides . For decTo better understand the clinical syndrome of HFpEF, we need to better characterize the transition from the asymptomatic pre-HFpEF stage to HFpEF itself. In a recent editorial, we coined the term “pre-HFpEF stage” to describe an asymptomatic patient (absent any signs or symptoms of HF) with preserved LVEF, incipient structural heart abnormalities (similar to those reported for HFpEF), and elevated biomarker surrogates of cardiac dysfunction. In contrast to “true HFpEF”, the key clinical component of pre-HFpEF is the absence of HF signs and symptoms . The evoThe present review focuses on non-left ventricular involvement in this transition with a mechanistic focus on the atria, right heart cavities, and the kidneys, and ultimately the currently accepted driver—systemic inflammation .By understanding each of these processes, we may be able to halt disease progression and eventually prolong the time a patient remains in the asymptomatic and pre-HFpEF stage.Atrial Failure is a new clinical entity defined as any atrial structural or functional abnormality  causing impaired heart performance and symptoms and worsening the quality of life or life expectancy . The impAtrial involvement in HFpEF is well recognized: Impaired atrial function, increased structural remodeling, and an excess of AF incidence are consistently observed in patients with HFpEF (and to a greater extent when compared with HFrEF) ,23,24,25Beyond its relationship to symptom onset, LA mechanical function has been consistently associated with higher adverse event rates in various cardiovascular populations ,31. LikeBesides mechanical LA impairment, several atrial failure-induced conditions might further promote adverse hemodynamics and prognosis. Electrophysiological phenomena, such as intra- and inter-atrial dyssynchrony, may play a relevant role in patients with HFpEF. Late LA activation, as observed in an advanced interatrial block, leads to delayed LA contraction, shortened LA emptying, decreased LA compliance, and increased filling pressure . Left atIn conclusion, atrial failure is associated with the onset of HF symptoms, stroke risk, hospitalizations, and increased mortality in patients with HFpEF. Therefore, efforts to obtain a more detailed, functional characterization of their atrial structure and function are crucial to refining the mechanistic and prognostic classification of patients with pre-HFpEF. Taken together, these observations suggest that HFpEF development is not limited to LV remodeling or dysfunction but also importantly involves the LA.One of the key pathophysiological phenotypes of HFpEF is the so-called “pulmonary hypertension (PH) and right ventricular dysfunction (RVD) phenotype” ,39,40. IThe exact mechanisms and risk factors causing RVD and pulmonary vascular (PV) disease in HFpEF are not fully elucidated and constitute a topic of great interest and ongoing research. In the classical paradigm, patients with HFpEF are at risk of PH because of elevated left pressure transmitted back to the pulmonary circulation, resulting in pulmonary venous hypertension. Notably, left ventricular diastolic dysfunction caused by deviation of the septum due to an increase in right ventricular pressure may also cause HFpEF. A chronic right pressure overload leads to RV remodeling and dysfunction. However, this classical paradigm does not fully explain the preponderance of RVD and PH across the whole spectrum of HFpEF patients. This process seems to be triggered and modulated by neurohormonal and inflammatory responses, in which comorbidities play an important and interrelated role ,40,42. IBeyond RV function and PV disease, the competency of the tricuspid valve is crucial. Functional tricuspid regurgitation (TR) is a consequence of maladaptive RV remodeling and PH, leading to further RV enlargement and dysfunction in a vicious cycle . FunctioThe spectrum of RVD and PH in HFpEF is very heterogeneous. Some patients do not present RH failure, whereas for others, right-sided dysfunction and PH are the dominant features . Do thesRV dysfunction can also progress over time. In a retrospective cohort of 271 patients with HFpEF, there was a 10% decrease in the RV fractional area change and a 21% increase in the RV diastolic area over a 4-year follow-up . InteresTherapies targeting the PH-RVD phenotype in HFpEF have shown disappointing results, and no therapy has yet demonstrated an improvement in prognosis. Several treatments are in the pipeline, and ongoing trials will shed light on this topic in the coming years . Besides2. Even mild-to-moderate renal dysfunction was found to be associated with echocardiographic indicators of diastolic dysfunction, although—notably—the associations remained significant only for men when stratifying for sex ..18].In LV myocardial biopsies of HFpEF patients, the coronary microvascular endothelial expression of adhesion molecules, such as E-selectin, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM), is upregulated. Adhesion molecules are also shed by endothelial cells into circulation where they become detectable as biomarkers. The elevation of circulating adhesion molecules in young adulthood was recently shown to predict stage B HFpEF two decades later . The expApart from attracting monocytes, endothelial dysfunction leads to the endothelial production of reactive oxygen species (ROS) through endothelial nitric oxide (NO) synthase (eNOS) uncoupling  and mitoLastly, as demonstrated by Schiattarella et al. , elevateIn conclusion, in the transition from the pre-HFpEF stage to overt HFpEF syndrome, we need to pay special attention to atrial failure, pulmonary hypertension, RVD, renal failure, and systemic inflammation above and beyond left ventricular hypertrophy. Specific targeting of each of these pathogenic mechanisms may halt disease progression and promote patient health and lifespan. The future perspectives within HFpEF management are exciting and immense; however, we will not succeed by stopping one wave at a time—we have to calm the ocean.


Sirt1 activity was measured in peripheral blood mononuclear cells (PBMCs). Angiotensin-Converting Enzyme 2 (ACE2) activity, Tumor Necrosis Factor-alpha (TNF-α) and Brain Natriuretic Peptide (BNP) levels were quantified in plasma. HFpEF showed lower Sirt1 and ACE2 activities than both HFmrEF and HFrEF (p < 0.0001), without difference compared to No HF controls. In HFmrEF and HFrEF a very strong correlation was found between Sirt1 activity and EF , and between ACE2 activity and Sirt1 . HFrEF showed the highest TNF-α levels without reaching statistical significance. Significant differences in BNP were found among the groups, with the highest levels in the HFrEF. Determining Sirt1 activity in PBMCs is useful to distinguish the HF patients’ phenotypes from each other, especially HFmrEF/HFrEF from HFpEF.Heart Failure (HF) is a syndrome, which implies the existence of different phenotypes. The new categorization includes patients with preserved ejection fraction (HFpEF), mid-range EF (HFmrEF), and reduced EF (HFrEF) but the molecular mechanisms involved in these HF phenotypes have not yet been exhaustively investigated. Sirt1 plays a crucial role in biological processes strongly related to HF. This study aimed to evaluate whether Sirt1 activity was correlated with EF and other parameters in HFpEF, HFmrEF, and HFrEF. Seventy patients, HFpEF ( Despite impressive advances in clinical/diagnostic tools and therapies, heart failure (HF) still represents a paramount public health problem being one of the most important causes of death and hospitalization worldwide . As a syThe most recent guidelines of the European Society of Cardiology (ESC) consider three patient’s categories, Heart Failure (HF) with reduced Ejection Fraction , HF with preserved ejection fraction , and HF with mid-range EF (between 40 and 49%) referred to as HFmrEF [Given the clinical implication, especially concerning comorbidities and therapy response, the correct characterization of the HF patients represents a crucial step for the management of this syndrome. One of the most important questions is if the HFmrEF represents a distinct phenotype or a transitional condition from HFrEF to HFpEF, or vice-versa . HoweverRecent studies have investigated the possible role in the HF of sirtuins, a family of NAD+ dependent deacetylases, among which Sirtuin 1 (Sirt1) is the best-characterized member . Sirt1 iThe overexpression of Sirt1 has been shown to favor the survival of cardiomyocytes, but to be also associated with cardiac hypertrophy and HF ,11. IndeSeventy patients with chronic HF in NYHA classes 2 and 3 were consecutively enrolled at the Cardiology Unit of the University Hospital of Salerno. Twenty-nine age-matched subjects without heart failure represented the control group (no HF controls). All the enrolled patients underwent a physical examination, blood chemistry tests, electrocardiographic and echocardiographic exams, and a 6-min walking test. Additionally, baseline demographic, clinical, echocardiographic, and functional data were collected on a predefined computerized datasheet. All subjects included in the study were in optimal medical therapy and managed according to ESC guidelines. All participants gave their written informed consent. The study was in accordance with the Declaration of Helsinki and its amendments and was approved by the local Ethical Committee (Comitato Etico Campania Sud Prot.n.4_r.p.s.o.).Transthoracic echocardiography (TTE) was performed following the ASE and ESC/EACVI recommendations using a Vivid E95 system with an M5S phased array and probe . All echocardiographic images were digitally recorded. The left ventricular (LV) end-diastolic diameter (LVEDD) and the LV end-systolic diameter (LVESD) were measured using M-mode by the parasternal long-axis view; the LV volumes  and the EF were calculated by the Simpson’s method using the apical 2-chamber and 4-chamber view. The LV diastolic function was characterized by the assessment of the ELV/e’LV ratio as a surrogate parameter of LV filling pressure. For the evaluation of the early-diastolic filling (E), the pulsed-wave Doppler sample volume was positioned at the tip of the tenting area of the mitral valve in the apical long-axis view. The mean of e’ was assessed in the basal inferoseptal and lateral LV region in the apical 4-chamber view using Tissue Doppler Imaging TDI. The left atrial (LA) volume index was calculated by biplane LA planimetry in the apical 2- and 4-chamber view. The right ventricular function resulted by the measurement of the tricuspid annular plane systolic excursion (TAPSE), and the pulmonary artery systolic pressure was estimated by tricuspid regurgitation velocity in the apical 4-chamber view and the right arterial pressure (RAP), derived from the inferior vena cava diameter and degree of respiratory collapse.The 6-min walking test (6MWT) was performed according to the American Thoracic Society (ATS) guidelines .The primary measurement was the total distance  walked. The patients were instructed to walk up and down a corridor of 30 m, covering as much ground as possible in 6 min without running. Blood pressure was recorded at the end of the test, and pulse and oxygen saturation. The latter was measured by using a handheld pulse oximeter  placed on the index finger of patients.® containing sodium EDTA . The separation of plasma and peripheral blood mononuclear cells (PBMCs) was obtained by Ficoll density gradient centrifuged at 3000 rcf spin for 30 min at room temperature. Aliquots of plasma and PBMCs were frozen at −80 °C until further analysis.Blood samples were collected in fasting conditions in the BD VacutainerTo measure SIRT1 activity, nuclear extracts (10 μL) were isolated by PBMCs using a nuclear extraction kit . Then, SIRT1 activity was determinate by a SIRT1/Sir2 Deacetylase Fluorometric Assay , following the manufacturer’s instructions. The values were reported as relative fluorescence/micrograms of protein (AU).® infinite 200 PRO) at an excitation wavelength of 320 nm and emission wavelength of 405 nm. Enzymatic activity was determined from a fluorescence rate increase over a 10–120 min time course, and the activity was reported as relative fluorescence units (RFU)/min.The ACE2 activity was measured as previously described  using flThe TNF-alpha evaluation was performed on plasma samples according to the manufacturer’s instruction .The measurements of Sirt1 and ACE2 activities and TNF-alpha levels were performed in a blinded fashion. All data are expressed as the mean ± SD of three independent experiments.p-value < 0.05.Data were analyzed using the SPSS (v 23.0) software package . The Shapiro–Wilk test was used to assess the normal distribution of data. Differences between multiple groups were evaluated by the analysis of variance (ANOVA) with the Bonferroni post hoc test and are presented as mean ± SD. The χ2 test was used to compare categorical variables. A multiple linear analysis was used to investigate the relationship between variables when appropriate. To explore the correlation between variables, Spearman’s correlation (r) was used. The statistical significance was established at a n = 9 per group was determined to provide sufficient power at 0.9 to detect a significant difference among groups.The sample size was calculated from similar studies where Sirt1 activity determination in PBMCs was assessed in healthy individuals and HF patients . We used an estimated standard deviation of 0.5 and the two-tailed alpha set at 0.05. An Then a total of 99 subjects  were included in the study.n = 23), HFmrEF (n = 23), and HFrEF (n = 24) categories defined according to the criteria of the ESC guidelines [p < 0.0001), with an increasing trend between the groups through EF reduction.The study population consisted of 99 subjects  including 29 No HF controls and 70 HF patients  belonginp < 0.0001) but no differences were found among the HF groups.From a functional point of view, all the HF groups showed a reduction in six-minute walking distance in comparison to the no HF controls (p = 0.042) and used more diuretics (p < 0.0001), beta-blockers (p < 0.0001), ACE-inhibitors (p < 0.0001), and statins (p = 0.004) in respect to the no HF controls. No differences were found in comorbidities and therapy among the HF groups.The HF groups were more affected by diabetes mellitus (p < 0.0001), without any difference compared to the no HF controls , and the use of beta-blockers .When a multivariate linear regression analysis was performed by using the EF as a dependent variable, after correction for the parameters statistically significant at the univariate analysis, the best predictors of EF were represented by Sirt1 activity . Then, because of the different characteristics of the HF in the three groups, to better explore the relationship between EF and Sirt1 activity, other multivariate regression analyses were performed. For each group, setting the EF as the dependent variable, the parameters statistically significant at the univariate analysis were identified, and then they were introduced in the multivariate analysis. In the no HF controls, the best predictors of EF were represented by the gender  and six-minute walking distance .The Sirt1 activity (used as dependent variable) was significantly associated to the HF groups .In the HFpEF group the EF was not associated to any variable. In the HFmrEF and HFrEF groups the best predictor of EF was represented only by the Sirt1 activity (for HFmrEF with 2 = 0.909) and HFmrEF (r2 = 0.899), but not in the HFpEF (r2 = 0.001). Moreover, a logistic regression analysis with the NYHA classes, as the dependent variable, showed that Sirt 1 activity represented the best predictor in the HFrEF  and HFmrEF  but not in the HFpEF (In he HFpEF B. In parThese findings suggest the possible role of Sirt1 as a marker useful to distinguish the HF phenotypes.p < 0.0001) without any difference compared to the no HF controls  and HFmrEF (r2 = 0.801), but not in the HFpEF (r2 = 0.149).In As shown in p = 0.434, r2 = 0.097, β = −0.026, 95%CI −0.104 0.052; for HFpEF p = 0.566, r2 = 0.024, β = −0.206, 95%CI −3.428 3.016; for HFmrEF p = 0.752, r2 = 0.025, β = −0.073; 95%CI −0.580 0.434; for HFrEF p = 0.388, r2 = 0.050, β = 0.126; 95%CI −0.175 0.427).At the univariate analysis, statistically significant differences in plasma levels of BNP were found among the groups, with the highest levels in the HFrEF group . An incrThe introduction in the recent ESC guidelines of the HFmrEF category  has giveThe processes and mechanisms involved in the cardiac failing phenotypes have not been exhaustively investigated, nonetheless elucidating the molecular card of the different HF patients might be of great help to better manage the disease and personalize the therapy.Sirt1 represents a good candidate in this field because of its involvement in cardiac pathophysiology . Indeed,Sirt 1 expression and activity are often measured in PBMCs, which represent a model helpful to provide a comprehensive overview of the cellular system status together with measurement of circulating serum or plasma markers . PBMCs a2 = 0.909) and HFmrEF (r2 = 0.899), while no correlation in the HFpEF and no HF control subjects was observed. Additionally, the higher Sirt 1 activity levels were significantly associated with the higher NYHA class in the HFrEF and HFmrEF but not in the HFpEF.Herein, we found that Sirt1 activity was much higher in PBMCs isolated from the HFrEF patients when compared to the HFmrEF and even more HFpEF. Importantly, the levels were so different making it possible to distinguish the HFmrEF/HFrEF from the HFpEF that, conversely, had Sirt1 activity very similar to the no HF control subjects. A very strong correlation between Sirt1 activity and EF values was found in the HFrEF  ,35,36. RIt has been demonstrated both in vivo and in vitro that resveratrol, a polyphenol able to activate Sirt1, leads to a decrease of angiotensin II receptor AT1 through Sirt1 activation . AnotherHere, we found an increasing trend of plasma TNF-alpha levels from the HFpEF through the HFmrEF to the HFrEF patients without reaching statistical significance.2 = 0.802) and the HFmrEF (r2 = 0.801) but not in the HFpEF (r2 = 0.149) was found (Our results show that the HFrEF group had levels of ACE2 activity significantly higher than those measured in the HFmrEF and even more in the HFpEF subjects, with the latter showing no difference when compared with the No HF controls. Notably, a positive correlation between Sirt1 activity and ACE2 activity in the HFrEF (ras found C.Altogether these findings suggest a role for Sirt1 activity as a biomarker to distinguish the three HF phenotypes.The highest Sirt1 activity in the HFrEF patients might reflect the high neurohormonal activation, including RAAS, which in turn characterizes systolic HF .As already stated, the HFmrEF remains insufficiently characterized compared with the other groups. We found that the HFmrEF and HFrEF patients have similar Sirt1 levels, both much higher than the HFpEF and the correlation between Sirt1 activity and EF values in the HFmrEF was as relevant as in the HFrEF, even if less strong. However, the intermediate mean value of the Sirt1 activity found in the HFmrEF group A seems tSirt1 activity induced by pharmacological and non-pharmacological activators has been demonstrated to ameliorate the health status of HF patients ,21,27. IIn our opinion, the most important result of the present investigation is the existence, observed for the first time, of the relationship between the EF and Sirt1 activity with a very strong correlation between Sirt1 activity and EF in the HFmrEF and HFrEF. Of note, this correlation does not exist in the HFpEF patients.This study is subject to some limitations. One of them is the lack of the cardiopulmonary stress test because it was performed not in all enrolled patients and no HF controls. Another limitation is the small number of patients included in each group.As discussed, the high levels of Sirt1 might reflect an adaptive activation of the sympathetic system and RAAS characterizing the systolic HF. The relationship between Sirt1 and circulating ACE2 activity found in the HFrEF and HFmrEF but not in the HFpEF patients corroborates this hypothesis. Measuring factors other than ACE2 activity involved in the neurohormonal modulation could be helpful to better classify patients with HF.Another limitation is the absence of Sirt1 activity levels definition in healthy subjects as a reference.Undoubtedly, further and larger studies are necessary to measure such and other inflammatory parameters, other than TNF alpha, and verify whether they correlate with Sirt1 activity.In our study, Sirt1 activity levels increased from the HFpEF through HFmrEF to the HFrEF.Sirt1 activity in the HFmrEF, showing an average value between the HFrEF and HFpEF subjects, suggests the hypothesis that the HFmrEF represents an intermediate phenotype. This is supported by the finding of the strong correlation between Sirt1 activity and EF values observed also in HFmrEF patients.The correlation between Sirt1 and ACE2 also reinforces the hypothesis that Sirt1 activity could be used as a biomarker to better differentiate the patients with different HF phenotypes, especially to separate HFmrEF/HFrEF from HFpEF.Further studies with a larger sample size are needed to confirm or deny these results and clarify whether monitoring Sirt1 activity levels can effectively help the management of the patients suffering from HF. Moreover, more trials should also be performed to better understand the mechanisms underlining the HF phenotypes that could explain the different Sirt1 activation and to define the range of Sirt1 activity levels associated with beneficial effects.


Tumor necrosis factor (TNF) and its receptors are widely expressed in non-small cell lung cancer (NSCLC). TNF has an established role in inflammation and also plays a key role in inflammation-induced cancer. TNF can induce cell death in cancer cells and has been used as a treatment in certain types of cancer. However, TNF is likely to play an oncogenic role in multiple types of cancer, including NSCLC. TNF is a key activator of the transcription factor NF-κB. NF-κB, in turn, is a key effector of TNF in inflammation-induced cancer. Data from The Cancer Genome Atlas database suggest that TNF could be a biomarker in NSCLC and indicate a complex role for TNF and its receptors in NSCLC. Recent studies have reported that TNF is rapidly upregulated in NSCLC in response to targeted treatment with epidermal growth factor receptor (EGFR) inhibition, and this upregulation leads to NF-κB activation. The TNF upregulation and consequent NF-κB activation play a key role in mediating both primary and secondary resistance to EGFR inhibition in NSCLC, and a combined inhibition of EGFR and TNF can overcome therapeutic resistance in experimental models. TNF may mediate the toxic side effects of immunotherapy and may also modulate resistance to immune checkpoint inhibitors. Drugs inhibiting TNF are widely used for the treatment of various inflammatory and rheumatologic diseases and could be quite useful in combination with targeted therapy of NSCLC and other cancers. Tumor necrosis factor (TNF) is a cytokine that plays a central role in inflammation, the immune response, and in cancer P value <0.05 in Gehan's test (P >0.05. (An examination of the the Cancer Genome Atlas (TCGA) database reveals that for the squamous cell subset of NSCLC lung squamous cell carcinoma (LUSC), TNF shows a trend as an oncogene, as patients with higher TNF mRNA levels are associated with shorter overall survival (OS), although this correlation does not reach statistical significance A. Moreovn's test B, and TNP >0.05. C. Gehan'P = 0.025, P = 0.07, P = 0.03, The adenocarcinoma subset of NSCLC (lung adenocarcinoma (LUAD)) includes patients with EGFR activating mutations, the prevalence of which ranges from 15% in Western populations to up to 50% in Asian populations Among the 500 TCGA-LUAD patients with RNAseq and OS data, there are 41 (8%) harboring EGFR activating mutation Data from the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial helps with some of these issues. The BATTLE trial included about 30 advanced-NSCLC (mostly LUAD) patients harboring active EGFR mutation who were treated with erlotinib after the first diagnosis n = 8) of ligands correlate with significantly shortened PFS with P <0.05, 50% (n = 10), having hazard ratio (HR) >1, consistent with oncogenic roles. Only 2 (10%) may prolong PFS with HR <1, suggesting tumor suppressive roles. Moreover, the majority of TNFRSFs also have HR >1 and paired with these 20 TNFSFs. Three TNFSFs with the top HR and strongest correlation to negative prognosis, TNFSF11/RANKL, TNFSF9/4-1BBL, and TNFSF7/CD70 are shown in P = 0.0108. By Gene Set Enrichment Analysis P < 0.001) enriched in oncogenes with higher HR associated with shorter PFS under erlotinib treatment . GBM is a devastating cancer with a dismal prognosis. Although there is substantial evidence that aberrant EGFR signaling is widespread in GBM TNF may also have an important role in mediating resistance to EGFR inhibition in NSCLC. About 10% to 15% of NSCLC patients in Western populations harbor EGFR activating mutations that render the tumors sensitive to EGFR inhibition, and EGFR TKIs are used as a treatment in this subset of patients. The remaining NSCLC tumors express EGFR wild type (EGFRwt) and do not respond to TNF inhibition exhibiting a primary resistance to EGFR inhibition. TNF upregulation was reported to be a universal and rapid early adaptive response to EGFR inhibition in experimental models of NSCLC regardless of EGFR status TNF may also mediate resistance to TKIs in renal cell carcinoma Immunotherapy with checkpoint inhibitors has been an exciting advancement for multiple cancer types including NSCLC. About 20% of patients with NSCLC respond to immunotherapy TNF has been intensively investigated for decades. Substantial progress has been made in understanding the structure of TNF and its receptors, and the signaling networks involved in TNF signaling. TNF is a key mediator of inflammation in rheumatologic and inflammatory diseases and anti-TNF treatments are highly effective. There is considerable information about the role of TNF in inducing cell death or cell survival, but often such information is derived from experimental models and needs validation in human tumors. A current important area of investigation is how TNF in tumors and TNF in the microenvironment of tumors influences the biology of cancers. TNF may also play an important role in mediating resistance to targeted inhibition, particularly in the context of EGFR inhibition in GBM and lung cancer. Whether TNF plays a role in resistance to targeting of other RTK nodes and/or in other tumor types is currently unknown. Future studies will elucidate these questions and also provide more insight into how anti-TNF therapy influences the response to immunotherapy.1.All primary NSCLC patients from TCGA-LUSC or TCGA-LUAD, the TCGA-LUAD EGFR-activating mutation was defined as harboring L858R or exon 19 deletion, but without T790M mutation. The recurrent NSCLC patients were excluded.2.The patients must have RNAseq data.3.The patients must have OS data.4.The number of patients was displayed in The recruitment criteria for TCGA data are as follows:1.Erlotinib treatment arm in the BATTLE trial, which is NCT00410059.2.From NCT00410059, the selection criteria are the same as our previous study 3.N = 25.The recruitment criteria of BATTLE trial data is as follows:https://www.cancer.gov/tcga.The results published here are in part based upon data generated by the TCGA Research Network: Ke Gong: Conceptualization, Data curation, Formal analysis, Visualization, Writing - original draft. Gao Guo: Data curation, Formal analysis. Nicole Beckley: Writing - review & editing. Yue Zhang: Formal analysis. Xiaoyao Yang: Formal analysis, Visualization. Mishu Sharma: Writing - review & editing. Amyn A. Habib: Conceptualization, Writing - original draft, Funding acquisition, Supervision, Resources.


Antibodies targeting the spike protein of SARS-CoV-2 present a promising approach to combat the COVID19 pandemic; however, concerns remain that mutations can yield antibody resistance. We investigate the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared following in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Importantly, escape mutants were not generated following treatment with a non-competing antibody cocktail. A promising approach to combat the COVID19 pandemic involves development of antiviral antibodies targeting the spike protein of SARS-CoV-2. The spike protein is a key mediator of viral infectivity required for attachment and entry into target cells by binding the ACE2 receptor , we used the VSV pseudoparticle system expressing the SARS-CoV-2 spike variants. Our top eight neutralizing antibodies maintained their potency against all tested variants , demonstNext, escape mutants were selected under pressure of single antibodies, as well as of antibody combinations, by using a replicating VSV-SARS-CoV-2-S virus . We rapiAnalysis of 22,872 publicly available unique genome sequences (through the end of May 2020) demonstrated the presence of polymorphisms analogous to two of the escape amino acid residues identified in our study, albeit at an extremely low frequency of one each. Thus, although natural variants resistant to individual antiviral antibodies were not widely observed in nature, these rare escape variants could easily be selected and amplified under the pressure of ongoing antibody treatment. Although these studies were conducted with a surrogate virus in vitro, one would expect that similar escape mutations may occur with SARS-CoV-2 virus in vivo under the selective pressure of single antibody treatment. While, the differential propensity of VSV and SARS-CoV-2 viruses to acquire mutations may impact the speed at which these escape mutants may arise, the likelihood of eventual escape remains high.Next, we evaluated escape following treatment with our previously described antibody cocktail (REGN10987+REGN10933), rationally designed to avoid escape through inclusion of two antibodies that bind distinct and non-overlapping regions of the RBD, and which can thus simultaneously bind and block RBD function. Attempts to grow VSV-SARS-CoV-2-S virus in the presence of this antibody cocktail did not result in the outgrowth of escape mutants . Thus, tIn addition to the above cocktail, we also evaluated escape following treatment with additional combinations (REGN10989+REGN10934 and REGN10989+REGN10987), this time consisting of antibodies that completely or partially compete for binding to the RBD – i.e., two antibodies that bind to overlapping regions of the RBD. Under selective pressure of these combination treatments, there was rapid generation of escape mutants resistant to one combination, but not the other . For an To functionally confirm that the spike protein mutations detected by sequencing are responsible for the loss of SARS-CoV-2 neutralization by the antibodies, we generated VSV-SARS-CoV-2 spike pseudoparticles expressing the individual identified spike mutations. These pseudoparticles were used in neutralization assays with single and combination antibody treatments, and IC50 values were calculated , and thus require the unlikely occurrence of simultaneous mutations at two distinct genetic sites for viral escape. A clinical candidate selection criterion for broad potency that includes functional assessment against naturally circulating sequence variants, as well as inclusion of multiple antibodies with non-overlapping epitopes, may provide enhanced protection against loss of efficacy. Future in vivo animal and human clinical studies need to pay close attention to possible emergence of escape mutants and potential subsequent loss of drug efficacy.


Background and Objectives: The selective kappa opioid receptor agonist U50,488 was reported to have analgesic, cough suppressant, diuretic and other beneficial properties. The aim of our study was to analyze the effects of some original chitosan-coated liposomes entrapping U50,488 in somatic and visceral nociceptive sensitivity in mice. Materials and Methods: The influence on the somatic pain was assessed using a tail flick test by counting the tail reactivity to thermal noxious stimulation. The nociceptive visceral estimation was performed using the writhing test in order to evaluate the behavioral manifestations occurring as a reaction to the chemical noxious peritoneal irritation with 0.6% acetic acid (10 mL/kbw). The animals were treated orally, at the same time, with a single dose of: distilled water 0.1 mL/10 gbw; 50 mg/kbw U50,488; 50 mg/kbw U50,488 entrapped in chitosan-coated liposomes, according to the group they were randomly assigned. Results: The use of chitosan-coated liposomesas carriers for U50,488 induced antinociceptive effects that began to manifest after 2 h, andwere prolonged but with a lower intensity than those caused by the free selective kappa opioid in both tests. Conclusion: In this experimental model, the oral administration of nanovesicles containing the selective kappa opioid agonist U50,488 determined a prolonged analgesic outcome in the tail flick test, as well as in the writhing test. Nanomedicine offers particularly valuable research and practical application tools in clinical medicine, improving the currently used methods of prevention, diagnosis, targeted therapy in various pathological conditions, from simple inflammatory illnesses to neoplastic diseases .The application of nanotechnology in the development of new drug formulations has opened a new area of researching the prolonged release of different pharmacologically active substances.The drug-carrying nanoformulations consist of solid biodegradable particles, with the size varying between 10 and 1000 nm, in which the active ingredient is dissolved, trapped, or encapsulated and/or in which the active substance is absorbed or attached . The mosGenerally, the nanoparticulate systems are used to achieve an extended release of active substances, to prolong drug effect in the targeted tissue, to reduce its adverse effects .Liposomes are systems with a high variation in shape and surface electric charge, but also in the composition of the lipid component, which determines the differences in permeability through biological membranes. The composition of the lipid bilayer has an essential role in modulating the fluidity of the vesicular membrane; this element contributesto the stability of liposomes, andalso to the kinetic model of releasing the encapsulated components ,5.VesiclKappa opioid receptors (KORs) are expressed in brain areas which express analgesia and affect cognition, but also in other areas throughout the body ,7. Like The trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide derivative U-50,488 is a selective KOR agonist, possessing analgesic, cough suppressant, and diuretic properties. It also suppresses the psychostimulant reward through the inhibition of dopamine signaling in the addiction pathways ,11,12, wThe purpose of our study was to evaluate the effects of some original chitosan-coated liposomes entrapping U50,488 in somatic and visceral nociceptive sensitivity in mice.The lipid used, L-α-phosphatidylcholine (L-α-lecithin), chitosan, chloroform and U50,488 were purchased from Sigma Chemical Co., Steinheim, Germany. We used chitosan with N-de-acetylation degree of 28%, apolydispersity index of 3.26, with average molecular mass Mn = 94,810, and average gravimetric mass Mw = 309,900. Chitosan was dissolved in 10% (w/w) acetic acid. To obtain chitosan solution with a 1% (w/w) homogeneity, the liquid was stirred at room temperature over 24 h and the excess of gas was removed by centrifugation at 1500 rpm for 30 min.Initially, the lipid (L-α-lecithin) was dissolved in chloroform; subsequently, a dry lipid film was achieved upon evaporation of the solution. A solution of U50,488 in distilled water was added for the film hydration, and continuous stirring at low rotation induced the formation of multilamellar vesicles. The lipid solution was sonicated, in order to convert the multilamellar vesicles to unilamellar vesicles . Then, aThe size distribution and Zeta potential were determined using Zetasizer Malvern Nano ZS Zen-3500 Apparatus . The transmission spectra were recorded using a Hewlett Packard 8453 UV–VIS spectrophotometer , to highlight the encapsulation of substances in vesicles and to establish the elimination curves. With a Nikon Ti Eclipse optical microscope , the obtained nanoparticles were visualized directly, the images being taken with a Coolpix 950 digital camera.For this study, white Swiss mice  were used. The animals were housed in standard laboratory conditions and received standard laboratory chow and water ad libitum. The study was approved (Protocol No. 19157/19.10.2009) by the Ethics Committee for Research of the University of Medicine and Pharmacy “Grigore T. Popa” from Iasi, Romania, in compliance with the regulations of the International Association for the Study of Pain regarding the handling of laboratory animals ,24.A total of 21 animals, assigned in 3 groups of 7 white Swiss mice , were used. At the same time, the animals were treated orally (using an eso-gastric device) with a single dose as follows:Group 1 (DW): distilled water 0.1 mL/10 gbw;Group 2 : 50 mg/kbw U50,488;Group 3 : 50 mg/kbw U50,488 entrapped in chitosan-coated liposomes.The influence on the somatic pain was assessed using tail flick test by counting the tail reactivity to thermal noxious stimulation (54 °C). The latency period of the response was measured before the experiment (baseline), at 15, 30, 60, 90 min, 2, 4, 6, 8, 10, 12 h after the administration of the substances. The recommended cut-off time of 12 s was used to prevent tissue damages. In this animal model, a prolongation of the response latency expressedthe analgesic effect, while a decrease in the reactivity time suggested the hyperalgesic effects of the tested substances ,26.To quantify the intensity of antinociception, the latency of the response was converted to percentage maximum possible effect (%MPE), according to the formula :%MPE = [The results obtained in tail flick test were presented as mean ± standard deviation (SD) of the mean latencies of the response, respective to the mean percentage maximum possible effect.For the experiment, 210 animals divided in 10 lots were used. Each lot consisted of identical 3 groups of 7 white Swiss mice (20–25 g): Group 1 (DW): distilled water 0.1 mL/10 gbw; Group 2 : 50 mg/kbw U50,488; Group 3 : 50 mg/kbw U50,488 entrapped in liposomes.The nociceptive visceral estimation was performed using the writhing test in order to evaluate the behavioral manifestations occurring as a reaction to the chemical noxious peritoneal irritation with 0.6% acetic acid (10 mL/kbw) .The tested substances were orally given at the same time at the beginning of the experiment. Acetic acid was intraperitoneally injected 5 min prior to each moment of the determination, according to the following scheme: lot I—half an hour; lot II—1 h; lot III—2 h; lot IV—3 h; lot V—4 h; lot VI—5 h; lot VII—6 h; lot VIII—8 h; lot IX—10 h; lot X—12 h after the administration of substances. The animals were placed in glass cages, and the number of writhes were counted for 5 min.In this experimental model, the diminution in the number of writhes was indicative of analgesia, while the increasein the behavioral manifestations number correspondedto a hyperalgesic effect of the tested substances.In addition, the antinociceptive effects of U50,488 free or entrapped in nanoparticles were assessed by calculating the pain inhibition percentage (PIP) :PIP = (NThe results collected in the writhing test were expressed as mean ± the standard deviations (SD) of the mean number of writhes, respective to thepercentage values of pain inhibition.p (probability) below 0.05, were considered statistically significant, compared to the control group.The data were centralized and statistically processed using the ANOVA  method and the Bonferroni test with the SPSS version 22.0. The Bonferroni correction, as a post-hoc test, was performed, in order to diminish the error rate when analyzing multiple data points. Values of the coefficient The obtained chitosan-coated liposomes entrapping U50,488 proved to have a mean size of 572 nm . The useThe measured mean Zeta potential of the vesicles containing U50,488 was +20.3 mV .p < 0.01) in the response latency, an effect lasting approximately 120 min after the substance administration (p < 0.05), 30 min (** p < 0.01), 60 min (** p < 0.01), 90 min (** p < 0.01) and 120 min (** p < 0.01) in the experiment. Its maximum possible analgesic effect was achieved at 90 min (%MPE = 56.1 ± 11.3%)  .p < 0.01), 4 h (** p < 0.01), 6 h (** p < 0.01) and 8 h (* p < 0.05) after administration ofthe substance. The manifested maximum possible antinociceptive effect of U50,488 entrapped in chitosan-coated liposomes was observed after 6 h in the experiment (%MPE = 51.7 ± 10.5%)  .p < 0.01) in the first 2 h and prolonged at 3 h (* p < 0.05) in the writhing test (p < 0.01), 1 h (** p < 0.01), 2 h (** p < 0.01) and 3 h (* p < 0.05), respectively, by the Bonferroni test. The maximum pain inhibition percentage was found after 2 h in the experiment (32.67 ± 7.5%)  .p < 0.05) within 3–5 h in this visceral pain model in mice (p < 0.01), 4 h (** p < 0.01), and 5 h (* p < 0.05) after the application of the chemical visceral stimulus. The maximum pain inhibition percentage was observed at 4 h in the experiment (19.23 ± 3.7%)  .Our previous research evidenced that the agent U50,488 was effectively incorporated in the inner aqueous layer of the vesicles. Physicochemical analysis also revealed that the dimensions of U50,488 vesicles varied from tens to hundreds of nanometers, with an average size of 572 nm. The addition of chitosan in lipid-U50,488 vesicle solution was associated with an increase in the nanoparticle dimensions and a stiffening of the bilayers. The vesicles had a mean Zeta potential of +20.3 mV, thus proving an incipient stability of the colloidal solution .Corroborating the transmission spectra of the agent released from the vesicles with the calibration curve of free kappa opioid, a high efficiency of trapping U50,488 in the chitosan-coated liposomes was shown. The analysis of the release curve from the vesicles showed that in the first 3 h, approximately 50% of the U50,488 incorporated amount was released, and after 7–8 h, 80% of the total amount of substance was found in the release medium .The anterior results, in concordance with the literature-communicated data, have shown significant analgesic effects of selective kappa opioid agents, exhibited in various experimental acute and chronic pain models in laboratory animals, and also in clinical trials ,32. U50,In vitro studies, as well as in vivo experiments, have suggested that various ionic channels are involved in the mediation of both somatic and visceral pain. Su X. et al. demonstrated that U50,488 substantially blocks the voltage-activated sodium current in colon sensory neurons in vitro and reduces the in vivo visceral behavioral manifestations due to mechanical noxious colorectal distention in rats .In vitro studies performed on isolated rat dorsal root ganglia neurons transfected with complementary deoxyribonucleic acid for enhanced green fluorescent protein proved antinociceptive activity, which was mediated by the inhibition of calcium channels and modulation of the calcium currents in sensory neurons involved in the nociceptive transmission ,41,42.So far, there are no communications regarding the design of nanoparticles entrapping the selective kappa opioid agonist U50,488, although the effects of this compound have been studied over time.We imagined an original method of incorporating the selective kappa opioid agonist U50,488 into vesicles that will transport and prolong drug release in the targeted area, depending on environmental conditions . The protocol for obtaining these nanoparticulate systems involves the use of association polymers based on the unmodified, biocompatible, and biodegradable polymer chitosan. Chitosan is a deacetylated product of chitin  insoluble in water and most organic solvents. Its derivatives have been developed as nanomaterials, such as nanoparticles, microspheres, hydrogels, and micelles ,44.Due to its low toxicity, biodegradability and biocompatibility, chitosan has been increasingly studied in a wide range of pharmaceutical, biomedical, and biotechnological fields, including gene therapy, bone regeneration, food industry, and agriculture ,46. ChitFor obtaining chitosan-based nanoparticles, several methods have been described: polyelectrolyte complexation, emulsion-droplet coalescence, emulsion crosslinking, ionotropic gelation, reverse micellization, and precipitation ,51.Because they have showed strong evidence for crossing the blood–brainbarrier , chitosaThe association of chitosan to the designed vesicles produced stiffening of the bilayer and stabilization of the particles, making them spherical and more confined; the obtained systems corresponding to the criteria of the colloidal solutions. In the experimental somatic pain model, the opioid kappa agonist U50,488 produced a significant and rapid antinociceptive effect, even after 15 min, which was manifested for 2 h. After this period, the effect was obviously reduced, reaching levels comparable to those found in the control group. The highest intensity of analgesic action (56.1 ± 11.3%) was observed at 90 min in the experiment. In the tail flick test, the administration of U50,488 incorporated into chitosan-coated liposomes was associated with a significant antinociceptive effect, which began after 120 min and persisted for up to 8 h, being the most intense  at 6 h in the experiment. After this time, the effects of U50,488 vesicles were similar to those highlighted in U50,488 and the control.In the experimental model of visceral pain, the administration of U50,488 caused a rapid antinociceptive effect, which persisted for 3 h. The strongest analgesic action of this opioid kappa agonist expressed after 2 h in the experiment, when a pain inhibition percentage of 32.67 ± 7.5% was highlighted. After 3 h, the effects of U50,488 were similar to those of the group that received distilled water in the writhing test. The use of U50,488 vesicles determined a significant antinociceptive effect, which manifested 3 h after the administration of the chitosan-coated liposomes and lasted for 5 h. The maximum intensity of analgesic action was found after 4 h in the experiment, when a maximum percentage of pain inhibition of 19.23 ± 3.7% was recorded. After 5 h, the effects of U50,488 incorporated into chitosan-coated liposomes were comparable to those of the free substance and to that of the control over the entire time interval up to 12 h.The oral administration of liposomes containing the selective kappa opioid agonist U50,488 was accompanied by prolonged analgesic effects inthe tail flick test, as well as in the writhing test; however, a latency period of occurrence of these effects was noted. In both cutaneous and visceral experimental pain models, the delayed onset of the antinociceptive effects, as well as the prolongation of the analgesic activity of the liposomes entrapping U50,488, can be attributed to the particularities of the release of the active substance from the chitosan-coated liposomes.The reference therapy for medium to severe pain is represented by opioid analgesics; thus, the opioid receptors are of utmost importance in modern medicine . Pain, bOriginal nanoparticles capable of incorporating U50,488 with high efficiency were developed. The use of chitosan-coated liposomes as carriers for U50,488 induced antinociceptive effects that began to manifest after 2 h, were prolonged, but had alower intensity than those caused by the free selective kappa opioid in the tail flick test, as well as in the writhing test.We can conclude that, in our experimental conditions, the administration of U50,488 entrapped in chitosan-coated liposomes presented the advantage of prolonged drug release, comparedwith non-entrapped substance in both somatic and visceral pain models in mice.


A nuclear role for the DEAD-box protein Dbp5 in tRNA export. Published 27, August 2019dbp5-L12A-R423A strain was generated at the same time as a number of other mutant strains and DNA sequencing of two independent isolates showed the correct genotype. These strains were then stored as glycerol stocks and used in the study. In follow on work after publication, we introduced the dbp5-L12A-R423A mutation in to an alternate genetic background and resulting strains were found to have unexpected phenotypes. This ultimately led to resequencing dbp5-L12A-R423A from the original glycerol stocks and it was found that the genotype was incorrect. We do know how this mistake arose, but suspect that strains were inadvertently mixed up at a point following sequencing and prior to the strains being frozen as glycerol stocks. It is our opinion that the true phenotypes of the dbp5-L12A-R423A mutant do not alter major conclusions made in the paper. We sincerely regret this error and apologize to anyone impacted by our mistake. We have corrected the manuscript through the following changes:We have identified a mutant strain used in this study that is not the correct genotype. Based on our records, the dbp5-L12A-R423A. Original and corrected text are shown below with new text indicated in bold.1. The manuscript text has been corrected to reflect figure changes and to report the correct mutant phenotype of Original:L12A/R423A is viable and there is an increased nuclear pool of Dbp5 as compared to R423A . Further, the untagged and integrated version of dbp5-L12A/R423A does not exhibit mRNP export or temperature-dependent growth defects . Using the double mutant, northern blotting was performed with probes against the tRNAIleUAU at 37°C . As compared to dbp5-R423A (2.2 ± 0.2), accumulation of the intron containing tRNA intermediate was reduced in the integrated dbp5-L12A/R423A (1.4 ± 0.3) strain to a level comparable to dbp5-L12A (1.4 ± 0.2), suggesting that increasing the nuclear pool of Dbp5 through inclusion of the L12A mutation largely rescued tRNA processing defects related to the R423A mutation. Combined, the SGA, northern, and in situ data employing the dbp5-L12A and dbp5-R423A mutations to alter Dbp5 nuclear levels are strongly supportive of Dbp5 functioning in tRNA export within the nucleus.Importantly, a strain expressing GFP-Dbp5dbp5-L12A/R423A double mutant (43 ± 7), returning to levels that were not significantly different from the dbp5-L12A mutant alone .This defect was largely rescued in the In any of these cases, introduction of the L12A mutation would rescue tRNA export defects, as was observed. This would occur through increased nuclear levels of Dbp5 above the threshold needed to support tRNA export, including a point where the frequency of binding events ensures the likelihood of binding being coupled to an ATPase hydrolysis event.Tyr determined by FISH at 25°C in integrated and untagged DBP5, dbp5-L12A, dbp5-R423A, and dbp5-L12A/R423A strains prior to (pre), during a 10 min starvation for amino acids (-AA), and 15 min after reintroduction of amino acids through addition of rich media (re-feed).Figure 5. Dbp5 supports re-export of mature tRNAs following nutritional stress. A. Localization of tRNACorrected:L12A/R423A is viable and there is an increased nuclear pool of Dbp5 as compared to R423A ; however, the untagged and integrated version of dbp5-L12A/R423A exhibited mRNP export defects at both 25°C and 37°C, and a strong temperature-dependent growth defect at 37°C . This result indicates that further limiting the pool of cytoplasmic Dbp5 in the context of the R423A mutation alters mRNP export. Using the double mutant, northern blotting was performed with probes against the tRNAIleUAU at 37°C . As compared to dbp5-R423A (1.5 ± 0.2), accumulation of the intron containing tRNA intermediate was similar in the untagged and integrated dbp5-L12A/R423A (1.9 ± 0.5), suggesting that depleting the cytoplasmic pool of Dbp5 through inclusion of the L12A mutation did not further impact tRNA processing, in contrast to what is seen with mRNP export. Due to the presence of mRNP export defects at 25°C, this mutant was not used to further interrogate the role of Dbp5 in tRNA export. Combined, the SGA, northern, and in situ data employing the dbp5-L12A and dbp5-R423A mutations to alter Dbp5 nuclear levels are strongly supportive of Dbp5 functioning in tRNA export within the nucleus, while facilitating mRNP export in the cytoplasmic compartment (e.g. at the cytoplasmic face of NPCs).A strain expressing GFP-Dbp5This sentence was removed.This sentence was removed.Tyr determined by FISH at 25°C in integrated and untagged DBP5, dbp5-L12A, anddbp5-R423A strains prior to (pre), during a 10 min starvation for amino acids (-AA), and 15 min after reintroduction of amino acids through addition of rich media (re-feed).Figure 5. Dbp5 supports re-export of mature tRNAs following nutritional stress. A. Localization of tRNAdbp5-L12A-R423A. Data from dbp5-L12A-R423A has been removed from Figure 5 panel A. We have elected to remove and not replace this data as the tRNA processing defects and poly(A)-RNA accumulation phenotypes of dbp5-L12A-R423A no longer make this a logical experiment. See below for original and corrected figures.2. Figure 4 panel D and Figure 4—figure supplement 1 panels B and C have been updated with data showing the correct phenotype for The corrected Figure 4 is shown here:The originally published Figure 4 is also shown for reference:The corrected Figure 4—figure supplement 1 is shown here:The originally published Figure 4—figure supplement 1 is also shown for reference:The corrected Figure 5 is shown here:The originally published Figure 5 is also shown for reference:The article has been corrected accordingly.


The sunflower sesquiterpene lactones 8-epixanthatin and tomentosin can bind to the hydrophobic pocket of sunflower KAI2 with an affinity much higher than for the exogenous ligand KAR.Sesquiterpene lactones (STLs) are secondary plant metabolites with a wide range of biological, such as anti-microbial, activities. Intriguingly, the STLs have also been implicated in plant development: in several Asteraceae, STL levels correlate with the photo-inhibition of hypocotyl elongation. Although this effect was suggested to be due to auxin transport inhibition, there is no structural–functional evidence for this claim. Intriguingly, the light-induced inhibition of hypocotyl elongation in Arabidopsis has been ascribed to HYPOSENSITIVE TO LIGHT/KARRIKIN-INSENSITIVE2 (HTL/KAI2) signaling. KAI2 was discovered because of its affinity to the smoke-derived karrikin (KAR), though it is generally assumed that KAI2 has another, endogenous but so far elusive, ligand rather than the exogenous KARs. Here, we postulate that the effect of STLs on hypocotyl elongation is mediated through KAI2 signaling. To support this hypothesis, we have generated homology models of the sunflower KAI2s (HaKAI2s) and used them for molecular docking studies with STLs. Our results show that particularly two sunflower STLs, 8-epixanthatin and tomentosin, can bind to the hydrophobic pockets of HaKAI2s with high affinity. Our results are in line with a recent study, showing that these two STLs accumulate in the light-exposed hypocotyls of sunflower. This finding sheds light on the effect of STLs in hypocotyl elongation that has been reported for many decades but without conclusive insight in the elusive mechanism underlying this effect. Sesquiterpene lactones (STLs) are secondary plant metabolites with a wide range of biological, such as anti-microbial, activities   of 8-epixanthatin and tomentosin in the hypocotyl, which continues until reaching a maximum at 72 h . Surprisingly, Arabidopsis KAI2 also strongly binds 8-epixanthatin and tomentosin with estimated Kd values of about 1 and 2 µM, respectively, compared with a Kd value for KAR1 of 57 µM, a significantly lower affinity than for the STLs. The latter value matches well with the experimentally obtained Kd of AtKAI2 for KAR1, which ranges from 4.6 to 148 µM (Sun et al. Molecular docking showed that 8-epixanthatin and tomentosin fit into the hydrophobic-binding pockets of HalKAI2a and b Fig. c, f withKAI2 has the catalytic triad residues, Ser95-Asp217-His246, capable of hydrolyzing butanolide substrates (Nakamura et al. With this study, we provide candidate ligands, consisting of known endogenous plant metabolites, that can bind to KAI2 with an affinity that is much higher than for the only known exogenous ligand KAR. This finding opens up a new path to elucidate the mysterious ligand of KAI2, an important open question in plant biology with large potential applications in agriculture. Our work supports the hypothesis of others that the STLs are a plant growth regulator involved in growth and development of sunflower. We show that this effect is likely due to the interaction with KAI2. Future experimental data should validate our computational work. Studies into metabolites, possibly sesquiterpenoid, with similar lactone ring structure in Arabidopsis and other flowering plants should further solve the long-standing enigma of the elusive KL in the rest of the plant kingdom.H.B. and M.R. designed the project, M.R. did the modelling, and M.R. and H.B. wrote the article.


Tumor necrosis factor alpha  is a critical regulator of immune responses in healthy organisms and in disease. TNF is involved in the development and proper functioning of the immune system by mediating cell survival and cell death inducing signaling. TNF stimulated signaling pathways are tightly regulated by a series of phosphorylation and ubiquitination events, which enable timely association of TNF receptors-associated intracellular signaling complexes. Disruption of these signaling events can disturb the balance and the composition of signaling complexes, potentially resulting in severe inflammatory diseases. TNF is a type II transmembrane protein that is expressed at the plasma membrane as a trimer . CleavagBinding of TNF to TNFR1 triggers receptor trimerization and leads to the assembly of the TNFR1-associated signaling complex (complex I) . Within A20 results in enhanced RIP1 ubiquitination and inflammation  leading to the formation of the necrosome . Unlike  article .In vitro and in vivo studies have suggested the critical role ubiquitin plays in TNF signaling, both in enabling signal complex formation and in protein degradation. For example, chronic proliferative dermatitis (cpdm) mice were originally characterized as a strain of C57BL/KaLawRij mice that developed eosinophilic dermatitis with epidermal hyperplasia, multi-systemic inflammation, and defects in lymphoid development  and, to a lesser extent, TNF in the patients’ fibroblasts. Similarly, the patients’ B cell responses to Toll-like receptor (TLR) 7 and 8 agonists, IL-1β, and CD40-ligand (CD40L) were also impaired. Interestingly, LUBAC deficiency had an opposing effect in monocytes. Specifically, monocytes had approximately four-fold increased IL-6 production following IL-1β stimulation and enhanced responses to TLR1 and 2 agonists  lymphoma   . Similarse (IBD) . This wase (IBD) . Howeveroduction , suggestoduction . Peripheoduction . TogetheIKBKG, the gene that encodes NEMO, are associated with both incontinentia pigmenti   . Loss ofout mice . The difTnf mRNA production and stability, develop Crohn’s-like ileitis that can progress to transmural and granulomatous inflammation and arthritis al cells . These mal cells . TNF sti in vivo . TogetheT300AATG16L1 polymorphism is associated with Crohn’s disease, and ATG16L1 has an important role in Paneth cell survival and function  mice are viable and healthy, even when aged to 18 months , while the data from the ulcerative colitis trial are still pending. GSK has also ventured into cancer trials with a different RIP1 inhibitor, GSK3145095  and Denali have tested their RIP1 inhibitors in clinical settings and reported that GSK2982772 and DNL104 were generally well tolerated in human subjects . Denali’K3145095 . HoweverK3145095 . This maK3145095 . Thus, aJW and DV wrote and contributed to this manuscript and approved this submission.JW and DV were employees and shareholders at Genentech-Roche.


It is generally believed that the inflammatory response in bone marrow mesenchymal stem cells (BMSCs) transplantation leads to poor survival and unsatisfactory effects, and is mainly mediated by cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α). In this study, we explored the mechanisms underlying the TNF-α-induced inflammatory response in BMSCs.We treated BMSCs with TNF-α (1 and 10 ng/ml) for 5 days. The expression levels of key inflammatory mediators were evaluated by Real-time PCR. Intracellular ROS level was measured by using a 2, 7-dichlorofluorescein diacetate (DCF-DA).We found that TNF-α treatment dramatically increased the expression levels of some key inflammatory mediators, including IL-6, IL-1β, IFN-γ and transforming growth factor β (TGF-β). Moreover, TNF-α induced intracellular oxidative stress by elevating intracellular reactive oxygen species (ROS) level, which is due to the increase of lipid peroxidation, the reduction of antioxidant Glutathione (GSH) levels and the inhibition of many antioxidant enzyme activities in BMSCs. Interestingly, 5 µM curcumin, a ROS scavenger, dramatically lowered the TNF-α-induced inflammatory response in BMSCs. In addition, TNF-α induced the activation of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38 and their down-stream transcription factors nuclear factor kappa B (NF-κB) pathway.ROS mediated the TNF-α-induced inflammatory response via MAPK and NF-κB pathway, and may provide a novel strategy to prevent the inflammatory-dependent impairments in BMSCs. Bone marrow mesenchymal stem cells (BMSCs) are characterized by their pluripotent differentiation and self-renewal capability, and now have been extensively applied in different fields, especially in BMSCs transplantation . TherefoInflammatory response in BMSCs transplantation is mainly mediated by cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α). TNF-α is a pro-inflammatory cytokine released by macrophages acute inflammation, and is responsible for the periodontal inflammation mediated bone loss . In TNF-via MAPK and NF-κB pathway. Despite the fact that TNF-α affects the bone formation has been reported previously, the underlying cellular and molecular mechanisms of TNF-α on BMSCs are largely unknown. In the present study, TNF-α-induced inflammatory response in BMSCs was investigated, and we found oxidative stress induced by ROS mediated this process Cell culture and treatmentBMSCs primary cells  were cultured to P2 generation in hmsc-bm medium , and P3 generation cells were incubated on 60 mm cell culture plate. The hmsc-bm medium contains 5 µM/l β-glycerophosphate, 50 ug/ml vitamin C, and 1×10-8 mol/l dexamethasone. Cells were exposed to a final concentration of TNF-α  for 5 days and then washed before conducting the bioassays. The reason to choose 1, and 10 ng/ml is based on the preliminary data about different concentrations of TNF-α treatments  in BMSCs proliferation by alamarBlue assay, showing that 0.1 ng/ml TNF-α treatment almost has no effect, and 10ng/ml TNF-α treatment has similar effect with 50, 100 ng/ml TNF-α.Cell proliferation activity assay5 cells/ml) seeded in 96-well plates were incubated with TNF-α  for 5 day. After the treatment, the cells were incubated with 10 % alamarBlue solution (1 mg/ml) for 4 hr, and the plates were then read by the plate reader  at wavelengths of 540 and 620 nm, respectively, to determine cell proliferation activity.Cell proliferation activity was evaluated by the alamarBlue method . In brieReal-time polymerase chain reaction (PCR) analysisIl-6: forward (AGC GCC TTC GGT CCA GTT GC) and reverse (TGC CAG TGC CTC TTT GCT GCT); Il-1β:forward (TGG CGG CAT CCA GCT ACG AA) and reverse (CCG GAG CGT GCA GTT CAG TGA); Ifn-r: forward (GAA ACG AGA TGA CTT CGA AAA GC) and reverse (GCT GCT GGC GAC AGT TCA); Tgf-β: forward (CAA GTA GAC ATT AAC GGG TTC AGT TC) and reverse (GGT CGG TTC ATG CCA TGA AT); β-actin: forward (TGG CAC CCA GCA CAA TGA A) and reverse (CTA AGT CAT AGT CCG CCT AGA AGC A ). The cycle threshold (Ct) was determined using the cycle at which the primary (fluorescent) signal crossed a user-defined threshold. Quantification was normalized by the Ct value of β-actin by using the 2−ΔΔCt formula.Total RNA were extracted from BMSCs using the TRIZOL  according to the manufacturer’s protocol. Total RNA (500 ng) was reverse-transcribed to cDNA and conducted RT-qPCR by using PrimeScript RT reagent kit , and PCR amplification was performed by SYBR Premix Ex Taq II kit . Real-time PCR was performed by using ABI PRISM 7900HT Fast PCR System (Applied Biosystems) according to the manufacturer’s instructions. Primers for human genes were designed and synthesized by Takara Co  as follows: Measurement of intracellular ROS level°C for 30 min. After staining, the DCF fluorescence was analyzed using a FACSCalibur cytometer (BD Biosciences). In this study, 5 µM curcumin was used to scavenge intracellular ROS level. Intracellular ROS level was measured by using a 2, 7-dichlorofluorescein diacetate (DCF-DA) detection kit according to manufacture’s instruction. Briefly, cells were washed twice with PBS buffer and digested with 0.25% trypsin. Then the cells were resuspended and incubated with 10 µM DCF-DA at 37 Measurement of intracellular lipid peroxidation, superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione reductase (GR) activities 5 cells/ml) were treated with TNF-α  for 5 day. After that, cells were scraped off the dishes with a silicon “policeman” and transferred into Eppendorf tubes. Cells were then lysed in ice-cold PBS by sonication followed by centrifugation at 15,000 g for 10 min at 4 °C. The resulting supernatants were used immediately for the following measurements.BMSCs  using MDA assay kit from Keygen Biotech, Co, Ltd. . SOD, GR, and GSH assay kits  were also used to determine the SOD activity, GR activity and measure the cellular reduced GSH, respectively. Preparation of protein extracts and Western blot assay °C for overnight. After incubation, the membrane was washed with TBST containing 0.1% Tween-20, then anti-mouse and anti-rabbit IgG (1:5000 dilution) were incubated with the PVDF membranes at room temperature for 1 hr. Signals were developed on X-ray film using an enhanced chemiluminescence system .The BMSCs were seeded in 10 cm plates with fresh medium, and then exposed to 1 and 10 ng/ml TNF-α for 5 days. RIPA lysis buffer was used to extract total protein and the protein concentration was determined by bicinchoninic acid (BCA) assay. SDS-PAGE was carried out in 10% gel with equal-loading amount of protein per lane. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% BSA for 1 hr, the membrane was incubated with a 1:1000 dilution of primary antibody in 5% BSA at 4 Primary antibodies of P-JNK (#9251), ERK1/2 (#9102), JNK (#9252), P-ERK1/2 (#9101), P38 (#9212) and P-P38 (#9211), IkBa (#4812), P-IkBa (#2859) were purchased from Cell Signaling Technology , NF-kB (C-20: sc-372), Nrf2 (H-300: sc-13032), heme oxygenase-1 (HO-1) (H-105: sc-10789), GST-glutathione-S-transferase 1/2 (GSTO1/2) (FL-241: sc-98560), β-actin (I-19: sc-1616), and corresponding secondary antibodies were all purchased from Santa Cruz Biotechnology .Statistical analysis P<0.05. Western blot band intensity was analyzed using imagine J and statistical analyses were conducted using SPSS software.All data represent the mean ±SD of three independent experiments. Differences were considered significant if TNF-α induced the changes of cell morphology and cell proliferation in BMSCsIncubation of BMSCs with TNF-α (1 and 10 ng/ml) for 5 days induced remarkable changes in cell morphology. Untreated BMSCs displayed a flat shape with round, triangular or spindle-shaped bodies and short processes, while TNF-α-treated BMSCs exhibited larger, more flattened or fibroblast-like morphology . Here, wTNF-α triggered inflammatory response in BMSCsIt is known that TNF-α treatment induced inflammatory response in BMSCs. To confirm this, we detected the expression levels of several key inflammatory mediators, including IL-6, IL-1β, IFN-γ and TGF-β by quantitative real-time PCR. We found that there was a quite similar change of gene expression profile of IL-6, IL-1β, IFN-γ and TGF-β after treating BMSCs with TNF-α for 5 days . ExposurTNF-α induces cellular oxidative stress in BMSCs To explore the mechanisms underlying the TNF-α-induced inflammatory response in BMSCs, we measured the intracellular ROS levels using DCFDA assay. As shown in ROS mediated the TNF-α-induced inflammatory response in BMSCs To investigate whether the oxidative stress is involved in the TNF-α-induced inflammatory response in BMSCs, here we used ROS scavenger (5 µM curcumin) to lower the intracellular ROS level and checked the inflammatory response in BMSCs after treatment with 10 ng/ml TNF-α. We found that intracellular ROS level was dramatically decreased by 5 µM curcumin . Next, wMAPK and NF-κB pathway involved in TNF-α-induced inflammatory response in BMSCs It is reported that MAPK pathway plays important roles in the regulation of several genes involving in immune and inflammatory responses through the regulation of transcription factors NF-κB and AP-1 . To inveSince NF-κB is also an important redox-sensitive transcription factor, we postulated that TNF-α might activate NF-κB signaling. In the canonical NF-κB pathway, NF-κB activation depends on IκBα phosphorylation and degradation. We thus examined the effect of TNF-α on NF-κB pathway in BMSCs. We found that TNF-α significantly increased NF-κB protein expression; TNF-α also enhanced IκB-α phosphorylation and decreased the levels of IκB-α , indicatTNF-α activates NRF2 pathway in BMSCsIn this study, we first observed the expression of NRF2 protein in BMSCs. As shown in via activation of MAPK and NF-κB pathway. This study provides a better understanding of the effects of TNF-α on BMSCs. This study explored the effect of TNF-α on cell proliferation and inflammatory responses in BMSCs as well as the underlying mechanisms. Although many experiments have observed that TNF-α could impair differentiation of MSCs, but the underlying mechanisms remain poorly understood. Here, we found TNF-α significantly increased intracellular ROS level, and ROS scavenger dramatically lowered the TNF-α-induced inflammatory response in BMSCs, indicating that oxidative stress mediated TNF-α-induced inflammatory response, probably Inflammation response is a series of cellular and molecular responses that defend the body from infections or other impairments , 17. It Previous studies have showed that TNF-α induces the generation of cellular ROS in different tissues through stimulating ROS biogenesis  and enhaOur results demonstrate that TNF-α promoted cell proliferation and induced inflammatory response by altering the expression of pro-inflammatory cytokines in BMSCs. Moreover, TNF-α induced oxidative stress via activating MAPK, NF-κB and NRF2 pathway in BMSCs, but which is main pathway need further study. These finding shed a new light on the study of molecular mechanism of inflammation-induced dysfunction of BMSCs.