Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases regulate production of reactive oxygen species (ROS) that cause oxidative damage to cellular components but also regulate redox signaling in many cell types with essential functions in the cardiovascular system. Research over the past couple of decades has uncovered mechanisms by which NADPH oxidase (NOX) enzymes regulate oxidative stress and compartmentalize intracellular signaling in endothelial cells, smooth muscle cells, macrophages, cardiomyocytes, fibroblasts, and other cell types. NOX2 and NOX4, for example, regulate distinct redox signaling mechanisms in cardiac myocytes pertinent to the onset and progression of cardiac hypertrophy and heart failure. Heart failure with preserved ejection fraction (HFpEF), which accounts for at least half of all heart failure cases and has few effective treatments to date, is classically associated with ventricular diastolic dysfunction, i.e., defects in ventricular relaxation and/or filling. However, HFpEF afflicts multiple organ systems and is associated with systemic pathologies including inflammation, oxidative stress, arterial stiffening, cardiac fibrosis, and renal, adipose tissue, and skeletal muscle dysfunction. Basic science studies and clinical data suggest a role for systemic and myocardial oxidative stress in HFpEF, and evidence from animal models demonstrates the critical functions of NOX enzymes in diastolic function and several HFpEF-associated comorbidities. Here, we discuss the roles of NOX enzymes in cardiovascular cells that are pertinent to the development and progression of diastolic dysfunction and HFpEF and outline potential clinical implications. Heart failure with preserved ejection fraction (HFpEF) is a complex, heterogenous, and highly prevalent multiorgan syndrome commonly associated with advanced age, obesity, and/or hypertension ,2. ThereDiastolic dysfunction, or the inability of the ventricles to effectively relax and fill with blood, is a key cause of heart failure in patients with HFpEF and results in abnormally high left ventricular filling pressures, despite the maintenance of adequate left ventricular ejection fraction ,12,13,14Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzymes control the regulated cellular production of ROS and have indispensable functions in cardiovascular physiology and disease ,37,38,39phox regulatory subunit for oxidase activity and protein stability [phox) require the translocation of additional cytosolic subunits to the oxidase complex for activation: NOX1 requires association with the p47phox or NOXO1 organizer subunit, p67phox or NOXA1 activator subunit, and the active (GTP-bound) Rho family small GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1), whereas NOX2 necessitates the interaction of p47phox, p67phox, p40phox, and active Rac1 or Rac2 with the NOX2:p22phox complex at the membrane to evoke superoxide production [There are seven NOX family enzymes (NOX1-5 and DUOX1-2), which are multi-pass transmembrane proteins that enzymatically generate superoxide via the transfer of electrons to molecular oxygen ,37,38. Ntability ,37,38. Noduction ,57. With regards to the cardiovascular system, NOX1, 2, 4, and 5 are expressed in cardiomyocytes, endothelial cells, and vascular smooth muscle cells, and NOX2 and NOX4 in fibroblasts . NOX2 isNOX2 and NOX4 are the best-studied NOX enzymes in the cardiovascular system and cardiomyocytes more specifically. While the generation of ROS by NOX2 is inducible by Rac1/2 GTPase activity such as downstream of angiotensin receptor (AT1R) activation by angiotensin-II (AngII) ,57,61, NGiven the paucity of animal models that faithfully recapitulate the multi-organ pathophysiology of HFpEF ,3 and thBasic science studies implicate NOX2, NOX4, and NOX5 in the deterioration of diastolic function and adverse cardiac remodeling in response to pathological stimuli . A globaMany studies have also revealed indispensable functions of NOX4 in cardiac biology and disease. The transgenic overexpression of Nox4 at modest levels in cardiomyocytes elicits elevated myocardial ROS production, fibrosis, and cardiomyocyte hypertrophy and apoptosis with advanced age . The carNOX4 also has critical roles in the inflammatory and wound healing response of the heart to ischemic injury. Although the transgenic overexpression of Nox4 in cardiac myocytes was sufficient to promote macrophage recruitment to the heart and polarization towards the M2 phenotype that was associated with a modest reduction in cardiac hypertrophy, fibrosis, and mortality following myocardial infarction , endogenAlthough not expressed in rodents, gain-of-function studies in mice suggest a role for the calcium-sensitive NOX5 enzyme in the adverse remodeling of the heart in response to RAAS activation or mechanical stress. Indeed, the cardiomyocyte-specific overexpression of human NOX5 in mice exacerbated cardiac hypertrophy, fibrosis, and oxidative stress in response to AngII infusion and cardiac pressure overload . Thus, NNOX enzymes are expressed in all cell types of the vasculature including the coronary vessels supplying blood to the heart ,37,38. V−/− mice [NOX4 is the most abundant NOX isoform in endothelial cells and a major source of ROS production in the vasculature and heart ,122. In −/− mice , while t−/− mice .−/− mice [Conversely, endothelial NOX1, NOX2, and NOX5 contribute to impaired vascular function, endothelial nitric oxide synthase (eNOS) uncoupling , and adv−/− mice . Most no−/− mice . The exp−/− mice , and the−/− mice , indicatNOX1, NOX4, and NOX5, and to a lesser extent NOX2, are expressed in vascular smooth muscle cells ,126,132.NOX4 in vascular smooth muscle cells plays critical roles in vascular aging, inflammation, and atherogenesis. NOX4 expression is upregulated in the atherosclerotic lesions of mouse models and humans ,138 and The calcium-dependent NOX5 enzyme is upregulated in the vascular smooth muscle cells of hypertensive patients and is required for AngII- and PDGF-induced ROS production ,140. NOXCardiac fibrosis is a major driver of cardiac pathology and diastolic dysfunction in HFpEF ,12. ResiNOX4 is the best-studied NOX isoform in cardiac fibroblasts and has important roles in the fibrotic remodeling of the heart. NOX4 is upregulated in cardiac fibroblasts in response to TGF-β and in heart failure and is required for TGF-β-induced superoxide production, Smad2/3 phosphorylation, myofibroblast transformation, and collagen production ,146. FutNADPH-dependent oxidase activity was originally discovered in neutrophils and was later found to be mediated by NOX2 ,148,149.NOX1 in peripheral monocytes correlate with the degree of diastolic dysfunction in patients with HFpEF [NOX4 generates ROS in peripheral monocytes and macrophages where it is upregulated in response to oxidized low-density lipoprotein to promote macrophage cytotoxicity , suggestth HFpEF , indicatNOX-dependent superoxide production by peripheral blood mononuclear cells is increased in patients with hypertension compared to normotensive controls and even further enhanced among patients with hypertension and left ventricular hypertrophy , suggestω-nitro-L-arginine (HFD/L-NAME) model of HFpEF [NOX2 and NOX4 have emerged as central regulators of the intracellular redox signaling landscape in cardiomyocytes . NOX2 isof HFpEF . NOX4 isof HFpEF ,67,157. Notably, both NOX2 and NOX4 activity promote the activation of hypertrophic gene expression in cardiomyocytes through distinct mechanisms that provoke oxidation-dependent nuclear export of histone deacetylase 4 (HDAC4), thereby derepressing the transcriptional activity of the hypertrophic transcription factors, myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) ,42,44,62Nuclear factor kappa B (NFκB) transcriptional activity regulates inflammatory gene expression programs and is enhanced in a NOX2-dependent manner in response to AngII ,158. IndNOX2 activity in cardiomyocytes promotes sarcoplasmic reticulum calcium cycling and contractility in response to AngII in part through the hyperphosphorylation of phospholamban , while mNOX4-generated ROS at cardiac mitochondria can influence redox signaling and promote mitochondrial oxidative damage and cardiomyocyte death ,64 Figu. Nox4 ovNitric oxide (NO), which is predominantly generated in the endothelium by eNOS, has potent vasodilatory activity in the coronary and peripheral vasculature via the activation of soluble guanylate cyclase (sGC) in vascular smooth muscle cells ,160 Fig. NOX-genX-box-binding protein 1 (Xbp1), that is required for the ER stress response and cardiac adaptation [Despite the elevation of ROS and the dampening of signaling downstream of NO  in HFpEF, paradoxically, NO and nitrosative stress seem to be nodal drivers of cardiac disease in HFpEF . NO can aptation ,170. Impaptation . Thus, tS-nitrosylation of histone deacetylase 2 (HDAC2) by neuronal NOS (nNOS) also plays a key role in HFpEF pathophysiology . nNOS exThe interplay between oxidative stress and NO production is complex but basic science studies and clinical data suggest that both NOX and NOS enzymes and oxidative and nitrosative stress play important roles in HFpEF. The NOX-mediated uncoupling of NOS enzymes in the vasculature could limit NO production and further promote the generation of ROS and reactive nitrogen species, oxidative tissue damage, and pathogenic redox signaling ,36,161. There is interest in targeting oxidative stress, and NOX isoforms in particular, as therapeutic avenues to delay adverse outcomes in HFpEF. As discussed above, NOXs play essential roles in cardiovascular oxidative stress and redox signaling in response to RAAS activation. NOX2- and NOX4-dependent ROS generation are specifically induced in the heart in response to elevated AngII, aldosterone, and catecholamines in the circulation in cardiovascular disease and can contribute to the deterioration of diastolic function. Unfortunately, for the most part, pharmacological strategies used to treat HFrEF have not been effective at reducing mortality or cardiovascular events in patients with HFpEF ,2,171. ASimilar to commonly prescribed heart failure therapies targeting the RAAS pathway, nitric oxide donor therapies have failed to improve HFpEF outcomes ,178. NeiAnother important caveat to NO donor therapy is that it could even promote pathogenic nitrosative stress in the heart and the upregulation of S-nitrosylation-dependent signaling mechanisms in cardiomyocytes that are central to HFpEF disease progression ,169 Fig. Indeed,Excitingly, recent clinical trials have demonstrated that sodium-glucose cotransporter 2 (SGLT2) inhibitors, which lower blood glucose by repressing glucose resorption in the proximal tubule of the kidney ,182, redFuture studies interrogating the regulation of NOX-mediated oxidative stress and redox signaling by distinct NOX isoforms in various cell types of the heart using pre-clinical models of HFpEF and diastolic dysfunction will facilitate the identification of novel targets to repress oxidant stress and pathogenic redox signaling. An examination of the phenotypic response, myocardial redox status, and redox signaling landscape of NOX isoform-specific conditional deletion mice in models of HFpEF  will help to uncover the mechanistic contribution of NOXs to HFpEF. Evidence from humans and animal models suggests that NOX-regulated oxidative stress and redox signaling mechanisms likely participate in HFpEF pathogenesis. Further investigation could suggest the development of isoform-specific NOX inhibitors, the targeting of regulators of NOX oxidase activity , or strategies to intervene with specific NOX-regulated redox signaling effectors as therapeutic targets for the treatment of diastolic dysfunction and HFpEF.


To investigate the severity and causes of gender imbalance in the counts of ophthalmology citations.Prog Retin Eye Res, Ophthalmology, JAMA Ophthalmol, and Invest Ophthalmol Vis Sci) between August 2015 and July 2020, and those that referenced these cited papers by 2021 July . The gender category of a given paper is defined by the gender of the first and last author . A generalized additive model to predict the expected proportion was fitted. The difference between the observed proportion and expected proportion of citations of a paper’s gender category was the primary outcome.The PubMed database was searched to identify cited papers that were published in four journals  papers slightly increased from 27% in 2015 to 30% in 2020. MM, FM, MF, and FF papers were cited as −9.3, −1.5, 13.0, and 23.9% more than expected, respectively. MM papers cited 13.9% more male-led (MM and FM) papers than female-led papers, and FF papers cited 33.5% fewer male-led papers than female-led papers. The difference between the observed proportion and expected proportion of MM citing papers within male-led and female-led cited papers grew at a rate of 0.13 and 0.67% per year.The high frequency of citations of female-led papers might narrow the gender gap in the citation count within ophthalmology. These findings show that papers by female-led are less common, so the gender gap might still exist even with their high citation count. Women have faced societal pressures and barriers associated with gender –3 comparDespite an increase in the proportion of female graduates in medical school  and the Assessment criteria for performance in academia include academic ranks, peer-reviewed publications, salary, and funding , 16, 17.Prog Retin Eye Res, Ophthalmology, JAMA Ophthalmol, and Invest Ophthalmol Vis Sci), which had the highest h-index  and one review journal (Prog Retin Eye Res: 152) with the highest h-index in the ophthalmology field in 2020  to each author whose name had at least 85% probability of belonging to someone labeled as male  according to the GenderAPI. We randomly selected 100 unique authors involved in the aforementioned dataset that we collected from PubMed for the manual gender verification and found that the accuracy of GenderAPI program was 98%  as Dworkin et al.  on the mage in R , which rp-value, or both. The CIs were computed by bootstrapping the cited papers . Randomization was conducted by probabilistically drawing new gender categories for each paper according to their estimated gender probabilities by GAM. The statistical significance was adjusted for multiple comparisons; p-values were corrected according to the Holm–Bonferroni method , a i method .Generally, the last author of a paper was considered the senior investigator. We defined a female-led (MF and FF) paper as the article in which the last author is female and a male-led (MM and FM) paper as the article in which the last author is male.In this study, we tested four hypotheses:Hypothesis 1: The citation rate of female-led papers is lower than expected.To verify this assumption, we first estimated the expected proportion of citations given to each category of authors. This expectation was calculated by summing the probabilities estimated by the GAM for all papers from 2015 to 2020. These values were compared by calculating the percentage difference between observed and expected proportions for each author’s gender group. If the hypothesis is true, the percentage difference of female-led papers will be less than 0.Hypothesis 2: The citation of female-led papers occurs to a fewer degree in MM papers.We used a similar method to those described above to test the second hypothesis. The primary difference is that, instead of calculating the observed and expected proportion by summing over the citations within all citing papers, we performed those summations separately for lists in papers with male-led papers and female-led papers. If this hypothesis is true, MM papers will be citing more male-led papers than female-led papers.Hypothesis 3: The proportion of MM citations of female-led papers will be decreasing more than that of male-led papers over time.The changes in male-led papers and female-led papers over time were estimated using linear regression. The CIs of this estimate was obtained using the article bootstrap procedure, and significance was assessed using the graph-preserving null model . If the Hypothesis 4: A relationship exists between local co-authorship networks and citation behavior.A co-authorship network of first and last authors is defined as where they established a connection by co-authoring a paper with another author before a given date. We examined how the citation behavior was affected by the co-authorship network. If the hypothesis is true, there will be a consistent citation behavior between with co-authorship networks and without networks.Prog Retin Eye Res (219 papers), Ophthalmology , JAMA Ophthalmol , and Invest Ophthalmol Vis Sci  from August 2015 to July 2020 (data from the PubMed database). From those, 3,813 were MM papers (47.17%), 1,950 were FM papers (24.12%), 1,209 were MF papers (14.95%), and 1,112 were FF papers (13.76%).Our data included 8,084 cited papers, which were published in Prog Retin Eye Res decreased from 35 to 18%; Ophthalmology hardly changed (from 28 to 27%); JAMA Ophthalmol increased from 30 to 34%, and Invest Ophthalmol Vis Sci increased from 26 to 29%. On the other hand, the overall proportion of articles that had females as first or last authors slightly increased from 50% in 2015 to 55% in 2020  that were published in the four ophthalmology journals. We found that these 5,864 papers had been cited by 53,962 times before August 2021. We then obtained the observed proportion in each gender category for all 5,864 papers [e.g., a given cited paper was cited by 97  citing papers, the observed proportion was 0.48, 0.28, 0.23, and 0.01, respectively; Hypothesis 1).We studied whether there were any relationships between gender and paper characteristics . We modeled the multinomial gender category  as a function of the above characteristics by fitting a GAM, by which we estimated the expected proportion of citing papers for a given cited paper . For allp < 0.001), FM papers were cited 0.5% less than expected , MF papers were cited 7.1% more than expected , and FF papers were cited 14.7% more than expected ; within female-led cited papers (TC: 16162), MM papers were cited 19.5% less than expected , FM papers were cited 4.3% less than expected , MF papers were cited 29.1% more than expected , and FF papers were cited 48.2% more than expected , whereas FF papers tended to cite more female-led papers.Among the abovementioned 5,864 cited papers that we screened, there were roughly 71% male-led papers and 29% female-led papers. In this section, after separating cited papers by gender, we found that, within male-led cited papers (TC: 37800), MM citing papers were cited 5.6% less than expected , whereas MM and FM papers cite less frequently than expected. One question is whether researchers are more likely to work with others of their own gender in the ophthalmology area as the findings by Ghiasi et al.  indicatef, we defined FF paper’s overrepresentation as the difference between the FF papers within f’s paper neighborhood and the overall proportion of FF papers within the network at the time of f’s publication. Specifically, the number of papers published before the given paper f is n, where the number of FF papers is FFall; the number of papers forming a co-author network with f is m, the number of FF papers is FFnet, and the FF paper overrepresentation means For a given paper p = 0.84), whereas they cited more FF papers by 2.4% for FM teams , 4.4% for MF teams , and 7.1% for FF teams , whereas they cited FF papers more by 2.2% for FM teams , 4.3% for MF teams , and 7.1% for FF teams . The citation behavior of women by other women remains after accounting for social networks (p = 0.93). These two results do support Hypothesis 4: There seems to be a relationship between local co-authorship networks and citation behavior.However, after the social networks have been accounted for, the gender citation patterns remain. Specifically, the median MM teams still cited FF papers less by around 0.38%  and one review journal (Prog Retin Eye Res: 152) with the highest h-index in the ophthalmology field in 2020. Furthermore, we collected more than 8,000 cited papers from these four journals, and more than 50,000 papers citing them across the whole PubMed database. Even though we believed that these datasets could be a good representation of the question we asked, we agree that an ideal way should be to consider all the journals. Further studies should explore the gender gap in citation behavior by examining more ophthalmology journals.Although our study reduces the confound of journal prestige, we still agree that it does not capture the entirety of the field as we selected only four journals. We aimed to evaluate the extent of gender differences in citations to the ophthalmology literature, so the h-index of the journal was the main index for selecting target journals , 36. BasThe previous studies on the gender bias in ophthalmology focus mainly on authors’ characteristics, such as the percentage of female authors , 27, femThere are multiple avenues by which future works could be undertaken based on our study. For example, the paper citation might differ in different ophthalmology subfields, such as retina, cataract, glaucoma, and strabismus. Therefore, it would be interesting to further assess the difference between the ophthalmic sections in the gender gap in citation counts. Limited by the resources we got from the PubMed database, we were not able to extract the keywords and classify the papers into these different categories. Future work may combine other databases into the GAM model to address it.In summary, despite the increase in the proportion of female-led papers from August 2015 to July 2020, the proportion was still found to be much lower than that of the male-led papers. Since the proportion of FF papers is much lower than that of MM papers, even if the FF papers cited more female-led papers, the gender gap in citation behavior might not be alleviated; this phenomenon might be related to social co-authorship networks.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SC and JZ designed the study. SC, YX, and WZ collected the data. SC, ZH, and JZ contributed to the interpretation of the results and critical revision of the manuscript for important intellectual content and approved the final version of the manuscript. All authors have read and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Heart failure (HF) with preserved ejection fraction (HFpEF) is a multi-organ, systemic syndrome that involves multiple cardiac and extracardiac pathophysiologic abnormalities. Because HFpEF is a heterogeneous syndrome and resistant to a “one-size-fits-all” approach it has proven to be very difficult to treat. For this reason, several research groups have been working on methods for classifying HFpEF and testing targeted therapeutics for the HFpEF subtypes identified. Apart from conventional classification strategies based on comorbidity, etiology, left ventricular remodeling, and hemodynamic subtypes, researchers have been combining deep phenotyping with innovative analytical strategies  to classify HFpEF into therapeutically homogeneous subtypes over the past few years. Despite the growing excitement for such approaches, there are several potential pitfalls to their use, and there is a pressing need to follow up on data-driven HFpEF subtypes in order to determine their underlying mechanisms and molecular basis. Here we provide a framework for understanding the phenotype-based approach to HFpEF by reviewing (1) the historical context of HFpEF; (2) the current HFpEF paradigm of comorbidity-induced inflammation and endothelial dysfunction; (3) various methods of sub-phenotyping HFpEF; (4) comorbidity-based classification and treatment of HFpEF; (5) machine learning approaches to classifying HFpEF; (6) examples from HFpEF clinical trials; and (7) the future of phenomapping  for the classification of HFpEF. Heart failure (HF) with preserved ejection fraction (HFpEF) is a multi-organ, systemic syndrome that involves multiple cardiac and extra-cardiac pathophysiological abnormalities –3. Becaun = 397), which they termed “phenomapping” ), although in most this is typically pulmonary venous hypertension; however, there are some HFpEF patients who have combined post- and pre-capillary PH. Treatment using specific drugs approved for pulmonary artery hypertension in unselected patients with HFpEF has yielded mixed results and is currently not recommended . StimulaDietary restriction in obese patients improves diastolic function , and a rAs mentioned earlier, multiple studies have now applied data-driven approaches (“phenomapping”) to HFpEF classification. These studies have used machine learning analytic techniques similar to our original phenomapping study and have shown that the HFpEF subtypes identified have different clinical features and outcomes 5). TheseDespite the rapid rise of machine learning and -omics studies in HF, including HFpEF, there are several challenges to these types of studies that must be considered when evaluating their clinical utility in the classification (sub-phenotyping) of HFpEF and in understanding underlying molecular mechanisms. Table Several ongoing and recently completed HFpEF clinical trials have taken advantage of sub-phenotyping of HFpEF in order to target therapeutics to particular HFpEF subtypes in an attempt to improve chances for HFpEF clinical trial success. For example, in the EMBARK-HFpEF trial (open-label proof-of-concept study of mavacamten [myosin inhibitor] in HFpEF [NCT04766892]), only patients with LVEF > 60%, LV hypertrophy, and elevated biomarkers (elevated natriuretic peptides or troponin) are allowed entry into the trial. These HFpEF patients are most similar to patients with genetic/familiar forms of HCM and therefore are hypothesized to be the patients most likely to benefit (and least likely to have adverse effects) from mavacamten therapy.In the SERENADE trial (NCT03153111), which is a randomized controlled trial of macitentan  in patients with HFpEF, elevated natriuretic peptides, cardiac remodeling (LVH or LA enlargement), and evidence of pulmonary vascular disease  are allowed entry into the trial. These patients are hypothesized to be those who will benefit most from macitentan. To further select patients who may benefit, SERENADE included a 4-week placebo run-in phase  and a 5-week macitentan run-in phase (to exclude patients who develop early fluid retention in response to macitentan). Only patients who qualify for the trial and make it through these two run-in phases were randomized in the trial.In the recently completed REDUCE LAP-HF II trial . In addition, advanced imaging, e.g., machine learning-based (magnetic resonance) image analysis and deep-learning algorithms for echocardiographic data may provide incremental prognostic value in HFpEF [Current phenomapping approaches for HFpEF are largely based on clinical data available from demographics and routine clinical parameters such as medical history, medication history, routine blood tests, as well as electrocardiographic and echocardiographic read-outs , 15. Futin HFpEF , 85. Metin HFpEF , 87. An in HFpEF .Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous, systemic, multi-organ syndrome that is rising in prevalence and is associated with high morbidity and mortality. While certain therapeutics  may be beneficial in the large majority of HFpEF patients, most treatments are not well suited for a one-size-fits-all approach. Unsupervised machine learning (phenomapping) combined with -omics analyses is a growing approach to the classification of HFpEF and likely represents the future of HFpEF precision medicine. Nevertheless, investigators and clinicians should understand the potential limitations of such approaches and should augment their initial phenomapping studies of HFpEF with follow-up studies to identify underlying molecular mechanisms with the hope of conducting successful precision medicine trials in the future.


Negative attitudes and prejudices towards people with a mental disorder are common across cultures and societies. The stigma associated with mental illness leads to a lower quality of life, given the discrimination and social exclusion suffered by people with this type of disorder. In the field of health, research has shown that doctors and nurses also manifest these types of stigmatizing behaviors and attitudes. The present study aims to create and apply an educational escape room for the purpose of training nursing students in mental health, promoting positive attitudes towards people who have a mental disorder. To do so, a pre-post study was conducted with an experimental group and a control group to determine whether the escape room was effective for the modification of stigmatizing behaviors compared to transmissive lecture class, and a third measurement was made at 6 months only to the experimental group to evaluate whether the changes produced by the escape room were maintained in the long term. The results indicate that the students participating in the study obtained better scores in sensitization and these remain better over time. It is concluded that the escape room used is suitable for the training and sensitization of future nursing professionals in the field of mental health, facilitating the learning of knowledge and positive attitudes towards severe mental disorder.The online version contains supplementary material available at 10.1186/s12909-022-03879-6. In recent years, there has been a significant increase in mental disorders worldwide, a trend that according to different sources and institutions will increase as a result of COVID-19 . Stigma The manifestation of these negative consequences, such as exclusion or social marginalization, would be are produced the position of power of the group that stigmatizes against the group that is stigmatized . DiffereThe origin and maintenance of these attitudinal barriers are currently an important challenge for professionals dedicated to mental health, given their negative effects both on the person who manifests the disorder and on their family environment. These stigmatizing attitudes and behaviors lead to difficulties in interpersonal relationships, work, or studies, which would condition a lower level of well-being and quality of life , 10..Although stigma toward a person with a mental disorder is common and frequent in various fields and professions, the review of the literature on the subject highlights the interest dedicated to the manifestation of these discriminatory behaviors by professionals and students related to the field of health . In thisCzechoslovakia 38-39: Borderlands. Both groups had an intervention with video games, although the experimental group had a video game with a sensitizing load, while the control group had a strong narrative related to it but did not address the topic explicitly. Their results show long-term between-group attitude change in micro explicit attitudes but not macro explicit attitudes, suggesting that this may owe to the historical fact addressed. They consider that video games can tell personal stories about particular issues that humanize certain concepts and allow them to have a real impact on people, favoring attitudes of awareness about the topics addressed in the playful narrative. On the other hand, Bujíc et al. [In the case of medical and nursing students, efforts have been made to design possible strategies to promote inclusive and non-discriminatory attitudes. Among these actions we find educational programs or courses that try to inform specifically about mental disorders , initiatc et al.  conducteAlthough there is still little evidence on the effectiveness of these playful tools in changing attitudes towards SMI , the resAmong the various types of video games, the escape room represents a highly valid tool to promote the change of attitudes because its dynamics have been proven to favor the learning of various skills, generating high motivation and interest in students . Escape To assess the escape room’s ability to reduce stigma for nursing students.To determine whether escape rooms have an impact on academic performance and cooperative skills among students.To assess whether the escape room has long-term effects on students and whether there are gender differences.After an analysis of the recent literature, it has been observed that there is a very limited number of investigations that use playful strategies, such as escape rooms, for awareness of SMI which also observe their long-term effect. This study aims to expand the use of the escape room as a strategy to promote learning and awareness of mental illness and evaluates the impact of the escape room compared to a transmissive lecture class on nursing students, as well as the long-term effect of the escape room. The specific objectives of this research were:This research consists of a quasi-experimental longitudinal study, with a control and experimental group. The intervention was carried out in the subject of mental health in the nursing degree program. Both the control and experimental groups attended their regular classes, however, the experimental group participated in an escape room with sensitizing material on SMI. Fig. The sample was selected using intentional non-probability sampling, as was convenience sampling, using the classes themselves as individual units. The sample initially included 6 classes from two universities in Andalusia (Spain).The sample was made up of a total of 253 students with a mean age of 22.77 (SD = 6.85) years, of which 58 were men and 194 women. The experimental group had 138 participants, 35 men, and 103 women. Regarding the control group, it was made up of 115 people, 24 men, and 91 women. A majority of women is observed in the sample because the nursing sector in Spain is dominated by women .Written and oral informed consent was gathered from the students. The methods used in this study were carried out in accordance with the Declaration of Helsinki. Ethics approval was obtained from the Research Ethics Committee of the University of Almería (UAL01/2021), Spain. Students were only excluded from the sample if they refused to give their informed consent to participate. The participants received neither economic nor academic incentives for taking part in the study.χ2 (165) = 3134.01, p < .001; * CFI = 0.942; * RMSEA (90% CI) = 0.040 (0.039-0.041); SRMR = 0.032.This questionnaire measures the 5 dimensions of collaborative learning, social skills (α = .74), group processing (α = .75), positive interdependence (α = .72), promoter interaction (α =. 76) and individual responsibility (α = .79). In addition, it displays good psychometric properties not only in the factors as seen previously, but it also obtains an α = .89 as a whole. The model was validated by confirmatory factor analysis with satisfactory results S-Bχ2 (193) = 1.326, p < .001; CFI = 0.983; RMSEA = 0.041 (0.039-0.041); SRMR = 0.042.2.2.2. Questionnaire on public stigma towards mental disorder AQ14 . This quTo evaluate this variable, the final exam score of a subject related to mental health was collected. The scores were established on a scale from 1 to 10, where the higher the score, the better the result.Escape rooms consist of a track game where players are locked in a room due to a narrative event and must solve a series of puzzles, unlock locks, and find hidden clues to escape at any given time, usually in less than an hour . The act“No Memories” is an escape room where players must remember both who they are and where they are to escape from the room. Throughout the escape room challenges, the participants eventually discover that their character is a person with SMI, leading a normal life, displaying the characteristics and difficulties present in people with SMI.Before starting the escape room, the students watched an explanatory tutorial video to understand how the platform works. The game design of the escape room follows a linear structure. Solving one clue will provide the object necessary to solve the next clue, and so on until the students escape. Tasks in a linear room must be solved in a particular order. The average duration of escape rooms is 60 minutes , so thisDue to COVID-19, the intervention was carried out online and synchronously through the university’s learning platform. The students had to solve the escape room in teams of 4 people. Each of these groups was in a separate chat where the teacher and the researchers could enter to answer questions or provide clues. The students were offered all the clues they requested. This decision was made because all students had to finish the escape room in order to have all the information about the content taught.After giving informed consent, the participants completed the questionnaires in an online classroom at the start of the course, while the researchers remained with the participants to administer the survey and to answer any questions. The researchers pointed out that all answers were anonymous and completely confidential. The students completed the questionnaires in a period of 20 minutes on average.Next, the “No Memories” escape room was applied to the experimental group. Once each team had concluded, the participants were again given the questionnaires.The participants in the control group attended and completed the subject on “Mental Health” in a traditional way (lectures and complementary work). They completed the questionnaires both at the beginning of the course and during the same period that the escape room was applied in the experimental group.Finally, the experimental group answered the questionnaires again 6 months after the intervention.The data analysis was guided by the proposed research objectives. First, the factors of the instruments used was calculated. A Student’s t-test for independent samples was continued in the previous tests to determine whether the groups  were statistically equivalent. This was followed with another Student’s t for samples not related to the post-test balances with the intention of verifying whether there were statistical differences just after applying the programs. To determine what happened in the experimental group longitudinally, two Student’s t-tests were carried out for related samples, comparing the pre-test composition with the monitoring pressures and the pre-test pressures with the post-test pressures. To determine whether the program affects sensitivity towards stigmatizing attitudes differently according to gender, a MANOVA was carried out with the total AQ14 score in the follow-up proportions and the total proportions of the AQ14 pretest were included as covariates.Finally, to measure the impact on academic performance, a Student’s t-test was conducted for independent samples with the score obtained in the subject in which the research was carried out.To facilitate the reading of the results, all the means and standard deviations of the investigation are reported in Table Guided by the objectives of the investigation, the first step was to determine whether the groups  were equivalent. For this purpose, a Student’s t-test was carried out for independent samples with the pre-test scores of both questionnaires. These results were reported in Table Continuing with the data analysis, another Student’s t-test was performed for independent samples with the post-test scores (those obtained immediately after treatment in the groups). These results were reported in Table However, for the “Stigmatizing Attitudes Questionnaire AQ14” the changes that occurred in the factors of this questionnaire are of a significant magnitude and are considered large effect sizes (d > .80), in this case there is a statistical difference in stigmatizing public attitudes in favor of the experimental group.In Table In order to know the evolution of the experimental group in terms of the factors of public stigmatization, a follow-up measure (Retest) was carried out, which was compared with the pre-test and post-test score of the experimental group. The results of these Student’s t-tests for paired samples are reported in Table p = .000) indicate that there are no differences by sex, which leads to the conclusion that the program affects both sexes in the same way.Finally, a MANOVA was conducted in order to determine whether gender is a variable to be considered when carrying out this type of program. With this aim, the total score of the post-test of the “Stigmatizing Attitudes Questionnaire AQ14” was utilized, along with the total score obtained in the pre-test of this same questionnaire. The results of the Pillai F Trace statistic . Throughout the escape room, various clues appear which allow players to pass the tests. After reading the tests, in the event that the team does not know how to solve it, they can ask the facilitator as many times as they need for assistance.


KO) mice that were stimulated with TNF, and the results were validated at the protein level by using multiplex cytokine assays and immunoblotting. After TNF stimulation, STAT3KO MEFs showed greater gene and protein induction of the inflammatory chemokines Ccl2, Cxcl1 and Cxcl10 than WT MEFs. These observations show that, by activating STAT3, TNF selectively modulates expression of a cohort of chemokines that promote inflammation. The greater induction by TNF of chemokines in STAT3KO than WT MEFs suggested that TNF induced an inhibitory protein in WT MEFs. Consistent with this possibility, STAT3 activation by TNFR1 increased the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in WT MEFs but not in STAT3KO MEFs. Moreover, enforced expression of Tnfaip3/A20 in STAT3KO MEFs suppressed proinflammatory chemokine expression induced by TNF. Our observations identify Tnfaip3/A20 as a new downstream target for STAT3 which limits the induction of Ccl2, Cxcl1 and Cxcl10 and inflammation induced by TNF.Tumour Necrosis Factor (TNF) potently induces a transient inflammatory response that must be downregulated once any invasive stimulus has resolved. Yet, how TNF‐induced inflammation is shut down in normal cells is incompletely understood. The present study shows that STAT3 was activated in mouse embryo fibroblasts (MEFs) by treatment with TNF or an agonist antibody to TNFR1. STAT3 activation was inhibited by pharmacological inhibition of the Jak2 tyrosine kinase that associates with TNFR1. To identify STAT3 target genes, global transcriptome analysis by RNA sequencing was performed in wild‐type MEFs and MEFs from STAT3 knockout (STAT3 Albert Baldwin , were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco) and 1× Penicillin−Streptomycin−Glutamine. Another WT MEF line was used in the validation experiments, which were a kind gift from the National Institutes of Health . HEK293T cells, a kind gift from Dr. Hassan Alaoui , were maintained in DMEM supplemented with 10% fetal bovine serum and 1× Penicillin−Streptomycin−Glutamine.WT and STAT3 knockout  according to the manufacturer's protocols. RNA quality control, library preparation and Illumina sequencing were performed by Novogene Corporation Inc. . Raw sequencing data preprocessing, mapping to the reference genome (mm10), and gene expression quantification were performed by Novogene. Differential gene expression analysis was performed by using the iDep tool.2.3KO MEFs was achieved by lentiviral transduction. Transfer plasmid containing the A20 insert was obtained from Origene . Packaging (psPAX2) and envelope plasmids (pMD2.G) were a gift from Dr. Hassan Alaoui. Transfer and packaging vectors were transfected into HEK 293T cells to produce lentiviruses using Lipofectamine 3000 reagent . Lentiviruses were harvested 48 h post transfection, concentrated using Lenti‐X concentrator , and used to infect STAT3KO MEFs in the presence of Transdux Max  reagent. Fresh medium containing puromycin  was added 24 h later and the cells were maintained and selected for 2 weeks. A20/Tnfaip3 overexpression in STAT3KO MEFs was confirmed by immunoblotting (data not shown).Stable A20 overexpression in STAT32.4MEFs were grown on 10 cm dishes until 80% confluent, serum‐starved for 24 h and then stimulated with TNFR1aab  for 4 h. Samples of the media were collected and analyzed in duplicate for cytokine secretion by using a multiplex fluorescent bead assay .2.5Cells were lysed with RIPA buffer (ThermoFisher Scientific), and lysate protein concentrations were measured using a Qubit protein assay kit (ThermoFisher Scientific). For Western blots, 30–50 μg of protein was fractionated in 4%–20% TGX gels  under reducing conditions and transferred onto a nitrocellulose membrane (Bio‐Rad Laboratories). The following primary antibodies were added after blocking for an hour with 5% non‐fat milk: STAT3 and Tyr705 pSTAT3 , GAPDH (ThermoFisher Scientific), and A20/TNFAIP3 . Horseradish peroxidase‐conjugated secondary antibody (Bio‐Rad Laboratories) was followed by visualization with FluoroChem digital imaging system . Relative quantification of protein bands was performed using ImageJ software (NIH).33.1705 phosphorylation , while 2 genes had lower induction in the STAT3KO MEFs (STAT3‐promoted genes) and 10 genes were similarly induced in response to TNF (STAT3‐independent genes) . The overlapping group of genes induced by TNF or the TNFR1aab in each MEF genotype were included in the TNF target gene list. Of the 35 TNFR1 induced genes, 23 genes were more greatly induced in the STAT3) Figure . Since m) Figure ,24, 23.3KO MEFs were treated with TNF or with the TNFR1aab for 4 h, and culture media were collected and analysed for cytokines and chemokines using a multiplex assay. Using a cut‐off of at least a two‐fold change, levels of Ccl2/Mcp1, Cxcl1/KC, and Cxcl10/IP10 were greater in STAT3KO versus WT MEFs in response to treatment with TNF or the TNFR1aab ‐A20/Tnfaip3 signalling pathway in MEFs. Most cells express low levels of A20/Tnfaip3, which is induced by TNF within 30 min by NF‐κBRicardo Antonia: Conceptualization ; data curation ; formal analysis ; investigation ; writing – original draft ; writing – review and editing . Eveliina Karelehto: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Kan Toriguchi: Investigation ; methodology ; writing – original draft ; writing – review and editing . Mary Matli: Investigation ; methodology ; writing – review and editing . Robert Warren: Funding acquisition ; writing – review and editing (supporting). Lawrence Pfeffer: Formal analysis ; resources ; visualization ; writing – original draft ; writing – review and editing . David Donner: Conceptualization ; formal analysis ; funding acquisition ; project administration ; writing – original draft ; writing – review and editing .The authors declare no potential conflicts of interest.Supplementary Figure 1 Transcription factor (TF) scores for differentially expressed genes (DEGs) in WT and STAT3KO MEFs at 4 h of TNF treatment. DEGs were subjected to transcription factor enrichment analysis using the ChEA3 tool26 in conjunction with the literature ChIP sequencing library.Click here for additional data file.


MTHFR gene as response markers to an anti-TNF-α therapy. A total of 81 patients were enrolled, 60% of whom responded to the therapy. Analyses showed that both polymorphisms were associated with a response to therapy in an allele dose-dependent manner. The association for c.665C>T was significant for a rare genotype (p = 0.01). However, the observed opposite trend of association for c.1298A>C was not significant. An analysis revealed that c.1298A>C, unlike c.665C>T, was also significantly associated with the drug type (p = 0.032). Our preliminary results showed that the genetic polymorphisms in the MTHFR gene were associated with a response to anti-TNF-α therapy, with a potential significance for the anti-TNF-α drug type. This evidence suggests a role for one-carbon metabolism in anti-TNF-α drug efficacy and contributes to further personalized RA interventions.Rheumatoid arthritis (RA) is an inflammatory autoimmune disease with a prevalence of 1%. Currently, RA treatment aims to achieve low disease activity or remission. Failure to achieve this goal causes disease progression with a poor prognosis. When treatment with first-line drugs fails, treatment with tumor necrosis factor-α (TNF-α) inhibitors may be prescribed to which many patients do not respond adequately, making the identification of response markers urgent. This study investigated the association of two RA-related genetic polymorphisms, c.665C>T  and c.1298A>C, in the  Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that symmetrically affects the polyarthritis of small and large joints, and causes joint and periarticular structural damage . RA has According to the American College of Rheumatology (ACR) and the European Alliance of Associations for Rheumatology (EULAR), the current therapeutic goal for RA treatment is to achieve remission or a low disease activity ,9 by folNUBPL (rs2378945), NCTN5 (rs1813443), PLA2G4A (rs12142623 and rs4651370) [LINC02549 (rs7767069), LARRC55 (rs717117G) [MED15 (rs113878252), MAFB (rs6065221) [CD84 (rs6427528 and rs1503860) [TNF (rs1800629), EYA4 (rs17301249) [PDZD2 (rs1532269) [CCL21 (rs2812378) [MTHFR genes c.665C>T  and c.1298A>C (rs1801131) and the occurrence risk of RA [Currently, there are five TNF-α inhibitor drugs, which present similar efficacy and safety profiles. These drugs include adalimumab, golimumab, infliximab, certolizumab, and etanercept . Althoug717117G) , MED15  , TNF  or with the degree of response . The characteristics of the patients are summarized in After the follow-up period, 81 patients who received anti-TNF-α therapy as a monotherapy or in combination with other phase I drugs  were selected and included in the further analyses. Patients who responded successfully to the first-line therapy, or who received non-TNF-α-targeting biologic DMARDs, or who did not complete the follow-up period, or with recorded data not passing the quality control were excluded. The average age of these patients was 56.24 ± 12.96 years and 67.9% of them were female. Among the patients, 4 did not respond to the first anti-TNF-α drug choice and received a second anti-TNF-α drug, which increased the record size to 85. Overall, 60% of the patients responded to therapy with an anti-TNF-α drug, and there was no significant difference between the monotherapy or combination therapy groups either with the overall response (GR+MR vs. NR) to the treatment (p = 0.79), age (p = 0.21), exposure to tobacco smoking (p = 0.78), or the presence of RF in serum (p = 0.52) or ACPA (p = 0.70).Among the studied parameters, no associations were detected between the response to the anti-TNF-α therapy and parameters such as sex . The RA cases bearing a CT heterozygous genotype showed a moderate trend towards an association with the response to therapy . Regarding the MTHFR c.1298A>C polymorphism, a similar but reversed allele dose-dependent trend of an association with a response to the anti-TNF-α therapy was observed. Among the responders, the AC heterozygous patients showed a trend towards a reverse association with a response to the therapy with TNF-α inhibitors  whereas the CC homozygous cases showed a 12-fold reduced chance of responding to therapy compared with AA homozygotes   .p = 0.14; TT homozygous: OR = 10.89, 95% CI 1.73–68.54, p = 0.01) compared with the MR group . Similarly, the level of response showed a very comparable association with c.1298A>C among the heterozygous subjects . However, due to the small sample size, it was not possible to compare the level of response in the CC homozygous subjects . No AC genotype effect was observed in those treated with TNF-α FP  (As both  = 0.69) . Due to MTHFR gene in an allele dose-dependent manner. Both c.665C>T and c.1298A>C variants have been reported to be associated with RA occurrence [MTHFR variants and a response to anti-TNF-α drugs.In the present study, we have provided evidence that in patients with RA, the response to anti-TNF-α therapy is influenced by common polymorphisms of the currence  but, to Notably, the minor allele of the c.665C>T variant was associated with a therapeutic response whereas the c.1298A>C variant was inversely associated. The finding of an opposite effect of the two studied variants was not surprising because of their close physical proximity and the consequent high linkage disequilibrium between them as well as the fact that their minor alleles are very rarely detected on the same chromosome .The MTHFR enzyme catalyzes the synthesis of 5-methyltetrahydrofolate and contributes to the removal of homocysteine by its remethylation to methionine . The C>TThe A-to-C transition at nt 1298 results in a glutamate-to-alanine substitution in the regulatory-binding domain of the enzyme, causing the reduced binding of S-adenosyl-methionine (SAM) and leading to a decreased enzyme activity , but to Both variants are associated with a higher expression of inflammation markers ,32 and tTNF, as the first released cytokine during injuries or stress , is secrp = 0.032). It is known that the distribution, pharmacological half-life, and degradation of anti-TNF-α drugs have several differences [MTHFR gene. If different polymorphisms and genotypes of MTHFR change the enzyme activity differently, it is possible to expect dissimilar consequences such as different contributions to an inflammatory status, which could be inferred by how slightly different drugs can act differently.TNF-α is initially produced in a precursor transmembrane (tmTNF) form on the cell surface; after cleavage by metalloproteinases, it is then released in a mature and soluble (sTNF) form, which mediates its biological activities through TNF-α receptors type 1 and type 2 (TNF-R1 and TNF-R2) . The fivferences ,50,51. Iferences ,52; unliferences ,54. ConsMTHFR gene and their association with drug ADME  as well as the response to different anti-TNF-α therapies in RA patients, a replication study with a larger sample size should be carried out. In addition, considering the strong linkage disequilibrium of these two SNPs, a diplotype analysis is of utmost importance; however, due to the limited number of cases, it was not possible to perform such an analysis.This study should be considered to be preliminary, and the results should be carefully interpreted as it was based on a small cohort of patients. In order to have an established concept of the effects of different polymorphisms on the MTHFR gene influenced the response to the anti-TNF-α therapy in RA patients, although in opposite directions and with a few differences depending on the drug type. Our results suggest a role of one-carbon metabolism in the efficacy of anti-TNF-α drugs, and may pave the way for more personalized interventions in the treatment of RA. In perspective, the development of an accurate prediction model based on a wide range of pharmacogenetic, metabolic [In conclusion, considering the failure of a phase I therapy in RA patients who have to initiate an anti-TNF-α regime as a phase II therapy, having efficient response-predictive markers such as pharmacogenetic markers could help patients know ahead of time whether the drug is likely to benefit and be safe for them without facing the complications associated with anti-TNF-α drugs. Our study showed that the c.665C>T and c.1298A>C polymorphisms in the etabolic , clinicaetabolic , circulaetabolic ,58, and etabolic  markers—A total of 528 RA patients included in the regional RA registry of the Emilia-Romagna region were enrolled from the Rheumatology units of five Italian hospitals, including the Sant’Anna University Hospital, Ferrara; the Rizzoli Orthopaedic Institute, Bologna; the S. Maria Nuova Hospital, Reggio Emilia; the Sant’Orsola-Malpighi University Hospital, Bologna; and the University Hospital, Modena. RA was diagnosed according to the ACR and EULAR criteria of 2010 . A peripMTHFR gene (including c.665C>T (rs1801133) and c.1298A>C (rs1801131)) was performed using pre-designed TaqMan 5′ exonuclease assays ; subsequently, the fluorescence signals of the probes were detected by an ABI 7300 Real-Time PCR System (Applied Biosystems), according to the supplier’s protocol.Genomic DNA was extracted from peripheral blood samples using Nucleon BACC1  or QIAamp DNA Blood Mini  kits, according to the manufacturer’s instructions. The DNA was titrated by a Qubit 2.0 fluorometer  using a Qubit dsDNA BR assay kit . The genotyping of the two SNPs in the p-value < 0.05 was considered to be statistically significant. All analyses were performed using Microsoft Excel (2016).The data were assessed for allele and genotype frequencies and the Hardy–Weinberg equilibrium. The odds ratio (OR) with a 95% confidence interval (CI) was calculated for four different comparisons, including the overall response vs. no response (GR+MR vs. NR), good response vs. no response (GR vs. NR), moderate response vs. no response (MR vs. NR), and good response vs. moderate response (GR vs. MR), assuming a co-dominant genetic model. The analyses were further assessed under dominant and recessive models. A


In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines  and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14 TNF-α) is an important proinflammatory cytokine characterizing the early inflammatory response  at Cornell University (IACUC protocol 2007–0079). Sampling procedures in cows were approved under IACUC protocol number 2017–0107.PBMC) using the forward primer AAGGATCCCAGGTCCTCTTCTCAAGCCTCAAGTAAC, including a BamHI restriction site (underlined), and the reverse primer ATATATAAGCTTTCACAGGGCGATGATCCCAAAGTAGACC, including a HindIII restriction site (underlined), and cloned into the mammalian IL-4 or IgG1 fusion protein expression vectors. Subsequently, TNF-α was expressed as a fusion protein with equine IL-4 or IgG1 in Chinese hamster ovary (CHO) cell lines as previously described (N-hydroxysuccinimide)-activated Sepharose column (Millipore Sigma) as previously described (3) followed by labeling with A647-conjugated goat anti-mouse IgG polyclonal antibody Fab2 fragment (ThermoFisher Scientific). For specificity testing of each clone, ExpiCHO cells transfected with IL-4 and an irrelevant protein were used. Hybridoma clones that detected TNF-α were expanded and purified using Protein G affinity columns . Protein concentrations were determined by bicinchoninic acid assay . The murine isotypes of these hybridoma clones were determined using mouse monoclonal antibody isotyping reagents (Millipore Sigma).Monoclonal antibodies specific to bovine TNF-α were produced as previously described for equine proteins . Brieflyescribed . The nucescribed . Immunizescribed . HybridoPotential cross-reactivity of the mAbs with other bovine cytokines was tested by flow cytometry against 3 recombinant bovine fusion proteins . Chinese hamster ovary cells transfected with the respective bovine fusion protein constructs or TNF-α/IgG1 as positive control were fixed with 2% paraformaldehyde and intracellularly labeled with the purified hybridoma clones as well as with isotype controls for mouse IgG1. Subsequently, cells were labeled with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse polyclonal antibody.Detection of native bovine TNF-α was tested in bovine whole blood and bovine PBMC. Blood samples were obtained from adult, lactating cows by venipuncture of the coccygeal vessels, and PBMC were separated by density gradient centrifugation as previously described .Escherichia coli LPS  or a mix of phorbol myristate acetate  and ionomycin  for 4 h at 37°C and 5% CO2 enriched atmosphere or left as an unstimulated control. All cultures contained brefeldin A  to prevent exocytosis of the native protein. After a 4-h incubation, cells were fixed with 2% paraformaldehyde. Cells were labeled in saponin buffer with purified, biotinylated mAbs and in parallel with mAbs against bovine CD14 conjugated with FITC  and a mixture of anti-bovine CD4 and CD8 mAbs both conjugated with A647 . Subsequently, cells were labeled with streptavidin R-phycoerythrin (ThermoFisher Scientific). Isotype-matched control antibodies were included in all experiments . All samples were analyzed with a FACS Canto II flow cytometer (BD Biosciences). For CHO cells, 10,000 events per sample, and for PBMC, 50,000 events per sample were acquired. All cells were gated as singlets according to their FSC height and area. The CHO cells were analyzed as a homogeneous group, whereas PBMC were gated by their expression of CD4/CD8 (T lymphocytes) and CD14 (monocyte subset).Bovine PBMC were stimulated with For the development of a fluorescent bead-based assay, purified mAbs were tested to identify pairs of coupling and detection antibodies using a TNF-α/IgG1 fusion protein standard . The TNFg and 4°C. In parallel, PBMC from 4 adult lactating cows were cultured at 37°C in a 5% CO2 enriched atmosphere, applying the same treatments and time points as for the whole-blood stimulation. Cell culture supernatants were collected by centrifugation for 10 min at 250 × g and 4°C. Samples were stored at −80°C until analyzed.Plasma samples were obtained from in vitro stimulation of whole-blood samples from 10 adult lactating cows. Whole blood was incubated with a mix of PMA/ionomycin or LPS (see above) or left unstimulated (control) for 2, 4, 8, 18, and 24 h at 38°C. Plasma was harvested by centrifugation for 10 min at 1,500 × In the initial screening of the hybridoma supernatants, mAb clones 65-2 and 197-1 detected the IL-4/TNF-α fusion protein by ELISA and flow cytometry (data not shown). The TNF-α mAb clones were expanded, purified, and further characterized by intracellular staining of different CHO cell transfectants followed by flow cytometry to both be of the IgG1 isotype. They did specifically detect transfectants expressing TNF-α/IgG1 while not reacting with transfectants of 3 other recombinant bovine proteins  or the equine recombinant IL-4/IgG1 fusion protein, indicating specific binding to recombinant bovine TNF-α and not the equine IgG1 portion of the fusion protein or the other bovine cytokines tested .Figure 1MFI) of 17,785 ± 898 (n = 11), as well as native protein in culture supernatants of PBMC stimulated with a mix of PMA and ionomycin  and low blank values . The range of TNF-α detection for the assay was 0.2 to 620 ng/mL  with a high mean fluorescence intensity (20 ng/mL . This is20 ng/mL , who use20 ng/mL . All ass20 ng/mL , demonstThe mAb pair was then used to detect native TNF-α in PBMC cell culture supernatants from 4 different cows, and plasma samples from 10 blood samples for whole-blood stimulation at 5 time points over 24 h , 2D. An + monocytes (+/CD8+ lymphocytes was expected because this cell population typically does not express LPS receptors (+ monocytes and in CD4+/CD8+ lymphocytes (Both mAbs were used in intracellular labeling to test for presence of cytoplasmatic TNF-α in bovine PBMC populations , 3B. Aftonocytes . Bovine onocytes . In thiseceptors . Stimulaphocytes , which iphocytes . Our objphocytes . It shouIn conclusion, the new bovine TNF-α mAb clones 65-2 and 197-1 specifically and accurately quantified soluble TNF-α in cell culture supernatants and plasma samples in a bead-based assay across a wide concentration range. In addition, both mAbs could differentiate cytoplasmatic TNF-α expression in bovine PBMC populations by flow cytometry. The newly developed mAb pair can be used as a tool to describe inflammatory responses in dairy cows in a variety of ex vivo– and in vitro–derived samples. Last, ideal stimulation times and stimuli concentrations for optimal TNF-α measurements can vary for whole blood and PBMC.


Notably, both LBBAP and BIVP significantly improved LVEF, LVEDD, NYHA class, and BNP compared with baseline. However, LBBAP significantly lowered BNP compared with BIVP  from baseline to 24-month follow-up. Moreover, patients who received LBBAP exhibited lower number of hospitalizations than those in the BIVP group (p = 0.019). In addition, we found that patients with moderately prolonged left ventricular activation time (LVAT) and QRS notching in limb leads in baseline ECG respond better to LBBAP for CLBBB correction. LBBAP might be a relative safe and effective resynchronization therapy and as a supplement to BIVP for patients with HF and CLBBB.Left bundle branch area pacing (LBBAP) has developed in an effort to improve cardiac resynchronization therapy (CRT). We aimed to compare the long-term clinical outcomes between LBBAP and biventricular pacing (BIVP) in patients with heart failure (HF) and complete left bundle branch block (CLBBB). Consecutive patients with HF and CLBBB requiring CRT received either LBBAP or BIVP were recruited at the Second Affiliated Hospital of Nanchang University from February 2018 to May 2019. We assessed their implant parameters, electrocardiogram (ECG), clinical outcomes at implant and during follow-up at 1, 3, 6, 12, and 24 months. Forty-one patients recruited including 21 for LBBAP and 20 for BIVP. Mean follow-up duration was 23.71 ± 4.44 months. LBBAP produced lower pacing thresholds, shorter procedure time and fluoroscopy duration compared to BIVP. The QRS duration was significantly narrower after LBBAP than BIVP (129.29 ± 31.46 vs. 156.85 ± 26.37 ms,  Heart failure (HF) is a major public health issue with high morbidity and mortality, resulting in considerable financial and service burdens to the health system . Left buHuang et al.  first prThe hospitalized patients, diagnosed with HF and CLBBB, were enrolled at the Second Affiliated Hospital of Nanchang University from February 2018 to May 2019. The inclusion criteria were QRS duration (QRSd) > 150 ms, ECG suggesting traditional CLBBB, NYHA function class II–IV, and optimized drug treatment for 3 months or more. The exclusion criteria were life expectancy was less than 1 year, or those with non-specific intraventricular conduction delay or right bundle branch block (RBBB). LBBAP was an alternative choice to failed BIVP or first choice in place of a CS lead. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Each patient signed an informed consent prior to enrolment  according to the methods described by Huang . HBP wasThe criteria of LBB capture followed by described previously : (1) pacFor BIVP implantation , the rigWe recorded fluoroscopy duration for LBBAP lead implantation and procedure time at implant. Lead parameters, including unipolar tip pacing thresholds and impedances of LBBAP or BIVP, were measured at implant and during regular follow-up of 1-, 3-, 6-, 12-, and 24-month. We measured QRS duration at implant and follow-up. We obtained echocardiography (LVEF and LVEDD) at preimplantation and regular follow-up of 1-, 6-, 12-, and 24-month. We also documented BNP concentration and NYHA class, and tracked complications and clinical outcomes, such as death and rehospitalization, during follow-up.t- or rank-sum tests. On the other hand, categorical data were presented as numbers and percentages, and analyzed using the chi-squared or Fisher's exact tests. Data followed by p < 0.05 were considered statistically significant.All statistical analyses were performed using SPSS software version 24.0. Continuous variables were presented as means ± standard deviations (SDs), and compared using two-tailed Student’s p = 0.041), but a lower incidence of chronic renal insufficiency . We observed no significant differences between the two groups with regards to the other clinical characteristics  and fluoroscopy duration  than BIVP. Additionally, capture thresholds of 3830 lead of LBBAP were lower than those of LV lead of BIVP at implantation, with these differences found to persist at 1-, 3-, 6-, 12-, and 24-monthduring follow-up (Table Comparisons in pacing parameters between patients in the LBBAP and BIVP groups are summarized in Table p < 0.001), while those of the 20 patients in the BIVP group significantly decreased from baseline 177.50 ± 16.99 to 156.85 ± 26.37 ms (p = 0.006) . Consistent with the above results, LBBAP resulted in a greater reduction in QRS duration compared with BIVP   between baseline and follow-up after LBBAP or BIVP revealed significantly narrowing of the 12-lead ECG QRSd in both groups Fig. A. In the06) Fig. B. Furthep < 0.001 and 31.40% ± 9.30 to 44.28 ± 14.26%, p = 0.003, respectively)  , NYHA Class  and BNP level  were significantly lower in the LBBAP than BIVP group, and these were consistent with improvement in LVEF    Fig. A. In add06) Fig. B, C. Morp = 0.007) , reduction in LVEDD , and NYHA class  compared with BIVP at 24-month follow-up from baseline, although these changes were not statistically significant  Fig. C. Moreovp = 0.001), indicating that LBBAP can achieve narrower QRSd compared with BIVP. Additionally, the rate of super response (LVEF ≥ 50%) in patients with full CLBBB correction (QRSd ≤ 130 ms) was significantly higher than that of patients with failed CLBBB correction (QRSd > 130 ms) in LBBAP group . Subgroup analysis suggested that full CLBBB correction was associated with improved LVEF.Among 21 patients, 13 achieved full correction of the CLBBB with a paced QRSd of ≤ 130 ms in the LBBAP group, while only 2 of 20 patients achieved a paced QRSd of ≤ 130 ms in the BIVP group. Obviously, the percentage of patients with a paced QRSd ≤ 130 ms was significantly higher in the LBBAP than BIVP group , while no significant difference between initial QRSd of CLBBB correction and failed CLBBB correction at baseline . Additionally, the sub-analysis at different follow-up periods, revealed that 13 patients with CLBBB correction exhibited higher LVEF, lower LEDD, lower BNP level and lower NYHA class compared to the 8 patients with failed CLBBB correction at 1-, 6-, 12-, and 24-monthfollow-up . In addition, we observed QRS notching in limb leads in 11 of 13 patients (84.62%) with CLBBB correction, while the notched R-wave occurred in only 2 of 8 patients (25.00%) with failed CLBBB correction (p = 0.018)  in patients with CLBBB correction was slightly shorter than that of patients with failed CLBBB correction , which suggested that LBBAP significantly reduced the incidence of rehospitalization compared with BIVP.We tracked complications and clinical outcomes, such as incidence of death and rehospitalization, in both groups during 24-month follow-up. Among 21 patients, 16 completed the follow-up of 24-month in the LBBAP group, while 15 out of 20 patients completed the whole follow-up in the BIVP group. Although no surgery-related complications occurred in both groups, one patient in each group died of HF (with LVEF lower than 35%). Four patients in the LBBAP group were re-admitted to the hospital because of worsening heart failure. On the other hand, one patient presented with symptoms of syncope because of being discharged after the pacemaker perceiving atrial fibrillation and was hospitalized twice. In the BIVP group, four patients were re-admitted to the hospital due to worsening heart failure, one was re-admitted three times, while another one patient (with LVEF less than 30%) was re-admitted six times. In total, the number of hospitalizations was significantly lower in the LBBAP than BIVP group  LBBAP is a feasible and safe approach for successful correction of CLBBB in patients with HF and CLBBB; (2) The long-term follow-up revealed that LBBAP significantly improved LVEF and NYHA functional class and further lowered BNP level and LVEDD; (3) LBBAP significantly shortened QRS duration and exerted better cardiac electrical resynchronization to relieve symptoms of HF, compared with BIVP; and (4) In the LBBAP group, patients with moderately prolonged LVAT and QRS with a notch in the limb leads in preoperative ECG, hence may benefit more from CLBBB full correction.Multiple studies have shown that LBBAP exerts fewer perioperative complications with no fatal adverse effects , 25–28. Although previous studies demonstrated that CRT therapy can significantly improve LVEF , only a Previous studies have reported successful correction of CLBBB by LBBAP, with high success rates achieved , 38. ForAs previously discussed, LBBAP might be advantageous over BIVP in treating patients with HF and CLBBB. We attempted to explore which patients with HF are best suited for LBBAP. Interestingly, we found that the preoperative 12-ECG lead V6 ventricular activation time (VAT) in patients with CLBBB correction was significantly shorter than in those that with failed CLBBB correction. Our results are consistent with previous studies which have reported that patients with CLBBB exhibited LVAT greater than 60 ms in leads V5 and V6 , 41, indThere have been some articles published about the comparisons between LBBAP and BIVP , 43, whiThere were several limitations in the present study. First, the small number of patients and non-randomized design may lead to inadequate power. LBBAP was in its early phase of clinical application in our hospital, and was only used in a small size of patients. Second, the definition of strict criteria for LBB capture was not used in the study. The characteristics of the ECG and the EGM in the LBBAP procedure, such as stim-LVAT, paced QRS morphology, and discrete component in the EGM, as the indirect criteria for LBB capture, were mainly used to distinguish LBBP from LVSP in this study. Indeed, it was difficult to distinguish them accurately in some cases. Currently, Wu et al.  proposedIn summary, LBBAP can significantly synchronize LV activation by correcting CLBBB and promote LV reverse remodeling. LBBAP is more effective in shortening QRS duration and enhancing echocardiographic and clinical responses than BIVP, therefore, may be a better candidate for resynchronization therapy for patients with HF and CLBBB.


Before HS, lentivirus harboring shRNA against MKK4 was injected into rats via the tail vein to knock down MKK4 expression. HS was induced by bloodletting via intubation of the femoral artery followed by resuscitation. The results showed that MKK4 knockdown reduced HS-induced apoptosis in the liver by decreasing Bax expression and the cleavage of caspase 3 and promoting Bcl-2 expression. Moreover, the generation of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) in the liver was promoted, while superoxide dismutase (SOD) activity was inhibited by HS. However, the effect of HS on oxidative stress was abrogated by MKK4 knockdown. Furthermore, MKK4 knockdown restored MMP and complex I and complex III activities and promoted ATP production, suggesting that HS-induced mitochondrial dysfunction in the liver was ameliorated by MKK4 knockdown. The inhibitory effect of MKK4 knockdown on the phosphorylation and activation of the JNK/c-Jun pathway was confirmed. Overall, MKK4 knockdown may suppress oxidative stress and subsequent apoptosis and improve mitochondrial function in the liver upon HS by inhibiting the JNK pathway. The MKK4/JNK axis was shown to be a therapeutic target for HS-induced liver injury in this study.Hemorrhagic shock (HS) triggers tissue hypoxia and organ failure during severe blood loss, and the liver is sensitive to HS. Mitogen-activated protein kinase kinase 4 (MKK4) activates the c-Jun NH2-terminal kinase (JNK) pathway, and its expression is upregulated in the serum of HS patients and mouse livers at 1 h post-HS. However, the function of MKK4 in HS-induced liver injury is unclear. The role of MKK4 was investigated  Hemorrhage is the leading cause of death after trauma , 2. Hemo2O2-induced vascular smooth muscle cell apoptosis, and MKK4/JNK signaling pathway inhibition reduces tributyltin chloride-induced apoptosis in hypothalamic neurons [2O2-induced oxidation by suppressing MKK4/JNK cascades [Mitogen-activated protein kinase kinase 4 (MKK4), belonging to the MAP kinase kinase family, is a kinase that directly phosphorylates c-Jun NH2-terminal kinase (JNK) . MKK4, w neurons , 20. Rea neurons . Baicalecascades . Moreovecascades . Additiocascades . In the cascades . Howeverin vivo and demonstrated that MKK4 knockdown plays a protective role in HS-induced liver injury by inhibiting JNK/c-Jun activation.MKK4 is essential for liver regeneration and damage repair. Based on these findings, we hypothesize that MKK4 plays a vital role in HS-induced liver injury by regulating apoptosis, oxidative stress, and mitochondrial function in hepatocytes, and this process may involve the JNK/pathway. This study used a rat model of HS to examine the function of MKK4 in liver injury A clinical study was conducted in 7 patients with severe HS after polytrauma from Wuxi 9th Affiliated Hospital of Soochow University. Thirty healthy participants served as the controls. The blood samples were collected from all subjects after obtaining written informed consent. The study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Wuxi 9th Affiliated Hospital of Soochow University.8 transduction units of lentivirus encoding short hairpin RNA (shRNA) against the MKK4 (LV-shMKK4) or negative control shRNA (LV-shNC). HS was induced by bloodletting via a femoral arterial catheter, and the mean arterial pressure (MAP) was maintained at 40-50 mm Hg for 1 h. Rats were resuscitated with lactated Ringer's solution for 1 h. Control rats underwent the same surgical procedure without bloodletting and resuscitation. One hour after resuscitation, all rats were sacrificed. Liver tissues and blood were collected for analysis  were allowed free access to food and water throughout acclimatization for 1 week. Three days before modeling, rats were injected through the tail vein with 1.0 × 10analysis . All aniΔΔCt- method and normalized to GAPDH. The primers were synthesized by Genscript Biotechnology Co., Ltd. , and the sequences were as follows: MKK4, 5′-CAACACTGGGATTTCACT-3′ (forward), 5′-ACTACTCCGCATCACTACA-3′ (reverse); hypoxia-inducible factor-1α (HIF-1α), 5′-CTATGTCGCTTTCTTGG-3′ (forward), 5′-TTTCTGCTGCCTTGTAT-3′ (reverse); and GAPDH: 5′-CGGCAAGTTCAACGGCACAG-3′ (forward), 5′-CGCCAGTAGACTCCACGACAT-3′ (reverse).Total RNAs in rat liver were isolated using Total RNA Isolation Kit  and reverse transcribed into cDNA with BeyoRT II M-MLV reverse transcriptase . Then, cDNAs were amplified with 2×Taq PCR MasterMix  and SYBR Green  under Exicycler™ 96 Real-time PCR System . The mRNA expression level was calculated using the 2α (1 : 500 dilution), p-JNK (Thr183/Tyr185) (1 : 1000 dilution), JNK (1 : 500 dilution), p-c-Jun (1 : 1000 dilution), c-Jun (1 : 500 dilution), Bax (1 : 500 dilution), cleaved caspase 3 (1 : 1000 dilution), Bcl-2 (1 : 500 dilution), and cytochrome c (1 : 500 dilution) overnight at 4°C. Antibody to GAPDH  or COX IV  was used for reference. Then, the membrane was incubated with the secondary antibody-horseradish peroxidase conjugate  and developed with an enhanced chemiluminescent (ECL) kit . Band intensities were quantified and normalized to GAPDH or COX IV.The liver tissues from the rats were homogenized in RIPA buffer , and total proteins were isolated. According to the manufacturer's instructions, liver tissues were cut into pieces, and the proteins in mitochondria were extracted using a mitochondrial protein extraction kit . Protein concentrations were quantified with a BCA kit . Protein lysates of liver tissues were fractionated on SDS-PAGE and transferred onto polyvinylidene fluoride  membrane. After being blocked in 5% milk  and dissolved in TBS-Tween for 1 h, the membrane was incubated with the primary antibodies purchased from Affinity Biosciences , including antibody to MKK4 (1 : 500 dilution), HIF-1μm. After rehydration, the liver sections were stained with hematoxylin  and eosin . Liver injury was examined by light microscopy . Apoptosis in the liver was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Paraffin-embedded liver sections were permeabilized in 0.1% Triton X–100 , labeled with a mixture of enzyme solution and label solution, and counterstained with 4′,6-diamidino-2-phenylindole  according to the instructions in the In Situ Cell Death Detection Kit . TUNEL-positive cells were observed under a fluorescence microscope .Liver tissues fixed in 4% paraformaldehyde were embedded in paraffin. The tissue block was cut at a thickness of 5 Enzyme-linked immunosorbent assay (ELISA) was used for the detection of IL-6 serum level. Serum was separated from the blood, and IL-6 serum level was examined with Rat IL-6 ELISA Kit  following the manufacturer's instructions. The levels of ALT, an indicator of liver damage, and LDH in serum were measured with an ALT kit  and a lactate dehydrogenase assay kit , respectively.ROS in the liver was assessed using the DCFH-DA method and a reactive oxygen species assay kit . Liver tissues were cut up for preparing liver cell suspension. Cell suspensions were centrifuged and resuspended in phosphate buffer saline (PBS). Cells were incubated with DCFH-DA, a fluorescent probe for ROS, for 30 min at 37°C. The fluorescence intensity of ROS was detected by a fluorescence microplate reader . The excitation wavelength was 490 nm, and the emission wavelength was 530 nm.MMP in the liver was assessed with mitochondrial membrane potential assay kit with JC-1 . Before detection, mitochondria in liver cells were isolated with Tissue Mitochondria Isolation Kit . Then, mitochondria isolated from cells were resuspended in PBS, incubated with JC-1 staining solution, and detected by a fluorescence microplate reader. MMP was expressed as the ratio of red fluorescence intensity to green fluorescence intensity.MDA level and SOD activity in the liver cells were examined using an MDA assay kit  and SOD assay kit , respectively. Liver tissues were homogenized in saline and centrifuged. Proteins in supernatant were quantified with a BCA kit . Optical density values of MDA and SOD were detected by ultraviolet-visible spectrophotometer  at 532 nm and 550 nm, respectively. MDA levels and SOD activity were calculated according to the standard curves.The liver tissues were homogenized in saline and centrifuged. Protein concentration in the supernatant was quantified with a BCA  kit, and ATP level was examined using an ATP assay kit . Optical density was measured under an ultraviolet-visible spectrophotometer at 636 nm. ATP level was calculated according to the standard curves. The activities of mitochondrial respiratory chain complex I and complex III were detected using the corresponding kit . Complex I and complex III were extracted using the extract buffer in the kit and quantified with BCA kit, respectively, before their activities were measured according to the instructions.P < 0.05 indicated a significant difference between different groups.The data were expressed as mean ± standard deviations (SD). GraphPad Prism 7.0 software was used for mean comparison and difference analysis. The values of different groups were compared with one-way analysis of variance (ANOVA) followed by Tukey's test. The serum samples were collected from 7 HS patients and 30 healthy participants. MKK4 was shown to be highly expressed in the serum of HS patients . DysreguTo explore the role of MKK4 in HS, a rat model of HS was established, and lentivirus-mediated MKK4 knockdown was carried out in vivo . The expH&E staining was performed to assess the severity of HS-induced liver injury. Cytoplasmic vacuolization and inflammatory cell infiltration occurred in the liver of HS rats, which was inhibited when MKK4 was knocked down . AdditioTUNEL staining and Western blot were performed to investigate MKK4 roles in HS-induced apoptosis in rat liver. The increased number of TUNEL-positive cells in the liver of HS rats compared to control rats showed that HS strongly triggered apoptosis in the liver of rats, while fewer apoptotic cells were detected in the liver of HS rats where MKK4 was knocked down . Bax, clHS- and resuscitation-induced oxidative stress in the liver was evaluated. Notably, ROS and MDA generation in rat livers was strongly enhanced, while SOD activity was suppressed by HS. When MKK4 was knocked down, the levels of ROS and MDA increased by HS were markedly reduced with elevated SOD activity Figures , suggestMMP in liver cells was examined with the JC-1 method. The decreased ratio of JC-1 red fluorescence to green fluorescence in HS rats indicated that HS and resuscitation significantly reduced MMP in the rat liver, which might trigger the reduction of ATP production. MKK4 knockdown partially restored MMP in the liver of HS rats . Consistα, which was downregulated by knockdown of MKK4 at both mRNA and protein levels (Figures α was positively correlated with that of MKK4. In this part, MKK4 knockdown was demonstrated to suppress the activation of the JNK/c-Jun and HIF-1α signaling pathway.To explore the pathway involved in the regulation of MKK4 to HS-induced liver injury, we analyzed the protein levels of JNK and c-Jun. Western blot showed that MKK4 knockdown decreased the phosphorylated JNK levels, which was dramatically increased by HS, without altering the total JNK level, indicating that MKK4 knockdown restricted the phosphorylation and activation of JNK Figures . The sam Figures . It seemIn this study, we revealed that MKK4 was upregulated in the liver when rats were subjected to HS and resuscitation, and MKK4 knockdown markedly abrogated the massive liver injury induced by HS. Subsequently, we analyzed apoptosis, oxidative stress, and mitochondrial damage in rat livers and found that these responses were significantly inhibited by MKK4 knockdown. Finally, we demonstrated that the JNK/c-Jun pathway in the liver was activated by HS and then inhibited by MKK4 suppression.HS affects the liver in a bimodal way through ischemia and reperfusion, which stimulates various cell signaling events, exacerbates oxidative stress and inflammatory responses, and ultimately results in hepatocyte death and liver dysfunction , 8. In tHowever, the mechanism by which MKK4 regulates the development of liver damage in response to HS is largely unclear. Cellular hypoxia during hemorrhage and the sudden increase in oxygen upon resuscitation is considered to be the cause of oxidative stress, thereby inducing mitochondrial damage and subsequent apoptosis . The rolα is critically involved in the pathogenesis of liver diseases [α functions as a key contributor to regulating homeostatic responses to hypoxia and oxidative stresses by activating transcriptional expression of genes that involves in inflammation and cell survival [α is related to ROS production and oxidative stress in hypoxia-induced rat liver [α expression or activation protected against experimental liver injury in vivo [α inhibitor rescued chronic liver failure through inhibiting cellular ROS production and mitochondrial dysfunction [α was upregulated at both mRNA and protein levels in liver tissues of rats after HS and resuscitation. Notably, knockdown of MKK4 resulted in a significant downregulation of HIF-1α. The expression of HIF-1α seemed to be positively correlated with that of MKK4 in HS rats. Suppression of hypoxia-induced activation of JNK signaling including MKK4 inhibited HIF-1α expression [α via activating transcription factor NF-κB [α downregulation in rat liver induced by HS. It is possible that MKK4 might function through regulating HIF-1α expression and its downstream transcriptional events or the relevant signaling pathways in HS. These deserved further investigation for comprehensive understanding of MKK4's role and mechanism in the future.Hypoxia is a major causative sensor and factor for deleterious oxidative stress in HS, and activation of HIF-1diseases . HIF-1α survival , 31. It at liver . Hypoxiaat liver . Inhibit in vivo , 35. HIFfunction . The exppression . MKK4/7-or NF-κB . In consin vitro.Overall, HS and resuscitation induce MKK4 expression and cause oxidative stress in the liver. On the one hand, massive amounts of ROS are produced during this process, enhancing the magnitude and duration of JNK activation by suppressing the activity of JNK phosphatases . Prolongin vivo. Except for the liver, the expression of MKK4 was examined in other organs including kidney and heart of HS rats after injection of LV-shMKK4 via tail vein. MKK4 expression was significantly increased in all these organs of HS rats and was efficiently downregulated after systemic delivery of LV-shMKK4. In the current research, the way of RNAi delivered systemically is widely used for the loss-of-function studies for a gene in experimental animal models, including HS or liver injury [A potential limitation of the current study is that the loss-of-function study for MKK4 was carried out with the use of lentivirus-mediated RNAi delivered systemically r injury –47. We hIn summary, our study identified MKK4 as a critical regulator of HS-induced liver injury. MKK4 knockdown prevented HS-mediated damage to the liver by inhibiting oxidative stress, mitochondrial dysfunction, and apoptosis. We demonstrated that MKK4 may play a protective role in liver injury in response to HS by inhibiting the JNK pathway. The MKK4-JNK pathway may be a therapeutic target for HS-induced liver injury.