Neuroinflammation in the rostral ventrolateral medulla (RVLM) has been associated with the pathogenesis of stress-induced hypertension (SIH). Neuronal mitochondrial dysfunction is involved in many pathological and physiological processes. However, the impact of neuroinflammation on neuronal mitochondrial homeostasis and the involved signaling pathway in the RVLM during SIH are largely unknown.The morphology and phenotype of microglia and the neuronal mitochondrial injury in vivo were analyzed by immunofluorescence, Western blot, RT-qPCR, transmission electron microscopy, and kit detection. The underlying mechanisms of microglia-derived tumor necrosis factor‐α (TNF-α) on neuronal mitochondrial function were investigated through in vitro and in vivo experiments such as immunofluorescence and Western blot. The effect of TNF-α on blood pressure (BP) regulation was determined in vivo via intra-RVLM microinjection of TNF-α receptor antagonist R7050.The results demonstrated that BP, heart rate (HR), renal sympathetic nerve activity (RSNA), plasma norepinephrine (NE), and electroencephalogram (EEG) power increased in SIH rats. Furthermore, the branching complexity of microglia in the RVLM of SIH rats decreased and polarized into M1 phenotype, accompanied by upregulation of TNF‐α. Increased neuronal mitochondria injury was observed in the RVLM of SIH rats. Mechanistically, Sirtuin 3 (Sirt3) and p-AMPK expression were markedly downregulated in both SIH rats and TNF-α–treated N2a cells. AMPK activator A769662 upregulated AMPK–Sirt3 signaling pathway and consequently reversed TNF-α–induced mitochondrial dysfunction. Microinjection of TNF-α receptor antagonist R7050 into the RVLM of SIH rats significantly inhibited the biological activities of TNF-α, increased p‐AMPK and Sirt3 levels, and alleviated neuronal mitochondrial injury, thereby reducing c-FOS expression, RSNA, plasma NE, and BP.This study revealed that microglia-derived TNF-α in the RVLM impairs neuronal mitochondrial function in SIH possibly through inhibiting the AMPK–Sirt3 pathway. Therefore, microglia-derived TNF-α in the RVLM may be a possible therapeutic target for the intervention of SIH.The online version contains supplementary material available at 10.1186/s12974-023-02818-6. Hypertension, which is the largest single contributor to the global burden of disease, has attracted great attention worldwide . A recen. summarized that mitochondrial homeostasis impairment exacerbated neuronal proteotoxicity in the diabetic aging brain [. revealed the role of mitochondrial dysfunction as a marker of neuronal death in ischemic stroke [The rostral ventrolateral medulla (RVLM) is the key central region that refers to the integration of vasomotor input and contains presympathetic neurons responsible for the regulation of sympathetic output –9. The ang brain . Jia et c stroke . Mitochoc stroke . Severalc stroke , 23. Ourc stroke . However. described that Sirt3 deregulation contributes to mitochondrial dysfunction and neuronal damage, which is associated with the pathogenesis of Alzheimer's disease [. proposed that neuron-specific knockout of Sirt3 reversed the protective effects of adiponectin receptor agonist on the mitochondria after traumatic brain injury [Sirtuin-3 (Sirt3), the deacetylase mainly located in mitochondria, participates in the regulation of mitochondrial functions, energy metabolism, and antioxidative defense systems , 25. Cum disease . Zhang en injury . Sirt3 on injury . In the The current work aimed to investigate the mechanisms involved in the effect of microglia-mediated neuroinflammation on neuronal mitochondrial function in SIH. The results demonstrated that microglia-derived tumor necrosis factor‐α (TNF-α) disrupted neuronal mitochondrial homeostasis in the RVLM though inhibiting the AMPK–Sirt3 pathway, and TNF-α suppression rescued neuronal mitochondrial injury and alleviated neuronal excitation, sympathetic outflow, and blood pressure (BP). Thus, the present study has implied that microglia-derived TNF-α could be exploited as a potential target for the prevention or treatment of SIH.Adult male Sprague–Dawley rats (n = 220) weighing 250–300 g and aged eight weeks old were maintained in separate cages at a 12 h light/dark cycle condition at a room temperature (23 °C ± 1 °C) with the humidity around 50–60% in the specified pathogen-free facility of Shanghai University. The experimental animals were allowed unlimited access to food and tap water. All efforts were made to minimize the number of animals used and their suffering. Figure 2/95% O2 at 37 °C. Mouse tumor necrosis factor‐α (TNF-α) was obtained from Novoprotein . The AMPK activator A769662 was purchased from MedChemExpress . N2a cells were treated separately for 24 h with TNF-α (30 ng/ml) or TNF-α (30 ng/ml) plus A769662 (30 and 60 μM).Mouse neuroblastoma N2a cell line was obtained from the Institute of Basic Medical Sciences of the China Science Academy. The cells were maintained in high glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in a humidified incubator with an atmosphere of 5% COThe BP and HR of conscious rats were continuously measured by the radiotelemetry system , as reported previously , 31. AftThe rats were anesthetized with inhaled isoflurane as above and the left kidney, renal artery, and sympathetic nerves were exposed through a retroperitoneal incision . The lef2 as an anticoagulant under anesthesia with isoflurane as above. The samples were centrifuged at 1000g for 15 min at 4 °C to isolate plasma. The plasma was collected for detection. The plasma norepinephrine (NE) concentration was measured by using NE ELISA kit  according to the manufacturer’s instructions. In brief, the standards and samples were added to a microplate pre-coated with the universal NE antigen, followed by the addition of biotin-conjugated anti-universal NE antibodies. After incubation, excess conjugates and unbound standards or samples were washed from the microplate, and the HRP-conjugated streptavidin was added into the wells. Next, the TMB substrate solution was added to each well after washing the microplate. The enzyme–substrate reaction was terminated by the addition of a sulphuric acid solution. Finally, the absorbance was assessed at 450 nm using a SpectraMax iD5 microplate reader . The results were presented as picogram per milliliter.Rat blood samples were obtained by cardiac puncture using EDTA-NaFor electrode implantation, rats were placed on the stereotaxic apparatus  under anesthesia with isoflurane as the above mentioned. Dental cement was used to embed electrode bundles  in the skull. These electrodes were implanted into the rostral ventrolateral medulla (RVLM)  according to the atlas of Paxinos and Watson . The eleImmunofluorescence staining was performed as described previously . The ratConfocal Z-stack images were taken using a confocal laser scanning microscope  at × 40 or × 63 magnification consisting of at least 12 images. Images of microglia were subjected to maximum intensity projection, and only cells whose branches were completely within the 3D Z-stack volume were quantified. We performed skeletonization analysis and Sholl analysis to quantify the morphological changes of microglia using Image J software. For mCherry-ONLY volume comparisons, the confocal images were transformed into 3D reconstruction files using Imaris 9.7 software. Numbers of EGFP puncta and mCherry puncta were determined using the spots module in Imaris 9.7. The mCherry-ONLY signal was quantified by zeroing the number in EGFP puncta and counting the remaining mCherry puncta. Other images were processed and analyzed using the Image J software.g for 5 min at 4 °C. The sediment was removed and the supernatant was transferred to another tube and further centrifuged at 12,000g for 10 min at 4 °C. Finally, the collected supernatant was used for Western blot analysis.The cytosol fraction was isolated using the mitochondria isolation kit  according to the manufacturer’s protocol. Briefly, the RVLM tissue was washed and homogenized using pre-cooled lysis buffer, and then was centrifuged at 600Total or cytosol protein extracted from RVLM tissue and total protein extracted from N2a cells were used to analyze protein expression by Western blot. Proteins were separated by SDS-PAGE in 4–12% gradient gel and transferred into the PVDF membranes . The membranes were blocked with QuickBlock™ Western  for 1 h prior to incubation at 4 °C overnight with primary antibodies including rabbit monoclonal antibody against iNOS , rabbit monoclonal antibody against CD86 , rabbit monoclonal antibody against Arg-1 , mouse monoclonal antibody against TNF-α , rabbit polyclonal antibody against p-AMPK , rabbit monoclonal antibody against AMPK , rabbit monoclonal antibody against Sirt3 , rabbit polyclonal antibody against ATP5A , mouse monoclonal antibody against UQCRC2 , rabbit monoclonal antibody against SDHB , mouse monoclonal antibody against TNFR1 , mouse monoclonal antibody against TNFR2 , and mouse monoclonal HRP-Conjugated β-actin antibody . After being washed with PBS and Tween 20 (PBST) buffer, the membranes were then incubated at goat anti-rabbit IgG  or goat anti-mouse IgG  for 1 h at room temperature. The amount of protein was assessed using super ECL detection reagents , and the immunostaining band was detected by an automatic chemiluminescence image analysis system . The data were normalized by developing the β-actin as loading control. Following the initial detection, as needed, we washed the targeted band protein by restore™ western blot stripping  for 30–60 min to remove the previous antibody. Subsequently, the PVDF membranes were reblocked for 1 h by QuickBlock™ Western after being washed three times with PBS. Finally, those membranes were incubated at 4 °C overnight with other primary antibodies. In this study, the membranes are reused at most once.® reagent  and converted into first-strand cDNA  in accordance with the manufacturer’s instructions. Quantitative real-time PCR was performed using the Hieff® qPCR SYBR Green Master Mix . Amplification and melting curves were recorded using a CFX96 Touch Real-time PCR Detection System. The sequences of primers are listed in Table 1. The relative quantification of gene expression was expressed as fold change via normalization against GAPDH.Total RNA from the RVLM or cell was extracted with TRIzolThe sample preparation for TEM was performed the same as in our previous publication . The RVLIntra-RVLM microinjection was performed as described in our previous study . BrieflyAccording to previous study , the ROSThe SOD and CAT activity levels in the RVLM and cultured N2a cells were determined using commercially available SOD and CAT kits  following the manufacturer’s instructions. The data were analyzed spectrophotometrically using a SpectraMax iD5 microplate reader .4 cells/well) were seeded into 96-well plates and cultured for 24 h followed by 24 h incubation with TNF-α (30 ng/ml) or TNF-α (30 ng/ml) plus A769662 (30 and 60 μM). Then, the medium was replaced with fresh medium. The CCK-8 working solution (10 μl) was added into each well and incubated together for 30 min. Optical density was measured at 450 nm using a SpectraMax iD5 microplate reader .The cell viabilities of N2a cells after TNF-α or TNF-α plus A769662 (AMPK Activator) treatment were estimated using CCK-8 assay kit . N2a cells . The N2a cells in different groups were covered with 500 µl JC-1 working solution and incubated at 37 ℃ for 20 min for full staining, and then washed with dyeing buffer twice. The fluorescence level was measured immediately using a laser scanning confocal microscope , where JC-1 aggregated in normal mitochondria to form polymers and emit red fluorescence, whereas unhealthy mitochondria can only exist in monomer form and produce green fluorescence due to the decrease or loss of membrane potential. The degree of mitochondrial depolarization can be detected by the ratio value of red to green fluorescence intensity.t test was used for experiments that contained two groups of samples. For comparison of multiple groups, one-way ANOVA with subsequent post hoc Bonferroni test was conducted. Sholl analysis data were analyzed by two-way repeated measures ANOVA. P < 0.05 indicated that the differences were statistically significant.Statistical analysis was performed using v 9.1 Graphpad Prism software. Data are presented as means ± standard error of the mean (SEM). The normality of the data was evaluated by the Shapiro–Wilk test. The data conformed to a normal distribution. Student’s unpaired Rats were subjected to electric foot shock stressors with buzzer noises for 2 h twice per day for successive 15 days. The results of hemodynamic parameters of rats under conscious and anesthetized conditions were shown in Fig. Activated microglia, exhibiting morphology changes, can be polarized to M1 or M2 phenotype . We initAs shown in Fig. Sirt3 deficiency has been found to be associated with mitochondrial dysfunction in many diseases . ImmunofThe biological functions of TNF-α are typically initiated by its engagement with TNF-α receptors (TNFR1 and TNFR2) . The RVLDouble immunofluorescent staining of neuronal activation marker c-FOS with TH-positive neurons in the RVLM was performed to study neuronal excitability. The result showed that TNF-α suppression could significantly decrease the proportion of c-FOS in TH-positive neurons in the RVLM of SIH rats Fig. A. SubseqThe main purpose of this study was to investigate the mechanism of microglial neuroinflammation affecting mitochondrial homeostasis in the rostral ventrolateral medulla (RVLM) neurons involved in the pathology of SIH. Our major findings were as follows: (1) Increased BP, HR, sympathetic outflow, and RVLM neuronal overactivity were exhibited in SIH rats; (2) Microglia were activated and particularly polarized to a M1 proinflammatory phenotype, accompanied by increased level of TNF-α in the RVLM of SIH rat; (3) The RVLM neuronal mitochondria were evidently injured in SIH rats; (4) Microglia-derived TNF-α resulted in neuronal mitochondrial dysfunction via the AMPK–Sirt3 signaling pathway and; (5) Microglia-derived TNF-α inhibition in the RVLM could rescue the injury of neuronal mitochondria and inhibit neuronal overactivation, sympathetic hyperexcitability, BP, and HR in SIH rats. Collectively, the present study revealed that microglia-derived TNF-α impaired RVLM neuronal mitochondrial function via the dysregulation of the AMPK–Sirt3 signaling pathway and that microglia-derived TNF-α might be a novel target for therapeutic intervention in SIH.. proposed TNF-α secretion from activated microglia triggered the increase of cerebellar neuronal excitability and modulated psychomotor behaviors [Neuroinflammation is implicated in the pathophysiology of many neurological diseases, including traumatic brain injury, mental illness, and neurodegenerative diseases . Microglehaviors . The micehaviors . In the Structurally damaged mitochondria are dysfunctional and unable to maintain the normal physiological activities of neurons . In this. reported that honokiol attenuated fluoride-induced mitochondrial injury and neuronal/synaptic impairment, which depended on promoting the expression of Sirt3 via an AMPK signaling pathway [. revealed that the protective actions of naringenin on mitochondrial dysfunction and cardiac damage following ischemia–reperfusion injury were abolished by the blockage of the AMPK–Sirt3 signaling pathway [. reported that R7050 reversed TNF-α-induced increase in NADPH oxidase activity in rats with obesity-related hypertension [Sirt3 has been substantiated to modulate almost all aspects of mitochondrial metabolism and function, protecting mitochondria from various injuries , 55. Cur pathway . Yu et a pathway . Activat pathway . The pre pathway . The blo pathway . Previou pathway . Ding etrtension . In the Chronic stress, which can be perceived by the brain, contributes to the development of SIH . Stress This study indicated that elevated TNF-α, which was derived from activated microglia, could cause neuronal hyperexcitation via disturbing mitochondrial homeostasis in the RVLM of SIH rats Fig. . The resAdditional file 1: Figure S1. Cell viability was determined using the cell counting kit-8  assay after treatment with TNF-α or TNF-α plus AMPK activator A769662 in N2a cells. Data were shown as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by post hoc Bonferroni test. n = 6 of independent cell culture preparations. #p < 0.05, ##p < 0.01 vs. TNF-α group.Additional file 2: Figure S2. The AMPK pathway activation rescued p‐AMPK and Sirt3 levels in TNF‐α-treated N2a cells. Representative immunoblot bands and quantitative analysis of Sirt3 and p-AMPK expression in control, TNF-α, and TNF-α plus AMPK activator A769662 groups. Data were shown as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by post hoc Bonferroni test. n = 3 of independent cell culture preparations. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. TNF-α group.


TNF-α inhibitors (TNFis) have revolutionized the treatment of certain chronic immune-mediated diseases, being widely and successfully used in rheumatic inflammatory diseases, and have also proved their efficacy in the treatment of inflammatory bowel disease (IBD). However, among the side effects of these agents are the so-called paradoxical effects. They can be defined as the appearance or exacerbation of a pathological condition that usually responds to this class of drug while treating a patient for another condition. A wide range of paradoxical effects have been reported including dermatological, intestinal and ophthalmic conditions. The causal mechanism of occurrence may implicate an imbalance of cytokines, but is still not fully understood, and remains a matter of debate. These paradoxical reactions often show improvement on discontinuation of the medication or on switching to another TNFi, but in some cases it is a class effect that could lead to the withdrawal of all anti-TNF agents. Close monitoring of patients treated with TNFis is necessary in order to detect paradoxical reactions. In this study we focus on reviewing IBD occurrence as a paradoxical effect of TNFi therapy in patients with rheumatological diseases .  Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that induces the activation of inflammatory cells, the production of cytokines, and the expression of adhesion molecules . TNF-α hHowever, as with most drugs, various adverse reactions have been reported with the use of TNF-α inhibitors, ranging from infections to the risk of malignancy ,10. The Several case series have reported an increased risk of de novo IBD, mainly CD, under treatment with anti-TNF agents . IFX andThe present review aims to describe the findings regarding the association between anti-TNF-α treatment and the occurrence of IBD as a paradoxical effect in patients with rheumatological diseases.A search of the published literature was conducted by exploring PubMed, Google Scholar, EMBASE and MEDLINE databases. The following search terms relating to the key question were set for the search including: “Inflammatory bowel diseases” AND “paradoxical effects” “biologic therapy” AND “paradoxical effects” “ulcerative colitis” AND “paradoxical effects”, “Crohn’s disease” AND “paradoxical effects”, “paradoxical IBD”, “anti-TNF-α/TNFi” AND “paradoxical effects”, “Etanercept” AND “paradoxical effects”, “Adalimumab” AND “paradoxical effects”, “Infliximab” AND “paradoxical effects”, “Golimumab” AND “paradoxical effects”, “Certolizumab” AND “paradoxical effects”. Studies evaluating any possible associations between biologic therapy and IBD as a paradoxical effect were identified. We reviewed the studies published between 2000 and 2023, excluding studies that were in a language other than English. Commonly cited published literature with high-quality research methodology/results and additional articles from bibliographies of recovered papers were examined and included where relevant.TNF-α is responsible for the synovial inflammation in RA, TNFis being successfully used in the treatment of the disease. Several authors describe the occurrence of IBD as a paradoxical reaction to anti-TNF-α treatment in patients diagnosed with RA . The assO’ Toole described the occurrence of IBD in nine patients diagnosed with RA, all presenting CD as a subtype of IBD and all being treated with ETN . In O’ToSome authors described isolated cases of IBD in RA patients treated with anti-TNF-α ,16,17,18Regarding PsA, only two cases of IBD occurrence as a paradoxical effect of anti-TNF-α therapy have been reported in the literature . ToussirTNF is important in the pathogenesis of AS, since TNF concentrations are elevated in the serum and synovial tissue of these patients ,21. TNF Braun et al. reviewed the data from nine trials (seven placebo-controlled) of patients with AS treated with anti-TNF agents, evaluating the flares or de novo IBD cases . There wUskudar et al. compared the occurrence of IBD in patients with AS treated with anti-TNF and in those without anti-TNF treatment . Seven pIn Braun’s study, the frequency of de novo IBD in patients with AS was evaluated at 0.8 per 100 patient-years with ETN and as 0.5 per 100 patient-years with placebo . In UskuAnother study compared 296 patients with spondyloarthropathy (of which 198 had AS) receiving anti-TNF-α agents (IFX/ADA/ETN) with 112 patients taking disease modifying antirheumatic drugs (DMARDs), to evaluate the paradoxical IBD development [On the other hand, in the study conducted by Braun, as well as in the one conducted by Fouache, no statistically significant difference was identified in new-onset IBD between patients treated with placebo and those treated with anti-TNF agents ,24.Toussirot et al. identified 12 patients with AS who developed IBD under anti-TNF treatment, 10 of whom were under treatment with ETN and 2 with IFX . The mosSong et al. also described three patients treated with ETN for active AS who developed a new onset of CD, while AS-related symptoms responded well to ETN . TypicalOther authors described isolated cases of IBD in patients with AS treated with anti-TNF-alpha ,37,38,39The most common symptoms for which patients presented were abdominal pain and diarrhea. Most of the reported cases were described in male patients, a fact probably explained by the more frequent occurrence of AS in men . IBD as Not only cases of de novo IBD were reported, but also flares of the disease. In Braun’s study, more flares of IBD than new onsets of IBD were reported . Marzo-ORegarding treatment with GOL, data in the literature did not report cases of de novo occurrence of IBD, but several cases of IBD flare were described ,39. BawaJIA is one of the most common rheumatological diseases in children. The prevalence varies between 16 and 150 per 100,000 children . The treThere is an increased interest regarding IBD in patients with JIA, since cases have been described of IBD onset upon treatment with anti-TNF-α, especially ETN. In general, ETN offers an acceptable safety profile in children with JIA, and provides significant and sustainable improvement in disease manifestations . AccordiMTX proved to be protective against IBD in JIA . In BrokRegarding the IBD subtype, most studies have described the predominant occurrence of CD. Van Straalen conducted a study on a group of 8942 patients, identifying 48 0.54%) cases of IBD: these included 13 cases (27%) of UC, 22 cases (46%) of CD and 13 cases (27%) of indeterminate colitis . Dalloch% cases oIn the published studies, the interval of IBD onset from the initiation of anti-TNF therapy varied. Dallochio described patients being treated with the TNFi for 7–78 months, while in Van Dijken’s study the interval varied between 9 days and 4.5 years ,50. In aVan Straalen observed that patients who developed IBD were significantly more often male, HLA-B27 positive and older at JIA onset than patients who did not develop IBD . On the Patients had never previously suffered from abdominal complaints, and they did not present other signs, suggesting that their arthritis could be a complication of a pre-existing subclinical IBD. The most common symptoms included abdominal pain, diarrhea, blood in stools, anorexia, fever and weight loss. The diagnosis was established on colonoscopy and biopsy findings. Clinical remission of IBD was obtained in all patients after discontinuation of ETN and initiation of IBD-specific therapy (including therapeutic switch to another TNFi) ,58,59,60Many reports indicate that during periods of intense immunosuppression with anti-TNF-α, IBD may develop or worsen while patients are taking the drugs used to treat these disorders. Thus, TNF inhibition seems to have paradoxical pro-inflammatory effects, in addition to the known anti-inflammatory ones. Increased numbers of unexpected paradoxical events involving TNFi therapy have been described, with an estimated incidence of more than 10% in patients receiving a TNFi ,61. PereSeveral controlled trials and sizable post-marketing studies describing paradoxical effects have been published recently, despite the fact that the majority of instances originate from retrospective research and individual case reports  . EvidencThere are few data available regarding the possible predisposing factors that could have precipitated the appearance of IBD under anti-TNF-α treatment. There are different mechanisms that may be involved in the pathogenesis ,64,65,66Prescott postulated the possibility of the occurrence of IBD as a result of the imbalance between TNF and IFN in susceptible individuals . ETN’s aIBD may be influenced by immune system dysregulation directed against microbial antigens located in the intestinal lumen. Increased T cell reactivity to microbial antigens or the inability of regulatory T cells to control normal responses may be the trigger for the disease onset . Cells iProof of an association of IBD with TNF treatment is strongest for ETN. O’Toole et al. performed a study on IBD provoked by ETN. They identified 443 cases of de novo onset of IBD and 43 cases  of flares of existing IBD reported in association with ETN therapy . A totalETN, in particular, is the main TNF-α inhibitor associated with the development of paradoxical IBD ,24,36 inOverall, the numerous case reports and case series describing new-onset IBD caused by ETN firmly indicate that ETN rarely induces IBD in susceptible patients. It is tempting to hypothesize that ETN, which has not been shown to have therapeutic benefits for CD, may result in evident mucosal inflammation more frequently than IFX or ADA, which are both very successful treatments for IBD. However, paradoxical induction of IBD by IFX and ADA is also likely, even though the evidence is weaker. The gastrointestinal reaction induced by biologics is considered to be uncommon, affecting more frequently the populations with AS, PsA or psoriasis, as an extra-articular manifestation of these diseases . It is kRegarding the IBD subtype, CD was most frequently identified ,3,4,24. The average age of diagnosis and the time interval between the initiation of anti-TNF treatment and the onset of IBD varies a lot in the data from the literature. In O’Toole et al.’s study, the average age was 38.4 (range 10–68 years) . In TousNSAIDs are frequently prescribed to patients with rheumatic diseases for their analgesic and anti-inflammatory effects, but they can also have an impact on the entire gastrointestinal system. The damage to the gastrointestinal system might create similarities in endoscopic and pathological characteristics with IBD. However, the absence of concentric diaphragmatic strictures and the presence of cobblestoning, longitudinal ulcers, or inflammatory polyps, along with histologic findings of granulomas, crypt abscesses, or crypt distortion, should indicate CD as the likely cause rather than NSAIDs . Even thRegarding the therapeutical approach, these paradoxical reactions often show improvement on discontinuation of the medication or on switching to another anti-TNF agent. If symptoms persist or worsen even upon treatment with a second inhibitor, alternative treatment options should be pursued . The manLimitations of our study consist in its retrospective design, the majority of instances originating from individual case reports . For this reason, we cannot have exact data regarding the incidence and prevalence of IBD in rheumatic patients treated with the TNFi. Furthermore, most cases reported lacked adequate detail for understanding the associated risk factors and natural history in greater detail. It is also plausible that these flares could be mere coincidences, and might not be directly connected to TNFi treatment, as no rechallenge was conducted. The presence of a flare upon reattempting the TNF inhibitor treatment would undoubtedly offer more compelling evidence to establish a causal relationship between the TNFi and the disease’s onset. Also, another limitation is bias in reporting of cases, and we cannot exclude the possibility that some cases were not declared and thus missed. However, we tried to collect all the data from the literature on this subject. Looking ahead, an ideal approach would involve the aggregation of a substantial number of IBD cases from diverse national and international cohorts or registries. By doing so, we could perform a comprehensive analysis utilizing multivariable techniques in a case-control study to confirm associations with risk factors, including drug therapy. This more extensive and rigorous investigation would enhance our understanding of the relationships between these factors and IBD, leading to more robust and reliable conclusions. Data from the literature suggest a possible link between the use of anti-TNF-α and IBD occurrence, as a paradoxical effect to these therapies in patients with genetic predisposition. However, the underlying mechanism is still not clear. Most of the reported cases or series of cases concern the occurrence of IBD in patients diagnosed with AS or JIA, the most frequent incriminating agent being ETN. As a subtype of IBD, CD may be more common. Further studies are needed to elucidate the complex association between the occurrence of paradoxical reactions and the use of biologic therapies.


Realgar-Indigo naturalis formula (RIF) exert therapeutic effects is only partially understood.Realgar has a long history ofuse in traditional medicines. However, the mechanism through which Realgar or In this study, 60 feces and 60 ileum samples from rats administered with realgar or RIF were collected to examine the gut microbiota.Bacteroidales was significantly altered after RIF administration, and it was predicted that these microorganisms contribute to the inorganic arsenic metabolic process.The results showed that realgar and RIF influenced different microbiota in both feces and ileum. Compared with realgar, RIF at low dosage (0.1701 g/3 ml) significantly increased the microbiota diversity. LEfSe and random forest analyses showed that the bacterium Bacteroidales in feces might participate in the inorganic arsenic metabolic process to exert therapeutic effects for realgar.Our results suggest that realgar and RIF may exert their therapeutic effects through influencing microbiota. The low dose of RIF had greater effects on increasing the diversity of microbiota, and  Minerals have been used within various traditional medicine systems since ancient times, and are still in use today . They ar2S2 and As4S4. Arsenic (As) is used mainly in the form of mineral arsenicals, including orpiment (As2S3), realgar (As4S4), and arsenolite . It is structurally different from sodium arsenate (As5+) and sodium arsenite (As3+), and is the form used in oral traditional medicines , but with relatively good oral safety profiles even on chronic administration, which gives realgar an advantage over ATO in treatments. Orally administered realgar was shown in clinical trials as highly effective and safe for patients with APL  was developed in the 1980s from a traditional medicine theory base, in which realgar is the principal element, and Indigo naturalis [Baphicacanthus cusia (Nees) Bremek], Salvia miltiorrhiza Bge, and Pseudostellaria heterophylla (Miq.) Pax are adjuvant components to enhance the effects of realgar. The main active components of RIF are realgar, tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T). RIF can reduce the mortality of murine promyelocytic leukemia in vivo by intensifying ubiquitination/degradation of onco-protein, enhancing G (1)/G (0) arrest, and inducing AQP9 to facilitate the transportation of other ingredients into tumor cells. Clinical trials showed that RIF was effective for certain APL, with a remission rate of over 90% and a 5-year overall survival rate of 87%  was provided by Anhui Tiankang Medical Technology Co., Ltd. The mineral medicine realgar was collected from Shimen, Hunan province in China. CMC-Na powder was provided by Shanghai McLean Biochemical Technology Co., Ltd.The solution composition included artificial gastric fluid, artificial intestinal fluid, and 50% ethanol prepared according to the Realgar-Indigo Naturalis Formula group (LRIF), high dose Realgar-Indigo Naturalis Formula group (HRIF), low dose Realgar group (LR), high dose Realgar group (HR), and control group (NC)]. Each group was further divided into six subgroups to protect the rats from excessive bleeding of blood sampling. For the single administration group, blood was collected after a single administration. For the first group, blood was collected at 0.5, 6, 24, and 64 h; for the second group, blood was collected at 1, 8, 30, and 72 h; for the third group, blood was collected at 1.5, 10, and 36 h; for the fourth group, blood was collected at 2, 12, and 42; for the fifth group, it was collected at 3, 16, and 48 h; for the sixth group, blood was collected at 4, 20, and 56 h. Approximately 0.7 ml of blood was collected from the carotid artery each time. A total of 5 ml blood was collected at the last time point (72 h) and divided into three groups, one 1 ml group, and two 2 ml groups. For the continuous administration group, rats were administrated for 8 consecutive days. The drug was administered at 9:00 a.m. every day, and 0.7 ml of blood was collected 3 h after administration. Rats were fed with food and water regularly during the period. Fasting for 8∼12 h before administration on the last day, blood was collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 30, 36, 42, 48, 56, 64, and 72 h, and about 0.7 ml of blood was collected from the carotid artery at each time point. At the last time point (72 h), 5 ml blood were collected and divide into three groups, one 1 ml group and two 2 ml groups. Then blood was transferred to a heparinized anticoagulation centrifuge tube and centrifuge immediately , then the upper plasma was collected and stored in a −20°C refrigerator for testing.A total of 180 specific pathogen-free (SPF) SD rats  were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. After 3 days of acclimatization in a laboratory environment at 20°C and 45–65% relative humidity with a 12-h light/dark cycle, SPF SD rats were randomly divided into five groups [low dose A total of 60 SPF SD rats  were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. . After 3Metagenomic DNA for each sample was extracted using the QIAamp DNA Feces Mini Kit . The quality of metagenomic DNA was then evaluated by 1% gel electrophoresis, followed by specific amplification using a pair of universal primers  targeting the V3–V4 hypervariable region of 16S rRNA gene. After PCR amplification, the quality of the products was accessed and purified using the AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions. Then, the purified amplicons were quantified by QuantiFluor-ST  and then sequenced under the MISEQ platform  of a commercial company  .PRJNA690686 (bacteria). Operational taxonomic units (OTUs) were clustered using uparse software (version 7.1),1 with a similarity of 97%. Each 16S rRNA gene sequence was analyzed with the RDP classifier (version 2.2)2 based on the Silva 16S rRNA database with a confidence threshold of 70%  were performed using the vegdist and adonis functions from the vegan package in the R programming environment (p < 0.05 and LDA score >4).All raw sequencing data were deposited in the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI) with the accession number d of 70% . Based oironment . Taxonomironment . Co-occuironment . Linear in vivo was further tested under different conditions. Rats were given single administration or continuous administration of a low dose of RIF (LRIF), high dose of RIF (HRIF), low dose of realgar (LR), high dose of realgar (HR), or empty control (NC), then As concentration in blood was measured. Continuous administration stabilized the concentration of blood As in rats compared with single administration  samples and 60 ilea  samples from 60 rats were collected . We thenR value of the unweighted in feces groups being R = 0.11 > 0 and p = 0.001 < 0.01, and that in ileum groups was R = 0.16 > 0 and p = 0.001 < 0.01. Thus, the grouping was reasonable , Bacteroidales (9.17%), and Ruminococcaceae (7.27%) at the genus level. Ileum samples had limited distributed microbiota that mainly consisted of Lactobacillaceae (80.75%) and Peptostreptococcaceae (8.61%) at the genus level (Prevotellaceae_NK3B31_group (7.35%), Prevotellaceae_NK3B32_group (3.64%), Prevotellaceae_ NK3B33_group (3.34%) at the species level while Lactobacillus intestinalis (60.23%) dominated Ileum samples at the species level  relRhodospirillaceae, Prevotella, and Barnesiella, while RIF and NC group shared Rhodospirillaceae, Sphingomonas, and Bacteroidales, suggesting that realgar and RIF influenced different microbiota  on the taxa that exhibited linear discriminant analysis (LDA) scores greater than four. As we expect, phyla Bacteroidetes and classes Bacteroidia among OTUs were among represented OTUs  in feces and Enterococcus (OTU 817) in ileum were annotated to the family of Bacteroidales and the genus of Enterococcus, respectively. The area under the ROC curve (AUG) was 0.83 and 0.96, respectively . A total of 24 functional categories and 4,792 functional descriptions were obtained in feces and ileum microbial communities. The relative abundances of functional profiles with bacterial taxa in each group were presented in 4S4). How realgar and RIF work in the human body to achieve their therapeutic effects is unknown. To the authors’ knowledge this is the first time the effects of realgar and RIF on gut microbiota have been studied and elucidated. This study showed that realgar and RIF can influence different microbiota. RIF at a low dosage significantly increased the diversity of microbiota compared with RIF at a high dose and realgar at a low or high dose. LEfSe and random forest analyses showed that the Bacteroidales in feces was important after RIF administration  and Bacteroidetes (42.49%) occupied the majority of the phyla population in feces, while Firmicutes (98.46%) was the most predominant in ileum. Clostridia (22.68%) and Bacteroidia (21.29%) constituted the most prevalent class phenotype in feces, however, in it was Bacilli (86.04%) in ileum. Clostridiales (22.68%) and Bacteroidales (21.29%) dominated the feces orders while Lactobacillales (85.76%) was the most abundant order in ileum. Ruminococcaceae (10.38%), Lachnospiraceae (10.28%), and Prevotellaceae (9.76%), and Bacteroidales (9.51%) were most prevalent families in feces, and Lactobacillaceae (80.75%), Peptostreptococcaceae (8.61%), and Enterococcaceae (4.47%) were the most abundant in ileum. Prevotellaceae (14.67%), Bacteroidales (9.17%), and Ruminococcaceae (7.27%) are the most dominant genus in feces. While Lactobacillus (80.75%), Peptostreptococcaceae (8.61%), and Enterococcus (4.47%) occupied the majority of the ileal genus. Bacteroidales and Ruminococcaceae were increased in LRIF and LR groups comparing to HRIF and HR groups, while Lachnospiraceae and Enterococcus were decreased dramatically in LRIF and LR groups. Enterococcus  has the highest weight among all OTUs in feces, while 26 Bacteroidetes, 24 Firmicutes, and 2 Proteobacteria in feces showed the highest degree.Arsenic accumulates in tissues especially in the heart, liver, spleen, lung, kidney, uterus and ovary, and the distribution and elimination of arsenic in the blood is slow. Drug metabolism studies showed that the renal excretion of realgar only accounts for 0.0367% of the dose, and the total arsenic excretion in feces accounts for 97.7% of the dose, indicating that realgar is mainly excreted through the gastrointestinal tract . SimilarBacteroidales is important. For example, Bacteroides belongs to Bacteroidales Gram-negative bacteria and is among the most abundant anaerobes in the human colon  can be found in the article/The animal study was reviewed and approved by the China Academy of Chinese Medical Sciences.LL: investigation, writing—original draft, writing—review and editing, formal analysis, funding acquisition, and supervision. CL: investigation, formal analysis, and writing—review and editing. NH and QW: formal analysis, writing—original draft, and writing—review and editing. ZhZ, CS, JS, and HY: formal analysis. MY and ZX: data analysis and organization. ZjZ: writing—review and editing, funding acquisition, supervision. All authors contributed to the article and approved the submitted version.


Diagnosing normal-pressure hydrocephalus (NPH) via non-contrast computed tomography (CT) brain scans is presently a formidable task due to the lack of universally agreed-upon standards for radiographic parameter measurement. A variety of radiological parameters, such as Evans’ index, narrow sulci at high parietal convexity, Sylvian fissures’ dilation, focally enlarged sulci, and more, are currently measured by radiologists. This study aimed to enhance NPH diagnosis by comparing the accuracy, sensitivity, specificity, and predictive values of radiological parameters, as evaluated by radiologists and AI methods, utilizing cerebrospinal fluid volumetry. Results revealed a sensitivity of 77.14% for radiologists and 99.05% for AI, with specificities of 98.21% and 57.14%, respectively, in diagnosing NPH. Radiologists demonstrated NPV, PPV, and an accuracy of 82.09%, 97.59%, and 88.02%, while AI reported 98.46%, 68.42%, and 77.42%, respectively. ROC curves exhibited an area under the curve of 0.954 for radiologists and 0.784 for AI, signifying the diagnostic index for NPH. In conclusion, although radiologists exhibited superior sensitivity, specificity, and accuracy in diagnosing NPH, AI served as an effective initial screening mechanism for potential NPH cases, potentially easing the radiologists’ burden. Given the ongoing AI advancements, it is plausible that AI could eventually match or exceed radiologists’ diagnostic prowess in identifying hydrocephalus. Hydrocephalus is a condition where there is an abnormal accumulation of cerebrospinal fluid (CSF) within the brain’s ventricles, resulting in an increased intracranial pressure. It is classified into two types; obstructive hydrocephalus, which occurs when there is a physical blockage in the cerebrospinal fluid (CSF) flow pathway, and communicating hydrocephalus, which is characterized by abnormal CSF accumulation in the ventricles and subarachnoid spaces due to defects in reabsorption. Normal-pressure hydrocephalus (NPH) is a subtype of communicating hydrocephalus, which is typically seen in older adults and can present with symptoms such as gait disturbance, urinary incontinence, and memory impairment. These symptoms can be mistaken for Alzheimer’s disease or other types of dementia, making NPH a challenging diagnosis .NPH is categorized into two types. The first is idiopathic NPH (iNPH), which is caused by an unknown reason that affects the reabsorption of cerebrospinal fluid back into the venous system. The second type is secondary NPH, which results from various factors such as bleeding in the brain’s cerebrospinal fluid, head trauma, infection, tumor, or complications of surgery. These factors can cause the accumulation of cerebrospinal fluid in the brain’s ventricles and subarachnoid spaces, leading to NPH symptoms such as gait disturbance, urinary incontinence, and memory impairment.Several studies have investigated the use of radiological parameters from non-contrast computed tomography (NCCT) of the brain for the diagnosis of hydrocephalus. Nevertheless, diagnosing NPH based on radiographic imaging remains challenging due to the lack of standardized measurement methods for these radiographic parameters . VariousWith the increasing application of technology in the medical field, artificial intelligence (AI) has emerged as a promising tool for improving diagnostic accuracy and reducing errors in radiology. AI can quickly analyze large amounts of medical data, such as imaging studies, and identify abnormalities that might be missed by human radiologists. By utilizing deep learning algorithms, AI can recognize patterns and anomalies in medical images, providing more accurate and efficient diagnoses .A typical pipeline for applying AI in NPH diagnosis involves three key steps. Firstly, it involves the identification of distinct regions, with a specific focus on the CSF and ventricular system in MRI  and CT  scans viSiriraj Hospital of Mahidol University is utilizing AI innovations for screening pulmonary tuberculosis in chest radiographs and ASPECT in patients with clinically suspected acute cerebrovascular ischemia. Currently, the hospital is conducting research to compare the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of AI to radiological parameters measured by radiologists in normal and NPH-diagnosed groups. This study aims to investigate how the typical AI pipeline can improve NPH diagnosis, and gain valuable insights into the potential benefits and limitations of using AI.A retrospective study carried out between December 2012 and August 2022, which included all patients over the age of 18 who had both clinical data and imaging available. NPH was confirmed in patients via the gold standard method for diagnosis: cerebrospinal fluid (CSF) tap test. Patients with intracranial mass, cerebral hemorrhage, and large cerebral infarction leading to anatomical distortion of the brain were excluded from the study.CT imaging was assessed for all patients at the time of initial diagnosis. The images were reviewed by two senior neuroradiologists with more than 20 and 10 years of working experience and a third-year radiology resident, who were blinded to the patients’ clinical status, using the department’s Picture Archiving and Communication System (PACS). Inter-observer agreement was evaluated between the two neuroradiologists and the resident. In cases of disagreement, the final judgement was made by consensus. The AI was also used to evaluate the same groups of patients and identify cases of NPH.Seven radiological parameters were used in this study, including Evans’ index, narrow sulci at high parietal convexity, dilatation of the Sylvian fissures, focally enlarged sulci, widening of temporal horns, callosal angle, and periventricular hypodensities. Each radiological parameter was separately converted into a point system with cut-off values based on earlier studies  and totaThe following radiological parameters were evaluated by two radiologists and a third-year radiology resident  by the maximal width of the inner table of the cranium in the same axial image .Narrow parietal sulci: At high-convexity and parafalcine region assessed in both axial planes in the most superior slices and coronal plane .Dilation of the Sylvian fissures: Reported as present or not present in the coronal plane compared with surrounding sulci .Focally enlarged sulci: Compared with surrounding sulci, usually found in coronal or axial planes .Temporal horns: Reported as mean width of the right and left side, measuring in the axial plane .Callosal angle: Angle between the lateral ventricles in the coronal plane through the posterior commissure perpendicular to the intercommissural plane .Periventricular hypodensities: Along the lateral ventricles graded as not present, present as a cap around frontal horns or confluently extending around the lateral ventricles .Our AI method comprises three main steps. Firstly, we trained a modified 2D U-Net model  for CSF The modified 2D U-Net model consists of four encoder blocks, a bottleneck, and four decoder blocks. It was trained using the sigmoid focal cross-entropy loss function  and was The extracted features were classified into two categories, namely global features representing entire brain region characteristics and local features representing entire brain region characteristics. The study aimed to analyze the impact of both global whole-brain volume metrics and local partition-brain metrics on NPH classification. The parameters for each feature in cerebrospinal fluid (CSF), White and Grey (WG) ratio, and standard deviation (Std) are shown below:CSF ratio all = CSF/WG ratio = CSF size = Brain size = Mean CSF ratio = Min CSF ratio = Min of Max CSF ratio = Max of Std CSF ratio = Mean CSF/WG ratio = Min CSF/WG ratio = Min of Max CSF/WG ratio = Max of Std CSF/WG ratio = This evaluation utilizes The extracted global and local volumetric features were used to train an NPH classification model. In particular, a logistic regression model was trained to perform a binary classification (1 = NPH and 0 = non-NPH) using stratified 5-fold cross-validation on 227 NPH and 110 normal data. The model was used a regularization parameter c = 10, and feature selection was performed using the chi-squared (chi2) method, resulting in the selection of 10 features: ‘CSF ratio_5’, ‘CSF ratio_4’, ‘CSF ratio_6’, ‘CSF ratio_9’, ‘Std CSF ratio_9’, ‘CSF ratio_3’, ‘CSF ratio_2’, ‘CSF ratio_1’, ‘CSF ratio_8’, ‘CSF_ratio_all’. The partitions 0–9 indicate different levels of the brain, with 0 being the lowest (closest to the neck) and 9 being the highest (at the top of the head).https://github.com/slundberg/shap accessed on 12 April 2023]. This allowed us to assess the impact of each feature when predicting the probability of NPH for each CT scan. Our analysis revealed that the three most influential features were CSF ratio_8, CSF ratio_5, and CSF ratio_4  curve between radiologists and AI. A binary logistic regression was used to determine the cut-off value for predicting whether the patient was normal, borderline, or had NPH.Clinical data and radiological findings are presented with descriptive statistics. Categorical data are present as numbers and percentages and compared using Pearson’s chi-squared test. Continuous data are reported as mean ± standard deviation (SD) and compared using independent p < 0.001) in the NPH group ; and 60 (57.1%) were men and 45 (42.9%) were women . ClinicaPH group . Univari< 0.0001 . Binary < 0.0001 .p < 0.001), and the ROC AI (p < 0.001). The narrow sulci of high parietal convexity, callosal angle, and periventricular hypodensities were omitted due to collinearity.The sensitivity for radiologists and AI was 77.14% and 99.05%, respectively, with a specificity of 98.21% and 57.14%, respectively, under the cut-off value of 5. NPV, PPV, and accuracy for radiologists were 82.09%, 97.59%, and 88.02%, respectively, while for AI, these values were 98.46%, 68.42%, 77.42%, respectively . The rece ROC AI  was 0.78The odds ratios (ORs) for Evans’ index, dilated sylvian fissures, focally enlarged sulci, and widening temporal horns were found to be statistically significant. Multivariate analysis revealed ORs of 3.49 (1.07–11.42) and 38.37 (6.04–243.56) for Evans’ indexes of 1 and 2, respectively. The OR for dilated Sylvian fissures was 3.07 (1.04–9.08), while for focally enlarged sulci was 7.88 (1.28–48.25), for widening temporal horn was 5.35 (1.88–15.16), and 12.55 (2.15–73.31) for the first and second grades, respectively .p < 0.001) in the NPH group. It should be noted that our study mainly focused on iNPH cases due to the small number of secondary NPH. The study findings suggest that NPH is increasingly being diagnosed in elderly patients undergoing brain imaging for other reasons. This is consistent with recent epidemiological surveys in Sweden that reported a 3.7% prevalence of iNPH among individuals aged 65 years old . NPH is Although various radiological parameters are used to help confirm the diagnosis, the cut-off values of each parameter are not well established. The morphologic features indicating the likelihood of morphologic features of NPH remain undefined. Furthermore, NPH commonly affects the elderly, who may have associated age-related brain atrophy. Therefore, the differential diagnosis between iNPH and cortical brain atrophy or small vessel disease is difficult . Our stuThere is no united agreement in standardized measurement for each radiologic parameter. For example, there are no specific images used in the measurements of Evans’ index, which is the most widely used parameter in ventricular width. Because CT of the brain provides numerous axial images, the maximal width of the frontal horns can be measured on the same or different images by each radiologist . MoreoveAlthough periventricular hypodensities are a supporting feature of NPH, it is difficult to separate between white matter ischemia, which is commonly found in elderly patients with small vascular disease and subependymal effusion resulting from NPH, and results in the exclusion of patients from further NPH evaluation . It can The use of our AI method presents a new paradigm compared to the existing NPH diagnosis methods. Bianco et al.  employedOur study found that radiologists had a better diagnostic specificity, PPV, and overall accuracy than AI. However, AI volumetric segmentation demonstrated higher sensitivity in detecting ventricular enlargement, indicating its potential as a screening tool. Moreover, the accuracy of AI can be improved through a learning process that involves measuring brain volumes in an increasing number of patients. As AI technology continues to advance, it may become a valuable tool for diagnosing and managing NPH.As our study is a retrospective review of 10 years’ worth of data, there is a risk of chronological bias. Furthermore, certain radiological parameters such as narrow sulci at the high parietal convexity, callosal angle, and periventricular hypodensities were omitted due to collinearity. Therefore, a large-scale prospective study is needed to further investigate these parameters and confirm our findings.Although our AI model did not perform as well as the consensus from three radiologists, it showed promise in the screening process by exhibiting higher sensitivity . This potential use case could lead to a reduction in the number of patients requiring confirmation by radiologists.Another aspect that this study has yet to explore is multicollinearity, which could impact the model’s interpretability, as indicated by the SHAP value. In our future research, we aim to address this issue by investigating more advanced models, like deep learning , that arOur study found that radiologists exhibited higher diagnostic sensitivity, specificity, PPV, and accuracy than AI. However, AI can serve as a screening tool in patients suspected of having NPH, reducing the workload on radiologists. Furthermore, AI’s accuracy can be enhanced through machine learning on an increasing number of brain volumetric measurements. In the future, AI may attain capabilities that are equivalent to or surpass those of radiologists in diagnosing hydrocephalus.


Heart failure with preserved ejection fraction (HFpEF) represents a syndrome involving multiple pathophysiologic disorders and clinical phenotypes. This complexity makes it challenging to develop a comprehensive preclinical model, which presents an obstacle to elucidating disease mechanisms and developing new drugs. Metabolic syndrome (MetS) is a major phenotype of HFpEF. Thus, we produced a rat model of the MetS-related HFpEF phenotype and explored the molecular mechanisms underpinning the observed pathological changes.A rat model of the MetS-related HFpEF phenotype was created by feeding spontaneously hypertensive rats a high-fat-salt-sugar diet and administering streptozotocin solution intraperitoneally. Subsequently, pathological changes in the rat heart and their possible molecular mechanisms were explored.β (GSK-3β) may stimulate cardiac hypertrophy, which was regulated by activated -RAC-alpha serine/threonine-protein kinase (AKT). Finally, the transforming growth factor-β1 (TGF-β1)/Smads pathway might regulate collagen production and fibroblast activation, promoting myocardial fibrosis.The HFpEF rats demonstrated primary features of MetS, such as hypertension, hyperglycemia, hyperlipidemia, insulin resistance, and cardiac anomalies, such as left ventricular (LV) remodeling and diastolic impairment, and left atrial dilation. Additionally, inflammation, myocardial hypertrophy, and fibrosis were observed in LV myocardial tissue, which may be associated with diverse cellular and molecular signaling cascades. First, the inflammatory response might be related to the overexpression of inflammatory regulators (growth differentiation factor 15 (GDF-15), intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial cell adhesion molecule-1 (VCAM-1)). Secondly, phosphorylated glycogen synthase kinase 3The HFpEF rat replicates the pathology and clinical presentation of human HFpEF with MetS and may be a reliable preclinical model that helps elucidate HFpEF pathogenesis and develop effective treatment strategies. HFpESo far, there are no well-established preclinical models of HFpEF. A certain amount of advancement has been achieved in developing animal models of HFpEF based on single risk factors related to the development and progression of HFpEF, including aging, obesity, hypertension, and diabetes . HoweverAs a result, in this study, a rat model mimicking the clinical features of the MetS-related HFpEF phenotype was established. Subsequently, the MetS and cardiac structure and function in HFpEF rats were assessed. Ultimately, we investigated the pathological changes in the rat heart  and their underlying pathophysiological mechanisms.2.2.1.STZ (Cat. no. S0130) was provided by Sigma-Aldrich . Total cholesterol (TC) assay kits (Cat. no. 643365), triglycerides (TG) assay kits (Cat. no. 625307), low-density lipoprotein cholesterol (LDL-C) assay kits (Cat. no. 590644), glucose (GLU) assay kits (Cat. no. 613975), and glycated serum protein (GSP) assay kits (Cat. no. 618474) were purchased from Roche Diagnostics Co. (Shanghai). Insulin assay kits (Cat. no. 20221110), atrial natriuretic peptide (ANP) assay kits (Cat. no. 20220221), B-type brain natriuretic peptide (BNP) assay kits (Cat. no. 20220217), soluble growth stimulated expression gene 2 protein (sST2) assay kits (Cat. no. 20220210BX), and galectin-3  assay kits (Cat. no. 20221118) were purchased from Huaying Biotechnology Research Institute . High-sensitivity C-protein (hs-CRP) assay kits (Cat. no. 20220305BX), interleukin-1β (IL-1β) assay kits (Cat. no. 20220315BX), and IL-6 assay kits (Cat. no. 20220312BX) were purchased from Xin Bosheng Biotechnology Co. .β , anti-P-GSK3β, anti-collagen type I (Coll I), anti-collagen type III (Coll III), anti-α-smooth muscle actin (α-SMA), anti-transforming growth factor-β1 (TGF-β1), anti-Smad2/Smad3, anti-P-Smad2/Smad3, and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The antibody characteristics, including antibody names, catalog numbers, and producers, were presented in The primary antibodies included anti-growth differentiation factor 15 (GDF-15), anti-intercellular adhesion molecule-1 (ICAM-1), anti-vascular endothelial cell adhesion molecule-1 (VCAM-1), anti-phosphoinositide 3-kinase (PI3K), anti-RAC-alpha serine/threonine-protein kinase (AKT), anti-phosphorylated AKT (P-AKT), anti-glycogen synthase kinase 32.2.n = 10 per group). Ten WKY rats served as the normal control group. The rats were allowed to acclimate to the laboratory environment for four weeks before the beginning of the experiment. All animal experiments were approved by the Animal Ethics Committee of Xiyuan Hospital, Chinese Academy of Traditional Chinese Medicine .Twenty SHR rats  and ten age-matched Wistar Kyoto (WKY) rats were purchased from Weitong Lihua Experimental Animal Technology Co. . All rats were housed at the experimental animal center  under standard laboratory conditions (23°C and a 12-hour dark-light cycle) with free access to food and water. Twenty SHR rats were randomly assigned to the SHR and HFpEF groups , followed by the current diet and intraperitoneal injection of STZ solution [25 mg/kg body weight (BW) per week] for eight weeks. The HFpEF group continued on their present diet for six weeks, while the WKY and SHR groups were provided with a normal maintenance diet until the end of the study. After the animal model was established, blood pressure was measured while the rat was awake. Echocardiography was done during inhalational anesthesia with isoflurane, and blood samples and heart tissue were collected.2.4.With a tail-cuff blood pressure device, blood pressure (systolic blood pressure (SBP) and diastolic blood pressure (DBP)) was measured non-invasively. At each time point, measurements were obtained three times, and the average result was analyzed.2.5.A high-resolution micro-ultrasound system  with an MX 250 transducer (21 MHz) was utilized to evaluate cardiac structure and function. Data analysis was conducted on Vevo LAB software version 5.7.0 . M-mode echocardiographic tracing was used to determine left ventricle (LV) Mass, LV end-diastolic anterior wall (LVAWd), LV end-diastolic posterior wall thickness (LVPWd), LV end-diastolic internal diameter (LVEDD), LV end-diastolic volume (EDV), and LV ejection fraction (LVEF). In a four-chamber view, the maximum left atrial (LA) anterior-posterior diameter (LAAPD) and LA left-right diameter (LALRD) were measured. Regarding LV diastolic function parameters, pulsed-wave Doppler parameters (isovolumic relaxation time (IVRT) and peak mitral inflow velocity during early diastole (E)) and tissue Doppler imaging parameters  and peak late diastolic mitral annular velocities (a′)) were assessed, and the E/e′ ratio was then calculated. All metrics were measured by an ultrasonographer who did not know which group each rat belonged to. Three consecutive cardiac cycles were analyzed, and an average value was determined for each metric.2.6.After echocardiography, blood samples were isolated to obtain serum and stored at −80 °C. TC and LDL-C were measured using an enzyme colorimetric method. TG was determined by a colorimetric method. GLU was measured with the hexokinase endpoint method. GSP was measured through a colorimetric fructosamine assay based on the nitro-blue tetrazolium reaction with ketamine. Insulin concentrations were tested with an enzyme-linked immunosorbent assay (ELISA). The index for insulin resistance (HOMA-IR) was calculated as fasting insulin (mIU/l) times fasting blood GLU (mmol/L) divided by 22.5. The fasting triglycerides and glucose (TyG) index was calculated as follows: TyG index = Ln [fasting TG (mg/dl)×fasting GLU (mg/dl)/2]. According to the manufacturer’s protocol, circulating hs-CRP, IL-1β, IL-6, ANP, BNP, sST2, and Gal-3 were determined using ELISA kits.2.7.n = 5 per group) were washed immediately with saline, then fixed in a 10% neutral formaldehyde solution at room temperature, dehydrated in an increasing series of ethanol baths, embedded in paraffin, and sectioned into 5-μm sections using a microtome. Hematoxylin & eosin (H&E) (n = 3 per group) and Masson’s trichrome staining (n = 2 per group) were utilized for histological and fibrosis analysis, respectively. Moreover, the cardiomyocyte cross-sectional area  and the left ventricular wall thickness (LVWT) at the midventricular level were measured to assess cardiac hypertrophy. To determine the degree of fibrosis, the collagen volume fraction (CVF) was measured with Image-Pro Plus 6.0 , with four distinct fields of view per section from the anterior, posterior, lateral, and septal walls of the LV; the perivascular fibrosis ratio (PFR) was determined by dividing the collagen areas deposited in the perivascular by the total vascular area.The heart tissues (2.8.n = 4 per group) were promptly frozen in liquid nitrogen and stored at −80°C. Total protein samples were extracted using a protein extraction kit . Protein concentration was determined using a bicinchoninic acid protein quantification kit , and absorbance was measured at 570 nm using an ELISA reader . Protein lysates were electrophoresed on a sodium dodecyl sulphate-polyacrylamide gel and transferred onto nitrocellulose membranes . After blocking, membranes were incubated successively with primary antibodies  and secondary antibodies of goat anti-mouse/rabbit IgG (H + L)-HRP. Signals from secondary antibodies were detected using a chemiluminescent reagent, and the image was captured using x-ray films. The results were quantified with TotalLab Quant software . Finally, the relative expression of proteins was standardized using GAPDH.The LV myocardial tissues (2.9.post hoc multiple comparisons were performed with the least significant difference (LSD) test. P-values < 0.05, 0.01, and 0.001 indicated statistical, significant, and highly significant differences, respectively.Statistical analyses were conducted using SPSS software (version 22). All data are expressed as mean ± standard deviations. One-way analysis of variance (ANOVA) was applied to analyze the data, and 3.3.1.P < 0.001, P < 0.001). The results demonstrated that HFpEF rats (induced by risk factors of hypertension and glucolipid dysregulation) developed a cluster of clinical features of the MetS, such as hypertension, hyperglycemia, hyperlipidemia, and insulin resistance.3.2.P < 0.01 or 0.001, P < 0.05, 0.01, or 0.001). Therefore, HFpEF rats exhibited cardiac remodeling, including increased LV mass, thickened LV walls, and enlarged LA and LV internal diameters.Structurally. Compared to WKY rats, parameters related to cardiac structure, such as LV Mass, LVAWd, LVPWd, LVEDD, EDV, LAAPD, and LALRD, were significantly increased in HFpEF rats . Thus, inflammatory regulators  play critical roles in chronic inflammation pathogenesis in HFpEF rats.In the present research, although serum inflammatory markers (hs-CRP and IL-6) in HFpEF rats were lower than in SHR rats , LVWT, and CSA compared with WKY and SHR rats . The results suggested that the PI3K/AKT pathway might promote myocardial hypertrophy by regulating GSK-3β phosphorylation in HFpEF rats.GSK-3β acts as a critical anti-hypertrophic regulator of cardiomyocytes. However, phosphorylation of the Ser9 site of GSK-3β, which is mainly regulated by the PI3K/AKT pathway, removes its inhibitory activity on hypertrophy and contributes to cardiomyocyte hypertrophy. In this study, PI3K protein expression, as well as the P-AKT/AKT and P-GSK-3β/GSK-3β protein ratios, were significantly higher in HFpEF rats than in WKY rats , serum Gal3 and CVF were dramatically higher than in SHR rats . Therefore, HFpEF rats exhibited significant myocardial fibrosis, as shown by massive collagen deposition in the perivascular and myocardial interstitium.Myocardial fibrosis is a well-recognized cause of LV diastolic dysfunction in HFpEF. In this study, serum biomarkers of fibrosis , PFR, and CVF were statistically higher in HFpEF rats than in WKY rats  are responsible for collagen synthesis and deposition. In this study, Coll I, Coll III, and α-SMA protein expressions were considerably higher in HFpEF rats than in WKY rats . The findings indicated that myocardial fibrosis in HFpEF rats might be associated with fibroblast activation and excessive collagen deposition.The cardiac collagen mainly consists of Coll I and Coll III . Activatβ1 activates the classical Smad signaling pathway, regulating downstream fibrosis-related genes, thereby modulating collagen synthesis and fibroblast activation, resulting in myocardial fibrosis , suggesting that activating the TGF-β1/Smads pathway might regulate fibroblast activation and collagen synthesis, thereby promoting myocardial fibrosis in HFpEF rats.TGF-fibrosis . The TGF4.β1/Smads pathway, respectively.HFpEF is a complex disorder with various underlying pathophysiologies and significant phenotypic heterogeneity. Thus, targeting therapy based on phenotype and pathogenic mechanisms may be an effective strategy . An opti4.1.To date, there is no optimal preclinical model due to the complexity and heterogeneity of HFpEF. It is well-recognized that an “ideal” preclinical model of HFpEF is supposed to accurately reproduce the clinical phenotypes and pathophysiological features of HFpEF patients –14. MetSThe HFpEF rat simulated the clinical features of MetS, such as hypertension, glucolipid metabolism disorders, and insulin resistance. Additionally, HFpEF is a complex syndrome involving cardiac remodeling and LV diastolic dysfunction . LV dias4.2.A systemic pro-inflammatory state might represent a bridge between various comorbidities and impaired cardiac structure or function in HFpEF . MetS-inInflammation involves diverse inflammatory mediators, such as cytokines, cell adhesion molecules, and chemokines. GDF-15 is an inflammatory cytokine secreted in response to inflammation and exerts an essential role in the pathophysiology of HFpEF . Cell ad4.3.Pathological myocardial hypertrophy, mainly defined by LV hypertrophy (LVH) and cardiomyocyte enlargement, represents an adaptive reaction of the heart to elevated hemodynamic pressure . LVH, prIncreasing evidence indicates that GSK-3β exerts an anti-hypertrophic regulatory role in cardiomyocytes. GSK-3β shows high activity in the basal condition  and inhibits various pathways that promote cardiac hypertrophy  . However4.4.Myocardial fibrosis, characterized by perivascular and interstitial fibrosis, is an essential pathological feature of HFpEF , 47. Hahα-SMA, are responsible for collagen production and deposition. We confirmed a significant increase in myofibroblasts and ECM production in HFpEF rats. The TGF-β1/Smads pathway serves as an essential modulator of collagen synthesis and fibroblast activation. TGF-β1 binds to its receptor and induces phosphorylation of the receptor-activated Smads (Smad2 and Smad3), which combine with the common-partner Smad (Smad4) to create trimeric complexes. The trimeric complexes migrate to the nucleus and then modulate fibrosis-associated gene transcription, including ACTA2, COL1A1, and COL3A1, which encode α-SMA, Coll I, and Coll III, respectively  , activation of the AKT/GSK-3β pathway, and activation of the TGF-


The issue's papers   that help scientist tackle some of these challenges  and later (through age 17). Much like earlier work in older youth, Special Issue papers demonstrate acceptable psychometrics across these developmental periods (Kirk et al., Findings on longitudinal outcomes in the Special Issue highlight the importance of early childhood irritability. Not only does this domain of psychopathology show continuity with similar forms of psychopathology during school age, but irritability relates to a broad array of problems. This includes symptoms of multiple internalizing disorders, involving expressions of anxiety or mood problems, as well as externalizing disorders, involving expressions of behavioral and neurodevelopmental difficulties. Such broad expressions of risk highlight the clinical importance of early irritability and dimensional approaches in young children and beyond. Not only is this domain important for the impact it has on young children's functioning, but it also can portend multiple areas of lasting difficulty.5Journal readers owe considerable gratitude to the authors of the Special Issue papers. These manuscripts draw attention to an important domain of psychopathology, irritability. Moreover, the papers highlight the measurement challenges raised by attempts to study the domain, and they illustrate novel techniques for meeting these challenges, such as IRT measurement techniques. Considerable other work suggests that these approaches could be useful in many areas of psychopathology. Finally, the Special Issue papers extend a focus on development that characterizes considerable research around mental health. Clearly, the importance of school age and adolescence has been recognized for decades in studies of many mental conditions. Moreover, researchers examining facets of neurodevelopmental disorders have long recognized the importance of studying young children. The Special Issue extends this focus applied to the neurodevelopmental disorders to a range of conditions, particularly those related to expressions of irritability with origins in the first 5 years of life.The authors declare that there are no conflict of interests.


Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes—MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox—to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy. Impaired muscle regeneration and atrophy of muscle fibers are observed in ageing and many chronic muscle disorders (Duchenne muscular dystrophy (DMD), facioscapulohumeral dystrophy (FSHD), cachexia, etc.) ,2,3,4. THigh doses of TNF impair myogenic differentiation in cultured myoblasts ,7,8,9. TROS regulate myogenic differentiation ,16,17; hThe features of ROS generation in skeletal muscles are described in detail in several reviews ,19,26. BMitochondria are one of the main sources of ROS in muscles . MitochoNon-mitochondrial sources of ROS, in particular NADPH oxidases NOX2 and NOX4, also participate in the regulation of myogenic differentiation ,32,33. IOxidative stress, like other stresses such as starvation, hypoxia, ER stress, etc., is a powerful stimulator of canonical macroautophagy. A common target of these stresses is the Unc-51-like kinase 1 (ULK1) complex, which triggers phagophore nucleation and the classic autophagy pathway involving Beclin 1 (BECN1), several autophagy-related genes (ATG), and LC3 ,40. AutoMitophagy plays an important role in myogenic differentiation and muscle regeneration ,44. PresHere, we investigated the mechanisms of mito-ROS-dependent disruption of myogenic differentiation under the influence of TNF. We focused on the study of the mitoROS-dependent effect of TNF on myoblasts at the stage of their preparation for fusion. To do this, we added TNF once, 24 h before changing the growth medium to the differentiation medium. Under these conditions, TNF disrupted the myogenesis of immortalized human MB135 myoblasts. TNF further enhanced the generation of mito-ROS and lipid peroxidation of mitochondria and mitophagy on days 0 and 1 of differentiation. The classic antioxidant Trolox and the mitochondria-targeted antioxidant SkQ1 prevented TNF-induced excess mito-ROS, lipid peroxidation, and mitophagy, and partially restored defects in myogenic differentiation. This indicated the key role of mito-ROS in the disruption of myogenesis at its early stage. AMPK/ULK1 signaling plays an important role in the induction of mitophagy during muscle regeneration ,56. We hSkQ1 was synthesized by G.A. Korshunova and N.V. Sumbatyan at the Belozersky Institute of Physico-Chemical Biology. All other reagents, unless otherwise noted, are from Sigma, USA. Culture plastic is produced by Costar, USA Cell cultures. Normal human immortalized myoblasts (MB135) derived from the muscle biopsy of a healthy individual  were a kTNF, antioxidants, and inhibitors treatment and myoblast differentiation. Cells were plated onto 12-well cell culture plates (2 × 105 cells per well), or 6-well plates (8 × 105 cells per well), or flux bottom confocal dishes  (8 × 105 cells per dish) and left overnight for attachment. The next day, the cells were treated with antioxidants . After 48 h, human recombinant TNF  was added. After another 24 h, cells were washed with DMEM twice, and differentiation was induced by replacing the Growth Medium with a low-serum Differentiation Medium composed of 98% DMEM  and 2% horse serum  supplemented with 10 ug/mL insulin, 5.5 ug/mL transferrin, 6.7 ng/mL sodium selenite . Autophagy inhibitor SBI-0206965 (5 μM), or activators rapamycin (100 nM) and AICAR (0.5 μM) were added 30 min or 24 h before TNF (denoted in the figures). Antioxidants were re-added to the differentiation medium.May–Grunwald Giemsa staining. After 3 days of differentiation, the cells were fixed with 4% PFA for 5 min . The wells with PFA-fixed cells were washed with phosphate-buffered saline (PBS), and stained with 200 μL of May–Grunwald dye for 5 min. Then, 1 mL of PBS was added to the wells without May–Grunwald dye removal and the cells were stained for additional 15 min in the diluted May–Grunwald solution. Afterwards, the wells were washed 3 times with distilled water, stained for 1 h with 1 mL of Giemsa stain (diluted 1:10 in PBS), washed with distilled water again and let dry. All procedures were performed at room temperature. The samples were observed and photographed using the Axio Imager microscope . Eight random fields of view were captured for each sample. Fusion Index (FI) was estimated using ImageJ by dividing the number of nuclei inside the myotubes by the total number of nuclei for each sample.® FlowSight® Imaging Flow Cytometer  kindly provided by the Moscow State University Development Program PNR5. The Amnis® FlowSight® Imaging Flow Cytometer was equipped with a 488 nm laser (60 mW) and an SSC laser (10 mW). Data were analyzed in Amnis® IDEAS®. Statistical analyses for MitoTrackerOrange CM-H2TMRos and MitoViewGreen stain results were performed for the medians of fluorescence distributions detected in the 560–595 nm or 480–560 nm channel, respectively. To analyze the oxidation of MitoCLox, the ratio of the medians of fluorescence distributions in the green (480–560 nm) and red (595–640 nm) channels was calculated for each sample. The data in the figures are presented as the means of these medians, normalized to the control.Flow cytometry. For flow cytometry, the cells  were incubated with MitoViewGreen probe , TMRM , mitoROS indicator MitoTrackerOrange CM-H2TMRos , or with ratiometric mitochondrial lipid peroxidation indicator MitoCLox (200 nM 4 h) ,53. ThenWestern blot analysis. Immunoblotting was performed as described previously . The celMitophagy detection. For mitophagy analysis, cells seeded in confocal dishes were transduced with a lentiviral vector containing mitoQC construction . Mito-QCp-values less than 0.05 (*), 0.01 (**), 0.001 (***) and 0.0001 (****) were considered significant. All experiments were performed in no less than 3 biological replicates. The number of biological replicates in each experiment (n) is denoted in corresponding figure legends.Statistical analysis. Statistical analyses were performed in GraphPad Prism 9 using ANOVA test with Dunnet’s correction for multiple comparisons or Mann–Whitney U test. Data are presented as mean ± SD. Pretreatment of MB135 myoblasts with 50 ng/mL TNF 24 h before induction of myogenesis resulted in a strong decrease in myoblast fusion and myotubes formation A,B, as wMyogenic differentiation of human MB135 myoblasts on day 1 after induction of differentiation was accompanied by a significant increase in the level of mitoROS, measured by MitoTrackerOrange CM-H2TMRos A,B, and TNF additionally increased the level of mitoROS both before (day 0) and after (day 1) the induction of differentiation A,B. MitoThe antioxidants Trolox  and SkQ1 , added Trolox and SkQ1 added first 48 h before TNF and then again in the differentiation medium partially restored the fusion defects A,B and MThe excess of cells containing ML in the MB135 cell population treated with TNF was decreased by Trolox and SkQ1 antioxidants . The effTo test this possibility, we analyzed the effects of autophagy inducers as well as an autophagy inhibitor on myogenic differentiation of MB135 myoblasts. Autophagy inducers rapamycin  and AICAR  added 24 h before differentiation induction caused a decrease in myoblast fusion and MYH2 expression on day 3 of differentiation A–C. On tExcessive or chronic inflammation is a common feature of various pathologies characterized by the loss of muscle mass. Proinflammatory cytokines such as TNF initiate intracellular signaling pathways leading to protein breakdown and muscle atrophy. These cytokines can also significantly impair myogenic differentiation essential for skeletal muscle regeneration ,7,8,9. TThe production of mitochondrial ROS accompanies myogenic differentiation and presumably correlates with the metabolic switch from glycolysis to oxidative metabolism . At the To study the possible role of mitochondrial ROS production in TNF-dependent myogenesis impairment, we used the mitochondria-targeted antioxidant SkQ1 . SkQ1 efIncreased generation of mito-ROS by dysfunctional mitochondria is characteristic of muscle pathologies ,16,17. EAntioxidant Tempol, but not SkQ1, reduces the thickness of the formed myotubes in MB135 culture . We haveMitochondrial ROS are able to stimulate selective autophagy of mitochondria (mitophagy), which is often considered as a mechanism to prevent the excessive generation of mitoROS by dysfunctional mitochondria ,63. We oMitochondria removal during muscle injury and after intense exercise is regulated by AMPK/ULK1 signaling ,56. ThisOverall, our results indicate that impairment of myogenesis by TNF is mediated by mitoROS-dependent excessive mitophagy that prevents myoblasts from completing their myogenic commitment/preparation for fusion. The delicate balance between mitophagy induction and prevention of mitophagy overstimulation is important for normal myogenesis.From a practical point of view, myogenesis is strongly dependent on autophagy; thus, the use of autophagy inhibitors to correct defects in myogenesis associated with excessive mitophagy seems highly questionable. At the same time, the mitochondria-targeted antioxidant SkQ1 does not affect normal mitophagy and myogenesis, but only suppresses excessive mitophagy and thus stimulates myogenesis impaired by the TNF inflammatory cytokine. SkQ1 and similar antioxidants may be potentially useful for the complex therapy of inflammatory muscle pathologies associated with impaired myogenic differentiation.