Tumor necrosis factor (TNF)-α is a potent mediator of inflammation and is involved in the pathophysiology of chronic kidney disease (CKD). However, the effects of TNF-α inhibition on the progression of kidney fibrosis have not been fully elucidated. We examined the effects of TNF-α inhibition by etanercept (ETN) on kidney inflammation and fibrosis in mice with aristolochic acid (AA) nephropathy as a model of kidney fibrosis. C57BL/6 J mice were administered AA for 4 weeks, followed by a 4-week remodeling period. The mice exhibited kidney fibrosis, functional decline, and albuminuria concomitant with increases in renal mRNA expression of inflammation- and fibrosis-related genes. The 8-week ETN treatment partially but significantly attenuated kidney fibrosis and ameliorated albuminuria without affecting kidney function. These findings were accompanied by significant suppression of interleukin (IL)-1β, IL-6, and collagen types I and III mRNA expression. Moreover, ETN tended to reduce the AA-induced increase in interstitial TUNEL-positive cells with a significant reduction in Bax mRNA expression. Renal phosphorylated p38 MAPK was significantly upregulated by AA but was normalized by ETN. These findings indicate a substantial role for the TNF-α pathway in the pathogenesis of kidney fibrosis and suggest that TNF-α inhibition could become an adjunct therapeutic strategy for CKD with fibrosis. Because the risk for cardiovascular events increases as CKD progresses2, preventing progression is essential for improving the disease prognosis. Kidney fibrosis, the final common pathway in CKD, is characterized by the replacement of functional renal tissue with extracellular matrix3, and provides a greater contribution to the risk for end-stage renal disease than glomerulosclerosis, which is another typical pathology of advanced CKD4. Kidney fibrosis is initiated by various tubular cell injuries, including ischemia, proteinuria, and exposure to toxic substances8. Injured tubular cells show cell cycle arrest, de-differentiation, senescence, and increased production of proinflammatory and profibrotic factors, resulting in activation and proliferation of myofibroblast and matrix secretion8. Several factors that contribute to the development of kidney fibrosis have been identified, however, they have not yet been translated into therapeutic agents.Chronic kidney disease (CKD) is increasing worldwide10, but inflammation also plays an important role in its progression12. Tumor necrosis factor (TNF)-α is a potent mediator of the inflammatory response produced by various cells, including macrophages, mesangial cells, and tubular epithelial cells13. The main pathways activated by TNF-α are centered on caspase, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK). The NF-κB and MAPK signaling pathways induce secondary responses by increasing the expression levels of several proinflammatory cytokines, further activating TNF-α14. High serum and renal levels of TNF-α have been reported in human CKD and experimental kidney disease, including unilateral ureteral obstruction (UUO), ischemia–reperfusion injury (IRI), and cisplatin-induced nephropathy models17. Furthermore, high serum levels of TNF-α are positively correlated with the severity of kidney injury15. Pharmacological and genetic inhibition of TNF-α ameliorates kidney injury in diabetic nephropathy and IRI models in which inflammation plays a critical role in the pathogenesis20. On the other hand, to elucidate the pathogenesis of the TNF-α pathway in the development of kidney fibrosis, several previous studies have employed the UUO model which is the most widely used mouse model of kidney fibrosis. In these studies, TNF-α inhibition using the pegylated form of soluble TNF receptor type 1 or genetic ablation of TNF receptors successfully had an anti-fibrotic effect against kidney fibrosis induced by the UUO model, indicating that the TNF-α pathway is a promising therapeutic target for kidney fibrosis25. However, the UUO model has potential limitations, such as the inability to assess changes in kidney function and the rapid progression to kidney fibrosis caused by hydronephrosis, which differs from the process of kidney fibrosis typically observed in human CKD26.The transforming growth factor (TGF)-β pathway is a master regulator of kidney fibrosis33. To investigate whether TNF-α inhibition prevents the development of kidney fibrosis, and to elucidate the underlying molecular mechanisms, we evaluated the effects of etanercept (ETN), which is a fusion protein competitively acting as a "TNF-α decoy receptor" to inhibit the binding of TNF-α to its cell surface receptor34, on kidney functional decline, inflammation, and fibrosis in a murine model of AAN.In this study, we employed aristolochic acid (AA)-induced nephropathy (AAN) as another model of kidney fibrosis to corroborate the findings reported for the UUO model. AAN is caused by direct cytotoxicity to renal tubular cells and is characterized by progressive tubular atrophy accompanied by chronic interstitial inflammation and fibrosisP < 0.001 and P < 0.001, respectively). There were no significant differences in heart to BW ratio among the three groups . There were no significant differences in BP and HR among the three groups , tissue weight, systolic blood pressure (BP), and heart rate (HR) are shown in Fig. P < 0.001 and P < 0.001; BUN, P < 0.001 and P < 0.001, respectively). In addition, creatinine clearance levels were significantly decreased in the AA and AA + ETN groups compared to the control group (both P < 0.001). There were no differences between the AA and AA + ETN groups in these parameters. Urinary albumin excretion in the AA and AA + ETN groups was significantly increased compared to the control group (both P < 0.001); however, urinary albumin excretion in the AA + ETN group was significantly decreased compared to the AA group (P = 0.047). In the pathological evaluation, the glomerular area in the AA group was significantly decreased, and that in the AA + ETN group tended to be decreased compared to the control group   Fig. b.Figure P < 0.001) . Renal mRNA expression of TGF-β1, a pivotal profibrotic cytokine9, was significantly and similarly increased in the AA and AA + ETN groups compared to the control group . In the pathological evaluation, AA administration caused severe kidney fibrosis, indicated by an increased area of picrosirius red (PSR) staining; however, ETN partially but significantly reduced kidney fibrosis . In a preliminary experiment in which we administered ETN at a lower dose , the expression of type I and III collagen and pathological kidney fibrosis were comparable between the AA and AA + ETN groups  Fig. a. Howevesis Fig. b. The quP < 0.001). Although there were no significant differences in renal TNF-α expression between the AA and AA + ETN groups, renal IL-6 and IL-1β expression was significantly decreased in the AA + ETN group compared to the AA group  . Immunohistostaining of F4/80 revealed obvious renal interstitial macrophage infiltration in the AA and AA + ETN groups  but was normalized by ETN (P = 0.034)  Fig. a. RegardP < 0.001) . Renal expression of Bax, a pro-apoptotic factor, was significantly increased in the AA group compared to the control group (P < 0.001) but was normalized by ETN (P < 0.001)  assay and evaluated apoptosis-related factors. Although the number of TUNEL-positive cells was significantly increased in the AA and AA + ETN groups compared to the control group (both 01) Fig. a-b, ETN 01) Fig. c. The anWe demonstrated that TNF-α inhibition by ETN attenuated kidney inflammation, fibrosis, and albuminuria in a mouse model of AAN. TNF-α inhibition suppressed the AA-induced increase in renal expression of inflammation- and fibrosis-related genes, including IL-1β, IL-6, and type I and III collagen. Moreover, TNF-α inhibition tended to reduce the AA-induced increase in renal interstitial TUNEL-positive cells and significantly reduced the AA-induced increase in renal Bax mRNA expression, a pro-apoptotic factor. These findings imply that the TNF-α pathway plays a substantial role in the development of kidney fibrosis, and that TNF-α inhibition may exert its anti-fibrotic effect by suppressing kidney inflammation and renal interstitial cell apoptosis.36. In this study, the AA-induced increase in renal phosphorylated p38 MAPK was normalized by ETN, suggesting that ETN attenuated the upregulated renal TNF-α pathway in the AAN model. This finding is consistent with the result that the AA-induced increase in renal expression of proinflammatory cytokines was significantly attenuated by ETN. Nonetheless, the renoprotective effect of ETN was limited to partial improvement of fibrosis and albuminuria. These results may be associated with the TGF-β pathway, macrophage infiltration, or the method of inhibiting the TNF-α pathway.p38 MAPK is downstream of the TNF-α signaling pathway, and its activation promotes apoptosis and the production of proinflammatory cytokines, including TNF-α, IL-1β, and IL-610. Therefore, the lack of a preventive effect of ETN on upregulation of the renal TGF-β pathway may explain its limited anti-fibrotic effect in the AAN model. Notably, previous studies on the roles of the TNF-α and p38 MAPK pathways in the development of kidney injury also demonstrated that inhibiting these pathways does not attenuate renal TGF-β expression38, indicating that the TGF-β pathway is less influenced by inhibiting the TNF-α pathway. No study has evaluated the effects of inhibiting both TNF-α and TGF-β signaling on kidney fibrosis, and future studies addressing this issue might make the anti-fibrotic effect of TNF-α inhibition shown in this study more prominent.In this study, the AA-induced increase in renal TGF-β expression was not affected by ETN. The TGF-β pathway is a master regulator of fibrosis that promotes the epithelial-mesenchymal transition and production of extracellular matrix protein via the Smad-signaling pathway39. Various strategies, including depletion or repletion of macrophages and blockade of chemokines or their receptors, have demonstrated a pivotal role for macrophages in kidney diseases46. However, in the present study, ETN did not affect renal interstitial macrophage infiltration induced by AA, and the renal gene expression levels related to macrophage infiltration, including MCP-1, F4/80, and CD68, were not affected by TNF-α inhibition. Previous studies on the effects of TNF-α inhibition in kidney injury models have also reported a lack of improvement of macrophage infiltration. For example, Meldrum et al. showed that administering PEG-TNFR1 has no effect on macrophage infiltration in a rat model of UUO25. Similarly, Misaki et al. reported that administering ETN reduces interstitial mononuclear cell infiltration 3 days after, but not 7 and 14 days after UUO47. The reason that ETN did not affect renal interstitial macrophage infiltration in the AAN model is unclear, but insufficient suppression of macrophage infiltration may explain the limited anti-fibrotic effects of ETN.Macrophages play a pathogenic role in the development of kidney inflammation and fibrosis22. In these studies, genetic ablation of TNF-α receptors had a certain anti-fibrotic effect (20–30% reduction) against UUO-induced kidney fibrosis, which seemed to be more prominent than the results of our study. Therefore, the limited renoprotective effect in this study might be associated with an insufficient ability of ETN to inhibit the TNF-α pathway.Genetic ablation of TNF-α receptors has been employed in several studies as another way to inhibit the TNF-α pathway29. Given that ETN had its renoprotective effect mainly in the renal interstitium, the partial reduction in AA-induced albuminuria may reflect the degree of improvement in renal interstitial damage by TNF-α inhibition.In this study, ETN partially but significantly attenuated the AA-induced increase in albuminuria, whereas it did not affect structural changes in the glomerulus. Because AA causes proximal tubular epithelial cell death without affecting the glomerulus, the AAN model exhibits mild albuminuria, which is considered tubular proteinuriaThis study has limitations. First, an ETN group in which mice received an i.p. injection of DMSO and an s.c. injection of ETN was lacking. Therefore, it is unknown whether TNF-α inhibition has any effect on normal kidneys. Second, because ETN did not improve the AA-induced reduction in creatinine clearance, it is unclear whether TNF-α inhibition has a renoprotective effect on kidney functional decline in the AAN model. This absence of an effect may be attributed to the experimental design such as the experimental period, doses of ETN, or the kidney injury model. Studies using longer and/or different doses of ETN and other kidney injury models, such as adenine-infusion and 5/6 nephrectomy, are needed to address this issue. Third, although the present study demonstrates the anti-fibrotic effect of ETN by suppressing inflammation and apoptosis and inhibiting p38 MAPK signaling, it is unclear which factor contributed more prominently.In conclusion, our findings show that TNF-α inhibition by ETN partially attenuates the development of kidney fibrosis and albuminuria by suppressing kidney inflammation and interstitial cell apoptosis in a mouse model of AAN. Our results support evidence of an anti-fibrotic effect of TNF-α inhibition on kidney fibrosis that has mainly been established by the UUO model. Collectively, these findings indicate a substantial role for the TNF-α pathway in the development of kidney fibrosis and imply that TNF-α inhibition could become an adjunct therapeutic strategy for CKD with fibrosis.This study was performed following the National Institutes of Health guidelines for the use of experimental animals and was reviewed and approved by the Animal Studies Ethics Committee of Yokohama City University. All experiments were performed according to the ARRIVE guidelines. Male C57BL/6 J wild-type mice were purchased from Oriental Yeast Co, Ltd. The mice were housed in a controlled environment with a 12:12 light/dark cycle and ambient temperature (25℃) and humidity. The mice were allowed free access to food and water. They were fed a standard diet .n = 8 mice per group)  and an s.c. injection of ETN (5 mg/kg)51. The ETN dose was determined from previous studies and our preliminary experiment  were randomly assigned to either the control group, AA group, or AA + ETN group , as described previously53. Mice were housed in metabolic cages for 2 consecutive days and given free access to food and water. A 24 h urine collection procedure was performed on day 2.A metabolic cage analysis was performed at the end of the experimental period, as described previouslyBlood samples were collected by cardiac puncture when the mice were euthanized in the fed state. Whole-blood samples were centrifuged at 3,000 rpm at 4 °C for 10 min to separate plasma, which was snap-frozen and stored at -80 °C until use. Plasma Cr, BUN, and urinary Cr were measured using an autoanalyzer . Urinary albumin was measured by an immunoturbidimetric assay .55. Briefly, mouse kidneys were fixed in 4% paraformaldehyde in PBS, incubated overnight at 4 °C, and embedded in paraffin. Sections (4 μm thick) were stained with periodic acid–Schiff (PAS) and PSR. The glomerular area was measured by tracing the outline of the glomerular tuft of at least 50 glomeruli in the cortical fields of PAS-stained specimens. Fibrotic areas were measured digitally using a fluorescence microscope  in the cortical fields of PSR-stained specimens. Immunohistochemistry was performed as described previously56. Sections were incubated with anti‐F4/80 antibodies . The TUNEL assay was conducted using an in situ apoptosis detection kit . Interstitial TUNEL-positive cells were counted in 10 randomly selected cortical fields (magnification: × 200). All measurements were blinded.Histological analyses were performed as described previouslyTotal RNA was extracted from kidney tissues using ISOGEN , and cDNA was synthesized using the SuperScript III First-Strand System , according to the manufacturer’s protocol. Real-time quantitative reverse transcription PCR analysis was performed using an ABI PRISM 7000 Sequence Detection System by incubating the reverse transcription products with the TaqMan PCR Master Mix and designed TaqMan probes . The TaqMan probes used for PCR were Bax, Mm00432051_m1; Bcl-2, Mm00477631_m1; CD68, Mm03047343_m1; collagen type I (Col1a1), Mm00801666_g1; collagen type III (Col3a1), Mm01254476_m1; F4/80, Mm00802529_m1; IL-1β, Mm00434228_m1; IL-6, Mm00446190_m1; MCP-1, Mm00441242_m1; TGF-β, Mm01178820_m1; and TNF-α, Mm00443258_m1. The mRNA levels were normalized to the 18S rRNA control.56. Briefly, kidney tissues were mechanically homogenized in SDS‐containing sample buffer with the complete protease inhibitor cocktail , and were denatured by heating at 95 °C for 5 min. Proteins were quantified using the RC DC protein assay kit . Equal amounts of protein extract were separated by 5–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane using the iBlot Dry Blotting System . The membranes were blocked with 5% skim milk for 1 h at room temperature and probed with specific primary antibodies to p38 MAPK , phosphorylated p38 MAPK . Horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies were added for 1 h at room temperature. The Immobilon Forte Western HRP substrate  was used for detection. The images were captured with auto-exposure, and automatically optimized using ChemiDoc Touch (Bio-Rad Laboratories).Western blotting analysis was performed as described previouslyP < 0.05 was considered significant.Statistical analyses were performed using Prism software version 9.0.1 . The results are presented as means ± SEM. Differences in multiple comparisons were assessed by two-way repeated-measures analysis of variance (ANOVA) for repeated measures data over time, including BW, BP, and HR, and one-way ANOVA was used for the other data. Post hoc analysis was performed using a Dunnett’s test and Tukey’s test when two-way repeated-measures ANOVA or one-way ANOVA indicated a significant difference, respectively. Supplementary Information.


Placentas from preeclamptic women display augmented tumor necrosis factor-alpha (TNF-α) levels with reduced expression of aquaporin 3 (AQP3). However, whether TNF-α modulates AQP3 expression remains to be elucidated. We hypothesize that elevated levels of TNF-α reduce AQP3 expression and negatively impact trophoblastic cell migration. Spontaneously hypertensive rats (SHRs) and Wistar rats (14–16 weeks) were divided into hypertensive and normotensive groups, respectively. Systolic blood pressure (SBP) was measured, and animals mated. In a third group, pregnant SHRs were treated with a TNF-α antagonist, etanercept  on days 0, 6, 12, and 18 of pregnancy. Placentas were collected on the 20th day of pregnancy. Human placental explants, from normotensive pregnancies, were incubated with TNF-α  and/or etanercept (1 μg/ml). Swan 71 cells were incubated with TNF-α (10 ng/ml) and/or etanercept (1 μg/ml) and subjected to the wound healing assay. AQP3 expression was assessed by Western blot and TNF-α levels by ELISA. SBP (mmHg) was elevated in the hypertensive group, and etanercept treatment reduced this parameter. Placental TNF-α levels (pg/ml) were higher in the hypertensive group. AQP3 expression was reduced in the hypertensive group, and etanercept treatment reversed this parameter. Explants submitted to TNF-α exposition displayed reduced expression of AQP3, and etanercept incubation reversed it. Trophoblastic cells incubated with TNF-α showed decreased cell migration and reduced AQP3 expression, and etanercept incubation ameliorated it. Altogether, these data demonstrate that high TNF-α levels negatively modulate AQP3 in placental tissue, impairing cell migration, and its relationship in a pregnancy affected by hypertension. Hypertensive pregnancy is a term commonly used to describe a broad spectrum of conditions, where patients present mild or severe elevations in blood pressure, along with multiple organ dysfunctions . HyperteThe most cited hypothesis about the pathogenesis of hypertensive pregnancies involves reduced placental perfusion in consequence of the inadequate trophoblastic invasion of the myometrium, resulting in poor placental blood supply (hypoperfusion) and diffuse maternal endothelial dysfunction  and the extravillous trophoblast (EVT). While the VT cells cover the chorionic villus and form a syncytium, acting in the fetal–maternal transport, the EVT cells differentiate into interstitial trophoblasts, which can migrate and invade the decidua and the myometrium to transform the maternal spiral arteries. As a result, the spiral arteries acquire the physiological properties necessary to establish adequate maternal blood flow to the developing uteroplacental unit , a pleiotropic cytokine that plays a central role in regulating inflammation, is involved in the pathogenesis of PE, where TNF-α levels are found to be raised in both serum and placental tissue  during placentation and its importance in fetal development have been recognized Female Wistar rats and spontaneously hypertensive rats (SHRs)  were used. The rats were maintained in the animal facility room, at 23 ± 2°C, with 12-h light/dark cycles, fed a standard commercial diet, and free water intake. Blood pressure (BP) was measured by tail-cuff plethysmography after 3 days of acclimatization. To mate, females were housed with Wistar males. Vaginal smears were taken daily, and the day on which spermatozoa were found in the vaginal smear was designated gestational day 0.n = 6) and normotensive group (n = 6), respectively. The third group was composed of pregnant SHRs (n = 8), treated with 0.8 mg/kg etanercept (Embrel®), on days 0, 6, 12, and 18 of pregnancy (DOP) via subcutaneous injection as performed previously  according to the manufacturer's instructions.Hospital Nacional Dr. Prof. Alejandro Posadas, Buenos Aires, Argentina. The investigation conforms to the principles outlined in the Declaration of Helsinki. The patients signed written consent forms before sample collection, and clinical data were collected.This study was approved by the Human Ethics Committee from the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires (EXP-FYB #0045449/2017) and the Ethics Committee from the n = 7) were obtained immediately after vaginal or cesarean delivery /F12 containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 32 mg/ml gentamicin at 37°C in a humidified gas mixture of 5% COExplants were placed in a 24-well plate in the proportion of five explants per well. These were then incubated with complete culture medium containing TNF-α at concentrations of 5, 10, or 20 ng/ml, etanercept at a concentration of 1 μg/ml or vehicle (culture medium) for 16 h.Explant viability was verified by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay. One explant from each well was removed and transferred to new wells and incubated with 0.5 mg/ml MTT solution (Sigma-Aldrich Corp.) at 37°C for 2 h. Each explant was transferred to a new well, and 100 μl of ethanol was added and incubated for 30 min with shaking at room temperature. Absorbance was measured at 595 nm wavelength.Explants' toxicity was verified by the release of the intracellular enzyme lactate dehydrogenase (LDH) into the incubation medium after 16 h. Aliquots of the culture medium (500 μl) from each explant were collected and centrifuged. The supernatant was taken (50 μl) and transferred to new wells where they were incubated with a lactate dehydrogenase (LDH) assay solution at room temperature for 30 min. A stop solution was added, and absorbance was measured at 590 nm wavelength.2 and 95% air. At confluence, cells were arrested in the corresponding medium supplemented with 0.5% FBS and incubated with 10 ng/ml TNF-α. Cell viability was assessed by the MTT assay as described previously . Proteins (50–60 μg) were loaded and separated by electrophoresis on a 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma-Aldrich). Non-specific binding sites were blocked with 5% skim dry milk in Tris-buffered saline solution with Tween-20  for 1 h at room temperature. Membranes were rinsed and incubated with an anti-AQP3 antibody  followed by incubation with a peroxidase-conjugated secondary antibody .Protein bands were detected using the ECL Plus Western Blotting Detection System  and then quantified using an image analysis software program .2 and 95% air. Once 90% of cell confluence was observed in the plate wells, a wound was made in each cell monolayer, across the center of the wells, using a 200-μl pipette tip. Then, cells were arrested in the corresponding medium supplemented with 0.5% FBS and incubated with 10 ng/ml TNF-α, 1 μg/ml etanercept, or vehicle (culture medium).Swan 71 cells were cultured in a 24-well plate in complete culture medium at 37°C for 24 h in a humidified gas mixture of 5% CO2) using an image analysis software and presented by percentage. The scratch distance at 0 h was considered 0% of gap closure. For migration speed, we measured the width of the wound as the mean distance between the edges of the scratch in each photograph, and this change in mean wound width was divided by the time spent on each migration.Wounded monolayers were examined at 0, 18, and 24 h and photographed using an optical microscope with a camera attached. Wound closure was measured by calculating the area . The significance of the results was analyzed by Student's p < 0.0001) and higher proportion of fetuses classified as SGA  compared to Wistar rats  and systolic blood pressure (SBP) was higher  in SHRs compared to Wistar rats. Interestingly, when we treated SHRs with etanercept, the SBP was found to be reduced but not normalized [163 ± 2 mmHg; p = 0.003  to evaluate AQP3 expression. Treatment with TNF-α did not affect explants' viability or toxicity . TNF-α rin vitro experiments demonstrated that incubation with 10 ng/ml TNF-α impaired wound healing  cells through inhibition of constitutive transcriptional activity of the AQP3 promoter . The arrangement of CTBs forms multinucleated syncytiotrophoblasts. During pregnancy progression, the CTB differentiates into the EVT, a cell type with migration properties that can invade the uterine wall. The reduced AQP3 expression observed in placental explants indicates that this event is happening before trophoblast differentiation into VT and EVT, probably being related to the most severe cases of PE, known as early-onset PE , on trophoblastic cell migration has been under study. IL-6, IL-8, IL-11, and IL-1β were found to stimulate in vitro trophoblast migration (Bauer et al., Evidence shows that AQPs may be involved in tumor cell migration (Cao et al., In conclusion, the present study demonstrates, for the first time, the harmful modulation of AQP3 by augmented TNF-α levels in placental tissue, affecting trophoblast cell migration. This relationship is also observed in placental tissue from hypertensive pregnancy. Altogether, these observations contribute to a better understanding of placental impairment during hypertensive disorders of pregnancy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Ethics Committee from the Faculty of Pharmacy and Biochemistry of the University of Buenos Aires. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Committee of Ethics in Animal from the Federal University of Mato Grosso.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Background: Muscle atrophy is a complex catabolic condition developing under different inflammatory-related systemic diseases resulting in wasting of muscle tissue. While the knowledge of the molecular background of muscle atrophy has developed in recent years, how the atrophic conditions affect the long non-coding RNA (lncRNAs) machinery and the exact participation of the latter in the mediation of muscle loss are still unknown. The purpose of the study was to assess how inflammatory condition developing under the tumor necrosis factor alpha (TNF-α) treatment affects the lncRNAs’ expression in a mouse skeletal muscle cell line. Materials and method: A C2C12 mouse myoblast cell line was treated with TNF-α to develop atrophy, and inflammatory-related lncRNAs mediating muscle loss were identified. Bioinformatics was used to validate and analyze the discovered lncRNAs. The differences in their expression under different TNF-α concentrations and treatment times were investigated. Results: Five lncRNAs were identified in a discovery set as atrophy related and then validated. Three lncRNAs, Gm4117, Ccdc41os1, and 5830418P13Rik, were selected as being significant for inflammatory-related myotube atrophy. Dynamics changes in the expression of lncRNAs depended on both TNF-α concentration and treatment time. Bioinformatics analysis revealed the mRNA and miRNA target for selected lncRNAs and their putative involvement in the molecular processes related to muscle atrophy. Conclusions: The inflammatory condition developing in the myotube under the TNF-α treatment affects the alteration of lncRNAs’ expression pattern. Experimental and bioinformatics testing suggested the prospective role of lncRNAs in the mediation of muscle loss under an inflammatory state. Skeletal muscle atrophy is a complex process resulting from an imbalance between anabolic and catabolic conditions. It is characterized by a decreased protein synthesis and enhanced proteolysis within the myocyte particularly caused by the hyperactivation of major cellular degradation pathways, including the ubiquitin-proteasome and autophagy-lysosomal systems ,2,3. TheThe pathogenesis of muscle atrophy is composite, but the corresponding hyperactivation of a systemic inflammatory response is a frequent event observed during chronic diseases. Thus, the elevation of pro-inflammatory cytokines’ level is thought to play an apparent role for muscle wasting . In partThe knowledge of muscle atrophy and its molecular background has developed in recent years. However, how the atrophic conditions affect the non-coding RNA (ncRNA) machinery and the exact participation of the regulatory network of the latter in the mediation of muscle wasting are still unknown. Among the ncRNAs, a group of molecules longer than 200 bases in length, called long non-coding RNAs (lncRNAs), seems to play an important role in the regulation of gene expression and management of cellular processes via different mechanisms, such as miRNA sponging or interaction with DNA and proteins. Even though lncRNAs do not encode proteins, their transcripts modulate diverse cellular processes, including cell differentiation, apoptosis, and autophagy ,14.According to recent findings, some lncRNAs are required for skeletal muscle formation and terminal differentiation of myoblasts, such as SRA and MUNC . The lncIn p < 0.001). Constructing chord diagrams, we visualized the network of the contribution between DElncRNA and the atrophy and non-atrophyclusters  lncRNAs were downregulated and 103 (58.6%) were upregulated in atrophy C2C12 myotubes treated with TNF-α comparing with control cells. The generated heat map illustrating DElncRNA(only 84 lncRNAs demonstrating at least 2-fold difference in expression between treated and untreated cells) with a hierarchical clustering between three untreated and three cell lines treated with TNF-α is demonstrated in clusters D. Additip < 0.001) (p < 0.001)  A. Second< 0.001) B.Top DElncRNAs were screened in order to discover a regulatory network and interaction with mRNA and miRNA with the application of GO and KEGG. First, we predicted the mRNA targets for the top DElncRNA with the aforementioned bioinformatics tools. Thereafter, the regulatory PPI network was constructed with the use of String and ShinyGO. Regarding the sponging ability of the top DElncRNA for miRNA, the role of the predicted small ncRNAs was investigated in the signaling pathways.p-value <1.0 × 10−16) (−26), gene expression (FDR = 1.48 × 10−22), and RNA transport (FDR = 2.07 × 10−16) werethe most enriched GO terms related to the biological processes, the structural constituent of ribosome (FDR = 1.03 × 10−35) for molecular function and mitochondrial ribosome (FDR = 3.13 × 10−53), ribosome (FDR = 3.43 × 10−48), and ribonucleoprotein complex (FDR = 2.56 × 10−46) for the cellular component. The top, most significantly enriched GO terms including biological process, molecular function, and cellular component arepresented in −28), RNA transport (FDR = 1.61 × 10−10), and spliceosome (FDR = 6.08 × 10−10) were three significantly enriched pathways (−7) and miRNA in cancer (FDR = 2.15 × 10−6). Interestingly, KEGG also emerged for FoxO signaling (FDR = 3.63 × 10−7) and TNF signaling (FDR = 3.0 × 10−3) as significantly enriched pathways interacting with the miRNA-467/669 family.Data derived from bioinformatics software predicted the mRNA of Npm1 (nucleophosmin 1), Thoc3 (THO complex 3), and Mrpl42  proteins as targets for Gm4117. Based on the prediction score, the PPI network was constructed. The PPI network that interacted with Gm4117 consisted of 43 nodes and 489 edges (enrichment × 10−16) A. Accordpathways C. Using p-value < 1.0 × 10−16) (−21) and transcription DNA-templated (FDR = 4.89 × 10−16); adenylate cyclase activity (FDR = 2.22 × 10−14) was a top GO term related to molecular function. Regarding the cellular component, the mediator complex (FDR = 2.35 × 10−52) and ubiquitin ligase complex (FDR = 4.15 × 10−16) were the most significantly enriched GO terms. A summary of the top GO enrichment terms is shown in −16) and GABAergic synapse (FDR = 3.17 × 10−9) were significantly enriched pathways (−7), MAPK pathway (FDR = 1.1 × 10−5), and proteoglycans in cancer (FDR = 1.1 × 10−5).Ccdc41os1 contributes to the regulation of the network between Cdk19 (cyclin-dependent kinase 19), Cgref1 (cell growth regulator with EF-hand domain 1), Bscl2 , and Reep1 (receptor accessory protein 1). The constructed PPI network consisted of 54 nodes and 648 edges (enrichment × 10−16) A. The mopathways C. A totapathways D. Ccdc41−30) and cellular protein modification process (FDR = 1.85 × 10−23), whereas response to stress (FDR = 7.87 × 10−3) and catabolic process (FDR = 1.93 × 10−11) were highly related terms for biological function. As for the cellular component, chromosome organization (FDR = 2.98 × 10−17) was the most significantly enriched GO term. Regarding KEGG terms, pantothenate and CoA biosynthesis (FDR = 2.29 × 10−3), central carbon metabolism in cancer (FDR = 5.90 × 10−3), and lysine degradation (FDR = 6.88 × 10−3) were significant terms for this analysis.According to the DIANA–LncBase Predicted v2 tool, 5830418P13Rik is especially capable to target the miRNA-466 and -669 families . A direct analysis of the lncRNA targets did not reveal significantly affected mRNAs. Considering the 5830418P13Rik miRNAs’ regulatory network, the top GO and KEGG enrichment terms were indentified. The top GO terms related to molecular function were cell differentiation  or 8W10E plates for cell viability measurement in ECIS® (Electric Cell-substrate Impedance Sensing). After reaching 80% confluence, DMEM with FBS was replaced by Differentiation Media (DM) containing 2% horse serum  and the abovementioned antibiotic mixture for 5 days to induce myoblast differentiation into myotubes. DM was changed every 48 h. After the differentiation. the cells in the atrophy model were treated with TNF-α and the control cells were treated with only free medium. Each study set was repeated in triplicate.The C2C12 mouse myoblast cell line was purchased from ATCC  and cultured in Dulbecco’s Modified Eagle Medium (DMEM)  containing 10% fetal bovine serum (FBS) , 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a humidified 5% CO5 cells/mL per well and again placed in an incubator space under appropriate conditions. After 24 h, the medium was replaced by a differentiation medium containing 2% donor horse serum and treated under different concentrations of TNF-α. During the experiment, the cells’ survival was monitored for 72 h with ECIS software. The difference between the C2C12 cell cultures’ (treated and untreated) viability was compared using a one-way ANOVA test.In the literature data, C2C12 myotube atrophy was established under different TNF-α concentrations and times of cell treatment. It mainly depended on cell confluence and the method on cell culturing: plates or flasks. Usually, the concentration of 10–100 ng/mL of TNF-α is used for atrophy model establishment with the treatment time ranging between 24 and 72 h. Prior to cells’ treatment with TNF-α, the myoblasts were cultured to about 80–90% confluence. In the discovery set, inflammatory-induced atrophy was performed by treating differentiated myotubes once with the addition of 100 ng/mL of mouse TNF-α recombinant protein  for 72 h. In the validation set, myotubes of the C2C12 cell line were treated with the following TNF-α concentrations: 10, 50, 100, and 200 ng/mL, respectively, for 72 h and under 100 ng/mL of TNF-α in 12- and 24-h time intervals, respectively, in order to monitor the dynamics in the lncRNA expression within the time interval and under different cytokine concentrations. The atrophy was established by a combination of cells’ degradation confirmed in microscopy examination, cell viability assay, and expression of muscle atrophy-related markers . Express−ΔΔCt algorithm with normalization of the raw data to housekeeping genes (set of 12 controls). All samples were analyzed in triplicates. A heat map of the lncRNA expression log values and the hierarchical clustering were conducted with the use of Morpheus software. To display statistical significance  versus fold change in order to identify key lncRNAs in the study set, the volcano plot was applied with the use of VolcaNoseR version2 software [p < 0.05 and fold log2 values < −2 and >2.RNeasy Micro Kit  was used for total RNA purification from the harvested cells. Prior to the molecular experiments, all RNA samples were measured to determine the concentration, yield, and purity. Samples that passed quality measurements were diluted to 100 ng/mL each. A combination of lncRNA PCR Profiler Array Mouse Inflammatory Response, Autoimmunity, Cell Development, and Differentiation  allowed us to determine the expression in a total of 176 lncRNAs in a StepOnePlus device . Data analysis was based on the 2software . ExpressThe lncRNAs selected from the discovery set were independently validated by qRT-PCR in a StepOnePlus device  to measure changes in their expression in time intervals and under the different TNF-α concentrations. Total RNA was purified both from a set of cell cultures treated with concentrations of 10, 50, 100, and 200 ng/mL TNF-α, respectively for 72 h and from a set of cell cultures after 12-, 24-, 48-, and 72-h treatment with 100 ng/mL TNF-α, respectively. For each experiment, cell lines were cultured in triplicates. One-way ANOVA test was used to compare differences in lncRNAs’ expression between C2C12 cell cultures (treated and untreated).Based on the mRNA–lncRNA and miRNA–lncRNA interactions, regulatory networks were searched with the available bioinformatics tools. IDs and sequences of previously selected mice DElncRNA were derived from RNAcentral and Ensembl databases. To predict the interaction between DElncRNA and mRNA, the ENCORI (the Encyclopedia of RNA Interactomes) and Mouse RGD tools were applied ,36,37. TDElncRNA-mRNA regulatory networks were built, and the protein–protein interaction (PPI) network between them was constructed. The String v11.5 is a tool used for visualizing and searching for interactions between predicted and known proteins’ sequences . Gene OnThe knowledge of the role of lncRNAsin the mediation of muscle loss is still limited because of the different mechanisms that can trigger muscle atrophy. Perhaps different atrophic conditions can selectively alter individual cellular pathways and change the expression pattern of ncRNAs. We are aware that our study is not free from limitations. We used a single factor to develop atrophy; however, atrophic conditions develop usually upon action of various factors. We also reliedon in vitro results, and the findings of the experiments and the bioinformatics analysis should be confirmed by in vivo models. Moreover, results should be confirmed by siRNA/knockdown experiments. Nevertheless, in our study we demonstrated that muscle atrophy developing under inflammation mediated by TNF-α affects the alteration in lncRNAs’ machinery managing muscle conditions, and novel lncRNAs exclusive for mediation of inflammatory-related muscle loss were selected. This observation encourages the further investigation of lncRNAs in order to estimate their role in muscle atrophy and to broaden our knowledge of their role in the same.


Social media has been extensively used for the communication of health-related information and consecutively for the potential spread of medical misinformation. Conventional systematic reviews have been published on this topic to identify original articles and to summarize their methodological approaches and themes. A bibliometric study could complement their findings, for instance, by evaluating the geographical distribution of the publications and determining if they were well cited and disseminated in high-impact journals.The aim of this study was to perform a bibliometric analysis of the current literature to discover the prevalent trends and topics related to medical misinformation on social media.The Web of Science Core Collection electronic database was accessed to identify relevant papers with the following search string: ALL=(misinformati* OR “wrong informati*” OR disinformati* OR “misleading informati*” OR “fake news*”) AND ALL= AND ALL=. Full records were exported to a bibliometric software, VOSviewer, to link bibliographic information with citation data. Term and keyword maps were created to illustrate recurring terms and keywords.Based on an analysis of 529 papers on medical and health-related misinformation on social media, we found that the most popularly investigated social media platforms were Twitter (n=90), YouTube (n=67), and Facebook (n=57). Articles targeting these 3 platforms had higher citations per paper (>13.7) than articles covering other social media platforms . Moreover, social media platform–specific papers accounted for 44.1% (233/529) of all identified publications. Investigations on these platforms had different foci. Twitter-based research explored cyberchondria and hypochondriasis, YouTube-based research explored tobacco smoking, and Facebook-based research studied vaccine hesitancy related to autism. COVID-19 was a common topic investigated across all platforms. Overall, the United States contributed to half of all identified papers, and 80% of the top 10 most productive institutions were based in this country. The identified papers were mostly published in journals of the categories public environmental and occupational health, communication, health care sciences services, medical informatics, and medicine general internal, with the top journal being the Journal of Medical Internet Research.There is a significant platform-specific topic preference for social media investigations on medical misinformation. With a large population of internet users from China, it may be reasonably expected that Weibo, WeChat, and TikTok (and its Chinese version Douyin) would be more investigated in future studies. Currently, these platforms present research gaps that leave their usage and information dissemination warranting further evaluation. Future studies should also include social platforms targeting non-English users to provide a wider global perspective. Public health information has been traditionally distributed to the public with the use of printed media, television, or radio. With the rise of participatory web and social media  and partWhile providing numerous empowerment opportunities, there lies a great potential for miscommunication and misinformation  within tVaxxed in 2016 [One of the most polarizing topics in recent years has been vaccination, following a scientific article from 1998 by Wakefield et al, which proposed a causative link between the measles, mumps, and rubella (MMR) vaccine and autism ,10. The  in 2016 . Similar in 2016 . This isAs medical misinformation is an increasingly relevant topic to study, the amount of available literature is growing. On this background, the aim of this study was to perform a bibliometric analysis of the current literature to discover the prevalent trends and topics related to medical misinformation on social media. Conventional systematic reviews have been published on this topic to identify original articles and summarize their methodological approaches and themes ,21. A biA bibliometric analysis is a study that applies mathematical and statistical methods to books and other media of communication, such as academic publications . SimilarThe “Analyze” function of WoS was used to provide initial descriptive statistics regarding the bibliographic data. The numbers of social media platform–specific papers were counted. The approach applied to Facebook is presented here as an example for the used evaluation strategy. In particular, we additionally searched with the following search string: ALL= Facebook* NOT (Twitter* OR Instagram* OR YouTube* OR Weibo* OR Whatsapp* OR Reddit* OR TikTok* OR WeChat*). When the original search string and this new search string were combined with the Boolean operator “AND,” the resulting papers mentioned Facebook but not the other referenced social media.We evaluated the publication and citation counts of contributors in terms of author, institution, country, and journal. We also computed the publication and citation counts of terms and keywords, and identified the top 10 most cited papers. The semantic content of the identified publications was analyzed in the following ways. Citations per paper (CPPs) were computed for terms occurring in the titles, abstracts, and keywords of the identified papers, and n-gram analysis was conducted to identify the most recurring metatext. These analyses aimed to answer the queries listed at the end of the Introduction. Further details are described below.The 529 identified papers were exported in full record with cited references to VOSviewer ,28 for sIn addition, keyword maps were produced for subsets of papers that were specific to Twitter, YouTube, and Facebook. For these maps, keywords with at least two appearances were included.Finally, an exploratory n-gram analysis was conducted with the online NGram Analyzer  that allOur search strategy identified a total of 529 scientific articles addressing medical misinformation on social media. The analysis of these papers revealed that the earliest papers on this subject could be traced back to 2010 and 2011, and the total publication and citation counts increased very rapidly, especially during the last 2 years . OriginaThe most productive author publishing in this subject area was Emily K Vraga from George Mason University . She started to publish on this topic in 2015 and accumulated a total of 13 papers, mostly with Leticia Bode and Melissa Tully. Leticia Bode and Joseph A Hill followed second in the list of the most productive researchers, with 9 papers each. Following them were 27 authors with 7 papers each. The top 10 most productive institutions, countries, journals, and WoS categories in which the analyzed works were published are listed in Social media platform–specific papers accounted for 44.1% (n=233) of all 529 identified papers . The mosA keyword map is shown in Keyword maps generated for the publication set that investigated Twitter, YouTube, and Facebook are shown in The top 10 most cited papers are listed in The exploratory n-gram analysis resulted in several meaningful 5-gram metatexts with at least four appearances as follows: 6 appearances, “as a source of information” and “the spread of fake news;” 5 appearances, “rumors stigma and conspiracy theories,” “the quality of health information,” and “content reliability and quality of;” 4 appearances, “health anxiety and health literacy,” “the relationship between message factors,” “intentions to trust and share,” “#PedsICU and coronavirus disease 2019,” “in low- and middle-income countries,” “actions for a framework for,” “interacted with perceived message importance,” and “verify and share the message.”Using bibliometric analysis, this study identified and quantitatively analyzed 529 papers on medical and health-related misinformation on social media, revealing the most popularly investigated social media platforms, prevailing research themes, most utilized journals, and most productive countries, institutions, and authors.This bibliometric analysis on 529 scientific articles concerning medical and health-related misinformation on social media revealed that the most heavily investigated platforms were Twitter, YouTube, and Facebook. This could be related to the finding that most of the top 10 productive countries were from North America and Europe where these social media platforms were dominant. The results also confirmed the hypothesis that the United States had the largest contribution in social media research. The total publication and citation counts increased very rapidly especially during the last 2 years, consistent with the trends identified by previous systematic reviews on this topic ,21. On tThough some social media platforms were not available in China, China still made it into the top 10 list of the most productive countries . With a The most productive author was found to be Emily K Vraga who is based in the United States. Her studies focused on how to correct health misinformation  dispersed on social media, particularly Facebook and Twitter ,41-43. TA very important role of social media is to provide peer-to-peer support, as investigated by some publications identified in this study see  and 5, fMeanwhile, from the perspective of policy makers, the large amount of information from social media can be monitored and used for the achievement of efficient outcomes. As seen from the results, “health policy services” was among the most recurring journal categories for the analyzed literature set  and “polAs mentioned in the Introduction, the fraudulent study linking the MMR vaccine to autism still has a lingering influence on social media, as it is still posted on the Facebook platform by antivaccine advocates, despite its retraction due to fraudulency . MoreoveAny publications in journals not indexed by WoS were missed in the current analysis. For example, there is a relevant paper investigating misinformation of COVID-19 on TikTok, which is not indexed by WoS . BesidesFor potential future research, artificial intelligence (AI) applications for social media content analysis would be an especially promising avenue. With increasing content and misinformation circulating on social media, it becomes practically impossible to manually determine and classify misinformation. AI or machine learning might be employed for such content analysis, which has the potential to achieve high accuracy . Yet, AIBased on an analysis of 529 papers on medical and health-related misinformation on social media, we found that the United States contributed to half of the papers, with 80% of the top 10 most productive institutions being based in this country. The papers were mostly published in journals belonging to the categories public environmental and occupational health, communication, health care sciences services, medical informatics, and medicine general internal. However, they were generally less cited than papers published in immunology, suggesting that more publications did not warrant more citations. Social media platform–specific papers accounted for 44% of all papers. The most popularly investigated social media platforms were Twitter, YouTube, and Facebook. They also had higher CPPs than other social media. Investigations on these platforms had different foci. Twitter-based research investigated cyberchondria and hypochondriasis, YouTube-based research investigated tobacco smoking, and Facebook-based research investigated vaccine hesitancy related to autism. COVID-19 was a common topic investigated across all platforms. An important implication of these findings is that often knowledge on specific themes related to medical misinformation relies on the predominant study of a single social media platform or limited number of platforms, and broader cross-platform studies could be a promising direction for future research. Future studies should also include social platforms aimed at non-English users to provide a wider perspective on global health misinformation.


Realgar has been used as a type of mineral drug that contains arsenic for thousands of years. Previous studies have shown that Realgar-induced acute kidney injury is associated with abnormal metabolism, but the underlying mechanism is poorly understood. The aim of this study is to investigate the metabolic changes in serum and kidney tissues of mice exposed to Realgar by using a metabolomic approach and explore the molecular mechanisms of acute kidney injury induced by Realgar. Forty mice were randomly divided into four groups: Control group, 0.5-, 1.0, and 2.0 g/kg Realgar group. After 1 week, the body weight and kidney weight of the mice were measured. The serum and kidney samples were used for LC-MS spectroscopic metabolic profiling. Principal component analysis (PCA), correlation analysis, and pathway analysis were used to detect the nephrotoxic effects of Realgar. Body weight decreased significantly in the 2.0 g/kg group, and the kidney weight index also showed a dose-dependent increase in Realgar. The PCA score plot showed the serum and kidney tissue metabolic profile of mice exposed to 2.0 g/kg Realgar separated from the control group, while the lower-doses of 0.5 g/kg and 1.0 g/kg Realgar shown a similar view to the Control group. Thirty-three metabolites and seventeen metabolites were screened and identified in the serum and kidney of mice in a dose-dependent manner. respectively. Correlation analysis showed a strong correlation among these metabolites. Amino acid metabolism, lipid metabolism, glutathione metabolism, and purine metabolism pathways were found to be mainly associated with Realgar nephrotoxicity. This work illustrated the metabolic alterations in Realgar-induced nephrotoxic mice through a metabolomic approach. Arsenic has long been known as a toxic compound and environmental carcinogen, which exposure causes oxidative stress, apoptosis, chromosomal abnormalities, and DNA damage . However4S4), has been widely used to treat infection, fever, and convulsion  of complex systems. This consists of the holistic thinking of TCMs and metabolomics techniques are extremely suitable for evaluating the toxicity of traditional Chinese medicines and exploring the mechanisms . RecentlIn this study, the nephrotoxic mechanism of Realgar was investigated by an untargeted metabolomic approach to analyze serum and kidney samples collected from control mice and mice treated with different doses of Realgar, which is expected to deepen the understanding of metabolic changes after exposure to Realgar.Realgar was provided and identified by the Shanghai Institute for Food and Drug Control  with 73.7% total arsenitc determined by Atomic Absorption Spectroscopy. Sodium carboxymethycellulose (CMC-Na) was purchased from Sinopharm . Mass spectrometry reagents including methanol, acetonitrile and formic acid were purchased from Sigma Chemical Co. .Male 6-week-old ICR mice weighing about 18–20 g were provided by the Experimental Animal Center of Shanghai Institute for Food and Drug Control . Mice were maintained at temperature (20–25°C) and 40–60% relative humidity for a 12-h light/dark cycle. They were fed freely water and diet, after 1 week of acclimatization, the mice were randomly divided into four groups for the treatment of 0.5% CMC-Na (Control group), Realgar suspended in 0.5% CMC-Na , respectively. The doses of Realar were referred to the basis of previously published literature . The detSerum samples were collected after following oral feeding Realgar for 30 min, then 50 μL of serum was mixed with 200 μL pre-cooled methanol and vortex mixed for 10 min. The mixture was then centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatant is dried with nitrogen and reconstituted with 20% methanol. The supernatant was centrifuged at 14,000 rpm for 15 min at 4°C and used to analyze by LC-MS. A mixture of equal quantities was extracted from all samples and used as quality control (QC) sample for the LC-MS analysis.The kidney tissues were dried by lyophilization and ground into powder. The powder (10 mg) was homogenized with pre-cooled 80% methanol (300 µL), mixed by vortexing, and subjected to sonication for 10 min on iced water (×3). The homogenate was then incubated at −20°C for 30 min and centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant is dried with nitrogen and reconstituted with 80% methanol and centrifuged at 14,000 rpm at 4°C for 15 min. The supernatant was added to the sample bottle for detection. A mixture of equal quantities was extracted from all samples and used as QC sample for the LC-MS analysis.® BEH C18 column , coupled with Orbitrap Fusion Lumos high-resolution mass spectrometer . The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) with running a gradient elution: 5% phase B for 4 min; 5–70% phase B for 7 min, 70–95% phase B for 15 min, 95% for 17 min, 95–5% phase B for 20 min at the flow rate of 0.2 ml/min. The injection volume was 5 μL. The MS signal acquisition was performed in positive and negative ion scanning modes respectively. One primary mass spectrometry scan resolution is 15,000 and secondary mass spectrometry scans resolution is 6,000, and both the first and secondary scanning ranges were 105 m/z—1,050 m/z. A QC sample was acquired for every six analytical samples to evaluate the stability of the analytical system and assess the reliability of the results.Kidney metabolic profiling adopted an UltiMate 3,000 ultra-high-performance LC system (Thermo Fisher Scientific) ACQUITY UPLCp < 0.05 was considered significant differences. The mass spectrometry-generated metabolomics data is in RAW file format. The quantitative analysis of the compound was conducted using Compound Discoverer Software 3.2 . The data was subject to principle components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using SIMCA-P version 14.1 software. The metabolites with VIP >1 and p < 0.05 were considered as potential biomarkers, and the online databases HMDB (http://www.hmdb.ca) were used to identify the compounds by matching the mass spectrum. Pathway enrichment was acquired according to the MetaboAnalyst 5.0  and the Kyoto Encyclopedia of Genes and Genomes  database.All data were presented as the mean ± SEM. The comparisons between two groups were performed with an unpaired two-tailed Student’s t-test using SPSS 23.0. The comparisons between several groups were performed with a one-way analysis of variance (ANOVA) using SPSS 23.0. Value of p < 0.05) and increased kidney weight index (p > 0.05).The body mass and kidney index of mice are shown in The PCA score plots shown good system stability of the analyses since the QC samples were tightly clustered in the serum and kidney samples , and allThe 2.0 g/kg dose group revealed a significant change of metabolites in comparison to the control group, so the OPLS-DA was used to monitor the changes of metabolites in the 2.0 g/kg dose group and the control group . R2X cu. As a vip value less than 0.05 is considered as a significant difference in the candidate metabolite between the Control group and Realgar groups; 3) online databases HMDB (http://www.hmdb.ca) was searched to characterize differential metabolites by matching the mass spectrum; and 4) an error of less than 5 ppm. Following the above criteria, then thirty-three metabolites from serum samples and twenty-three from kidney samples were identified considered as potential biomarkers  the variables with VIP value more than the one is an important screening parameter; 2) the omarkers . In ordeomarkers . To furtomarkers . The treTo further understand the relationships between metabolites, Pearson’s correlation analysis was performed and shown in To further clarify the possible metabolic pathways that were affected by Realgar, the metabolites in serum and kidney tissues were integrated and imported into Metaboanalyst 5.0 for pathway analysis, and the pathway was filtered by the parameter Impact >0. As shown in The traditional toxicity evaluations showed that Realgar treatments elicited nephrotoxicity, as characterized by elevated blood urea nitrogen, creatinine levels in serum, and kidney histopathologic alterations. These have been confirmed in our previous studies, and Realgar at a high dose induced more severe acute kidney injury than exposure to a low dose . In thisHigh-resolution mass spectrometry combined with metabolomics was used to analyze metabolite alterations in serum and kidney of Realgar mice. Thirty-three endogenous metabolites in serum and seventeen endogenous metabolites in the kidney were selected as toxic biomarkers, which are mainly involved in amino acid metabolism, Lipid metabolism, Glutathione metabolism, and Purine metabolism. Amino acid metabolism, Glautathione metabolism, and Purine metabolism were the same metabolic pathways between serum and kidney, and Lipid metabolism might be partly attributed to oxidative stress caused by Realgar. Therefore, these four pathways were of great attention to us. The mechanism of toxicity was proposed by analyzing their formation and transformation in the pathway .Taurine has many different biological functions, bile acid binding, antioxidant, etc., which are important for organism health and disease. Taurine has been reported to have a protective effect against drug-induced nephrotoxicity . In RealHistidine, Citrulline, Ornithine were only detected in serum samples, and the disruption of amino acid metabolism was observed mainly in serum samples. Histidine is one of the 22 proteinogenic amino acids and can be catabolized to glutamate, which has antioxidant properties . In this2+ concentration and induce ROS production to initially induce oxidative stress , an important intracellular regulatory metabolite, is involved in several reaction systems, especially in defense against oxidative stress. In our study, pathway analysis showed that metabolites in serum and metabolites in the kidney simultaneously regulate glutathione metabolism. As shown in Histidine metabolic disruption has been observed in this study, and its metabolite AICAR activates the purine metabolic process . Most ofThis study provided more comprehensive information about the toxicity of Realgar through testing both serum and renal metabolic profiling. However, limitations of this study need to be further addressed. Firstly, serum metabolites come from multiple organs, such as kidney, liver, intestine and so on. Therefore, this result is not specific for reflecting the nephrotoxicity of Realgar. This is one of the tasks that our research group is currently preparing to carry out. Secondly, some metabolites may be not associated with Realgar, but due to acute kidney injury. Acute kidney injury animal model will be established to screen out specific metabolites by comparing Realgar-induced acute kidney injury with other causes-induced acute kidney injury. In addition, we are also trying to find clinical samples of acute kidney injury patients.This work aimed to study Realgar-induced acute kidney injury through using a high-resolution mass spectrometry technology combined with a metabolomics approach. LC-MS analysis of mice serum and kidney highlighted some disturbances in the endogenous metabolite profile, which may be related to disturbances in the biochemical pathways of acute kidney injury induced by Realgar. In the study, amino acid metabolism is most affected by Realgar. Lipid metabolism, Glutathione metabolism, and Purine metabolism were also affected by Realgar. The high-resolution mass spectrometry combined with metabolomics can capture and explore the metabolic alterations induced by traditional Chinese medicines in toxicological effects and can be used to further investigate the toxicity mechanisms of traditional Chinese medicines.


The mean age of participants was 38.85 ± 11.56 years. The mean duration of snuff use was 20.43 ± 12.79 years and the common site for Naswar placement was the lower vestibule . TNF-α levels among SD were 9.6 ± 3.3 pg/mL, which were significantly higher than levels in NSD, 5.2 ± 3 pg/mL (p < 0.05). The number of cellular micronuclei in SD was 30.7 ± 7.8, which was comparatively higher than in NSD, which was 9.2 ± 3.3 (p < 0.05). The duration of snuff use was positively correlated to TNF-α levels (p = 0.048) rather than the micronuclei number (p = 0.97). SD showed higher levels of TNF-α and cellular micronuclei compared with NSD (control subjects); a positive correlation was shown with the duration of snuff use. We conclude that TNF-α and micronuclei are potential salivary biomarkers for an oral biological effect in snuff (Naswar) users.The aim of the study was to investigate the unstimulated whole saliva (UWS) tumor necrosis factor alpha (TNF-α) and cellular micronuclei in snuff dippers (Naswar) compared to healthy control subjects. The case control study was conducted over 9 months at a tertiary care center. Sixty patients were divided into two groups: Snuff dippers (SD) (Naswar) and non-snuff dippers (NSD) (control subjects). The included self-reported SD used Snuff twice daily for more than 12 months. UWS was collected and salivary TNF-α assessment was performed using enzyme-linked immunosorbent assay (ELISA). For cellular micronuclei, buccal mucosa was brushed to obtain cells in Naswar users, fixed with a dibutylphthalate polystyrene xylene (DPX) mounting to view micronuclei. Means and standard deviations were compared using the t-test and outcomes were related using Pearson correlation, considering  Oral cancer includes lesions of the oral cavity, oropharynx, and salivary glands. Oral squamous cell carcinoma (OSSC) is the most frequent oral cancer, comprising more than 90% of all oral cancers . The morNaswar is a niche powdered snuff, frequently used in South and Central Asia regions and countries including Pakistan, India, and Sri Lanka . FrequenThe literature has stated that a high level of salivary tumor necrosis factor alpha (TNF-α) acts as an early indicator for the diagnosis of OSCC in contrast to histopathological review . TNF-α iRegardless of recent advances, the diagnosis of OSCC is often reported in the late stages of cancer, which reduces the survival rate and compromises prognosis . Thus, sThe protocol for the study was designed under the standards of the Helsinki declaration (1964) and approved by the Prime Foundation institutional review board, Peshawar, Pakistan (Prime/IRB/2020-258). Subsequent to the approval of the protocol, the participants were presented with informed consent for their voluntary participation. Each patient was given the right to withdraw without any consequences. The study was reported using the STROBE guideline .The analytical case control study was conducted over a period of 9 months in 2019 at the outpatient department of a tertiary care institute. A total of 60 patients, age ranging from 18 to 62 years without any history of oral cancer was recruited to participate in the study. The patients were divided into two groups based on the history of snuff dippers (Naswar) and non-snuff dippers (NSD) (control subjects).After noting previous abuse history, medical status, and clinical examination, patients were categorized into groups based on snuff dipping. Self-reported SD were using snuff (Naswar) twice daily for more than 12 months without any mucosal lesions and systematic disease. The self-reported SD group participants had an intact oral mucosa i.e., without premalignant and frank malignant lesions. Self-reported NSD were individuals who did not habitually use snuff in the last 12 months (control subjects). Excluded individuals included patients with a history of abuse except for snuff, chronic periodontitis, alcoholics, workers associated with occupational hazards, and those with incomplete consent forms. In addition, patients with a history of oral lesions, pathological dry mouth syndrome or medications such as antihistamines, antihypertensives, anticholinergics, antidepressants, and bronchodilators were also excluded from the study due to a hindrance in the ability to collect sufficient unstimulated saliva samples.All included subjects completed a questionnaire to gather critical information prior to clinical examination. The questionnaire included information to be completed by patients self-reporting on their age, gender, abuse history (duration of snuff use and the site of snuff placement), and medical history, including drug use. Furthermore, an experienced senior clinical consultant (W.M.) undertook an oral examination, including assessment of oral hygiene and periodontal screening using the community periodontal index (CPI) (WHO standard) to assess the presence or absence of gingival bleeding, calculus, or deep pockets . The orag for 10 min to filter debris. Saliva was transported to the pathology laboratory and stored at −80 °C.Patients were requested to perform a mouth rinse (3 min) with distilled water before proceeding with the saliva and epithelial cells collection from 10:00 a.m. to 12:00 p.m. After 10 min, the patient’s saliva (2‒3 mL) was collected in a sterile Falcon tube of 5 mL, under non-stimulatory conditions. The process included swallowing first, followed by a forward head tilt, and spitting out all UWS into the sterile tube through a plastic funnel. The collected UWS was centrifuged at 4000× g for a minute followed by preparation of working solution at 500 pg/mL using 1 mL standard and sample diluents for reference and left for 10 min. Dilutions were made in 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 0 pg/mL sequence. Biotinylated diluents  were used to prepare the concentrated solution of 1x from a 100x diluted solution. Similarly, a 100x concentrated HRP conjugate working solution was diluted to 1x concentration with its diluents.The TNF-α assay was performed using the DeQuantoTM Human TNF-α ELISA kit, (# QT 4001) according to the manufacturer’s instructions. The ELISA technique used in the study was the sandwich method. After thawing the tube for each sample, two 0.5 mL aliquots were made that were stored at −30 °C for the ELISA assay. All reagents were maintained at room temperature with microplate preheated for 15 min (96-well microplates). To prepare a 750 mL solution, a 30 mL concentrated wash buffer was prepared with 720 mL distilled water. The prepared solution was centrifuged at 10,000× Well plates were incubated at 37 °C for 90 min. After filtering the liquid, a biotinylated detection antibody at 100 µL was added and incubated for 1 h at 37 °C. Subsequently, the solution aspired was washed thrice and added to the 100 µL HRP conjugate , followed by incubation at 37 °C for 30 min. An amount of 90 µL of substrate reagent  was added and incubated for 15 min at 37 °C. The plate’s outcome was read at 450 nm and calculated following the addition of stop solution (50 µL of 0.16 M sulfuric acid).For cellular analysis, the buccal mucosa was brushed 10 times using a sterile cytobrush  to obtain cells from buccal vestibule in SD and NSD subjects. Each dipping site of Naswar was brushed to compare the obtained cells with the control subjects. The obtained specimen was spread on the prepared glass and fixed with 80% methanol after drying for 20 min; 3 slides were prepared per patient. Subsequently, Giemsa staining was performed with a 10% solution for 20 min. After washing and drying, the DPX mounting medium  was poured and pressed on the slide, followed by storage at room temperature for microscopic analysis . The cytological smears were observed microscopically with a consultant oral histopathologist (A.M.). The slides were viewed under oil immersion lens at 1000x magnification to identify and then count the micronuclei per cell. Careful counting existed to avoid overlapping of fields and repeated counting of same cells. A number of 1000 cells were counted for each case and Tolbert et al. criterion was followed for identification and scoring of micronuclei [p ≤ 0.05 as significant.Data were analyzed using a statistical package for social sciences . For categorical variables, frequencies and percentages were computed. Quantitative variables were measured and compared using means and standard deviations. An independent sample t-test was applied to compare the mean difference among the study groups in terms of TNF-α and micronuclei. Pearson correlation was employed to compare the difference between snuff and non-snuff users regarding age and duration of snuff use and considering n = 30), SD and NSD . The upper vestibule was the site for snuff dipping in 36.6% of subjects (n = 11). A number of 8 (26.6%) snuff users showed poor oral hygiene and 22 (73.3%) had fair oral hygiene. Conversely, 18 control subjects (60%) had fair oral hygiene and 12 control subjects (40%) had relatively good hygiene. Overall, good, fair, and poor oral hygiene was observed in 20% (n = 12), 66.6% (n = 40), and 13.3% (n = 8) of study subjects (p = 0.071). The presence of calculus between SD and NSD was 12.6% and 8.8%, respectively, (p = 0.135). Finally, the CPI index scores among SD and NSD were 2.13 (0–4) and 1.75 (0–4), respectively, (p = 0.444).All sixty participants recruited in the study were men with a mean age of 38.85 (± 11.56) years, divided into two groups  (p < 0.05)  . Similar < 0.05) . Figure The photomicrograph of NSD buccal cells is presented in p ≤ 0.01). The levels of TNF-α were observed to be directly proportional to the micronuclei number. Thus, an increase in the TNF-α value simultaneously showed an increase in the micronuclei nuclei number, which indicates a positive correlation. The study aimed to establish a correlation between age, duration of snuff use, TNF-α, and micronuclei . Comparip-values, 0.454 and 0.099, respectively) (p = 0.048) (p = 0.007)  . However= 0.048)  along wi= 0.007) . This poThe present study aimed to determine the UWS TNF-α levels and cellular micronuclei numbers in snuff dippers (SD) compared with NSD (control subjects). The study showed that irrespective of age, the users (SD) displayed high levels of UWS TNF-α and micronuclei in the buccal vestibule. Moreover, the duration of use was found to be significantly associated with raised levels of TNF-α and cellular derangement overtime. Thus, the null hypothesis, that there is no difference in TNF-α and micronuclei numbers among SD and NSD, was rejected. Naswar is a combination of crushed tobacco leaves, slaked lime, and ashes, which is either inhaled or dipped in the vestibule of either side. After 5 min, the nicotine-associated products seep into the inner mucosa and exhibit a slight burning and numbing sensation . The effTo date, biopsy and histopathological review are considered a gold standard practice; however, these tools are not sufficient in the screening of premalignant lesions and cancers. Each year, thousands of OSCC cases are reported at advanced stages despite the invention of advanced examination tools and technology . Thus, lIn the present research, a positive correlation with the duration of substance use was established with the TNF-α levels and micronuclei numbers. The literature suggests that the prolonged use of betel nuts and Naswar maintains consistent contact with the mucosa, resulting in slow release of nitrosamine . MethylnHowever, the rising levels of micronuclei were identified to be dependent from TNF stimulation. According to previous studies, areca nuts have the potential to stimulate the production of TNF-α in saliva, which positively correlates with time and dose ,27. It wOutcomes of the present investigation showed that increasing age was not associated with an increase in TNF-α levels and micronuclei numbers. Bruunsgaard et al.  reportedThe present study proved the clinical significance of TNF-α and micronuclei in oral cancer screening of high-risk patients. Inflammatory mediators, including TNF-α, are induced in chronic inflammatory conditions such as periodontitis and gingivitis. Although periodontitis patients were excluded, the presence of plaque may have induced gingival inflammation, acting as a confounding factor for raised TNF-α levels . In addiSnuff dippers showed significantly higher levels of USW salivary TNF-α and cellular micronuclei compared to non-snuff dippers (control subjects), irrespective of age. TNF-α and cellular micronuclei presented a positive correlation with the duration of snuff use. We conclude that TNF-α and micronuclei are potential salivary biomarkers for an oral biological effect of snuff (Naswar) users. Further investigations are recommended to validate the use of TNF-α and cellular micronuclei as salivary biomarkers for early diagnosis of oral squamous cell carcinoma.


Inflammation appears as a cardinal mediator of the deleterious effect of early life stress exposure on neurodevelopment. More generally, immune activation during the perinatal period, and most importantly elevations of pro-inflammatory cytokines levels could contribute to psychopathology and neurological deficits later in life. Cytokines are also required for normal brain function in homeostatic conditions and play a role in neurodevelopmental processes. Despite these latter studies, whether pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF) impact neurodevelopmental trajectories and behavior during the immediate postnatal period remains to be elucidated. To address this issue, we have injected mouse pups daily with recombinant TNF from postnatal day (P)1 to P5. This yielded a robust increase in peripheral and central TNF at P5, and also an increase of additional pro-inflammatory cytokines. Compared to control pups injected with saline, mice injected with TNF acquired the righting and the acoustic startle reflexes more rapidly and exhibited increased locomotor activity 2 weeks after birth. Our results extend previous work restricted to adult behaviors and support the notion that cytokines, and notably TNF, modulate early neurodevelopmental trajectories. TNF is expressed as a 27 kDa transmembrane form (mTNF) that can be cleaved into a soluble 17 kDa form (sTNF) released in tissues and blood (Early life stress (ELS) exposure enhances susceptibility to neurodevelopmental disorders. Inflammation appears as a cardinal mediator of the deleterious effect of ELS on neurodevelopmental trajectories . More gend blood . mTNF sind blood . TNF andnd blood . Cells ond blood .In animal studies, immune activation induces a surge in both peripheral and central pro-inflammatory cytokines, including TNF . This isTnf-knockout (KO) mice have shown that maternal TNF determined the fear response of the adult offspring . Pregnant dams were housed into individual cages and remained in the same cages until the end of the experiment. The day and time of birth were recorded and only pups born on E19.5 were used. The day of birth was considered as postnatal day 0 (P0). At P1, pups were sexed and only male pups were used for the experiments described here forth. Since both epidemiological and animal studies suggest sex differences in responses to early adversity and a higher susceptibility of males to neurodevelopmental disorders . The study was in accordance to procedures approved by the Ministère de l’Enseignement Supérieur et de la Recherche (APAFIS#19129-201902071212672). Timed-mated OF1 female mice [Charles River ] were purchased at 15.5 days of gestation [corresponding to embryonic day (E) 15.5 post-conception]. Mice were housed in a temperature (22–24°C) and hygrometry (70–80%)-controlled room with a 12 h light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.) with isorders , only maEnvironmental factors, maternal effects, and litter effects are acknowledged confounding variables in developmental and behavioral studies. To overcome the maternal prenatal effect, litters of 12–14 male pups from distinct mothers were randomly formed and fostered to new single mothers. Each litter was housed individually with its newly reassigned mother. Also, the conditions tested (TNF doses or vehicle treatment), were represented in each individual litter. To keep track of the treatment group, pups were labeled on the back according to their group with distinct colors using ink odorless markers. Marking was renewed every other day to guarantee clear identification over the course of the experiments. In doing so, we obtained both TNF-treated and control pups bred by the same mother, thus excluding the confounding effect of differential maternal care or milk quality, that could contribute to developmental or behavioral differences. In the pilot experiment used to determine the optimal TNF dose to be used, pups were injected intraperitoneally  with either carrier- and endotoxin-free recombinant murine soluble TNF  diluted in sterile PBS, or PBS as vehicle for the control group. We administered 5 μL of solution per pup, with the concentration of 0.25, 1, 5, and 20 μg/Kg adjusted to a standard pup weight for each day of the treatment. Pups were treated once a day from P1 to P5. At P5, body mass (BM) was recorded and pups were sacrificed 3 h post TNF injection by decapitation, then blood and brain were collected.In further experiments, we used the highest TNF dose of 20 μg/Kg and three independent cohorts were generated and subjected to behavioral testing. Again in this set of experiments, to control for the litter effect, newly formed litters were randomly assigned to new mothers and half of each litter was randomly assigned to the two treatment groups (TNF or vehicle). To keep track of treatment group, each pup was labeled on the back according to its treatment group. Altogether 46 pups were injected with TNF and 47 with PBS, once a day for 5 days, from P1 to P5.The acquisition of developmental milestones and reflexes ontology as well as early life behavior were assessed using procedures derived from Fox’s battery of tests and Wahlsten’s adaption of Fox’s tests , as descAmbulation was assessed every day between P6 and P12 to monitor the acquisition of walking proficiency. Each pup was placed on a flat, hard surface and the walking pattern was assessed over 1 min, according to previous scoring criteria : 0 = no The day of the opening of the ear canal, defined as a fully detached outer ear membrane, was recorded in pups aged P3–P5. A fixed score of 0, 1 or 2, according to the number of ears everted per animal was assigned.The day of the eyelid opening, defined as any visible break in the membrane covering the eye, was recorded in pups aged P12–P16. A fixed score of 0, 1 or 2, according to the number of eyelids opened per animal was assigned.Every 2 days between P2 and P6, each pup was placed on its back on a flat, hard surface and kept immobile for 5 s. The pup was then released and the time taken to return to the upright position was recorded. Animals unable to perform the righting after 1 min were assigned a score of 60 s.Pups aged P10–P14 were examined daily to determine the day of reflex acquisition. A cell counter was used to generate an auditive stimulus and the presence (score: 1) or absence (score: 0) of a startle response was recorded.At P6, individual pups were placed on a cotton-padded dish into a thermo-controlled (26°C) soundproof chamber. USV were recorded for 5 min using the UltraSoundGate Condenser Microphone and 116 USB Audio device (Avisoft Bioacoustics), as described . SonograEach pup was separated from its litter and placed into the center of a rectangular-shaped transparent plastic box divided into three zones: the “maternal” zone on one side, the central zone and the “clean” zone on the opposite side from the maternal zone. The maternal zone contained freshly sampled maternal bedding, while the clean zone contained clean bedding. A Plexiglas odor separator was placed above the central zone to prevent the maternal smell from permeating. The movement of the pup was video-recorded over a period of 10 min. The ANY-maze video tracking software (reference) was used to determine: the latency to the first entry in the maternal bedding zone and the time spent in each of the three zones.At P13, each pup was individually placed into a rectangular-shaped transparent plastic box. The movement of the pup was recorded over a period of 10 min and the videos were analyzed using the ANY-maze videotracking software to determine the total distance traveled and the time spent mobile. The exploratory index was then computed by dividing the distance by the time mobile.g, 4°C, 20 min) and serum was transferred to clean 0.5 ml Eppendorf tubes, immediately frozen in liquid nitrogen and stored at −80°C until further analysis. Whole brains were removed from the skull, parted in hemibrains, immediately snapped-frozen in liquid nitrogen, and stored at −80°C until further analysis.Pups were sacrificed by decapitation at age P5 in the pilot cohort used to determine the optimal TNF dose  and one 2 1.25 mM, Triton 0.5%) containing protease and phosphatase inhibitors  and centrifuged . The protein content of the resulting supernatant was dosed using the Bradford method . Cytokines were measured in serum samples diluted to 1:4 and in 200 μg of brain proteins using either the V-PLEX® Mouse TNF Kit for TNF single measures or the V-PLEX® Pro-inflammatory Panel 1 Mouse Kit for multiplex measures , according to the manufacturer’s instructions . Briefly, eight serial dilutions of standards and buffer only (in duplicates) were run together with samples run in singlicate using the Sector Imager 2,400 plate reader . Concentrations of cytokines in each sample were interpolated from standard curves generated with a five-parameter logistic regression equation in Discovery Workbench 3.0 software . IL-2, IL-4, and IL-12p70 were below the Lower Limit of Detection (LLOD) in the majority of the samples and were therefore not considered.Hemibrain samples were homogenized using a Dounce potter in a lysis buffer  with post-hoc Sidak’s correction for multiple comparisons was used. Statistical significance was set at p-value (p) lower than 0.05. All univariate analysis was performed using GraphPad Prism version 6.00 for Windows . Details regarding statistical analyses outcomes are reported in Data normality was assessed using the Kolmogorov-Smirnov test. For normal data, the two-tailed unpaired Student’s To account for correlations between repeated measures on the same individual, and random effects due to the cohort and developmental time of measurement, we used the generalized estimating equations (GEE) approach to model each outcome, considering treatment, cohort and time as covariates . GEE canTo determine the optimal dose of TNF to use in the study, we performed a pilot experiment in which mouse pups were injected with TNF doses ranging from 0.25 to 20 μg/Kg . Even whAt P5, pups injected with TNF (20 μg/Kg) exhibited increased serum levels of TNF, IL-1β, IL-6, IL-10, and CXCL1, as compared to vehicle-injected pups, while IL-5 and IFN-g levels were not impacted . This waAt P16, vehicle- and TNF-injected pups exhibited similar levels of TNF, IL-1β, IL-6, CXCL1, IL-5, and IFN-γ in both serum and brain samples . In contThese results suggest that perinatal injections of TNF from P1 to P5 induce an immediate acute systemic inflammation, that spreads to the brain. However, by P16, inflammation has resumed and only the anti-inflammatory cytokine IL-10 appears overexpressed.To monitor the behavioral consequences of perinatal systemic inflammation induced by TNF treatment, pups injected with TNF (20 μg/Kg) or vehicle from P1 to P5 were subjected to a battery of tests . There wHowever, TNF treatment had a significant effect on the acquisition of early reflexes over time . Both thWe further studied the impact of perinatal TNF treatment  on commuIn the olfactory orientation test, TNF- and vehicle-injected pups performed equally well as measured by the latency to first reach maternal bedding  and the Regarding locomotor activity, TNF-injected pups spent more time mobile  and travOur study is based on gain-of-function experiments in which mouse pups are injected intraperitoneally with recombinant TNF daily from P1 to P5. We show that this yielded a robust increase in serum TNF levels at P5. Concomitantly, we observed an increase in the serum levels of the pro-inflammatory cytokines IL-1β and IL-6, the chemokine CXCL1, and the anti-inflammatory cytokine IL-10. This suggests that exogenous TNF injected peripherally triggers the secretion of other peripheral cytokines.via microglia, as the magnitude of IL-1β increase in the brain (threefold) is higher than in serum (1.27-fold). Upon TNF injections, the lack of elevation of other cytokines in the brain at P5 (including pro-inflammatory cytokines) suggests that their transfer from the periphery to the brain or their local production in the brain relies on distinct regulatory processes, independently of TNF or IL-1β.The effects of TNF injections appear more selective in the brain. Indeed, we show that, at P5, TNF-injected pups exhibit increased brain levels of both TNF and IL- 1β, while other cytokines are unimpacted. Since it was demonstrated in 3-days old rat pups that radiolabeled soluble TNF injected peripherally crossed the BBB using a saturable transport system , it is lThe increases in pro-inflammatory cytokine levels are transient, as, by P16, the levels of all these cytokines have returned to control levels in brain or serum. By P16, the only cytokine exhibiting higher levels is the anti-inflammatory cytokine IL-10. It is possible that IL-10 elevation is consecutive to the transient increase in pro-inflammatory cytokines TNF and IL-1β in the brain at P5. Indeed, IL-10 is a potent anti-inflammatory cytokine secreted to resume inflammation notably by inhibiting TNF and IL-1β expression by activated immune cells and microglia .via release of anti-inflammatory cytokines such as IL-10 and low levels of pro-inflammatory cytokines  , in contvia TNFR1. Also, IL-1β is increased in the brain at P5 in TNF-injected pups. Since IL-1β receptor IL1R1 is expressed in endothelial cells, neurons and at very low levels in astrocytes , and does not involve visual inputs since eyelids are not opened or vestibular inputs, as the vestibular system is not mature yet at P2 . The certhe body . It may the body . This conucleus reticularis pontis caudalis (PnC), located in the pons of the brainstem. Finally, the PnC axons synapse to spinal motor neurons to induce the startle , the first acoustic neurons of the central nervous system . The CRN startle . It is pAt P13, pups have hardly opened their eyelids and spatial information processing is still immature . At thisWhether the changes we observed reveal a beneficial or negative neurodevelopmental impact of TNF remains to be determined. On one hand, a precocious acquisition of reflexes and increase in exploratory behavior may reflect developmental precocity and could confer selective advantages to TNF-injected pups. On the other hand, faster reflex acquisition can reflect an inappropriate neurodevelopmental timing which translate into hyperactivity in TNF-injected pups. On the whole, our data support the notion that the pro-inflammatory cytokine TNF selectively impacts neurodevelopmental trajectories.in utero to MIA exhibit USV communication deficits and hypoactivity, and elevated TNF levels in serum at P16 , increased circulating levels of IL-1β and TNF-α . This wam at P16 . Hence, Genetic, environmental factors, maternal effect and litter effects are acknowledged confounding variables in developmental and behavioral studies. We accounted for environmental and maternal effects by adapting our experimental procedures, notably by pooling pups of different litters to form new litters and including TNF- and vehicle-treated pups in the same litters. Our experimental model is the OF1 outbred stock obtained from “a closed population of genetically variable animals that is bred to maintain maximum heterozygosity” as defined in To account for the effect of variables  which we could not experimentally control, but which, besides TNF treatment, could impact developmental and behavioral outcomes, we used the Generalized Estimating Equations (GEE) model. This modeling strategy is particularly adapted to the study of outcomes measured repeatedly over time on the same individuals and subjected to confounding effects. In place of more classical regression methods which estimate the effect of changing one or more covariates on a given individual, GEE focuses on estimating “population-averaged” effects . GEE canFirst, our study is restricted to males. A large body of evidence suggests that males are more at risk than females for neurodevelopmental disorders . LikewisOur study revealed the selective early life impact of perinatal TNF on reflex acquisition, as well as locomotor activity. This suggests that TNF can modulate early developmental trajectories and complement previous behavioral studies performed in adult animals. Future work is required to understand the astrocytic, microglial or neuronal pathways underlying the effects of TNF on early development, and whether they converge on neurodevelopmental pathways generally impacted by ELS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Ministère de l’Enseignement Supérieur et de la Recherche.LD and NG designed the project. CP carried out the experiments. CP, SB, and LD analyzed and interpreted the data. JV and PG advised on experimental design and contributed methods. LD and CP wrote the manuscript. CP, JV, SB, and NG revised the manuscript. All authors contributed to the article and approved the submitted version.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.