Chronic inflammation is a typical characteristic of polycystic ovary syndrome (PCOS), in which, tumor necrosis factor (TNF)-α plays an important role. We investigated whether anti-TNF-α therapy can alleviate the core phenotypes of PCOS. In pubertal female Wistar rats, release pellets of letrozole (LET) were administered continuously for 90 days to induce PCOS-like phenotypes, followed by treatment with etanercept (ETA), a TNF-α inhibitor. ETA significantly inhibited increases in body weight and androgen, TNF-α, and MCP-1 levels, excessive recruitment of lipid droplets, altered levels of pre-adipose differentiation markers, and abnormal development of follicles. In addition, TNF-α and testosterone (T) levels in the rat sera were significantly positively correlated. Further experiments were performed to investigate the relationship between TNF-α and androgen. Persistent exposure of the RAW 264.7 cell line to low doses of testosterone significantly enhanced TNF-α expression and activated the NF-κB signaling pathway, which were blocked by ETA. Furthermore, treatment with TNF-α promoted the production of testosterone in KGN granulosa cells by reducing CYP19A1 expression, whereas ETA treatment blocked this process. In conclusion, anti-TNF-α therapy with ETA may be an efficient method to alleviate PCOS, whose underlying mechanism may be associated with its ability to reduce excessive androgen levels. Polycystic ovary syndrome (PCOS) is a common endocrine disease that affects 6–21% women of reproductive age, approximately 75% of whom experience infertility due to anovulation ,4. AlthoChronic inflammation is considered as an important contributor to the pathogenesis of PCOS ,13, whicSeveral chemicals, most of which are androgen and its derivatives, have been used in mouse and rat to induce phenotypes that mimic those of PCOS ,20. Howein vitro experiments on mouse macrophage cells (RAW 264.7 cell line) and ovarian granulosa cells (KGN cell line) to investigate the direct association between androgen and TNF-α, and explore the potential underlying mechanisms. Furthermore, ETA was employed to attenuate the possible abnormal alterations induced by androgen or TNF-α.The direct relationship between androgen and inflammatory factors in PCOS has not been elucidated yet. In some studies, androgen seemed to be an efficient protective agent against the negative influences of TNF-α ,26, whicSince chronic inflammation is a key contributor to the pathogenesis of PCOS, the current study applied anti-TNF-α therapy using ETA in a letrozole-induced PCOS rat model. In addition, direct interaction between TNF-α and androgen and their roles in physiological activities were also investigated.Fifteen female Wistar rats aged 21 d were purchased from Dashuo Experimental Animal Co. Ltd. , and housed (five rats per cage) under controlled conditions . The study was approved by the Animal Ethics Committee of Sichuan University (grant number: WCSUH-2018-32). The rats were implanted subcutaneously with 90-day continuous-release pellets . The pellets for polycystic ovary syndrome (PCOS) model rats released 200 μg letrozole (LET) per day, while the control pellets lacked the bioactive molecule. The PCOS model rats were treated with experimental drug etanercept  8 weeks post implantations. All the rats were sacrificed after 3 weeks of ETA treatment . In the Rats were weighed weekly on electronic scales during induction of polycystic ovary syndrome (PCOS), before the beginning of the treatments with the medicines, and at the end of the treatments. Testosterone (T) level in the rat serum, measured by GC-MS before and after the treatments, was used to represent the androgen levels. On the final day, the rats were fasted overnight before oral administration of glucose (2 mg/kg body weight), and blood glucose levels were measured using ACCU-CHEK  at 0, 15, 30, 60, and 120 min post-gavages.Multi-inflammatory factors in rat serum, including TNF-α, MCP-1, IFN-γ, IL-1α, IL-1β, IL-2, IL-6, IL-10, and IL-17, were analyzed using Merck Millipore suspension array kit. The inflammatory factors secreted by RAW 264.7 cell line induced by lipopolysaccharide (LPS) were also analyzed using the same suspension array kit, and TNF-α induced by testosterone (T) was determined using ELISA kit .2. RAW 264.7 cells were treated with different doses of testosterone  for 3 d. Etanercept (ETA) was used to treat the inflammation of RAW 264.7 cells induced by testosterone (100 nM) and LPS (1 μg/mL).RAW 264.7 and KGN cell lines were provided by Dr. Ma Yaxian , and Dr. Wang Fei , respectively, who purchased them from ATCC. RAW 264.7 cells were maintained in DMEM medium  supplemented with 10% FBS , while KGN cells in DMEM/F12 medium  supplemented with 10% FBS. Both of them were incubated at 37°C in a humidified atmosphere with 5% COAfter overnight fixing with 4% PFA, fresh tissue samples were dehydrated by gradually exposing to 50%, 70%, 80%, 90%, and 100% (twice) ethanol, and dimethylbenzene (twice) at room temperature. Then, they were embedded with paraffin at 60°C and cut into sections. Paraffin sections were dewaxed by following the reversed steps of embedment, and then washed twice with distilled water before staining. The sections were stained with hematoxylin for 5 min, treated with the differentiation fluid  for 30 sec, washed with tap-water for 15 min, and then stained with eosin for 2 min. Lastly, the stained sections were dehydrated, and sealed with coverslips and imaged under microscope.Before immunofluorescence, sections were incubated in boiling 0.01 M citrate buffer for 20 min to fix the antigens after dehydration. Cell slides were then fixed by overnight incubation with 4% PFA. Subsequently, the samples were sealed with 3% BSA, incubated overnight at 4°C with primary antibodies, followed by incubation with fluorescent secondary antibodies, and then with DAPI, and finalized. The primary antibody for P65 (1:100 dilution) was purchased from Proteintech Group  and for CYP19A1 (1:100 dilution) from Boster Biotech Co. Ltd. .-ΔΔCt method, and actin was used as the exogenous control gene. The primers were designed using Primer Premier 5.0  and synthesized by Chengdu Diqiao Biotechnology Co., Ltd. .Total RNA was extracted using Trizol , according to the manufacturer’s instructions, and the concentration was measured using Nanodrop 2000 . The RNA was reverse transcribed using reverse transcription kit  and a Thermal cycler , and real-time qPCR was performed using the SYBR Green system  in an ABI 7500 , as per the manufacturer’s instructions. Expression of target genes was measured using the 2To obtain the protein, tissue samples and cells were lysed using RIPA lysis buffer and centrifuged at 12,000×g for 20 min to collect the supernatant without lipid. Protein concentration was measured using the BCA protein assay . Equal amounts of protein were loaded onto an SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) at room temperature for 1 h, and then incubated with primary antibodies at 4°C for overnight. Each membrane was washed with TBST three times for 15 min followed by incubation with an HRP-conjugated secondary antibody  at room temperature for 1 h. Finally, each membrane was developed using an enhanced chemiluminescence (ECL) detection kit  and visualized using X-OMAT BT film . The antibodies for TNF-α, TNFR1, and TNFR2 were purchased from Santa Cruz , the primary antibodies for AR, IκB, NF-κB, and P65 from Proteintech Group , TNF-α from CST , and β-actin, and GAPDH from Zenbio Biotech Co., Ltd. .TNF-α ELISA kits for both mouse and rat species were purchased from Neobioscience Biotech Co. Ltd. . Testosterone ELISA kits for rat and human were obtained from Cusabio . The levels of TNF-α and testosterone were measured according to the manufacturers’ instructions.t-test, whereas, more than two groups were compared by One-way ANOVA, in which each pair was evaluated by post-hoc analysis. Statistical significance was set at P<0.05.The data are presented as mean ± SD. Two groups were compared by Student’s The PCOS model of rat was established by implanting continuous-release letrozole pellets for 8 weeks, and subsequent ETA therapy for 3 weeks . Overwei2 levels, which were reported to be tightly associated with follicular development, induced by LET, were also attenuated after ETA treatment  were exposed to different doses of T  for 3 d. ELISA results showed that high level of T (100 nM) significantly increased the TNF-α levels at day 3 (A significant positive correlation was found between the levels of TNF-α and testosterone (T) in rat sera . Therefoat day 3 . Howeverat day 3 . The latCytokine profiling revealed the increased expression of certain inflammatory factors, including IL-1α, IL-2, and IL-6, and decrease in the levels of other cytokines in PCOS model . TherefoTNF-α activates the NF-κB signaling pathway. Therefore, we examined the effect of T exposure on the downstream pathway and on the effect of ETA. Although short-term exposure (day-1) to T did not influence TNF-α levels or NF-κB signaling pathway significantly, persistent exposure significantly promoted expression of AR, TNF-α, TNFR1, and TNFR2 proteins, and activated NF-κB signaling pathway by enhancing the expressions of P65, NF-κB, and IκB proteins . LipopolExcess TNF-α secretion has been considered to be a characteristic of PCOS . ImmunofBased on the above results, we propose a potential mechanism for the effect of ETA in the PCOS model .Although chronic inflammation plays a key role in the pathogenesis of polycystic ovary syndrome (PCOS), few investigations on the underlying mechanisms, or on the detailed roles of inflammatory factors in PCOS progression, have been made. TNF-α, a key Th-1 related inflammatory factor, has attracted a lot of attention in a variety of diseases, and numerous studies have confirmed its roles in physiological activities –32, whicIn the current study, we first tried to apply anti-TNF-α therapy in the treatment of PCOS in a rat model induced by letrozole. Letrozole is a nonsteroidal aromatase inhibitor that inhibits the activity and alters the expression of CYP19A1, which converts androgens to estrogen in the ovary, thereby efficiently decreasing the estrogen/androgen ratio in follicular fluid . Comparein vitro as well as in vivo. Mechanistically, ETA can significantly attenuate the inflammatory condition by decreasing the expression of p65 in the NF-κB signaling pathway induced by lipopolysaccharide (LPS) or testosterone.ETA, a fusion protein of the TNF receptor and IgG1 Fc, and a widely used clinical TNF-α inhibitor in rheumatoid arthritis, was used in our model to block the effects of TNF-α. ETA showed excellent ability to scavenge TNF-α molecules Elevated secretion of androgen by the ovary is a key feature of PCOS. Our results indicated that excessive androgen is an important contributor to the chronic inflammation induced during PCOS. TNF-α affects the secretion of androgens, which explains our observation that anti-TNF-α therapy significantly decreased the elevated androgen level in the PCOS model . In termin vitro experiment may partially explain the unknown influences of androgens on inflammation, including short-term exposure of low-dose T that significantly decreased the secretion of TNF-α, transforming mouse macrophage cells (RAW 264.7) to the M2 type, and long-term exposure that aggravated the TNF-α expression and activated the NF-κB inflammatory signaling pathway, transforming mouse macrophage cells to the M1 type. Furthermore, testosterone, but not DHT was used in in vitro model, therefore, our result cannot exclude the possibility that altered estrogen level could also be involved in the regulation, which need further clarification.Although chronic inflammation has been found to be related to other phenotypes, such as hyperandrogenism, the factors that induce inflammation are still not elucidated. It is widely known that excessive androgen level is a common complication of PCOS and can partially recapitulate the multiple features of PCOS. Dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and many other androgens had been previously used to induce the phenotypes of PCOS in a variety of animal models. In the rat model of this study, serum testosterone (T) level was found to be positively correlated with serum TNF-α level. Thus, excessive androgen level was considered as one of the major causes of chronic inflammation. However, it was still not determined whether androgen could cause a significant inflammation in different environments or diseases. Several investigations reported that androgen could alleviate the serious inflammatory condition and protect cells from the damages inflicted by inflammatory factors under certain conditions. Nevertheless, the results of our Obesity is a classic phenotype of PCOS and is closely related to chronic inflammation. ETA suppressed the excessive lipid recruitment in the PCOS model. It is noteworthy that some relevant clinical studies have reported that anti-TNF-α therapy using ETA in obese patients effectively improved fasting glucose levels and changed the adiponectin composition ,34. We bIn conclusion, ETA could efficiently alleviate the clinical features of PCOS. It efficiently decreased the elevated body weight and serum androgen, TNF-α and MCP-1 levels, and the size of lipid vacuoles, inhibited the expression of pre-adipose differentiation markers, and alleviated the abnormal ovarian condition in PCOS. The results indicated a successful application of anti-TNF-α therapy against PCOS in a rat model, and it is thus speculated that anti-TNF-α therapy could be further explored to be successfully applied in clinical settings or in combination with the conventional treatments in the future.S1 File(RAR)Click here for additional data file.


Turnip mosaic virus (TuMV) have been shown to be efficient tools for antibody sensing with a very high sensitivity if adequately functionalized with the corresponding epitopes. Taking advantage of this possibility, TuMV virus-like particles (VLPs) have been genetically derivatized with a peptide from the chaperonin Hsp60, a protein described to be involved in inflammation processes and autoimmune diseases. Antibodies against the peptide have been previously shown to have a diagnostic value in at least one autoimmune disease, multiple sclerosis. The functionalized Hsp60-VLPs showed their significant increase in sensing potency when compared to monoclonal antibody detection of the peptide in a conventional immunoassay. Additionally, the developed Hsp60-VLPs allowed the detection of autoantibodies against the Hsp60 peptide in an in vivo mouse model of dextran sodium sulfate (DSS)-induced colitis. The detection of minute amounts of the autoantibodies allowed us to perform the analysis of their evolution during the progression of the disease. The anti-Hsp60 autoantibody levels in the sera of the inflamed mice went down during the induction phase of the disease. Increased levels of the anti-HSP60 autoantibodies were detected during the resolution phase of the disease. An extension of a previously proposed model for the involvement of Hsp60 in inflammatory processes is considered, incorporating a role for Hsp60 autoantibodies. This, and related models, can now be experimentally tested thanks to the autoantibody detection hypersensitivity provided by the functionalized VLPs.Nanoparticles derived from the elongated flexuous capsids of  Turnip mosaic virus (TuMV), a virion with an elongated and flexuous structure, 700 nm long and 12 nm wide, with ca. 2000 copies of the coat protein in each particle, from which multifunctional VNPs can be obtained by genetic fusion and/or chemical conjugation to the coat protein (CP)  or overnight at 4 °C for mice sera (diluted 1:100 in the same buffer). Then, secondary antibodies, diluted 1:1500 in the same buffer, were added and incubated for 1 h at room temperature. Color was developed by alkaline phosphatase reaction and detected after addition of p-nitrophenylphosphate. Absorbance was measured at 405 nm (TECAN Genios Pro). Differences between both coating-VLPs were calculated, since this would be the measure attributable to autoantibodies against Hsp60. The normalization process was done as follows. A serum pool was made using the sera from healthy mice and this pool was included in all the plates in order to calculate the background of each plate. The normalized values of autoantibodies directed against Hsp60 in the DSS serum samples were obtained by taking into account the mean value of the healthy serum pool in each plate and the mean value of the healthy serum pool from all the plates, in order to minimize the effects of the variation in the background signal. The data obtained were analyzed based on the non-parametric Mann–Whitney U test.Indirect enzyme-linked immunosorbent assays (ELISA) were performed in order to evaluate the sensitivity provided by the VLPs, in comparison with the whole Hsp60 protein and free peptide at equal peptide amounts, and also to measure autoantibody levels in peripheral blood. Plates (Nunc MaxiSorp) were coated with 1 μg purified VLPwt or Hsp60-VLPs, resuspended in 50 mM sodium carbonate buffer, pH 9.6; or equivalent amounts of Hsp60 protein or peptide, resuspended in the same buffer and incubated overnight at 4 °C. Plates were washed intensively, and incubated for 1 h with commercial antibodies [Hsp60 (D307) Antibody 4870S, CellSignal and Anti-Hsp60 antibody (ab46798), Abcam; different dilutions in phosphate-buffered saline, pH 7.4, 0.05% Tween 20 , an ELISA was developed with a pool of sera from healthy, non-inflamed mice, in order to determine physiological levels of anti-Hsp60 autoantibodies. Unmodified VLPs  were used as a negative control. The results showed that only with the Hsp60-VLPs could the anti-Hsp60 autoantibodies be detected above the background of the plate and negative controls in healthy mice sera. Therefore, the high ability of Hsp60-VLPs to detect autoantibody levels, undetectable by conventional procedures such as ELISA, was demonstrated. This result indicates that VLPs can be used as a tool to detect variations in the levels of anti-Hsp60 autoantibodies in mice sera when the traditional methodologies based on the complete Hsp60 protein cannot be detected, even at basal autoantibody levels in non-inflamed mice.Due to the proven ability of Hsp60-VLPs to detect low levels of autoantibodies in the sera samples, potential diagnostic applications were assessed. Since Hsp60 is deeply involved in inflammatory processes ,25,47, aThe evolution of Hsp60 autoantibody levels in the inflammatory model induced by DSS was studied. The progress of the pathology, as well as the recovery after removing the chemical agent, was determined by monitoring the body weight and granulocytes levels in peripheral blood, as shown in The results show a significant decrease in body weight of mice treated with DSS, significant at day 7 and 9. This decrease continues until the DSS was withdrawn, after which the mice recovered weight and reverted to a non-pathological state. Regarding granulocyte levels, there was an increase in blood, in this case significantly at 5 days. In this case the reversal begins later, several days after withdrawing DSS.To evaluate the potential of Hsp60-VLPs as a novel tool in the knowledge of the inflammatory pathology, an ELISA was performed. Autoantibody levels measured with the complete Hsp60 protein were not detectable, as shown in The results showed a marked decrease in anti-Hsp60 autoantibody levels following the progress of inflammation at 5 and 10 days (significant according to the Mann–Whitney U nonparametric test), maintaining higher levels in healthy individuals and the basal levels at day 0 in the DSS group. These results demonstrate TuMV functionalized VNPs as a hypersensitive tool in the progression analysis of the inflammation, being able to detect levels of autoantibodies in cases where conventional methodology is not capable.In the diagnosis of autoimmune pathologies, current techniques face two main problems, especially when diagnosis is made by detecting specific autoantibodies in serum—background and low autoantibody levels. The high background is due to numerous substances present in serum, and low autoantibody levels occur at early stages of pathology development, a time in which a proper diagnosis would allow early treatment, and a better patient prognosis ,49. ThisTo address these challenges, the use of nanoparticles as a multimeric presentation system is an alternative approach, which should provide high sensitivity and solve the epitope–adhesion problem ,9,51,52.To test TuMV VNPs as a new tool for autoantibody detection, Hsp60 was chosen as an antigen. This protein is involved in numerous inflammatory mechanisms, also related to autoimmune pathologies ,35,38,40According to these results, and because this peptide was chosen due to its diagnostic potential in autoimmune pathologies , an evalThanks to the high sensitivity shown by Hsp60-VLPs, it was possible to evaluate the evolution of autoantibody levels with disease progress in a murine model. In this case, and against expectations, autoantibody levels decreased concomitantly with the inflammatory process, as shown in All the ideas raised would place Hsp60 as a study target. In this case, TuMV VLPs allowed the identification of possible markers in inflammatory pathologies, being able to generate diagnostic systems of high sensitivity and specificity, useful to identify new diagnostic and therapeutic targets. In addition, considering cases such as Hsp60, where autoantibodies can act as regulatory agents, functionalized VLPs could be used not only as a diagnostic tool, but also in therapy for immunization, where the great immunogenic power of VNPs from TuMV has been demonstrated . This woThis study lays the groundwork for the development of new theranostic tools, based on viral nanoparticles functionalized with specific epitopes involved in autoimmune pathologies, allowing the creation of new systems for autoantibody-based specialized diagnosis and for targeted treatments.


Patients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation.TNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 μg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for μCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes.TNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling.This study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised.The online version of this article (10.1186/s13075-019-1962-3) contains supplementary material, which is available to authorized users. Patients with inflammatory arthritis experience extra-articular manifestations, including osteoporosis and muscle wasting, which closely correlate with measures of disease activity –4. GlucoIt remains unclear what the effects of GCs on bone and muscle are, when used to treat new onset inflammatory arthritis, in particular, whether the beneficial effects of controlling articular and systemic inflammation on bone and muscle outweigh their direct catabolic actions in these tissues.Murine models of polyarthritis have proven a powerful tool in examining the pathophysiology of inflammatory diseases, such as RA. The TNF-tg mouse is a murine model of polyarthritis driven by the transgenic overexpression of the pro-inflammatory cytokine TNFα that proved valuable in the initial validation of anti-TNFα biologicals . We haveProcedures on animals were performed under guidelines by the Animal (Scientific Procedures) Act 1986 in accordance with the project licence (P51102987) and approved by the Birmingham Ethical Review Subcommittee (BERSC). The TNF-tg model of chronic inflammatory polyarthritis, obtained courtesy of Dr. George Kollias , were maintained on a C57BL/6 background and compared to WT littermates . At day n = 4) were recruited prior to elective joint replacement surgery. Trabecular chips of approximately 400–600 mg were excised and placed in PBS prior to culturing. Reagents were obtained from Sigma  unless otherwise stated. Trabecular bone chips from patient samples were cultured in osteoclast growth media to facilitate release of osteoblasts , patients with hip osteoarthritis (OA) . Monocytes were cultured in selective survival media  from healthy donors, obtained from the Scottish National Blood Transfusion Service (approved by Glasgow NHS Trust-East Ethics Committee), were isolated via Ficoll-paque PLUS  density gradient centrifugation and CD14Primary myotubes were generated from tibialis anterior as previously described . In brie−Δct)].Gene expression in cells and tissues was assessed using TaqMan® Gene Expression Assays (ThermoFisher Scientific). Tissues were homogenised in liquid nitrogen with a sterile pestle and mortar. mRNA was isolated using an innuPREP RNA Mini Kit  as per the manufacturer’s instructions. One microgramme of RNA per sample was reverse transcribed using Multiscribe™ using the manufacturer’s protocol (ThermoFisher Scientific) to generate cDNA. Alp, Bglap, Redd1, Foxo1, Trim63 and Fbxo32 were determined using species-specific probe sets by real-time PCR on an ABI7500 system . Final reactions are listed in Additional file Serum IL-6 , P1NP and CTX-1  and conditioned media pro-collagen I α1  were determined using a commercially available ELISA assays in accordance with the manufacturer’s instructions.Histochemistry was performed on paraffin-embedded 10-μm sections of hind paws and quadriceps of WT and TNF-Tg animals following staining with haematoxylin and eosin. Pannus size at the metatarsal-phalangeal joint interface was determined using Image J software as previously reported . SectionFront paws and tibias from mice were imaged using a Skyscan 1172 micro-CT scanner (Bruker) using X-ray beam settings of 60 kV/167 μA with a 0.5-mm aluminium filter. Projections were taken every 0.45° at 580-ms exposure. Image volumes were reconstructed using the Feldkamp algorithm  having applied beam hardening correction. Trabecular bone parameters (bone volume to tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N)) of the tibia were analysed using CTAn software. One millimetre of bone (150 sections) in the metaphyseal region beneath the growth plate was analysed, and regions of interest (ROI) were selected by drawing around the trabecular network for each cross-sectional slice. Front paws were reconstructed, and MeshLab 1.3.2 was used to generate meshes which could then be scored for bone erosions as described previously .Briefly, muscle were homogenised in 10-fold volume excess of ice-cold sucrose lysis buffer  using either an unpaired Student’s t test or two way ANOVA with a Bonferroni correction Tukey post hoc analysis where a Gaussian distribution is identified.Statistical significance was defined as P < 0.005; TNF-tg vehicle, 119.8 ± 29.9, TNF-tg CORT, 485.1 ± 43.7 ng/ml; P < 0.005)    . Serum IL-6 was potently upregulated in vehicle-treated TNF-tg animals and strongly suppressed in animals receiving corticosterone. These data demonstrate that corticosterone administered at 100 μg/ml in drinking water over 3 weeks is sufficient to markedly suppress disease activity and joint destruction.TNF-tg mice received drinking water containing vehicle or corticosterone at 100 μg/ml for 3 weeks. Daily oral water intake of corticosterone was calculated per mouse and was shown to be 22.0 ± 0.83 and 23.2 ± 2.0 μg/g body weight/day and did not vary significantly between groups    Fig. b. Analys05) Fig. c. In con01) Fig. d. TogethP < 0.0001, TNF-tg/vehicle, 269.7 ng/ml ± 27.2 vs TNF-tg/corticosterone, 32.3 ng/ml ± 7.5; P < 0.0001) , a comparable suppression of gene expression was apparent in both groups receiving corticosterone relative to vehicle  .To delineate the actions of corticosterone on bone turnover in TNF-tg mice, we examined systemic markers of bone formation (P1NP) and resorption (CTX-1) and modelled therapeutic GC treatments in human cultures of osteoblasts and osteoclasts in combination with TNFα. Whilst TNF-tg animals had significantly lower P1NP levels at day 53 relative to WT counterparts, both groups developed a comparable suppression of P1NP in response to corticosterone      and significant reduction in large fibres (2200–2600 μm2) relative to vehicle-treated controls   , anabolic and anti-anabolic myokines and signalling , muscle differentiation  and inflammatory myokines and signalling  Additional file 2:Figure S1. (a) body weights (g), (b) daily corticosterone intake (μg/g body weight/day) and (c) serum corticosterone determined by ELISA (ng/ml) in in WT and TNF-Tg animals receiving either vehicle or corticosterone (100 μg/mL) in drinking water over 3 weeks. Values are expressed as mean ± standard error of six per group for weight and at least three animals per group for steroid intake and serum measurement. Statistical significance was determined using two-way ANOVA with a Tukey post hoc analysis. * P < 0.05, **P < 0.005, ***P < 0.001. (TIF 435 kb)Additional file 3:Figure S2. Representative images of human primary culture osteoclast activity assessed on mineral-coated plates at day 14 treated with vehicle, cortisol (1000 nmol/l), TNFa 10 ng/ml) or a combination of both. Images were acquired using EVOS FL Auto Cell Imaging System (Life Technologies). (TIF 1090 kb)Additional file 4:Figure S3. (a-p) Gene expression of Foxo1, Fbxo32, Trim63, Ube3a, Igf1, Igf2, Mstn, Redd1, Myog, MyoD, Myf5, Myf6, Tnfa, Il6, Cxcl1 and IkBa were determined by RT qPCR in quadriceps for either WT mice and TNF-Tg animals receiving either vehicle or corticosterone (100 μg/mL) in drinking water over 3 weeks. Values are expressed as mean ± standard error of six animals or primary cultures derived from four separate animals. Statistical significance was determined using two-way ANOVA with a Tukey post hoc analysis. *P < 0.05, **P < 0.005, ***P < 0.001. (TIF 1454 kb)


Lettuce is often implicated in human norovirus (HuNoV) foodborne outbreaks. We identified H-like histo-blood group antigens (HBGAs) on lettuce leaves as specific binding moieties for virus-like particles (VLPs) of HuNoV GII.4/HS194/2009 strain. The objective of this study was to determine whether HuNoV-lettuce binding is mediated through the virus HBGA binding sites (HBS). Toward this objective, VLPs of historical HuNoV GII.4 strains  with known natural mutations in their HBS, two newly generated VLP mutants of GII.4/HS194/2009 (D374A and G443A) and a VLP mutant (W375A) of GI.1/Norwalk/1968 along with its wild type VLPs, which displays distinct HBS, were investigated for their binding to lettuce. ELISA revealed that historical GII.4 strains binding to lettuce was dependent on their HBGAs profiles. The VLP mutants D374A and G443A lost binding to HBGAs and displayed no to minimal binding to lettuce, respectively. The VLPs of GI.1/Norwalk/1968 strain bound to lettuce through an H-like HBGA and the binding was inhibited by fucosidase digestion. Mutant W375A which was previously shown not to bind to HBGAs, displayed significantly reduced binding to lettuce. We conclude that the binding of HuNoV GII.4 and GI.1 strains to lettuce is mediated through the virus HBS. Caliciviridae family [Human norovirus (HuNoV) continues to be the leading cause of foodborne disease outbreaks in the United States (US) . It is ee family . Human ne family . Infectie family . Currente family ,8,9. Howe family .In the US, analysis of HuNoV foodborne outbreaks that could be attributed to a single food category showed that fruits and vegetables accounted for 51% of the outbreaks . SpecifiHuman HBGAs are complex glycans expressed on the surface of red blood cells and mucosal surfaces of secretor individuals or can be present as free antigens in the biological fluids of secretor individuals such as saliva, milk and intestinal contents . In humaThe objective of this study was to determine whether HuNoV binding to lettuce carbohydrates is mediated through the virus HBS. To accomplish this objective, we have assessed the binding of some of the historical GII.4 VLPs  to lettuSf9) with the recombinant baculovirus according to standard protocols described in our previous publications [Sf9 cells with the recombinant baculovirus, cells and media were harvested at 7–10 post-inoculation days (PID). The VLPs were pelleted through sucrose cushions and were further purified by CsCl gradient ultracentrifugation. The protein concentration was quantified using the Bradford method and the morphology of the VLPs was examined by transmission electron microscopy (TEM) after staining with 3% phosphotungstic acid (pH 7.0) [A recombinant baculovirus carrying the VP1 gene (ORF2) of the Hu/NoV/GII.4/HS194/2009/US strain ) was generated previously by our laboratory [ications ,18. Brie(pH 7.0) ,24,28. T(pH 7.0) .CCTTTGAAACTCACC-3′ and 5′-GGTGAGTTTCAAAGGCATTTTCTGTATCAGTACC-3′, respectively. To mutate G (Glycine) to A (Alanine) at amino acid 443 (G443A), the forward and reverse primers were 5′-GCCCGGATGCAGCGCGTATCCCAACAACATGG-3′ and 5′-CCATGTTGGGATACGCGCTGCATCCGGGC-3′, respectively. After confirmation by sequencing, LR recombination was performed with the entry vector and BaculoDirectTM Linear DNA to generate the recombinant baculovirus DNA carrying the mutant VP1 gene of HS194. The insect Sf9 cells were transfected by the recombination reaction products and the cells containing the recombinant baculovirus DNA were positively selected by ganciclovir. The expression of mutated HS194 capsid proteins in the Sf9 cells and culture supernatants were examined by immunoblot using the guinea pig antiserum against HS194 strain. The recombinant baculoviruses were propagated to prepare virus stocks with high titers for routine VLP expression. The production, purification and examination of GII.4.HS194 VLP mutants were performed as referenced above [The two recombinant baculoviruses for the expression of VLP mutants (D374A and G443A) of the HuNoV GII.4/HS194/2009/US strain were generated in this study. The Gateway entry vector (pENTRTM/SD/D-TOPO-HS194) containing the major capsid protein VP1 gene of WT HS194.2009 strain was used as a template. The QuikChange II XL site-directed mutagenesis kit  was used to make the site mutation in the VP1 gene in the entry vector for the mutants. To mutate the D (aspartic acid) to A (Alanine) at amino acid 374 (D374A), forward and reverse primers were: 5′-GGTACTGATACAGAAAATGed above .g. The resulting pellet was subjected to a series of ethanol washes (80% twice and 100% once) and was then stirred for 2 h at 4 °C in 60 mL methanol: chloroform . The pellet was collected by filtration using Whatman® glass microfiber filters grade GF/A , washed with ice-cold methanol: chloroform and finally with ice-cold acetone. The pellet was vacuum dried overnight at RT and the resulting CWMs were stored at 4 °C in a tightly sealed bottle under dark conditions.Extraction of lettuce leaves CWM was performed at 4 °C as described in our previous work . Mature g for 2 min and the resulting supernatant was used to coat MaxiSorpTM 96-well plates . The plates were incubated at 4 °C for 18 h. Following three washes with PBS-T, blocking was performed with 200 µL of PBS supplemented with 2% nonfat dry milk (blocking buffer) for 1 h at 37 °C. Following blocking and washing, 50 µL of HuNoV VLPs diluted in PBS (10 µg/mL) were added and the plates were incubated for 1 h at RT. For all the historical GII.4 VLPs and mutants, the primary and secondary antibodies used were rabbit anti-GII.4 VLPs and a goat anti-rabbit IgG HRP-conjugated  at 1:1000 and 1:2000, respectively. The rabbit anti-GII.4 norovirus polyclonal sera was generated from a rabbit hyperimmunized with HuNoV VLP cocktail including GII.4 1987, 2002, 2006b and 2009. This antibody was able to detect all GII.4 VLPs from 1987 to 2009 used in this study. This antibody was generated as described previously [The CWM-ELISA protocol was followed as previously described ,18. Brieeviously . For theb and Ley antigens [Purified porcine gastric mucin (PGM) type III  was used as a positive carbohydrate control since it was previously reported to contain primarily A, H and less of Leantigens ,18,28,34antigens . For ELIFor assessing whether the two mutations at position 374 and 443 of GII.4.HS194 affected its HBGA binding profile, binding to PGM or B-type HBGA human saliva was tested. First, to determine whether the primary antibody (rabbit anti-GII.4 VLPs) can still detect the mutant VLPs, an ELISA plate was coated with 2 µg/mL of each of the WT and mutant VLPs for 4 h at RT. Following an overnight standard blocking and washing steps, the primary antibody was serially diluted (1:10), added in duplicate to each VLP-coated wells and allowed to bind for 1 h at 37 °C while the secondary antibody (goat anti-rabbit IgG HRP-conjugated) was added at 1:2000 as described above. The primary antibody bound to WT and mutant VLPs without significant differences at all the tested dilutions; however, the binding signal for WT and mutant VLPs started to steadily decrease beyond the 1:8000 dilution (data not shown). Second, ELISA plates were coated with PGM at 5mg/mL or with a human blood type B saliva at 1:500 dilution. Briefly, 16 µg/mL of each of the two mutants and the wildtype VLPs were serially diluted (1:2), added in duplicate to the wells and allowed to bind for 1 h at 37 °C. Following standard washings, blocking and incubations as described above, the primary antibody (rabbit anti-GII.4 VLPs) was added at 1:8000, while the secondary antibody (goat anti-rabbit IgG HRP-conjugated) was added at 1:2000. The ELISA plates were stopped and read at A450nm.2), dehydrated in 30 mL of gradient ethanol series (70% (v/v), 90%, 95%, 100% and 100%) for 6 h and embedded in paraffin blocks. Five-micrometer sections were cut using a microtome and collected on glass slides . Slides were dried at 37 °C for 2 h, deparaffinized in xylene for 15 min and rehydrated in 30 mL gradient of ethanol series . Slides were incubated with cellulase R-10  at a final concentration of 1 µg/µL at 37°C for 1 h. Following washing three times with 300 mL of PBS containing 0.05% Tween-20 (PBS-T), blocking was performed with 200 µL of PBS supplemented with 1% bovine serum albumin (BSA). For fucosidase digestion, each slide was digested with 20 U α-1,2 fucosidase  at 37 °C for 1 h. For inhibition with HBGA monoclonal antibody or lectins, a 200 µL of anti-H type 1 HBGA monoclonal antibody BG4  diluted in PBS were added to the slides and incubated for 1 h at 37 °C. Plant lectins DBA (Dolichos biflorus), LEA (Lycopersicon esculentum), BS-I (Bandeiraea simplicifolia) and LcH (Lens culinaris), which recognize GalNAc (type A HBGA), GlcNAc, α-D-Gal (type B HBGA) and α-D-Man/α-D-Glc, respectively, were diluted in TBS buffer  to a final concentration of 100 µg/mL added to the slides and incubated for 1 h at 37 °C. Subsequently, the slides were incubated with 200 µL of HuNoV VLPs  at RT for 1 h. The bound VLPs on lettuce tissues were detected using guinea pig anti-GII.4 NoV VLPs or rabbit anti-GI.1 VLP antisera (1:1000) and Alexa Fluor 488-conjugated goat anti-guinea pig or anti-rabbit IgG (Invitrogen) at 1:700 dilution . Negative controls used included slides treated as above but without the VLPs. The slides were washed three times with 300 mL PBS-T after each incubation step. The plant chloroplast autofluorescence was shown in red while nuclei were stained with 4’,6-diamidino-2- phenylindole (DAPI) and shown in blue. All pictures were taken using the same exposure time.The VLP-lettuce tissue IFA protocol was followed as previously described ,18. Brie® version 5  was used for statistical analyses. One-Way ANOVA followed by Tukey’s post hoc test was used to determine significant differences between the different treatments. Each treatment in each ELISA assay was run in triplicate and the ELISA was repeated at least twice to determine assay consistency. The VLP mutants (D37A and G443A) dose response ELISA with B-type saliva and PGM were only performed once with duplicate wells but with multiple VLP concentrations. The Alexa Fluor 488 green fluorescent intensity of all IFA images was measured by ImageJ software (https://imagej.nih.gov/ij/). The mean fluorescent intensity for WT VLPs (GII.4 or GI.1) was transformed to 100% and the intensity values of the different treatments were adjusted accordingly. Differences in means were considered significant when the p-value was <0.05. Data were represented as mean ± standard errors. Significantly different means were denoted by different alphabetical letters. The binding of VLPs to PGM or lettuce CWM was considered positive when the A450 signal was significantly higher than the highest A450 of control negative wells.GraphPad PrismThe binding of our previously characterized strain GII.4/HS194/2009 to PGM was not significantly different from that of the GII.4 historical strains of 1987, 1997 and 2006 A. The GIA450 values of ≤ 0.4. In contrast, the WT VLPs binding to PGM  under TEM as those of the GII.4.HS194.2009 WT VLP A. Besideg to PGM B or the g to PGM C increasMutation of WT GII.4.HS194.2009 at amino acid residue 374, abolished VLP binding to PGM, as its binding signal was similar to that of negative control wells A. HoweveThe binding of GI.1 to lettuce was visualized using IFA Aa. Pre-tHuNoV GI.1 VLPs bound to PGM, while its mutant W375A exhibited significantly reduced binding A. The biy antigen and less efficiently to H3 , while others (residues 389–395—named site 2) are temporally evolving . BindingAlthough HuNoV GII.4 is the predominant strain globally and is often implicated in leafy green outbreaks ,39,40,41In conclusion, we have shown that the binding of HuNoV GII.4 and GI strains to lettuce is mediated through the virus HBGA binding sites. HuNoV GI.1 was shown to bind to lettuce in a similar fashion to GII.4, that is, through H-like HBGA antigen. The binding of historical HuNoV strains to lettuce correlated with their ability to bind to HBGAs. The lettuce binding profiles of historical GII.4 HuNoV strains which mimicked their PGM  binding profiles as well as the mutants of GI.1 and GII.4 which lost HBGA binding resulting in loss or significant reduction in binding to lettuce provided multiple evidence to support our hypothesis. Overall, our research suggests that integrating epidemiological investigation during norovirus foodborne outbreaks with genotyping data will allow for a better assessment of the relationship between circulating/emerging norovirus genotypes and lettuce and the impact on foodborne disease.


In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability  Endothelial cells (ECs) are connected by different junctional proteins that mediate vascular barrier function and intercellular communication5. The reorganization of tight and adherens junctions has been frequently linked to microvascular dysfunction and hyperpermeability, often induced by TNF and other inflammatory mediators8. However, little is known on the role of connexins (Cxs) and their channels, i.e. gap junction channels (GJCs) and hemichannels, in the pathogenesis of SIRS. GJCs consist of two hemichannels, each containing 6 Cx proteins. The latter are composed of four transmembrane regions, two extracellular loops, a cytoplasmic loop and cytosolic N- and C-terminal tails. Hemichannels provide a passageway between the intra- and extracellular environment, while GJCs connect the cytoplasm of two neighbouring cells, providing a route for direct cell-cell communication. Both hemichannels and GJCs allow the passage of ions and small molecules (≤1.5 kDa) such as adenosine trisphosphate (ATP) and inositol trisphosphate (IP3)10. Cx43 is the most abundant and widespread Cx in mammals, including in vascular ECs12. Cx43 hemichannels are primarily closed under physiological conditions14; however, they are reported to open upon various, mostly pathological stimuli such as ischemic or inflammatory conditions16. Given the large conductance of Cx43 hemichannels, excessive opening of these channels is expected to have deleterious consequences for the integrity of the cell due to dissipative electrical current flow, disturbed cellular ion homeostasis, Ca2+-entry and loss of crucial metabolites such as e.g. ATP or glutamate18.During TNF-induced SIRS, an exaggerated production and secretion of pro-inflammatory cytokines (e.g. interleukin 6 (IL-6), IL-1) and chemokines (CXCL-1) into the circulation results in the activation and increased permeability of the endotheliumin vivo by making use of TAT-Gap1919 and TAT-CT922, respectively known to block or promote Cx43 hemichannel opening . Gap19 and CT9 both have their targets inside the cell, at the C-terminal tail and cytoplasmic loop of Cx43, respectively, and are therefore fused to a translocation sequence (TAT) to improve their membrane permeation in vivo. Combined, our in vivo and in vitro approaches demonstrate that Cx43 hemichannels, through TNF-induced Ca2+-increases, lead to renal vascular permeability and animal mortality in SIRS.Our aim was to investigate whether selectively targeting Cx43 hemichannels could rescue animals from TNF-induced SIRS, particularly from vascular leakage. We targeted Cx43 hemichannels 25. To address the role of Cx43 hemichannel opening, we injected TNF intravenously (i.v.) in C57BL/6J mice in the absence or presence of peptides modulating Cx43 hemichannel function. Prophylactic administration of Gap27 (25 mg/kg i.v.), a peptide mimicking a conserved domain on the second extracellular loop of various Cxs and known to first block hemichannels followed by delayed GJC block26, significantly protected 50% of the mice against TNF-induced hypothermia and mortality  and obtained significant protection against hypothermia and mortality  sensitized mice to TNF, leading to 100% mortality and stronger hypothermia , reporting cell lysis, and pro-inflammatory cytokines and chemokines such as IL-6 and CXCL-1 were analysed 6 hr post-TNF. Increased levels of LDH, IL-6 and CXCL-1, as well as of P-selectin, marking EC activation, were observed  of ~220 pS, consistent with the opening of Cx43 hemichannels  at ~7.31 ng/ml TNF (Hill coefficient = 3) 31, that may modulate Cx43 hemichannel opening32. Accordingly, Ca2+-imaging experiments on HeLa-Cx43 cells demonstrated Ca2+-oscillations in response to 100 ng/ml TNF  (p < 0.001)  Fig. . Moreove 3) Fig. . TNF is 01) Fig. .2+]i played a role in activation. At normal resting 50 nM [Ca2+]i, unitary currents only appeared at +60 mV but at higher [Ca2+]i (250 or 500 nM), unitary currents additionally appeared at more physiological negative membrane potential 34, and found that it did not protect mice against mortality, but significantly ameliorated body temperatures  was found to significantly protect against hypothermia, visible after 6 hr and maintained until 24 hr, and against mortality  decreased TNF-induced renal vascular leakage, suggesting that CaMKII may be implicated in this process , making use of carbenoxolone39, which inhibits both GJCs and hemichannels, but also affects many other targets43. Here, we demonstrated a significant impact of selective Cx43 hemichannel blockade via TAT-Gap19 on renal vascular permeability. Other studies linked an increased ex vivo Cx43 expression in rat kidneys upon lipopolysaccharide treatment to a pro-inflammatory effect44, and showed an upregulation of Cx43 in chronic kidney disease, which was associated with increased pro-inflammatory signalling45. However, in these studies, Cx43 hemichannel functionality was not addressed.Cx43 channels have previously been implicated in vascular permeability alterations based on 46. However, the TAT-Gap19 doses used were very high (up to 120 mg/kg compared to 5 mg/kg in this study).Circulating peptides are rapidly cleared from the blood suggesting that the protective effect of TAT-Gap19 on mortality occurring over a time span of several days involves an early and crucial event. Of interest, in a model of mild CLP in Balb/c mice, Gap26 and TAT-Gap19 exhibited opposite effects, with Gap26 being protective and TAT-Gap19 worsening mortality21, was used to contrast the TAT-Gap19 effects, and was shown to sensitize the TNF-induced phenotype in vivo. To understand the opposite effects of TAT-Gap19 and TAT-CT9, we investigated the modulating effect of TNF on hemichannel activity through electrophysiological recording and found that TNF rapidly and dose-dependently stimulated Cx43 hemichannel opening, via [Ca2+]i-elevation. These findings demonstrate that Cx43 hemichannel opening is an early event that may crucially affect the outcome, as exemplified by the strong protective effect of a single injection of TAT-Gap19 on long lasting outcome measurements such as mortality.TAT-CT9, which enhances hemichannel opening47, which subsequently induces [Ca2+]i -elevation in neighbouring cells via paracrine purinergic signalling49. In fact, hemichannel-linked ATP-release plays a major role in cell-to-cell propagation of Ca2+-waves37, which have been demonstrated to facilitate spread of inflammation along the vascular endothelium. Interestingly, PPADS protected mice against TNF-induced mortality and hypothermia, suggesting that purinergic receptor activation is contributing to TNF-induced pathology. BAPTA-AM on the other hand only protected mice against TNF-induced hypothermia and renal vascular permeability, but could not rescue against mortality, presumably because BAPTA-AM does not only inhibit pathological Ca2+-signalling, but also interferes with normal cell function. As a result, organ function may be disturbed by BAPTA-AM thereby compromising the function of heart, liver, kidney, brain and others, which may tip the mortality balance and obscure the protective potential.It is well known that Cx hemichannels are also involved as a pathway for ATP-release8. However, different strategies for ameliorating microvascular leakage in several mouse models of sepsis were associated with better survival outcome8. Interestingly, these protective effects were not associated with lower inflammatory cytokine levels, as found here and in another study, investigating the role of receptor interacting protein kinase (RIPK) 1-dependent necroptosis in TNF-induced SIRS3. Although the mechanisms by which vascular permeability is induced are still not fully understood, it is conceivable that excessive opening of hemichannels upon TNF-induced changes in [Ca2+]i could lead to detrimental outcomes in cells, via a cellular imbalance of ionic/molecular gradients18 and a subsequent osmotic water flow50. Indeed, the contribution of open hemichannels to cell death induction has been debated in various studies46 and has been reported in retinal ECs under ischemic conditions41. However, in our study, the TNF challenge did not promote renal cell death, as quantified by TUNEL staining, suggesting it was not causative for the observed TNF-induced renal permeability. In contrast, in the liver, TNF-induced endothelial permeability was shown to be induced by RIPK1-dependent necroptosis3. Another pathway underlying permeability can be attributed to Ca2+ and CaMKII function38. CaMKII has been linked to myosine light chain (MLC)- and MLC kinase-phosphorylation51, which are importantly involved in vascular endothelial barrier dysfunction53. Our finding of decreased renal vascular leakage with KN-93 CaMKII inhibition points to involvement of a Ca2+/CaMKII-dependent cascade, which is a central hub in the signalling cascade leading to endothelial barrier leakage38.The causal relation between vascular permeability and mortality is still a matter of debatein vivo efficacy and that the TNF-induced SIRS mice model has limited relevance for the complex condition of human SIRS/sepsis. As a result, further studies are needed to unravel the potential role of Cx43 targeted therapies in the clinical setting of SIRS treatment.These findings emphasize the specific contribution of Cx43 hemichannels in inducing vascular permeability and blocking these channels protects against TNF-induced vascular leakage, hypothermia and eventually mortality. Limitations of the present study are that the peptides used have a short life-time and low 54, were cultured in Dulbecco’s modified eagle medium , supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 2 mM glutamine, 10 µg/ml streptomycin, 10 U/ml penicillin, 0.25 µg/ml fungizone  and 1 µg/ml puromycin . HeLa-WT cells were grown in the medium without puromycin. Mouse GJA1 gene was cloned into the EcoRI/BamHI restricted cloning site of the expression vector pMJgreen. Cytomegalovirus promoter was used. The vector also contains a puromycin N-acetyl-transferase (Pac) gene encoding region. EA.hy926 human ECs, a hybrid clone generated by fusing HUVECs with A549 cells, were cultured in DMEM, supplemented with 10% FBS, 1% L-glutamine, 0.4% sodium pyruvate and 2% HT-supplement . All cells were cultured at 37 °C in a 10% CO2 atmosphere and were tested for mycoplasma on a regular basis.HeLa cells, stably transfected with Cx43Escherichia coli and purified (>95%) in the VIB Protein Core . The biological activity of murine TNF (in vivo) and human TNF (in vitro) was 7.94 × 109 IU/mg and 6.8 × 107 IU/mg respectively, and was determined using an MTT -2,5-diphenyltetrazolium bromide] assay. Peptides (purity > 95%) were purchased from Pepnome (China) and Genosphere Biotechnologies . Endotoxin contamination was checked by a Limulus Amebocyte Lysate assay and was <1.0 EU/mg. Gap27 (SRPTEKTIFII), TAT-Gap19 (YGRKKRRQRRR-KQIEIKKFK), Gap19 (KQIEIKKFK), TAT-CT9 (YGRKKRRQRRR-RPRPDDLEI), CT9 (RPRPDDLEI) and TAT (YGRKKRRQRRR) were diluted in endotoxin-free phosphate-buffered saline (PBS) or pipette solution for in vivo or in vitro use respectively. BAPTA was purchased from Invitrogen (B1212). BAPTA-AM  was dissolved in dimethylsulfoxide (DMSO) (+0.01% pluronic® (Invitrogen)) and further diluted in endotoxin-free PBS for in vivo use. Pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid , and KN-93  were dissolved in DMSO and further diluted in endotoxin-free PBS for in vivo use. Fluorescein isothiocyanate (FITC)-dextran  was used in vascular permeability studies.Recombinant mouse TNF and human TNF were produced in 2+ and Mg2+ and under conditions of K+-channel blockade with Cs+, Ba2+ and TEA+. Cells were bathed in a recording chamber filled with a modified Krebs-Ringer solution, consisting of (in mM): 150 NaCl, 6 CsCl, 2 MgCl2, 2 CaCl2, 5 glucose (10 for EA.hy926 cells), 5 HEPES, 1 BaCl2 and 2 Pyruvate, with pH adjusted to 7.4. The standard whole-cell recording pipette solution was composed of (in mM): 130 CsCl, 10 NaAsp, 0.26 CaCl2, 5 HEPES, 2 EGTA, 5 TEA-Cl and 1 MgCl2, with pH adjusted to 7.2. For experiments on EA.hy926 cells, 5 mM MgATP was additionally added to the pipette solution. A baseline free intracellular Ca2+  of 50 nM, as well as other concentrations , were calculated with Webmax Standard software (http://www.stanford.edu/~cpatton/webmaxcS.htm). In experiments where cells were challenged with TNF, the pipette EGTA concentration was 2 mM for HeLa-Cx43 cells and 0.1 mM for EA.hy926 cells; for Ca2+ chelation experiments aimed at suppressing [Ca2+]i changes, EGTA was changed to 10 mM BAPTA. Gap19 and CT9 peptides (100 µM) were added to the pipette solution. Cells were exposed to recombinant human TNF via a fast local perfusion system. An EPC 7 PLUS patch-clamp amplifier  was used for single channel recordings. Data were acquired at 6 kHz using a NI USB-6221 data acquisition device from National Instruments  and WinWCP acquisition software . All currents in whole-cell configuration were filtered at 1 kHz (7-pole Besselfilter). For single channel analysis, holding currents were subtracted from the recorded current traces, giving traces that only contained unitary current events. Unitary conductances were calculated from the elementary current transitions Δi as: γ = Δi/Vm. From these data, we constructed all-point conductance histograms that displayed one or more Gaussian distributions. These were fit by a probability density function assuming independent channel opening56. Channel activity was quantified from the charge transfer Qm associated with unitary currents; this was done by integrating the unitary current traces over the duration of the voltage step as: Qm = ∫idt. Unitary events in the tail currents were counted manually.Subconfluent cultures of HeLa-Cx43 cells and EA.hy926 cells were seeded on 13-mm-diameter glass coverslips  and experiments were performed at subconfluency the next day. All recordings were performed in the presence of extracellular Ca2, 0.81 mM MgSO4, 13 mM NaCl, 0.18 mM Na2HPO4, 5.36 mM KCl, 0.44 mM KH2PO4, 5.55 mM d-glucose, and 25 mM HEPES) for 45 min at 37 °C. After 45 min, coverslips were washed and then left for an additional 15 min at 37 °C in HBSS-HEPES to allow for de-esterification. Cells were thereafter transferred to an inverted epifluorescence microscope  equipped with a superfusion system allowing fast solution changes. Superfusion was switched off during the registration of oscillatory activity. Images were taken every second with a 40x oil-immersion objective and an electron-multiplying CCD camera . We used a Lambda DG-4 filterswitch  to deliver excitation at 482 nm and captured emitted light via a 505-nm long-pass dichroic mirror and a 535-nm bandpass filter (35-nm bandwidth). Recordings and analysis were done with custom-developed QuantEMframes and Fluoframes software written in Microsoft Visual C++ 6.0. Oscillatory activity was recorded in a 10 min observation window.HeLa-Cx43 cells were seeded onto 18-mm-diameter glass coverslips , and experiments were performed at subconfluency the next day. The cells were loaded with 5 µM fluo3-AM in HBSS-HEPES . All mice experiments were organized in accordance with institutional, national, and European animal regulations, and got approved by the animal ethics committee at Ghent University (ECD 2013–064).All mice were bred in specific pathogen-free or in conventional animal facilities, and were males between 8–12 weeks during all the experiments. As connexin expression levels can be altered by the hormonal cycle, male mice were used to limit variability related to the hormonal status. Mice were housed in air-conditioned, temperature-controlled rooms with 14/10-hour light/dark cycles. Food and water were provided 57. Blood and tissue samples were collected at different time points after injection as outlined in the results section. Blood was collected by cardiac or retro-orbital puncture. Vascular permeability was evaluated as previously described58.Peptides were diluted in endotoxin-free PBS (Sigma-Aldrich) and injected i.v. in a volume of 200 µl at 5 or 10 mg/kg for TAT-Gap19, 5 or 10 mg/kg for TAT, 10 or 15 mg/kg for TAT-CT9, and 25 mg/kg for Gap27, 15 min before i.v. injection of murine TNF (7.5–15 µg/20 g) in 200 µl endotoxin-free PBS (pH 6.8) (prophylactic administration). Therapeutic administration consisted of i.v. injection of 5 mg/kg of TAT-Gap19 30 min after TNF. BAPTA-AM (2 mM in DPBS (6% DMSO)) and KN-93 (10 mg/kg in DPBS (6% DMSO)) were injected i.v. respectively 30 min and 60 min before TNF injection. Control injections consisted of an equal amount (i.v.) of DPBS (6% DMSO). PPADS (50 mg/kg) was injected intraperitoneally (i.p.) 15 min before injection of TNF. Survival and body temperatures were monitored until 5 days post TNF-injection. Rectal body temperatures were recorded with an electric thermometer , and dead mice (25 °C) are included in all body temperature graphs, as was previously implementedin situ cell death detection kit; Roche) in accordance with the manufacturer’s instructions, 6 hr after TNF injection. Sections were counterstained with Hoechst (Sigma-Aldrich). Micrographs were acquired using a Zeiss Axioscan Z.1 slide scanner  at 20x magnification, with a Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics), and illuminated with an HXP 120V light source. Analysis was performed using VolocityⓇ  and QuPath (GitHub) softwares.Tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned at 5 µm and dewaxed before staining procedure was initiated. Cell death was identified by terminal deoxynucleotidyl-transferase dUTP nick-end labeling (TUNEL) staining  ligand 1 (CXCL-1) were determined with Luminex magnetic bead-based assays . Plasma P-selectin levels were measured through a Quantikine ELISA kit .*for p < 0.05; **for p < 0.01; ***for p < 0.001 or ****for p < 0.0001.Statistical analysis was performed in GraphPad Prism (v.7.0). All Kaplan-Meier survival curves were analysed by the log-rank Mantel-Cox test. Body temperatures and vascular permeability assays were analysed by a two-way ANOVA with Tukey’s multiple comparisons test to compare more than two groups, and by a two-way ANOVA with Sidak’s multiple comparisons test to compare two groups. A Shapiro-Wilk normality test indicated normally distributed data. All immunohistochemical and serum parameters were compared with a one-way ANOVA with a Bonferroni post-test to compare all pairs. In the electrophysiology recordings, comparisons between two groups were done with a two-tailed t-test and comparison of more than two groups was performed with a one-way ANOVA and Bonferroni post-hoc test. Significant results are indicated with Supplementary Figures and Legends


This process can contribute to the imbalance of bone remodeling, which ultimately leads to osteoporosis. Therefore, reducing the ROS generation during osteogenesis of BMSCs may be an effective way to reverse the impairment of osteogenesis. Melatonin (MLT) has been reported to act as an antioxidant during cell proliferation and differentiation, but its antioxidant effect and mechanism of action during osteogenesis of MSCs in the inflammatory microenvironment, especially in the presence of TNF-α, remain unknown and need further study. In our study, we demonstrate that melatonin can counteract the generation of ROS and the inhibitory osteogenesis of BMSCs induced by TNF-α, by upregulating the expression of antioxidases and downregulating the expression of oxidases. Meanwhile, MLT can inhibit the phosphorylation of p65 protein and block the degradation of IκBα protein, thus decreasing the activity of the NF-κB pathway. This study confirmed that melatonin can inhibit the generation of ROS during osteogenic differentiation of BMSCs and reverse the inhibition of osteogenic differentiation of BMSCs in vitro, suggesting that melatonin can antagonize TNF-α-induced ROS generation and promote the great effect of osteogenic differentiation of BMSCs. Accordingly, these findings provide more evidence that melatonin can be used as a candidate drug for the treatment of osteoporosis.Accumulation of reactive oxygen species (ROS), which can be induced by inflammatory cytokines, such as tumor necrosis factor-alpha (TNF- ROS can be formed as a natural byproduct of cellular oxygen metabolism, mainly in the mitochondria. It also can be derived from nicotinamide adenine dinucleotide phosphate oxidase (NAPDH oxidase/NOX), peroxisome, xanthine oxidase, and lipolytic enzyme [α) can lead to large production of large amounts of ROS [Reactive oxygen species (ROS) are oxygen-containing chemically active substances. They include the hydroxyl radical (OH), hydroxyl ion  play an important role in bone formation. Generally, BMSCs can differentiate into osteoblasts, chondrocytes, and adipocytes . Severalignaling . Accordiα.Melatonin (MLT) is a hormone produced mainly by the pineal gland. It has been shown to have a wide range of effect, including the inhibition of tumor growth, immunomodulation, and regulation of circadian rhythms . In 1995α. The aims of this research are to further confirm the importance of melatonin as an antioxidant during the osteogenesis of BMSCs as well as to provide additional support for the use of melatonin as a candidate drug for the treatment of osteoporosis.In this study, we investigated whether melatonin can decrease the ROS generation and thus protect osteogenic differentiation of BMSCs in an inflammatory microenvironment induced by TNF-κB signaling protein , goat anti-mouse IgG, and goat anti-rabbit IgG secondary antibodies were from Cell Signaling Technology Inc. . Abs against GAPDH was from Proteintech Group Inc. . Melatonin was purchased from Sigma-Aldrich , and recombinant human TNF-α was purchased from R&D Systems . The osteogenic differentiation medium was purchased from Cyagen Biosciences Inc. .Antibodies against runt-related transcription factor-2 (RUNX2), osteopontin (OPN), NADPH oxidase1 (NOX1), and NADPH oxidase2 (NOX2) were purchased from Abcam . Cu/Zn superoxide dismutase (Cu/Zn SOD or SOD1), catalase (CAT), NF-n = 3) as described previously [2. After 48 h, nonadherent cells were removed by changing the medium. Thereafter, the medium was changed every three days. When the cells reached 85%–95% confluence, they were trypsinized, counted, and plated again. Cells from passages 3–6 were used for the experiments.The study was approved by the Ethical Committee of Sun Yat-sen University, and written informed consent was obtained from all participants. MSCs were isolated from bone marrow obtained from healthy volunteer donors (eviously . Brieflyα (10 ng/mL), and melatonin (100 μmol/L). The osteogenic medium was composed of low-glucose DMEM, dexamethasone (0.1 μmol/L), 10% FBS, ascorbic acid (50 μg/mL), and β-glycerol phosphate (10 mmol/L). The medium was changed every 72 hours.BMSCs were cultured in a 6-well plate with complete medium. After reaching 80% confluence, the cells were cultured with osteogenic medium and treated with or without vehicle , TNF-2DCFDA; Invitrogen, Waltham, MA, USA). Adherent cells in the 6-well plate were incubated with DMEM containing CM-H2DCFDA (10 μmol/L) for 25 min at 37°C in darkness. Cells were washed with PBS three times, and fluorescence from the plate was photographed using the microscope . Suspension cells  were resuspended in DMEM containing CM-H2DCFDA (10 μmol/L) and incubated for 25 min at 37°C in darkness. After washing with PBS three times, fluorescence intensity was quantified at 488 nm excitation and 525 nm emission by flow cytometry . Each sample contained 30,000 cells. Data was calculated by FlowJo .ROS production in BMSCs was detected and measured by a fluorescence probe, 2′,7′-dichlorodihydrofluorescein diacetate  for 10 min after 14 days of osteogenesis. After removing the fixing solution, the cells were stained with Alizarin red S (ARS) solution (Cyagen Biosciences Inc.) for 10 minutes at room temperature. After three washing with PBS, the cells were photographed. Calcium deposits are shown in red. Stains were measured at 405 nm to quantify the amount of Alizarin red. Each experiment was performed in triplicate.RUNX2, OPN, NOX1, NOX2, SOD1, and CAT. The expression level of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as a reference. The Ct value of the GAPDH was subtracted from the Ct value of the target gene (ΔCt), and the average ΔCt value of the triplicates was recorded. The relative expression levels of each gene were determined using the 2-ΔΔCt method. Primer sequences used in this study are listed in Total RNA was extracted from cells in different treatment groups using a RNAiso Plus reagent  and then converted to cDNA using PrimeScript RT Master Mix (TaKaRa) according to the manufacturer's protocols. qPCR was performed on a Light Cycler 480 Real-Time PCR Detection System  using SYBR Green I Master Mix (Roche). Expression levels of the following genes were analyzed: Cells were rinsed twice with ice-cold PBS and lysed in RIPA lysis buffer  containing protease inhibitor cocktail . Cell lysates were centrifuged at 12,000 g for 15 min at 4°C, and the protein concentration was quantified using the BCA protein assay kit (Beyotime). Equal amounts of each sample were subjected in 10% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to PVDF transfer membranes . Membranes were blocked with 5% nonfat dry milk for 1 h at 25°C and then incubated with the designated antibodies at 4°C overnight. Antibody-specific labeling was observed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies for 1 h at room temperature, and the results were visualized with the ECL kit (Millipore). The bands were quantified by ImageJ software  and normalized to GAPDH as the loading control.P < 0.05 was considered statistically significant.All quantitative data were given as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc test for multiple comparisons. All statistical analyses were conducted with the SPSS 20.0 statistical software package . α during osteogenesis of BMSCs, we induced the human BMSCs to undergo osteogenesis and BMSCs were treated with or without vehicle, TNF-α (10 ng/mL), and melatonin (100 μmol/L). As shown in α treatment alone increased the ROS fluorescence on days 3, 7, and 14. And melatonin supplementation decreased the ROS fluorescence induced by TNF-α during the osteogenic differentiation. Flow cytometry revealed that TNF-α treatment rendered the ROS levels higher than those in the vehicle group, whereas melatonin cotreatment decreased the generation of ROS , and melatonin (100 μmol/L). After 14 days of osteogenic differentiation, Alizarin red S (ARS) staining revealed that TNF-α supplementation alone reduced the calcium deposits induced by osteoblast and melatonin can counteract their interference mineralization , and melatonin (100 μmol/L). RT-qPCR and Western blot showed the levels of expression of oxidative enzymes, including NOX1 and NOX2, to be enhanced in the presence of TNF-α, while the addition of melatonin to TNF-α significantly reduced the expression of NOX1 and NOX2 , and melatonin (100 μmol/L). RT-qPCR and Western blot indicated that TNF-α decreased the level of expression of SOD1 and CAT on days 3, 7, and 14. Melatonin treatment significantly upregulated the expression level of these genes , and melatonin (100 μmol/L). Western blot showed that TNF-α supplementation alone can significantly increase the phosphorylation of p65 protein and the degradation of IκBα on days 3, 7, and 14 over levels observed on day 0. The addition of melatonin to TNF-α attenuated the effect  and inhibited the osteogenic differentiation of BMSCs  can catalyze H2O2 into oxygen and water. TNF-α treatment can reduce the expression of SOD1 and CAT, which indicated that there is an imbalance between the oxidase system and the antioxidase system during ROS production in the presence of TNF-α.The generation and elimination of ROS depend mainly on the balance between oxidase and antioxidase. NADPH oxidative enzymes, such as NOX1 and NOX2, are among the major sources of ROS. The presence of inflammatory cytokines upregulates the expression of NADPH oxidative enzymes in bone and joint disease and consequently enhances the proliferation of ROS . In our α . Moreoveα requires further confirmation. According to previous studies [μmol/L, the proosteogenic, anti-inflammatory, and antioxidative effects of melatonin can be maximized. In the present study, we induced BMSCs, which have a stronger osteogenesis potential than other sources of MSCs, to undergo osteogenic differentiation, and we found that melatonin can decrease the generation of ROS—which had been significantly increased by TNF-α supplementation—to a remarkable degree (β) [Melatonin has been recognized as an antioxidative molecule. In addition to directly scavenging oxygen free radicals, melatonin can also upregulate the expression of antioxidase and downregulate the expression of oxidase. It has been reported that melatonin can be classified as a mitochondria-targeted antioxidant . Recentl studies , 19, whee degree , throughe degree  and upree degree . Then, we degree , as had gree (β) .κB signaling pathway is recognized as the main downstream target of TNF-α. A growing evidence has revealed that the activation of NF-κB signaling contributes to the TNF-α-inhibited osteogenesis of MSCs [α can activate the NF-κB signaling and upregulate the expression of NADPH oxidases, which contribute to a high level of ROS. In this study, we examined the expression and phosphorylation level of the NF-κB pathway protein at different points in time during BMSC osteogenic differentiation. Results showed that the NF-κB pathway is continuously activated after TNF-α treatment (α-induced ROS generation can subsequently cause the continued activation of NF-κB signaling and promote TNF-α expression, constituting a positive feedback loop and forming a vicious cycle [κB signaling activity was blocked (κB signaling might be relative to the crosstalk between TNF-α and ROS and that melatonin might break this cycle. Accordingly, we concluded that melatonin may exert its antioxidative effects at least partially through inactivation of the NF-κB pathway.The NF- of MSCs , 33. Moe of MSCs  reportedreatment , contribus cycle . After m blocked . We conjα, to induce ROS production and impair osteogenic differentiation of BMSCs. This unilaterally demonstrated that melatonin can reverse the inhibition of osteogenic differentiation of BMSCs caused by TNFα-induced ROS. Whether melatonin can antagonize the ROS induced by other inflammatory cytokines, such as IL-1 and IL-6, still needs further investigation. Second, our research on underlying mechanisms was not sufficiently complete. We found that after TNF-α treatment, the NF-κB pathway was activated, with increased IκBα degradation and p65 phosphorylation. Melatonin supplementation could significantly reduce this. However, the role and underlying mechanisms of melatonin on specifically inhibiting the activation of NF-κB signaling induced by TNF-α also need further study. At last, our study was in vitro research only. Future studies should focus on the experiments in an animal model that could provide in vivo evidence to verify the antioxidative role of melatonin on osteogenesis of BMSCs.Our study had some limitations. First, we used only one of the inflammatory cytokines, TNF-α, is involved in the inhibition of osteogenesis differentiation of BMSCs; melatonin can protect against the oxidant damage by suppressing oxidant enzymes and improving antioxidant enzymes. This process may be associated with the activation and inactivation of the NF-κB signaling pathway. Our previous studies had already showed that melatonin plays a vital role in promoting osteogenesis of BMSCs. For this reason, we consider that through its antioxidative, anti-inflammatory, and proosteogenic effects, melatonin may have a bright future as a candidate drug for maintaining the osteogenic ability of BMSCs and treating osteoporosis.In conclusion, the present study showed that the generation of ROS, which can be induced by TNF-


Heart failure with preserved ejection fraction (HFpEF) is a difficult disease with high morbidity and mortality rates and lacks an effective treatment. Here, we report the therapeutic effect of dapagliflozin, a sodium-glucose cotransporter 2 inhibitor (SGLT2i), on hypertension + hyperlipidemia-induced HFpEF in a pig model.HFpEF pigs were established by infusing a combination of deoxycorticosterone acetate (DOCA) and angiotensin II (Ang II), and Western diet (WD) feeding for 18 weeks. In the 9th week, half of the HFpEF pigs were randomly assigned to receive additional dapagliflozin treatment (10 mg/day) by oral gavage daily for the next 9 weeks. Blood pressure, lipid levels, echocardiography and cardiac hemodynamics for cardiac structural and functional changes, as well as epinephrine and norepinephrine concentrations in the plasma and tissues were measured. After sacrifice, cardiac fibrosis, the distribution of tyrosine hydroxylase (TH), inflammatory factors (IL-6 and TNF-α) and NO-cGMP-PKG pathway activity in the cardiovascular system were also determined.Blood pressure, total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) were markedly increased in HFpEF pigs, but only blood pressure was significantly decreased after 9 weeks of dapagliflozin treatment. By echocardiographic and hemodynamic assessment, dapagliflozin significantly attenuated heart concentric remodeling in HFpEF pigs, but failed to improve diastolic function and compliance with the left ventricle (LV). In the dapagliflozin treatment group, TH expression and norepinephrine concentration in the aorta were strongly mitigated compared to that in the HFpEF group. Moreover, inflammatory cytokines such as IL-6 and TNF-α in aortic tissue were markedly elevated in HFpEF pigs and inhibited by dapagliflozin. Furthermore, the reduced expression of eNOS and the PKG-1 protein and the cGMP content in the aortas of HFpEF pigs were significantly restored after 9 weeks of dapagliflozin treatment.9 weeks of dapagliflozin treatment decreases hypertension and reverses LV concentric remodeling in HFpEF pigs partly by restraining sympathetic tone in the aorta, leading to inhibition of the inflammatory response and NO-cGMP-PKG pathway activation. With the world population aging, heart failure (HF) has become a very important cause of death in older people , 2. MoreDapagliflozin, a sodium-glucose cotransporter 2 inhibitor (SGLT2i), is a newly developed oral antidiabetic drug that blocks glucose reabsorption via SGLT2 inhibition in the kidney and thus reduces glucose levels in an insulin-independent manner . Some stHence in this study, we established a HFpEF pig model with a combination of deoxycorticosterone acetate (DOCA), angiotensin II (Ang II) and Western diet (WD) feeding, that manifests major features of clinical HFpEF patients to a large extent , 8, 23, Thirty-nine-week-old female landrace pigs weighing 30–40 kg were used in this study, ten of which served as normal controls , and the remaining twenty pigs were used to establish the HFpEF model. A flow diagram of the experiment is shown in Fig. A standard transthoracic echocardiogram was performed using a commercially available echocardiographic system . Standard 2D imaging and M-mode imaging were performed to measure the interventricular septum thickness at end-diastole (IVSd), left ventricular posterior wall thickness (LVPW), LV mass index, and left atrium (LA) diameter and left ventricle (LV) diameters at end-diastole (LVEDD) and systole (LVESD). A pulse-wave Doppler was used to evaluate the isovolumetric relaxation time (IVRT), deceleration time (DT), mitral inflow E wave and mitral inflow A wave. A tissue Doppler was used to measure the median mitral annular early diastolic velocity (e′), followed by the calculation of E/e′ and E/A. The stiffness index β, an index of the elastic property of the arterial wall, was calculated from the blood pressure and the diameter of the aortic root as follows: Stiffness β = Dd/(Ds−Dd) × ln(Ps/Pd), where Ps and Pd were the systolic and diastolic blood pressures, and Ds and Dd were the systolic and diastolic inner diameters of the aortic root, respectively. At least three consecutive beats were taken for each measurement. All analyses were completed off-line in a blind fashion on a workstation .The invasive hemodynamic study was conducted under general anesthesia at baseline and at the 9th and 18th weeks to asseess cardiac function. The LV ± dp/dt, end-diastolic pressure–volume relationship (EDPVR), end-systolic pressure–volume relationship (ESPVR), left ventricular ejection fraction (LVEF) and cardiac output (CO) were measured with a 7-Fr combined catheter-micromanometer  through the femoral artery. All pressure–volume loop data were recorded at least 8 to 10 beats at end-expiration from the raw LV pressure and conductance volume data using commercially available software . Right heart catheterization was performed to investigate pulmonary hemodynamic changes using a Swan-Ganz catheter  conducted under complete aseptic precautions via the right internal jugular vein.Plasma brain natriuretic peptide (BNP) and Ang II were determined to assess myocardial stretch and renin–angiotensin–aldosterone system (RAAS) system activity, respectively, using available enzyme-linked immunosorbent assay (ELISA) kits . The levels of plasma epinephrine (E) and norepinephrine (NE) were measured to analyze the sympathetic response with commercially available ELISA kits . NE concentrations in the LA, LV, aorta and kidney tissues were also determined . The levels of IL-6, TNF-α and cGMP in the aorta were assessed via available ELISA kits . Plasma total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), HDL and glycosylated hemoglobin (HbA1c) were measured with specific kits according to the manufacturer’s instructions . Urinary electrolyte and osmolality levels were measured by the autoanalyzer CX5 . Urinary glucose and uric acid levels were measured by Cobas Mira autoanalyzer .The aortic tissue homogenates were mixed with a 2 × solution of sample buffer  SDS, 25% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue) and heated at 95 °C for 3 min. Next, 40 μg of protein sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes . Membranes were blotted for 1 h in 5% (w/v) fat free milk and incubated overnight at 4 °C with primary antibodies against eNOS , p-eNOS , PKG1 , and β-actin . The bound antibody was detected using a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Bands were visualized by enhanced chemiluminescence  and analyzed using ImageJ . All protein expression was normalized to that of β-actin, which served as an internal control.Hematoxylin–eosin (HE) and staining Masson’s trichrome staining were performed to evaluate general morphology and collagen formation by conventional methods. The free wall of the LV was divided into three layers, which were cut at 10 μm thickness parallel to the apical-basal axis for Masson’s trichrome and perpendicular to the apical-basal axis for TH immunofluorescence staining. The aorta was cut perpendicular to its long axis for TH immunofluorescence staining. We also cut the LV subendocardium and LA tissue perpendicular to the cardiomyocyte long axis for HE staining to assess the cross-sectional area (CSA) of cardiomyocytes. Five randomly selected fields of each section were assessed in a blinded fashion and one representative image was selected.For TH immunofluorescence staining, tissue sections were deparaffinized, rehydrated, antigen retrieved and blocked in 10% normal goat serum , followed by incubation with a primary antibody against TH  overnight at 4 °C. The bound antibodies were detected with Alexa Fluor-conjugated secondary antibodies . The fluorescence images were obtained using a fluorescence microscope  at excitation and emission wavelengths of 543 nm and 573 nm, respectively, and measured with Axiovision software .All data are expressed as the mean ± standard deviation (SD). We employed one-way analysis of variance (ANOVA) or two-way repeated measures ANOVA followed by the Bonferroni post hoc test. SPSS 22.0  was used for the statistical analyses. A p value < 0.05 was deemed statistically significant.+ excretion. As expected, the plasma BNP, E, NE and Ang II concentrations in HFpEF pigs were strongly increased when compared with the Normal group. Moreover, the plasma levels of E and NE were significantly reduced in the DAPA group. Furthermore, we measured the NE concentration in LV subendocardium, LA, aorta and renal tissues. Similarly, in the HFpEF group, NE was significantly increased as compared with the Normal group, but more importantly, dapagliflozin treatment significantly decreased the NE concentration in the aortic tissues of HFpEF pigs. Data of plasma measurement results from baseline and from the 9th week are presented in Additional file As shown in Table p < 0.01). Importantly, the SBP was significantly lower in the DAPA group than in the HFpEF group from the 14th to the 18th week, while the DBP showed comparable results from the 15th to the 18th week. In the DAPA group, when comparing the blood pressure in the 18th week to that in the 9th week, both SBP and DBP were decreased after dapagliflozin treatment, suggesting that dapagliflozin could prevent the development of hypertension in HFpEF pigs.During the study, the blood pressure of experimental pigs was measured weekly. After 1 week of dapagliflozin administration, both the systolic Fig. a and diaTo explore structural alterations in the left heart, several key parameters were measured by echocardiography. Representative images of echocardiographic M-mode Doppler imaging are shown Fig. a. Pigs iTo further investigate LV fibrosis in HFpEF pigs and the effect of dapagliflozin, Masson’s trichrome staining was performed. As shown in Fig. Invasive left cardiac catheterization was carried out to determine LV contractility by calculating the values of ESPVR, LV + dp/dt and LVEF. As depicted in Fig. Additionally, invasive right cardiac catheterization was performed to investigate the alteration in pulmonary pressure under the conditions of HFpEF and dapagliflozin treatment. PASP and PCWP were significantly higher in HFpEF pigs compared with the Normal group, especially at the 18th week. Interestingly, both of PASP and PCWP them were significantly decreased after 9 weeks of dapagliflozin treatment Fig. d, f. TheWe measured LV function  and compliance by echocardiography and invasive cardiac catheterization. As shown in Fig. As TH is a pivotal enzyme involved in catecholamine synthesis and usually reflects sympathetic tone, we measured TH expression in tissues from the LV and aorta by immunofluorescence staining. As shown in Fig. The NO-cGMP-PKG signaling pathway in endothelium cells is postulated to be one of the most important factors associated with vasodilation, resulting in a reduction in blood pressure. As presented in Fig. The data in the current study provide a deeper understanding of cardiac structure remodeling, cardiac function and sympathetic tone alterations, and the molecular mechanism involved in HFpEF. With regard to the therapeutic effect of dapagliflozin on HFpEF, the following issues were addressed in this work: (1) Dapagliflozin treatment could decrease both SBP and DBP and prevent the progression of LV concentric hypertrophy remodeling and LA dilation in non-diabetic HFpEF pig models with favorable safety and effectiveness; (2) With echocardiography and an invasive hemodynamic assessment, no discernible effects of 9 weeks of dapagliflozin treatment on LV fibrosis or diastolic function were observed in HFpEF pigs; (3) Dapagliflozin treatment significantly decreased plasma adrenal medullary hormone levels in HFpEF model, and strongly mitigated the sympathetic tension of aortic tissue but not that of the LV myocardium; in the aorta, the inflammatory response and the NO-cGMP-PKG signaling pathway were deteriorated, and both were improved by dapagliflozin treatment; and (4) It is pertinent to note that dapagliflozin presented a negative regulatory effect on PASP and PCWP.Dapagliflozin is a newly developed oral antidiabetic drug that enhances renal glucose excretion or glycosuria and reduces hyperglycemia by the highly selective inhibition of SGLT2 . Previou2+ sensitization and/or activating Ca2+-activated K+ channels to reduce the intracellular Ca2+ concentration [Dapagliflozin was also found to improve arterial stiffness, endothelial dysfunction and vascular smooth muscle dysfunction and reduce circulating markers of inflammation in type 2 diabetic mice , which antration , 32. Macntration  describentration , 10. Forntration , no signntration  also fouSchwarzl reported a HFpEF model induced by DOCA  and a WD containing high content of salt (4%), cholesterol (4%), crude fat 25%), cholate (0.5%), and sugar 26%) for 12 weeks in landrace pigs % for 12 . In addi%, cholatFurthermore, we noted that the response of TH expression to dapagliflozin treatment was more sensitive in the aorta than in the LV. The underlying mechanism of this disparity remains elusive. We speculate that the experimental period and the plasma concentration of dapagliflozin should be considered. Owing to the limited data on dapagliflozin in the HFpEF pig model, more work in this area is sorely needed. In addition, we found that LA was dilated, and thus, PASP and PCWP were increased in HFpEF pigs, consistent with prior studies. Interestingly, the effect of dapagliflozin on attenuating the detrimental PASP and PCWP in HFpEF pigs was significant , 40. SinThe results presented here demonstrate that dapagliflozin might decrease blood pressure and prevent the progression of LV concentric hypertrophy in the HFpEF pig model by mitigating sympathetic nerve tone and the inflammatory response in the aorta, leading to reactivation of the NO-cGMP-PKG pathway. These data are important in elucidating the potential mechanisms of the dapagliflozin-mediated improvement in cardiac function and provide a basis for the further development of innovative therapeutic strategies.Additional file 1: Table S1. Detailed ingredient contents of Standard diet and Western diet.Additional file 2: Table S2. Characteristics and biochemical indicators of pigs at baseline and at the 9th week. Values are expressed as the mean ± SD. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni post hoc test. ap < 0.05 vs. the Normal group at the same time point.Additional file 3: Table S3. Data of renal excretion in pigs at the 18th week. Values are expressed as the mean ± SD. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni post hoc test. ap < 0.05 vs. the Normal group, bp < 0.05 vs. the HFpEF group.Additional file 4: Fig. S1. Dapagliflozin had no significant effect on cardiomyocyte CSA or LA fibrosis in HFpEF pigs. The cardiomyocyte CSA of the LV and LA was calculated (A and B). Representative images of Masson’s trichrome staining of the LA posterior wall are shown (C). The area percentages of LA fibrosis were calculated (D). Values are expressed as the mean ± SD. n = 10 pigs per group. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05 vs. the Normal group.Additional file 5: Fig. S2. Dapagliflozin had no significant effect on CO, LVEDD or LVESD in HFpEF pigs. CO, cardiac output; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension. Values are expressed as the mean ± SD. n = 10 pigs per group. Statistical analyses were performed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05 vs. the Normal group.