Background: Increased permeability of pulmonary capillary is a common consequence of sepsis that leads to acute lung injury. In this connection, ulinastatin, a urinary trypsin inhibitor (UTI), is used clinically to mitigate pulmonary edema caused by sepsis. However, the underlying mechanism of UTI in alleviating sepsis-associated pulmonary edema remains to be fully elucidated. As tight junctions (TJs) between the pulmonary microvascular endothelial cells (PMVECs) play a pivotal role in the permeability of pulmonary capillary, this study investigated the effect of UTI on expression of junctional proteins in PMVECs during sepsis.Methods: Male adult Sprague Dawley rats were subjected to cecal ligation and puncture (CLP) and divided into sham, CLP, and UTI+CLP groups. UTI was administered every 8 h for 3 days before CLP. At 48 h after surgery, Evans blue (EB) was administered to evaluate the pulmonary vascular leakage. Histological staining was used for evaluation of lung injury score. Using immunofluorescence staining and Western blot, the expression of junctional proteins  in pulmonary endothelia was assessed. In vitro, PMVECs were divided into control, lipopolysaccharide (LPS), and UTI+LPS groups for examination of expression of junctional proteins and TNF-α as well as inhibitor of NF-κB (IκB), p38 mitogen-activated protein kinases (p38 MAPKs), c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) signaling pathways. Additionally, the expression of various junctional proteins was determined in PMVECs of control, LPS, and TNF-α receptor antagonist-LPS groups. PMVECs were also treated with TNF-α and TNF-α receptor antagonist and the expression of various junctional proteins was assessed.Results: Compared with the CLP group, UTI markedly decreased EB leakage and lung injury score. The expression of occludin, claudin-5, and ZO-1 was decreased in both CLP rats and LPS-treated PMVECs, but it was reversed by UTI and TNF-α receptor antagonist. TNF-α expression was vigorously elevated in the lung of CLP rats and in LPS-challenged PMVECs, which were suppressed by UTI. In addition, TNF-α also reduced occludin, claudin-5, and ZO-1 expression in PMVECs, but these effects of TNF-α were antagonized by pretreatment with TNF-α receptor antagonist. Furthermore, UTI inhibited LPS-induced activation of NF-κB and mitogen-activated protein kinases (MAPKs) pathways in PMVECs.Conclusion: UTI effectively protects TJs and helps to attenuate the permeability of pulmonary capillary endothelial cells during sepsis through inhibiting NF-κB and MAPKs signal pathways and TNF-α expression. Mortality from sepsis is primarily attributed to injury and dysfunction of multiple organs . Among tPulmonary capillary endothelial cells or pulmonary microvascular endothelial cells (PMVECs) are tightly held with each other, thus forming a single and continuous layer as a barrier between blood and the pulmonary interstitium. Transcellular and paracellular routes are involved in the transport of macromolecules and fluids across the endothelial barrier , whereasThe inter-endothelial tight junctions (TJs) play a critical role in the paracellular permeability of the endothelial barrier . TJs prein vitro and in vivo , a potent multivalent Kunitz-type serine protease inhibitor derived from human urine, has been used clinically to treat acute pancreatitis and acute circulatory failure . It has  in vivo  due to i in vivo . Recentl in vivo , such as in vivo , 2016. F in vivo . HoweverIn the handling and use of rats for CLP, ethical guidelines as stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals were followed. All experimental protocols and use of animals were approved by the approval authority at Jinan University (No. 20180225016), and all efforts were made to minimize the number of rats used and their suffering.Table 1) weighing 250–300g were obtained from the Experimental Animal Center of Jinan University. They were kept in a 12 h light–dark cycle-controlled room, set at room temperature at 22–25°C and humidity (55 ± 15%), and fed with standard laboratory diet and water ad libitum.A total of 96 adult male Sprague Dawley rats   every 8 h via an indwelling vein needle . The rats in the CLP group were given an equal volume of saline. In the sham group, rats were given neither normal saline (NS) nor UTI treatment. Vital signs  were recorded every 6 h. The clinical features of temperature and respiratory rate were recorded every 3 h till the rats were euthanized at 48 h after CLP.g for 30 min and supernatants assayed for EB concentrations. The dye absorbance value in the solution was then detected by a microplate reader , compared with a series of standard EB dilutions in formamide, and converted to mg of EB/mL of the solution. Based on this value, the amount of EB contained in the wet lung samples was then determined. All experiments were repeated at least in triplicate.Protein extravasation, indicative of capillary permeability, was evaluated by measuring the extravasated Evans blue (EB). This method has previously been described by Table 2, the LIS was obtained by the sum of each of the six independent variables: neutrophils in the alveolar spaces and/or in the interstitial spaces, presence of hyaline membranes, proteinaceous debris filling the airspaces, alveolar septal thickening, and alveolar congestion. This sum was weighted according to the relevance ascribed to each feature by the American European Consensus Committee  and the right middle lobe tissue was isolated and fixed by immersion in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. Following hematoxylin and eosin staining, pathological changes in lung tissues were examined under a light microscope. Lung injury score (LIS) was quantified by an investigator blinded to the treatment groups using recently published criteria . As showommittee  and then2 culture flasks with completed medium composed of Dulbecco’s Modified Eagle Medium  supplemented with 20% fetal bovine serum , 1% antibiotic , and heparin (90–100 U/ml) at 37°C in humidified 5% CO2/95% air. The medium was partially changed every 48 h for 10–14 days till the cells grew to confluence. For Western blotting and immunofluorescence labeling, cells were plated in six-well plates (3.5 × 105 cells/well) and grown to confluence for experimental treatments.Primary rat PMVECs  were maintained in 75 cm4 cells/well) and cultured overnight. PMVECs were then treated with UTI  (ranging from 2500 to 20000 U/ml), lipopolysaccharide  (ranging from 0.5 to 32 μg/ml), rat tumor necrosis factor alpha  (ranging from 0.5 to 32 ng/ml) for 3, 6, 12, and 24 h in triplicates. After removal of medium, 90 μl of new basic medium and 10μl of CCK8 were added to each well and the plate was incubated for additional 3 h . The optical density (OD) was then read at 450 nm using a microplate reader. The assays were performed in triplicate.Viability of endothelial cells was assessed by Cell Counting kit-8 . To determine the cytotoxic effect of UTI, LPS, and TNF-α, PMVECs were plated into 96-well microplates  for 1 h and then incubated with basic medium + LPS (1 μg/ml) for 24 h, whereas the cells in LPS group were subjected to basic medium + LPS (1 μg/ml) for 24 h. In the control group, cells were incubated with basic medium for 24 h without any treatment. For TNF-α and signaling pathway determination, cells were pretreated with UTI at the same concentration for 1 h, but they were only incubated with LPS for 6 h and 30 min–1 h, respectively. The cells were then harvested for protein extraction or immunofluorescence microscopy (6-well plates). To further investigate the role of TNF-α in destruction of the tight junctional proteins, PMVECs were randomly divided into control, LPS, and TNF-α receptor antagonist (An) + LPS group. The cells in control and LPS group were prepared as mentioned above, whereas in An+LPS group, the cells were subjected to pretreatment with TNF-α receptor antagonist (0.2μM)  for 30 min  followedTo detect the tight junctional proteins given treatment of TNF-α and TNF-α receptor antagonist, PMVECs were randomly divided into control, TNF-α, and An+TNF-α group. PMVECs in TNF-α group were incubated with basic medium + TNF-α (10 ng/ml) for 18 h. In An+TNF-α group, the cells subjected to pretreatment with TNF-α receptor antagonist (0.2 μM) were incubated for 30 min followed by incubation with basic medium + TNF-α (10 ng/ml) for 18 h. In the control group, cells were incubated with basic medium for 18 h without any treatment. They were then harvested for protein extraction or immunofluorescence microscopy (6-well plates). All experiments were repeated at least in triplicate.A total of 20 rats were used for Western blotting analysis. The left lung obtained from each group was frozen in liquid nitrogen and stored at −80°C. Tissue samples from various groups were homogenized with protein extraction reagent  containing protease inhibitors. For PMVECs of different groups, after the medium was discarded, the cell samples were rinsed more than twice with 1× PBS; the cells were lysed with the lysis buffer. The treated cells were mechanically scraped off with a rubber scraper and centrifuged at 13,000 rpm for 25 min and the supernatant was collected.Protein concentrations of both tissues and PMVECs were determined by using Pierce TM BCA Protein Assay Kit . Samples of supernatant containing 40 μg protein of PMVECs were heated to 95°C for 5 min and then separated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis in 10% gels. Protein bands were electroblotted onto polyvinylidene difluoride (PVDF) membrane and blocked with 5% non-fat milk for 1 h at room temperature .The membranes were incubated with primary rabbit polyclonal antibody occludin , claudin-5 , ZO-1 , and TNF-α , as well p38 MAPK Rabbit mAb , Phospho-p38 MAPK Rabbit mAb , p44/42 MAPK (Erk1/2) Rabbit mAb , Phospho-p44/42 MAPK (Erk1/2) Rabbit mAb , JNK Mouse mAb , Phospho-JNK Mouse mAb , IκB Mouse mAB , and Phospho-IκB Goat pAB . Primary antibodies were diluted in 5% BSA overnight at 4°C before the membranes were incubated with the secondary antibodies, either with anti-rabbit IgG, HRP-linked antibody  or anti-mouse IgG, HRP-linked antibody . The immunoblots were detected by a SuperLumia ECL Kit . The band intensity was quantified in Image J software . All experiments were repeated at least in triplicate.Table 3.A total of 17 rats from various experimental groups were used for double immunofluorescence labeling. Following deep anesthesia with 6% sodium pentobarbital, the rats were sacrificed by perfusion with 2% paraformaldehyde in 0.1 M phosphate buffer. The lung was removed, and paraffin was embedded. The inferior lobe of the right lung was sectioned at 7μm thickness on a microtome . For blocking of non-specific binding proteins, tissue sections were incubated in 5% normal goat serum diluted in PBS for 1 h at room temperature (22 to 24°C). The sections were then incubated in a humidified chamber with the above mentioned primary antibodies diluted in PBS overnight at 4°C. Following washing in PBS, sections were incubated, with the respective fluorescent secondary antibodies: Cy3-conjugated secondary antibody and FITC-conjugated CD31 for 1 h at room temperature. After three rinses with PBS, the sections were mounted with a fluorescent mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) . Colocalization of various biomarkers was confirmed and images were captured using a confocal microscope . The details of the antibodies used are given in 5 cells/well) for 10–14 days till they grew to confluence. The medium was partially changed every 48 h. After different treatments, the PMVECs were fixed in 4% paraformaldehyde in 0.1 M PBS for 15 min at room temperature followed by blocking with 10% donkey serum for 1 h after rinsing with 1× PBS for 3 times at 5 min each. Subsequently, the coverslips with adherent cells were incubated in a humidified chamber with the respective primary antibodies as previously described overnight at 4°C, followed by the FITC/Cy3-conjugated secondary antibodies incubation for 1 h at room temperature. The coverslips were then mounted in DAPI containing the mounting medium after rinsing in 1× PBS for 3 times at 5 min each. Images were captured using a confocal microscope. All experiments were repeated at least in triplicate.Pulmonary microvascular endothelial cells of various groups were seeded on coverslips in a 6-well plate  at 450 nm.A total of 17 rats from various experimental groups were used for ELISA. TNF-α concentration of homogenates from the right lung in different groups was measured by TNF-α ELISA kit  according to the manufacturer’s protocol. The reaction plates were read within 30 minutes in an ELISA plate reader  followed by multiple comparison of Tukey test was used to determine the statistical significance of different groups. All experiments were conducted at least in triplicate from different cell or tissue samples. The difference was considered statistically significant when Figures 1B-1,B-2). Examination of hematoxylin and eosin-stained lung sections showed drastic histopathological changes caused by CLP as evident from the distinct thickening of alveolar septa, massive lymphocyte and neutrophils infiltrates in the interstitial spaces, hemorrhage in the stroma, pronounced alveolar collapse, and high LIS. However, in rats that were given UTI pretreatment, inflammatory infiltration in the lung tissue was reduced; additionally, there was diminution in destruction of lung structures and a significant decline in LIS when compared with the control rats that had not received UTI pretreatment .Of the 76 rats  that underwent CLP, 52 of them survived the surgical operation and were therefore considered as successful sepsis model for subsequent experimental analysis. Twenty four of them died within 48 h after operation and were excluded from the experimental analysis. As in clinical scenarios, the surviving CLP-induced sepsis rats presented with significantly raised temperature and respiratory rate compared with the sham group. UTI decreased the clinical indices in CLP-induced sepsis rats. In gross lung preparations, massive vascular exosmose of EB inundated the lung tissues after CLP. Following UTI treatment, the extravasated EB was markedly reduced , to LPS at the concentration of 100–1000 ng/ml for 36 h , and to TNF-α in the range of 1–1000 ng/mL for 24 h , respectively. Only when cultured with UTI at 15000 U/ml for 36 h or at 20000 U/ml for more than 12 h, the viability of cells was significantly decreased . At 10 μg/mL LPS for 36 h or 20μ g/mL LPS for 12 h, significant cell death was detected . Considering the above, we have used UTI at concentration of 10000 U/mL for 24 h, LPS at 1 μg/mL for 24 h, and TNF-α at 10 ng/mL for 18 h for all subsequent in vitro analysis.The cytotoxicity data were obtained by the MTS assay for the effect of UTI, LPS, and TNF-α on PMVECs. Cell viability remained relatively unchanged when PMVECs were exposed to UTI at 5000–10000 U/ml within 36 h  and PMVECs . By Western blotting and immunofluorescence labeling, intense occludin expression was detected in the lung tissue of sham rats. In rats subjected to CLP, occludin expression was hardly detected . On the other hand, in CLP rats that were given UTI treatment, occludin expression was restored and enhanced by almost twofold . Expression changes of occludin in PMVECs followed that in the rat lungs. Thus, in comparison with the matching controls, occludin immunofluorescence and its protein expression level as determined by Western blot was diminished or decreased in LPS-treated cells. The decline was significantly reversed by a regain of 40–50% increase in UTI pretreatment cells .Expression of various junctional proteins  was assessed in CLP rat lungs  and ZO-1  was noticeably decreased in the lungs of CLP rats. A striking feature after UTI pretreatment was the pronounced increase in claudin-5  and ZO-1  immunofluorescence. Additionally, claudin-5 and ZO-1 protein levels rose by 50–100% as shown by Western blot when compared with the respective CLP group in the absence of UTI . In PMVECs of UTI+LPS group, immunofluorescence intensity of claudin-5  and ZO-1  was evidently increased when compared with the respective LPS group; increase in claudin-5 protein level by Western blot was about 50% when compared with the LPS group ; ZO-1 intensity was also substantially increased to approximately 100% when compared with the LPS group .In parallel to occludin, expression of claudin-5 . Upregulated TNF-α expression was confirmed by Western blot which showed an increase by more than twofold in comparison with the control . Interestingly, TNF-α expression was suppressed by as much as 50% when PMVECs were given UTI pretreatment .Lipopolysaccharide treatment elicited a vigorous TNF-α expression in PMVECs as revealed by immunofluorescence  concentration was increased by more than double in sepsis-induced group compared with the sham group; in UTI pretreatment group, it was suppressed by about 40%.Because TNF-α concentration in the rat lung homogenates was too low to be detected, we had used the TNF-α ELISA kit for more accurate analysis. TNF-α , claudin-5 , and ZO-1  was significantly decreased in LPS group by 40–80% when compared with the control; it was significantly augmented when pretreated with TNF-α receptor antagonist (An+LPS group).Western blot in PMVECs showed the expression level of occludin , whereas it was markedly augmented by TNF-α receptor antagonist in An+TNF-α group . Similar changes were determined for Claudin-5  and ZO-1 . In PMVECs as assayed by Western blot, the protein expression level of tight junctional proteins including occludin , claudin-5 , and ZO-1  was significantly decreased by 50–80% following TNF-α treatment compared with the control group. Very strikingly, it was markedly elevated to a level comparable to that of the control cells when pretreated with TNF-α receptor antagonist .Immunofluorescence intensity of occludin in TNF-α treatment group was significantly decreased in comparison with PMVECs control .Compared with the control group, the ratio of p-P38/P38, p-IκB/i-κb, p-JNK/JNK, and p-ERK/ERK in PMVECs was increased significantly at 30 min and 60 min following LPS treatment; it was markedly depressed when given UTI treatment  by intraperitoneal injection, or intravascular injection. Furthermore, UTI administration decreased the exosmose of Evans blue, which otherwise would have inundated the lung tissues in CLP-induced rats as revealed by gross examination and pathological LIS based on light microscopy . It is well known that the vascular endothelium possesses some of the properties of inflammatory cells and could be activated by cytokines or endotoxin caused by infection, injury, or cancer  that the expression of tight junctional proteins such as occludin, claudin-5, and ZO-1 was decreased in the lung tissues of CLP-induced sepsis rats when compared with the sham group. Remarkably, UTI pretreatment reversed such changes; in other words, the expression of the above mentioned tight junctional proteins was restored to a varying extent. To confirm this, we have extended the study to rat primary cultured endothelia cells challenged with LPS. Results in vitro  were consistent with those in vivo. It is, therefore, suggested that during lung infection, activated endothelial cells undergo morphological changes along with biochemical changes of TJs, which conceivably would contribute to endothelia barrier disruption and inflammatory leakage. In the lung, endothelial cells serve as a semi-permeable barrier between the vascular contents and the pulmonary airspaces and play a critical role in regulating the tissue fluid homeostasis and the inflammatory response. There is supporting evidence that TJs play a critical role in the integrity alveolar-endothelia barrier . TNF-α, a key early signaling molecule in the inflammatory cascade, is a cytokine released not only by the activated immune cells such as monocytes and macrophages but also by the activated endothelial cells  that the suppression or detrimental effects of LPS on production of tight junctional proteins were abrogated following administration of TNF-α antagonist into LPS stimulated PMVECs. This stands to reason that, among others, TNF-α is specifically involved in the process. Secondly, we have shown that PMVECs stimulated with TNF-α produced similar destruction effects on tight junctional proteins caused by LPS. More importantly, TNF-α receptor antagonist can increase the expression of tight junctional proteins  in TNF-α-treated PMVECs. Hence, it is justified to conclude that in the present CLP and PMVECs models, UTI acts to inhibit the expression of TNF-α in the endothelial cells, which would then arrest the degradation of junctional proteins and integrity of TJs. Consequently, it prevents the disruption of endothelia barrier and ultimately reduces the inflammatory edema of lung blood vessels following septic insult. The present results have shown that the effect of UTI on TJs was shared by TNF-α receptor antagonist, which suggested that UTI might exert its TJs-protective effect through anti-TNF-α by inhibiting the action of endogenic TNF-α and extrinsic TNF-α. While pulmonary endothelial cells have been regarded as a kind of inflammatory cells that produce TNF-α, massive pulmonary macrophages of M1 phenotype tend to accumulate in the septic lung tissues which may serve as the main source of TNF-α. In consideration of this, it is suggested that the decreased effects of TNF-α on TJs may be attributed to polarization of M1 macrophages into M2 phenotype with decreased production of TNF-α by UTI. Supporting this argument is the fact UTI has recently been shown to decrease the inflammatory response through polarization of macrophages from M1 into M2 phenotype , p38 mitogen-activated protein kinases (p38 MAPKs), c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) in PMVECs subjected to different treatments were followed. IκB is the cytoplasmic inhibitory protein bounded to the transcription factor nuclear factor-kappa B (NF-κB) to keep it in an inactive state. The phosphorylation of IκB and its subsequent degradation allows the activation of NF-κB. p38 MAPK, JNK, and ERK all belong to a class of MAPKs. Both NF-κB and MAPKs are involved in directing cellular responses to diverse stimuli like proinflammatory cytokines and regulate cell functions of proliferation, gene expression, differentiation, cell survival, and apoptosis . A commoFigure 7, compared with the control group, the phosphorylated form of IκB, p38, JNK, and ERK was increased significantly at 30 and 60 min following LPS treatment, whereas UTI treatment markedly depressed LPS-induced IκB, p38, JNK, and ERK phosphorylation. The present results are consistent with others as demonstrated in LPS-induced animal or human umbilical vein endothelial cells  and in vitro (PMVECs and primary culture) experiments including immunofluorescence, Western blot analysis, measurement of lung injury score etc. Y-YD and D-MY assisted with the cell work. BX performed the CLP-model. MF and W-HZ prepared the first draft of the manuscript. All authors participated in discussion and editing, and approved the final manuscript.H-DW and H-KZ conceived and designed the experimental project. MF, W-HZ, and W-LS carried out the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Carthamus tinctorius L, attenuated bleomycin-induced pulmonary fibrosis in mice. This study is to investigate the effect of HSYA on the proliferation and inflammatory level of human fetal lung fibroblasts (MRC-5 cells) induced by tumor necrosis factor-α (TNF-α) and explore the underlying mechanisms.  Hydroxysafflor yellow A (HSYA), an effective ingredient of the Chinese herb α, HSYA, or/and etanercept  antagonist, 500 ng/mL) before cell proliferation was detected. The laser confocal microscope was used to observe the role of HSYA in binding of TNF-α and its receptor. Co-immunoprecipitation was used to detect the binding of TNFR1 and TAK1-TAB2 complex. Real-time quantitative RT-PCR and western blot were used to detect the expressions of inflammation-related cytokines and proteins related with the NF-κB pathway. Luciferase reporter gene assay and chromatin coprecipitation method were used to detect the interaction between AP-1 and TGF-β1 promoter.  MRC-5 cells were treated with different concentrations of TNF-α (5 ng/mL) was used to induce inflammation and proliferation in MRC-5 cells. HSYA can partially suppress the stimulation of TNF-α on proliferation and inflammatory response of MRC-5 cells. HSYA could compete with TNF-α to bind with TNFR1 and hamper the binding of TNFR1 to TAK1-TAB2 complex. In addition, HSYA could also inhibit the activation of the NF-κB signal pathway and suppress the binding of TGF-β1 promoter with AP-1.  TNF-α-induced proliferation and inflammatory response of MRC-5 cells through the NF-κB/AP-1 signaling pathway, which may provide theoretical basis for HSYA treatment in pulmonary fibrosis. Evidence in this study suggested that HSYA affects TNF- After quantification, RNA was then reversely transcribed into cDNA. Further, the PCR reaction system was configured according to fluorescence-quantitative PCR kit  instructions. The real-time quantitative RT-PCR experiment was carried out by ABI7500 quantitative PCR instrument  with the reaction conditions of predenaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 34 s. RT-PCR primers were as follows: IL-1μL/50 mL culture flask on ice for 30 min. Subsequently, under the condition of 12,000 rpm at 4°C, lysis buffer was then centrifuged for 10 min. The supernatant was collected and split into 0.5 mL centrifuge tubes for storage in −20°C or quantification using a BCA kit. Next, 6 × SDS loading buffer was added at 100°C for protein denaturation. The protein was then separated by SDS electrophoresis, and the membrane was transferred by a precooled transfer buffer at 4°C for 1.5 h. The membrane was then blocked with 5% nonfat milk in TBST buffer for 1 h. TBST configured primary antibodies of IL-1β , TGF-β1 , IL-6 , Phospho-IKKα/β , IKKβ , Phospho-NF-κB p65 , NF-κB p65 , IκBα , and β-actin  were separately incubated at 4°C overnight before TBST wash for 3 × 10 min. After that, goat anti-rabbit IgG or goat anti-mouse IgG was used for incubation for 2 h at room temperature , followed by TBST wash. Expression levels of IL-1β, IL-6, TGF-β1, and β-actin was determined after color development.The MRC-5 cells treated for 48 h were washed with precooled PBS buffer for 3 times and then added with protein extraction lysis buffer in a 100 β1 promoter-TA-luc fragment and TGF-β1 promoter mutant fragment were cloned and bound to the Promega vector . The constructed vector and Tag-AP-1 plasmid  were transfected into TNF-α stimulated MRC-5 cells, followed by treatment with or without HSYA (45 μmol/L). After 48 h, fluorescence intensity of each group was measured using a luciferase kit .The designed TGF-α (2.5 ng/mL) stimulated MRC-5 cells were treated with/without 45 μmol/L HSYA for 8 h. Then, cells were transfected with WT-TGF-β1-TA and Tag-AP-1 plasmids, and 48 h later, cells were fixed by 37% formaldehyde to a final concentration of 1%. The fixation reactions were terminated by adding 10 ml of glycine. Then, cells were exfoliated with cell scraping solution containing protease inhibitor, centrifuged, precipitated, and resuspended with 1 ml precooled SDS lysate. The prepared sample was subjected to ultrasonic fragmentation   to break chromatin into 200∼1000 bp on ice, with ultrasonic interval of 40 s each time. AP-1 antibody  or negative control IgG antibody and positive control c-jun polyclonal antibody  were added to chromatin centrifuge tubes after elimination of nonspecific antibody for overnight at 4°C. Then, 60 μL of Protein G was added for incubation on a shaker at 4°C. When IgG/DNA, c-jun/DNA, and AP-1/DNA complexes were eluted using eluent, 8 μl of 5 mol/L NaCL was added overnight in 65°C water bath to de-crosslink. DNA was purified by using the ChIP kit , and the purified DNA was used for quantitative PCR detection. PCR products (10 μl) were subjected to agarose gel electrophoresis with mass fraction of 2.0%. Input DNA, used as positive control, is de-crosslinked and purified genomic DNA after nonspecific antibodies were eliminated.TNF-T test. While one-way analysis of variance (ANOVA) test was used to measure multiple comparisons. Statistical significance was regarded as a P value of <0.05.Data were analyzed by SPSS 18.0 software  and GraphPad Prism 6.0. The statistical significance between two sets of data was calculated using α at concentrations of 0, 2.5, 5, 7.,5 and 10 ng/mL. After 48 h, the effects of different concentrations of TNF-α on the proliferation of MRC-5 cells were determined. The results showed that the 5 ng/mL TNF-α treatment could markedly promote the proliferation of MRC-5 cells (P < 0.01), as well as increasing the mRNA and the expression levels of IL-1β, IL-6, and TGF-β1 , compared with 0 ng/mL TNF-α treatment. The 2.5 and 7.5 ng/mL TNF-α could slightly promote MRC-5 cell proliferation, while 10 ng/mL TNF-α treatment could not promote the proliferation of MRC-5 cells . As shown in α treatment could markedly promote mRNA and protein expression levels of inflammatory factors IL-1β, IL-6, and TGF-β1 . Taken together, 5 ng/mL TNF-α was used for subsequent experiments.MRC-5 cells were stimulated by TNF-α induced proliferation and inflammatory response of lung FB. The analysis result of HSYA by high-pressure liquid chromatography is presented in P > 0.05) as well as the mRNA and protein expression levels of inflammatory factors IL-1β, IL-6, and TGF-β1 in cells subjected to 5, 15, and 45 μmol/L HSYA treatment , which proved that HSYA had no toxic or side effects on normal cells. Compared with the TNF-α group, the TNF-α + HSYA (5 μmol/L) group has no obvious effect on cell proliferation, while the TNF-α + HSYA (15 μmol/L) group and TNF-α + HSYA (45 μmol/L) group can significantly inhibit cell proliferation (P < 0.01), indicating that HSYA could suppress TNF-α induced proliferation of MRC-5 cells in a dose-dependent manner.Next, this paper explored the effect of HSYA on TNF-α group, the mRNA and protein expression levels of IL-1β, IL-6, and TGF-β1 in the TNF-α + HSYA (5 μmol/L) group had no distinct difference, while the mRNA and protein expression levels of IL-1β, IL-6, and TGF-β1 in the TNF-α + HSYA(15 μmol/L) group and TNF-α + HSYA(45 μmol/L) group were significantly reduced . These results indicate that HSYA can inhibit TNF-α induced proliferation and inflammatory response in MRC-5 cells.Quantitative RT-PCR and western blot experiments showed that compared with the TNF-α stimulated MRC-5 cells proliferation and expression of inflammatory factors by competing with TNF-α to bind with its receptor TNFR1. The experimental results showed that, compared with the TNF-α + ENCP group, no obvious difference on OD value and mRNA and protein expression levels of inflammatory factors IL-1β, IL-6, and TGF-β1 in the TNF-α + HSYA (45 μmol/L) + ENCP group was found  group was significantly lower than that in the FITC-TNF-α group (P < 0.01). In contrast with the FITC-TNF-α + ENCP group, the colocalization level of FITC-TNF-α and TNFR1 in the FITC-TNF-α + HSYA (45 μmol/L) + ENCP group was slighted suppressed, although failed to achieve any significant difference (P > 0.05). All in all, the above results showed that HSYA inhibits the proliferation and inflammatory level of MRC-5 cells induced by TNF-α by competing with TNF-α to bind with TNFR1. After TNFR1 antagonist was added, HSYA was not able to inhibit cell proliferation and inflammatory response of MRC-5 cells induced by TNF-α.Next, in this step, we surmise that HSYA regulates TNF- Figures . Comprehα to bind with TNFR1 and also inhibits the binding of TNFR1 with TAK1-TAB2 complex, Co-IP was performed to detect the expressions of TAK1 and TAB2 in TNFR1 complexes. Co-IP was carried out using TNFR1 antibody, and TAK1 or TAB2 antibodies used for western blot. The results manifested that, compared with the TNF-α group, TAK1 and TAB2 signals were undetectable in the HSYA (45 μmol/L) group. The protein expressions of TAK1 and TAB2 in the TNF-α + HSYA (45 μmol/L) group were clearly decreased. Meanwhile, corresponding protein bands of TNFR1, TAK1, and TAB2 were detected in total cell lysis (P < 0.01), which indicated that HSYA can significantly inhibit the binding of TNFR1 and TAK1-TAB2 complexes, thus affecting TNF-α induced proliferation and inflammation response in MRC-5 cells.To investigate whether HSYA competes with TNF-α induced proliferation and inflammatory response in MRC-5 cells via the NF-κB signaling pathway. Those findings revealed that, compared with the TNF-α group, the levels of IKK and p65 phosphorylation in TNF-α + HSYA (45 μmol/L) group were clearly decreased, while the protein levels of IκBα were markedly increased, indicating that HSYA can inhibit the activation of the NF-κB signal pathway .We then sought to determine whether HSYA affects TNF-β1 promoter and AP-1. TGF-β1 promoter-TA-luc plasmid (TGF-β1-TA) plasmid was constructed, and the experimental results showed that the fluorescence signal intensity in the TNF-α + HSYA (45 μmol/L) group was significantly enhanced (P < 0.01) compared with the TNF-α group. Meanwhile, the binding of AP-1 protein and TGF-β1 promoter was inspected by chromatin immunocoprecipitation and the content of TGF-β1 promoter by quantitative RT-PCR. The experiment clearly indicated that the expression of TGF-β1-TA was not detected in the group supplemented with IgG antibody. The content of TGF-β1-TA was higher in the group with AP-1 antibody and the group with c-jun antibody (P < 0.01). The expression of TGF-β1-TA in the TNF-α + HSYA (45 μmol/L) group was clearly decreased compared with the TNF-α group (P < 0.01). The results were verified by agarose gel electrophoresis experiment , and the study has confirmed that TNF-α promotes the expression of TGF-β1 in FB by activating AP-1 [β1 gene promoter contains two AP-1 binding sites [β1 gene. In our study, we confirmed that HSYA inhibits the binding of TGF-β1 promoter to AP-1, thereby suppressing the expression of TGF-β1, indicating HSYA could block AP-1 pathway. Consistent with the results of our study, HSYA was proved to suppress TGF-β1 expression and thus attenuates airway remodeling in chronic obstructive pulmonary disease (COPD) [TGF-diseases . Transcring AP-1 , 22. TGFng sites , and anye (COPD) .α, binding to TNFR1, can facilitate multiple signal transduction pathways, including mitogen-activated protein (MAP) kinases and NF-κB pathways [κB pathway. Studies have shown that, in the process of lung fibrosis, NF-κB can mediate pulmonary fibrosis and alveolar inflammation by promoting gene transcription of cytokines such as TNF-α, IL-8, IL-1β, and TGF-β1 [κB activation [κB signal pathway related proteins showed that HSYA can inhibit the activation of NF-κB pathway and suppress the expressions of IL-1β, IL-6, and TGF-β1. Therefore, HSYA suppress TNF-α induced inflammatory response of FB by inhibiting the activation of NF-κB pathway. In our research, we have found that HSYA could block the binding of TGF-β1 promoter with AP-1, thus inhibiting the expression of TGF-β1. Taking together, HSYA affects TNF-α-induced inflammatory and proliferation of MRC-5 cells via blocking the activation of the NF-κB/AP-1 signaling pathway.Also, previous findings elucidate that TNF-pathways . Next, wd TGF-β1 , 26. In tivation . Detectiα induced proliferation and inflammatory response of FB through inhibiting the NF-κB/AP-1 signaling pathway. However, further investigation is necessary to reveal these results and expound the precise mechanisms whereby HSYA suppresses the downstream signal transduction pathway.To sum up, experiments of this study revealed that HSYA may be used for a therapeutic agent for IPF treatment based on the finding that HSYA suppresses TNF-


Tumor necrosis factor (TNF) inhibitors (TNF-i) are effective in the treatment of entero-Behcet’s disease (BD). However, there is no objective tool for assessment of disease activity in entero-BD; therefore, it is not easy to evaluate treatment effectiveness in the clinical setting. In addition, because corticosteroid (CS) is considered for standard therapy, the effectiveness of TNF-i without CS has not been well examined. In this retrospective study, the effectiveness of CS without TNF-i and the effectiveness of TNF-i with or without CS therapy were investigated and compared.n = 22; TNF-i group: n = 49 ). All patients had active ulcerative lesions. The primary endpoint was the ulcer cure rate evaluated by lower gastrointestinal endoscopy. Secondary endpoints were ulcer improvement rate, disease activity improvement based on the quantitative disease activity index for intestinal Behcet’s disease (DAIBD), and CS-sparing effect.This study included 71 patients with entero-BD who were followed up for 1 year (CS without TNF-i group: P < 0.01). There were no statistically significant differences between the TNF-i with CS group and the TNF-i without CS group in any of the endpoints.Ulcer cure rates were 13.6% in the CS without TNF-i group, 60.0% in the TNF-i with CS group, and 44.8% in the TNF-i without CS group. Ulcer improvement rates were 27.2% in the CS without TNF-i group, 60.0% in the TNF-i with CS group, and 51.7% in the TNF-i without CS group. The multivariate analysis revealed that TNF-i was an independent predictive factor for cure of the ulcerative lesions. The DAIBD and concomitant CS dose were significantly decreased in both the CS without TNF-i group  and the TNF-i group (DAIBD 64.7 → 21.1. CS 18.7 → 3.88 mg/day). The ulcer cure and improvement rates were significantly higher in the TNF-i group. In addition, the proportion of concomitant CS dose less than 7.5 mg was significantly higher in the TNF-i group (CS without TNF-i group 18.2% vs. TNF-i group 85%, This study demonstrated that compared to CS alone, TNF-i improve disease activity and possess a higher ulcer healing effect and CS tapering effect with or without concomitant CS. Behcet’s disease (BD) is a relapsing, systemic inflammatory disease that affects the skin, mucosa, eyes, genitourinary organs, joints, blood vessels, central nervous system, and gastrointestinal system . Entero-The present study included 71 patients diagnosed with entero-BD according to the revised diagnostic criteria proposed by the Behcet’s Disease Research Committee of Japan (2003) and the criteria for diagnosis of BD recommended by the International Study Group , 15. AllIn the present study, we investigated the effectiveness of 1-year treatment with TNF-i and compared the results to those achieved with CS without TNF-i. We also investigated effectiveness of TNF-i without CS and compared to that of TNF-i with CS.Remission induction therapy mainly with CS but without TNF-i (22 patients) and remission induction therapy with TNF-i  were newly introduced at our hospital. Study patients were followed up for 1 year after the introduction of the treatments at our hospital and affiliated institutions, as shown in Fig. The DAIBD  is a quantitative disease activity index proposed by Cheon et al. in 2011 , 15. PriU test, and the Wilcoxon signed-rank test. The patients were classified into groups with cure of intestinal ulcerative lesions or not, or with achieving CS less than 7.5 mg/day group or not. In addition, univariate and multivariate logistic regression analyses were performed on these groups. In the multivariate analysis, the proportion of cure of intestinal ulcerative lesions or achieving CS less than 7.5 mg/day at year 1 were used as a dependent variable. All reported P values were two-sided. The level of significance was set at P < 0.05.Data are expressed as mean ± standard deviation (SD). Differences between the groups and between baseline data and data measured at 1 year were compared using Fisher’s exact test, the Mann–Whitney All analyses were conducted using JMP version 10.0 . For statistical analysis, data from TNF-i discontinued cases were complemented using the last observation carried forward method.Table P = 0.3984)  (Table The ulcer cure rate at 1 year was 13.6% (3 of 22 patients) in the group with CS without TNF-i. In the same group, the ulcer improvement rate was 27.3% (6 of 22 patients). In contrast, the ulcer cure rates at 1 year were 60.0% (12 of 20 patients) in the group with TNF-i and CS, and 44.8% (13 of 29 patients) in the group with TNF-i without CS. The ulcer improvement rates were 60.0% (12 of 20 patients) in the group with TNF-i and CS and 51.7% (15 of 29 patients) in the group with TNF-i without CS. Although the ulcer cure and improvement rates were significantly lower in the group with CS without TNF-i than in the group with TNF-i, there were no significant differences in these outcomes between the TNF-i with CS group and the TNF-i without CS group  DAIBD was 85.2 ± 29.6 at baseline and 40.5 ± 44.7 at 1 year, showing a significant decrease. In the group with TNF-i, the mean (± SD) DAIBD was also significantly decreased, from 64.7 ± 34.9 at baseline to 21.1 ± 28.9 at 1 year. In the group with TNF-i and CS, the mean (± SD) DAIBD was 70.7 ± 38.4 at introduction and 20.8 ± 29.3 at 1 year, demonstrating a significant decrease. Likewise, the mean (± SD) DAIBD was 60.5 ± 32.2 at introduction and 21.4 ± 25.5 at 1 year, showing a significant decrease in the group with TNF-i without CS  dose of concomitant CS was 32.3 ± 16.4 mg/day at baseline and 20.6 ± 18.3 mg/day at 1 year, demonstrating a significant decrease. Likewise, the mean (± SD) dose of concomitant CS in the group with TNF-i and CS was 18.7 ± 20.9 mg/day at baseline and 3.88 ± 3.87 mg/day at 1 year, showing a significant decrease Fig. . As the P = 0.1920). The status of 3 patients who newly started CS in the group with TNF-i without CS was classified as “Discontinuation.” The reasons for discontinuation are summarized in Fig. The retention rates up to month 12 were 90.0% (18 of 20 patients) in the group with TNF-i and CS and 75.9% (22 of 29 patients) in the group with TNF-i without CS, demonstrating no significant differences  except entero-BD; some of the patients were under treatment with CS or under dose reduction of CS for entero-BD. For these patients, although we newly introduced TNF-i, choices regarding use and dose of CS were made at the physicians’ discretion. We considered that as one of the reasons of the difference at the baseline concomitant CS. On the other hand, although no evident predictive factor achieving CS less than 7.5 mg/day was found in our study Tables  and 5, tP = 0.3114, by Fisher’s exact test). Based on these results, although the activity of entero-BD was sufficiently controlled and maintained at low levels with TNF-i therapy without CS in some patients, there may be other patients for whom concomitant CS use is necessary. In addition, the present study failed to identify any background factors that could predict which patients would be non-responders or experience relapse in the TNF-i without CS group. Furthermore, we did not find any additive effect of MTX on TNF-i (Tables The TNF-i group was further classified into two groups according to the presence or absence of concomitant CS use (TNF-i with CS group and TNF-i without CS group) to compare the effectiveness of TNF-i. The TNF-i formulations and the necessity of concomitant CS use were, in principle, determined at the discretion of the treating physicians. However, no significant differences in background factors were observed between the two groups at the time of TNF-i introduction Table . Under ti Tables  and 7. ILimitations of the present study include a small sample size (49 patients) and a short retrospective observation period (1 year). In the future, studies with a larger sample size and a longer observation period are warranted to assess the long-term efficacy of TNF-i in controlling disease activity and to identify predictive factors to detect patients who do not require concomitant usage of CS.In the present study, we administered TNF-i to 49 patients with refractory entero-BD and demonstrated its high efficacy based on both endoscopy results and a quantitative disease activity index. In particular, comparison between the TNF-i group and the CS without TNF-i group revealed that TNF-i is highly effective in curing ulcers and reducing concomitant CS doses and identified as an independent predictive factor for cure of the intestinal ulcerative lesions. Furthermore, TNF-i demonstrated sufficient efficacy in both the TNF-i with CS group and the TNF-i without CS group to control disease activity without concomitant CS in some patients. In contrast, TNF-i therapy with CS was associated with concerns regarding infection, and TNF-i therapy without CS with concerns regarding non-responders and relapse.


Macrophages stand in the first line of defense against a variety of pathogens but are also involved in the maintenance of tissue homeostasis. To fulfill their functions macrophages sense a broad range of pathogen- and damage-associated molecular patterns (PAMPs/DAMPs) by plasma membrane and intracellular pattern recognition receptors (PRRs). Intriguingly, the overwhelming majority of PPRs trigger the production of the pleiotropic cytokine tumor necrosis factor-alpha (TNF). TNF affects almost any type of cell including macrophages themselves. TNF promotes the inflammatory activity of macrophages but also controls macrophage survival and death. TNF exerts its activities by stimulation of two different types of receptors, TNF receptor-1 (TNFR1) and TNFR2, which are both expressed by macrophages. The two TNF receptor types trigger distinct and common signaling pathways that can work in an interconnected manner. Based on a brief general description of major TNF receptor-associated signaling pathways, we focus in this review on research of recent years that revealed insights into the molecular mechanisms how the TNFR1-TNFR2 signaling network controls the life and death balance of macrophages. In particular, we discuss how the TNFR1-TNFR2 signaling network is integrated into PRR signaling. Tumor necrosis factor-alpha (TNF) is a highly pleiotropic cytokine that affects practically any type of cell. It triggers cellular responses reaching from the induction of inflammatory gene expression programs, over the stimulation of cellular proliferation and differentiation to the activation of cellular suicide programs such as apoptosis and necroptosis .Tumor necrosis factor-alpha is the name giving and prototypic ligand of the TNF superfamily (TNFSF). It is expressed (i) as a type II single spanning transmembrane protein and (ii) as a soluble variant which is released from the transmembrane form by proteolytic processing in the stalk region which separates the characteristic TNF homology domain (THD) from the transmembrane and the intracellular domain . Since tTNFR1 and TNFR2 belong to different subgroups of the TNFRSF. TNFR1 is a death receptor (DR) and harbors a death domain (DD) in its cytoplasmic part . The DD After binding of soluble or membrane-bound TNF, the DD-containing cytoplasmic proteins TNFR1-associated death domain (TRADD) and receptor interacting protein kinase-1 (RIPK1) recruit to TNFR1 due to DD–DD interactions . DeficieL heterodimers, which have a limited enzymatic activity, cleave RIPK1 and RIPK3 and prevent so complex IIb triggered formation of necroptosis-inducing oligomeric RIPK1/RIPK3 aggregates  and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B(Akt) pathway . The latIn sum, TNFR1 and TNFR2 are differently activated by soluble and membrane TNF, induce production of their own ligand in some cells, engage receptor-specific but also common pathways and the various TNFR1/2-associated signaling pathways are, last but not least, interconnected by regulatory circuits. TNF and its receptors therefore constitute a complex signaling network what is reflected by the fact that additive, synergistic or even antagonistic effects have been reported for the two TNF receptor types.in vitro infection with mycobacteria (S)  . Worth mignaling .Studies with SMAC mimetics (IAP antagonists) depleting cIAP1/2 and the cIAP1/2-related XIAP molecule as well as evaluation of XIAP and cIAP1/2 knockout cells revealed that these molecules are crucial for the survival of primary murine bone marrow-derived macrophages . The surin vivo effects of these pathogenic factors can therefore be difficult to define.In accordance with the established anti-necroptotic activity of caspase-8, robust/maximal necroptosis induction by TNF in macrophages requires caspase-8 inhibition . In mostIn accordance with the fact that PRRs strongly stimulate TNF production by macrophages, it has been described that induction of TNF and TNFR2-mediated TRAF2 depletion contribute to macrophage necroptosis induced by certain TLRs, including TLR4 and TLR3 . TNF-indLipopolysaccharide-induced TLR4 signaling has not only the potential to trigger necroptosis via induction of endogenous TNF but also adjusts the necroptotic sensitivity of macrophages for TNF in a complex manner . On the Studies with human-induced pluripotent stem cells (hiPSCs) showed that differentiated RIPK1-deficient macrophages progressively undergo autocrine TNF-dependent cell death . Thus, RBesides the classical NFκB pathway, the type I interferon pathway is a second major signaling pathway which is regularly engaged by PRRs . The cenNoteworthy, there is not only evidence that type I interferons contribute to TNF-induced necroptosis but vice versa also that TNF- and/or necroptosis-associated signaling contribute to the induction of type I interferons. TNF induces in macrophages not only NFκB-regulated genes but also, with delay, typical signal transducer and activator of transcription-1 (STAT1)- and interferon response proteins such as MX1, IRF7 and STAT1 itself . The stiBesides apoptosis and necroptosis, pyroptosis is a third form of programmed cell death which is of particular relevance in macrophages . PyroptoYersinia pestis for example not only activates the NLRP3 inflammasome but also the pyrin inflammasome (Most pathogens activate in macrophages several types of inflammasome complexes. ammasome . The senammasome . TNF andammasome . Thus, tammasome . Indeed,ammasome .in vivo. Noteworthy, the pyroptotic GSDMD p30 fragment is able to trigger mitochondrial ROS production (Just recently two studies demonstrated that intrinsic, thus mitochondria-dependent apoptosis in bone marrow-derived macrophages is accompanied by activation of the NLPR3 inflammasome and IL-1β activation . Noteworoduction  and the oduction  and, as oduction . Thus, tin vivo conditions which of the various individual TNF-related signaling mechanisms gain dominance. Thus, future studies must show whether there are key factors that determine the quality of TNF signaling on macrophage viability. It appears particularly important to learn more about the crosstalk of concomitantly occurring signaling paths engaged by TNF and other inducers of macrophage cell death. Last but not least, it will be important for the understanding of the role of TNF for macrophage biology to learn more about the systemic immunological net-effects triggered by timely limited “immunogenic” death versus persistent inflammatory activation of macrophages.There are a considerable number of high quality publications addressing the role of TNF in the life death balance of macrophages. In sum, these studies show that the effect of TNF on macrophage viability not only depends on the integrated and complex activity of the TNFR1-TNFR2 signaling network but also from its crosstalk with other, equally complex signaling systems engaged by PRR-, inflammasomes and interferons. It is thus not really surprising that the precise net-effects of TNF on macrophages in infection diseases and cancer are still poorly predictable. Indeed, it is not even clear whether and if yes to which extend, the reported effects of TNF on macrophages are generalizable to all types of macrophages. Likewise, it is unclear under which All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α. Necroptosis is a non-caspase-dependent mechanism of regulated cell death . Its iniLike necrosis and apoptosis, necroptosis is closely related to the occurrence, development, and prognosis of diseases, such as inflammation , ischemiHowever, it is unknown whether necroptosis is involved in TNF-α-induced osteoblast death. If yes, then these cells could be protected by treatment with Nec-1, which could be an effective clinical strategy for treating orthopedic diseases caused by TNF-α. Therefore, we established a TNF-α-treated osteoblast model to determine whether necroptosis plays a role in TNF-α-induced mouse osteoblast cell death.The MC3T3-E1 osteoblast cell line was provided by the Institute of Basic Medical Sciences (China). Cells were incubated in α-MEM medium  containing glutamine, 10% fetal bovine serum  and 1% penicillin/streptomycin . Recombinant mouse TNF-α was purchased from Peprotech . Nec-1 was purchased from MedChemExpress . Z-IETD-FMK, a specific inhibitor of caspase 8, was purchased from Selleck . The primary antibodies used to detect p-MLKL (ab196436) and RIPK3 (ab56164) were purchased from Abcam (UK). The primary antibodies used to detect MLKL (37705S), cleaved caspase 3 (9661s), and caspase 3 (9662s) were obtained from Cell Signaling Technology (USA). β-actin (TA-09) and goat anti-mouse/rabbit IgG secondary antibodies were purchased from ZSGB-BIO (China).MC3T3-E1 cells were inoculated into 96-well culture dishes. When the cells grew to approximately 60% confluency, the culture medium was renewed, and the cells were treated with TNF-α  for 12 and 24 h. Next, the medium was renewed, 100 µL of 10% CCK-8  medium was added to each well, and cells were incubated at 37°C for 3 h. The absorbance was measured at a wavelength of 490 nm using a microplate reader . The same method was applied to measure the absorbance of cells after treatment with inhibitors. Specifically, the cells were treated with Z-IETD-FMK (40 µM), Nec-1 (50 µM), or Z-IETD-FMK (40 µM) and Nec-1 (50 µM) for 1 h, after which TNF-α was added  and the cells were incubated for another 24 h. Cell viability was calculated from a formula based on the absorbance according to the CCK8 manufacturer's instructions: Cell viability (%) = (A (dosing) - A (blank)) / (A (0 dosing) - A (blank)) × 100%, where A (dosing) is the absorbance of cells treated with CCK8 and drugs; A (blank) is the absorbance of medium and CCK8 solution without cells; and A (0 dosing) is the absorbance of cells treated with CCK8 but without drugs. The values on the y-axis indicate the ratio of the number of viable cells to the blank control group, which indirectly indicates the effect of the drugs on cell proliferation.MC3T3-E1 cells were inoculated into 24-well culture dishes. Next, the cells were treated with Z-IETD-FMK (40 µM), Nec-1 (50 µM), or Z-IETD-FMK (40 µM) and Nec-1 (50 µM) for 1 h, after which they were treated with TNF-α (20 ng/mL) for an additional 12 or 24 h. Before analysis, the cells were washed twice with PBS and resuspended in binding buffer. Antibody staining was performed to detect Annexin-V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) using an apoptosis kit according to the manufacturer's instructions . Briefly, 5 µL of Annexin-V-FITC and 5 µL of PI were added to each tube. The mixture was incubated in the dark at room temperature for 15 min. Cell death was analyzed by flow cytometry .g for 5 min at 25oC. Then, 120 μL of the supernatant from each well was transferred to a new 96-well plate and mixed with 60 μL of LDH test solution  at 25°C for 30 min in the dark. The absorbance was then measured at 490 nm . The percentage of LDH that was released compared with the total LDH was calculated by subtracting the absorbance of the blank control from the measured absorbance for each group according to the following formula: (absorbance of drug-treated cell group – absorbance of sample control group) / (absorbance of sample maximum enzyme activity control group – sample control group absorbance) × 100.MC3T3-E1 cells were inoculated into 96-well culture dishes. When the cells reached approximately 60% confluency, the culture medium was removed and changed to 1% serum culture medium. After treatment for 6, 12, or 24 h, the dishes were centrifuged in a multiwell plate centrifuge at 400 The cells were treated with Z-IETD-FMK (40 µM), Nec-1 (50 µM), or Z-IETD-FMK (40 µM) and Nec-1 (50 µM) for 1 h, after which they were treated with TNF-α (20 ng/mL) for an additional 12 or 24 h, and protein lysates were extracted in radioimmunoprecipitation (RIPA) lysis buffer containing 4% protease inhibitor, 4% phosphorylase inhibitor, and 1% PMSF. The protein concentration was determined using the BCA assay. Equal amounts of protein lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories), which were blocked with 5% fat-free milk in PBS-Tris buffer for 2 h and then incubated overnight at 4°C with primary antibodies. TBST was used to wash away excess antibodies. The membranes were then incubated with the secondary antibody for 2 h at room temperature. Next, TBST was used to wash away excess secondary antibodies. Protein detection was performed using ECL reagent . The results are reported as ratios of expression relative to that of β-actin.MC3T3-E1 cells were treated as described above, and total RNA was extracted using Trizol reagent . cDNA was synthesized using Roche reagent . Real-time PCR analysis was performed using a C1000 thermocycler and an ABI7500 real-time PCR system  with a Roche Fluorometric Quantitation Kit ).-ΔΔCT method and were normalized to the house-keeping gene β-actin. The primer pairs are presented in The amplified products were measured using amplification curve analysis. All data were analyzed using the 2Data are reported as means±SD from at least three independent technical replicates. Data were analyzed using SPSS 22.0 software .When MC3T3-E1 cells were treated with TNF-α, their viability decreased as the TNF-α concentration increased. When cells were treated with a single concentration of TNF-α, their viability decreased over time. Treatment with 20 ng/mL TNF-α significantly reduced cell viability. Taken together, these results demonstrated that TNF-α inhibited MC3T3-E1 cell viability in a dose- and time-dependent manner .When cells were treated with 20 ng/mL TNF-α, Nec-1, and Z-IETD-FMK, the cell viability trends were different. Nec-1 did not significantly increase cell viability, while the caspase-8 inhibitor Z-IETD significantly increased viability (P<0.05). These results suggested that the cell death induced by treatment with TNF-α was primarily mediated by apoptosis, not necroptosis. However, when the cells were treated with TNF-α plus Z-IETD, Nec-1 significantly enhanced cell viability compared with Z-IETD treatment alone , P<0.05.Flow cytometric analysis showed that treatment with TNF-α resulted in a rapid increase in the percentage of AV+PI− and AV+PI+ cells. To distinguish between late apoptotic cells and necrotic cells in the AV+PI+ population, cell death was assessed at two time-points (12 and 24 h). As shown in At the first time-point tested (6 h), treatment with TNF-α resulted in a significant increase in LDH release. When the cells were treated with Nec-1, LDH release did not decrease compared with treatment with TNF-α alone, while treatment with Z-IETD-FMK alone significantly decreased LDH release (P<0.05). Treatment with Z-IETD-FMK plus Nec-1 resulted in a significant decrease in LDH release over time compared with treatment with TNF-α , P<0.001Treatment with TNF-α alone significantly increased the expression of cleaved caspase 3 . When thTreatment with TNF-α significantly increased RIPK3 <0.01 andIn this study, we demonstrated for the first time that necroptosis was involved in the regulated cell death of mouse osteoblasts treated with TNF-α. This conclusion is supported by several independent lines of evidence. First, TNF-α plus Z-IETD triggered necroptosis in MC3T3-E1 cells. Second, Nec-1 inhibited a core element of the necroptosis pathway, RIPK1, and down-regulated the expression of RIPK3 and MLKL phosphorylation. Third, treatment with Nec-1 prevented necroptosis when the cells were treated with TNF-α plus Z-IETD, as shown by the increased cell viability and reduced percentage of necrotic cells.In addition, cell viability decreased as the TNF-α concentration increased, and in cells treated with the same concentration of TNF-α, cell viability decreased over time. Treatment with 20 ng/mL TNF-α significantly inhibited cell viability. Taken together, these results demonstrated that TNF-α induced MC3T3-E1 cell death in a dose- and time-dependent manner. This phenomenon has also been observed by Zheng, et al.  in osteoTreatment with TNF-α plus Nec-1 did not reduce cell viability and increase LDH release with significance. However, the population of A+PI− cells and the expression of cleaved caspase 3, the specific protein in apoptosis pathway, increased significantly, suggesting that Nec-1 may induce apoptosis. TNF-α plus Z-IETD-FMK significantly increased the expression of protein RIPK3 and MLKL phosphorylation, indicating TNF-α plus Z-IETD-FMK induced necroptosis. These results may confirm Galluzzi et al. findings, which suggest that apoptosis and necroptosis may occur simultaneously, and that necroptosis can even be converted to apoptosis under specific conditions .Differently from other types of cells, Z-IETD plus TNF-α decreased LDH release in the osteoblast cell line. The result may indicate that TNF-α induced osteoblast cell death mostly in the manner of apoptosis and subsequent late apoptosis, whereas necroptosis induced by Z-IETD plus TNF-α only played a relatively limited part. Therefore, Z-IETD plus TNF-α protected from LDH release more than increased LDH release by inducing necroptosis.Simultaneously blocking necroptosis and apoptosis provided greater protection to cells treated with TNF-α than blocking either cell death route individually. Treatment with Nec-1 and Z-IETD-FMK increased cell viability and cell survival and decreased LDH release more effectively than treatment with Nec-1 or Z-IETD-FMK alone. This is because Nec-1 inhibits necroptosis, which is induced by Z-IETD-FMK plus TNF-α. Thus, when TNF-α-induced apoptosis is blocked by Z-IETD-FMK, necroptosis still occurs.Some of our results are unclear and require further study. For example, the changes in RIPK3, MLKL, and caspase 3 mRNA levels were not consistent with the changes in the corresponding protein expression levels. This may be because we detected the active or functional forms of the proteins. Thus, future studies should explore the exact relationship between mRNA and nonfunctional protein expression levels.Our findings may lead to the development of an effective strategy for the treatment of osteonecrosis after bone cement implantation. Osteonecrosis has been confirmed in vertebral bone tissue sections after PMMA (polymethyl methacrylate) implantation . We prev


Bionanoparticles such as microorganisms and exosomes are recognized as important targets for clinical diagnostic and therapeutic applications as well as for food safety and environmental monitoring. Other nanoscale biological particles including liposomes, micelles, and functionalized polymeric particles are widely used in nanomedicine. The recent development of microfluidic and nanofluidic technologies has enabled lab-on-a-chip platforms for separating and analyzing bionanoparticles; however, many challenges exist before these platforms are widely adopted. For example, the complex composition of biological fluids creates a high background for detection. The small dimension and often low concentration of target species require significant amplification of the sensing signal. This special issue collects some recent discoveries and developments of micro- and nanofluidic strategies for the processing and analysis of biological nanoparticles. Eight papers are published in this special issue and cover the design of microfluidics for bioseparation ,3,4,5,6,The collection of papers describe a wide spectrum of separation and sensing mechanisms. For example, Wang et al.  incorporWe appreciate all of the authors who submitted papers to this special issue. We would also like to thank all of the reviewers for dedicating their time to help improve the quality of the submitted papers. The collection of papers will hopefully bring out more innovative ideas and fundamental insights to overcome the hurdles faced in the separation and analysis of bionanoparticles.


TNF is generally believed to be a master pro-inflammatory cytokine, and anti-TNF therapy has become the mainstream treatment for some autoimmune diseases. However, experimental evidence indicates that TNF preferentially activates Tregs, resulting in their proliferation, and enhancing their phenotypic stability and suppressive capacity. This effect of TNF is mediated by the cell surface receptor TNFR2, which is highly expressed by both human and mouse Tregs –4. FurthUrbano et al. from Radboud University sheds some new light on this important topic. The results from their in vitro studies indicate that, through epigenetic regulatory pathways, the autocrine TNF-TNFR2 feedback loop promotes the stability of a highly suppressive phenotype for Tregs, namely one that expresses high levels of TIGIT, FOXP3, Helios, and EZH2. The study by He et al. from the University of Macau in turn provides both in vitro and in vivo experimental evidence that P38 MAPK is a component of the signaling pathway, which leads to TNFR2 mediated expansion of Tregs in response to TNF stimulation, as demonstrated by using small molecule inhibitors of known TNFR2 signaling pathways in T cells. The implications of the existence of such signaling pathways in disease are discussed by Yang et al. from Sun Yat-sen University, summarizing prior knowledge on the signaling pathways pertaining to TNF-TNFR1 and TNF-TNFR2 interactions in the context of autoimmune diseases and tissue regeneration. The main conclusion was that targeting TNFR1 or TNFR2 signaling pathways has significant therapeutic implications.Although there is compelling evidence that TNF signaling through the TNFR2 receptor preferentially stimulates Tregs, the molecular mechanism had remained largely unknown. The study of human Tregs by Kampan et al. from Monash University. Furthermore, this study found that IL-6 present in malignant ovarian cancer ascites is responsible for the up-regulation of TNFR2 expression and the expansion of highly suppressive Treg subsets. This finding may help to devise novel immunotherapies aiming to eliminate indirectly tumor-associated immune-suppressive Treg activity by inhibiting IL-6. Salomon et al. from Centre d'Immunologie et des Maladies Infectieuses discussed both historic and current understanding of the puzzling role of TNF as well as anti-TNF therapy in immune and inflammatory responses, leading up to our current understanding of the TNF-TNFR2 pathway and its decisive role in controlling the activation of Tregs. Through the analysis of recent reports on the therapeutic use of agonistic TNFR2-targeting agents in graft vs. host disease (GvHD), this review article raises the possibility of targeting of TNFR2-expressing Tregs using such agents, as potentially a safe and efficient approach to enhance anti-tumor immunity. In addition to its expression on immunosuppressive Tregs, TNFR2 can also be expressed by some tumor cells. Sheng et al. from Zhengzhou University summarized recent research regarding the role of TNFR2 in the promotion of carcinogenesis, cancer immune evasion and tumor growth. They concluded that TNFR2 was an ideal candidate for targeted tumor therapy. Moreover, this article clearly explains bi-directional signaling via TNFR2, given membrane-bound TNF (mTNF) preferentially binds to and activates TNFR2, and in addition to the forward signaling of mTNF → TNFR2, reverse signaling  can also occur. In this case, mTNF acts as a receptor that can transduce activating intracellular signaling when interacting with either sTNFR2 or membrane-bound TNFR2 on the surface of cells. Qu et al. from Tianjin Medical University further takes up this discussion into the possible role of forward and reverse crosstalk between mTNF and TNFR2 in an immunosuppressive tumor microenvironment. Indeed, these reviews leave open the question of whether reverse signaling by TNFR2 expressing Tregs may substantially affect mTNF-expressing immune cells and tumor cells.The tumor microenvironment is often rich in both TNF, as well as in TNFR2-expressing immune cells, specifically immunosuppressive Tregs or myeloid-derived suppressor cells (MDSCs) , 7. HighPegoretti et al. from University of Groningen discussed the possibility of selectively modulating the individual TNF receptors, TNFR2 or TNFR1, for the treatment of multiple sclerosis (MS), a neurodegenerative autoimmune disease which is currently resistant to anti-TNF treatment. Mancusi et al. from the University of Perugia discussed the need to evaluate whether Treg activation via TNFR2 could be used to practically enhance the yield, purity, and/or efficacy of Tregs used for cell therapy. Furthermore, they propose that such an approach has the potential for quick clinical translation in HSCT trials, since it is reported that Tregs have the capacity to prevent GvHD and promote immune reconstitution. Ahmad et al. from Universiti Sains Malaysia analyzed the role of TNF-TNFR2 interactions in immune tolerance to allergens and concluded that targeting TNF-TNFR2 interactions may represent a novel strategy for the treatment of allergic inflammatory responses. Based on the idea that genetic variation in the promoter of the TNFRSF1B gene could have a major impact on disease susceptibility, as well as potential responsiveness to TNFR2-targeting therapies, Li and Anderson from National Cancer Institute at Frederick proposed a key transcription factor binding site that may have significant effects on TNFRSF1B promoter activity, and suggested that it should be considered in future studies.The significant role that TNFR2-expressing Tregs can play in the pathogenesis and treatment of other types of diseases are also included in this Research Topic. + Tregs, TNFR2 also plays a key role in modulating the activity of other type of immunosuppressive cells, such as myeloid-derived suppressive cells (MDSCs), Mesenchymal stem cells (MSCs) and CD8+ Treg cells. In this Research Topic, Chavez-Galan et al. from University of Geneva studied the role that mTNF may play a role in promoting accumulation and enhancing function of MDSC in the pleural cavity during an acute mycobacterial infection. They found that the interaction of mTNF expressed by MDSCs and TNFR2 expressed by CD4 T cells is required for protection against the lethal inflammatory responses, which are sometimes associated with pleural mycobacterial infection. However, Schmid et al. from the University of Regensburg did not observe significant effects on MDSCs after TNFR2 agonist treatment in vivo. In addition to CD4 Tregs, some TNFR2-expressing CD8 T cells also have suppressive capacity. Ye et al. from Huazhong University of Science and Technology analyzed the functional consequences of TNFR2 expression by CD8+Foxp3+ Tregs and by CD8+ Teffs, and concluded that a TNF-TNFR2 mediated coordinated complex events ultimately result in strong CD8+ T cell-mediated immune responses. Mesenchymal stem cells (MSCs) have immunosuppressive properties which may be therapeutically harnessed in the treatment of autoimmune diseases. Yan et al. from the University of Macau discusses the role of TNF signaling through TNFR1 and TNFR2 on the biology of MSCs. The effect of TNF in MSC-based therapy for autoimmune and inflammatory diseases is also discussed.In addition to conventional CD4Mohamud et al. from Monash University reported that PS50G treatment preferentially stimulates the expansion of highly suppressive TNFR2+ Tregs, which express high levels of Ki-67, LAP, and CTLA-4. This is likely caused by the induction of CD103+ DCs in mice treated with PS50G. This property of engineered nanoparticles may prove to be useful in the treatment of inflammatory human diseases. Urbano et al. from Radboud University reported that the combination treatment with rapamycin and a TNFR2 agonist antibody was able enhance hypo-methylation of the FOXP3 gene, and consequently promote the stability of Tregs. Clear therapeutic potential has spurred the development of agonistic or antagonistic TNFR2-targeting biological agents in the recent years. Zou et al. from the University of Macau provides an overview regarding the latest progress in the study of TNFR2-targeting pharmacological agents and their therapeutic potential through the up- or down-regulation of Treg activity. In addition to protein drugs, this review suggests that small molecule inhibitors of TNFR2 may also have therapeutic value. Shaikh et al. from University of Macau performed a virtual screening of 400,000 naturally occurring small molecule compounds against TNF-binding sites of TNFR2. Their results indicate that a number of compounds could block the ligand-binding site of TNFR2. In vitro and in vivo studies are now needed to verify the results of this virtual study. Progranulin (PGRN) is a protein with immunosuppressive properties, which purportedly inhibits TNF-induced TNFR1/2 signaling by directly binding to TNFR1 and TNFR2. However, Lang et al. from University Hospital of Würzburg didn't observe a direct interaction between PGRN and TNFR1/2 in cellular binding studies.Synthetic glycine coated 50 nm polystyrene nanoparticles (PS50G) are able to inhibit allergic airway inflammation. Taken together, 19 primary research reports and review articles in this Frontiers Topic further support and substantiate the decisive role of TNF-TNFR2 interactions in promoting the activation, expansion and phenotypic stability of Tregs, as well as other immunosuppressive cells which may further include CD8 Tregs, MDSCs and MSCs. The critical role of TNFR2 signaling in helping maintain immune homeostasis, in promoting an immunosuppressive tumor microenvironment and in dampening autoimmune or allergic responses, is highlighted across these articles, as is an exploration of the molecular mechanisms that underlie this interaction. Emerging trends in the development of TNFR2-targeting therapeutics are further highlighted. We thus believe this article collection will be helpful for investigators performing fundamental research, as well clinical researchers. Given the substantial potential that targeting TNF-TNFR2 signaling offers for treatment of multiple diseases, we hope this collection of articles will spur further research, and eventually lead to new useful treatments.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arthrobacter sp. strain ATCC 21022 were sequenced. The phages all have relatively small genomes, with each genome containing 15,556 bp. All 8 phages are closely related to previously described cluster AN Arthrobacter phages .Eight siphoviral phages isolated from various soil types and locations in southwestern Pennsylvania using  Arthrobacter spp. are soil bacteria useful for their ability to protect themselves from heavy-metal toxicity and to survive long periods under stressful conditions induced by starvation, temperature shifts, ionizing radiation, oxygen radicals, and toxic chemicals , with coding sequences predicted by GeneMark v2.5p (Sequencing, assembly, and finishing of the genomes were performed according to Russell . Phage grk v2.5p  and GLIMrk v2.5p , and 26 rk v2.5p . Phamerark v2.5p  was usedrk v2.5p  or tRNAsrk v2.5p . See TabVibrio cholerae O1 (Each genome contains 26 putative genes. Given that the assignment of gene products to Phams was based on amino acid sequences similarity , all of lerae O1 , 17. TheGenBank accession numbers are provided in


In vitro, we found that AT-Lipoxin A4 markedly suppressed protein expression in TNF-α signals in human pulmonary microvascular endothelial cell, such as tumor necrosis factor receptor-associated factor 2 (TRAF2), TNF-R1-associated death domain (TRADD), receptor-interacting protein (RIP), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Moreover, AT-Lipoxin A4 inhibited downstream signals activated by TNF-α, including NF-κB/p65, JNK/MAPK, and ERK/MAPK. In vivo, AT-Lipoxin A4 significantly decreased pathological scores of the pancreas and lungs and the serum levels of IL-6 and TNF-α. Immunofluorescence, western blotting, and real-time PCR assay showed that AT-Lipoxin A4 significantly attenuated the expression of TNF-R1, TRADD, TRAF2, and RIP in the lungs of SAP rats. In addition, the activation of NF-κB was also downregulated by AT-Lipoxin A4 administration as compared with SAP rats. AT-Lipoxin A4 could inhibit the production of proinflammatory mediators and activation of TNF-α downstream signals such as NF-κB and MAPK. Downregulation of TNF-α signals by AT-Lipoxin A4 may be a significant mechanism in the attenuation of SAP-associated lung injury.Our previous studies verified the potent anti-inflammatory effects against severe acute pancreatitis (SAP) of AT-Lipoxin A4 and their analogues. However, the anti-inflammatory effects of AT-Lipoxin A4 on SAP-associated lung injury are not thoroughly known. We used western blot, polymerase chain reaction (PCR), and immunofluorescence to investigate the downregulation of TNF- A challenge exists in the early clinical treatment of severe acute pancreatitis (SAP) in terms of how to control the occurrence of pancreatitis-induced multiple organ dysfunction syndrome (MODS), as a common end-stage pathophysiological process of SAP and a major cause of early death of patients with SAP . MODS ofAs a barrier between blood circulation and organization, the vascular endothelium plays a pivotal role in the occurrence and development of inflammation. Pulmonary microvascular inflammation, which involves the production of cytokines and upregulation of adhesion molecules on endothelial cells, is one of the early stage manifestations during the pathogenesis of ALI . Studiesα is generally derived from activated fixed-tissue macrophages and circulating monocytes. There are various kinds of inflammatory factors, among which TNF-α is discovered earlier and playing an important part in the development of SAP and the occurrence of systemic complications [α is thus considered a proximal mediator that regulates the synthesis of several cytokines, chemokines, and adhesion molecules in endothelial cells and neutrophils [α plays a central role in SIRS and is associated with lung injury caused by acute necrotizing pancreatitis. The TNF-α pathway is divided into apoptosis pathways and proinflammatory pathways, including the TNF-R2 (P75) and TNF-R1 (P55) paths [α works by binding to TNF-R1, subsequently docked by adaptor proteins such as TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP) and then triggers a series of intracellular events that ultimately results in the activation of a major transcription factor, nuclear factor κB (NF-κB) [κB can in turn enhance the transcription of the TNF-α gene, thereby forming a vicious feedback loop that is able to amplify the early inflammatory signals and aggravate the initial inflammatory effects [TNF-ications . In the trophils . TNF-α p5) paths . TNF-α w (NF-κB) , 10. The effects .α. The anti-inflammatory effects of LXA4 may be dependent on the inhibition of NF-κB/p65 and p38/MAPK pathways, both of which are downstream signaling of TNF-α and its receptor 1 pathway. Using of LXA4 to inhibit inflammation and protect the target organ is a new strategy for the treatment of acute necrotizing pancreatitis, although the specific mechanism is not fully elucidated.Lipoxins (LXs) are synthesized locally from arachidonic acid at an inflammatory site by transcellular biosynthesis and considered to act as an “inflammatory braking signal” in inflammation by limiting the trafficking of leukocytes to the inflammatory site , 13. Lipα/TNF-R1 pathway.As a more stable analog of LXA4, AT-Lipoxin A4 shares many anti-inflammatory activities with the native LXA4 . Howeverμg/mL streptomycin  at 37°C in a humidified atmosphere with 5% CO2. For the experiments, HPMECs were seeded onto 96-well plates, 6-well plates, or 60 mm culture dishes.HPMECs were purchased from American Type Culture Collection (ATCC) and maintained in PRMI-1640  supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 α/TNF-R2 (p75) expression in the HPMECs. Two groups were set up for the cell transfection: the control group without the transfection and the TNF-α/TNF-R2 group with lentivirus target sequence interference. The p75-specific shRNA-encoding pLenti6.3-GFP vector, pGag-pol vector, pRev vector, and Pvsvg vector  were cotransfected into the 293T cell line using ViraPower™ Lentiviral Expression Systems  to produce lentivirus stocks with a negative construct as a negative control. The 293T cells were harvested after transfection for 48 hours, and the cell debris was removed by centrifugation at 4°C. The crude viral extract was filtered, and centrifuge enrichment was conducted.In this study, a lentiviral vector was constructed to specifically silence TNF-A total of 48 healthy male Sprague-Dawley (SD) rats  were provided by the Laboratory Animal Center of Wenzhou Medical College. The rats were bred and housed in standard shoe-box cages in a climate-controlled room with an ambient temperature of 20–22°C and a 12 h light-dark cycle. They were treated in accordance with the protocols approved by the Institutional Animal Committee of Wenzhou Medical College. All animals received care in accordance with the Guide for the Care and Use of Laboratory Animals.N = 16), the SAP group (N = 16), and the AT-Lipoxin A4 group (N = 16). Rats in the AT-Lipoxin A4  group were injected with 0.1 mg/kg of AT-Lipoxin A4  through the femoral vein 30 minutes following the procedure, while those in the other two groups were give normal saline in the same manner. In order to reduce the influence of alcohol on the experimental results, we used a self-made low-flow liquid nitrogen device to wash AT-Lipoxin A4  in a sterile experimental table. AT-Lipoxin A4 (100 μg) in normal saline (1 mL) was dissolved in an EP tube and rinsed with liquid nitrogen rinsing for about 3 minutes. Eight rats from each group were randomly submitted for sacrifice after 12 and 24 h following the induction of SAP.The SAP rat model was established according to Zhao et al. . BrieflyThe surgical procedure for sham rats was identical to those for the other groups, including bile duct cannulation. The blood of the inferior vena cava was collected and centrifuged at 4°C, and the serum was stored at -80°C for further studies. Fresh pancreatic and lung tissues were removed, fixed with 4% formaldehyde or frozen in liquid nitrogen overnight, and then stored at -80°C.Formaldehyde-fixed pancreatic and pulmonary tissue blocks were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). All microscopic sections were examined by two independent pathologists who were blinded to the experimental protocol. Pancreatitis severity was scored according to a protocol described by Schmidt et al. . The patα were determined by ELISA using kits  and performed following the instructions provided by the manufacturer. Concentrations of IL-6 and TNF-α were expressed in picograms per milliliter of the serum.Rat serum cytokines including interleukin-6 (IL-6) and TNF-Total RNA was isolated from tissue samples using a TRIzol Kit and was subjected to reverse transcription using oligo-dT-primed RT following the manufacturer's instructions. Quantitative real-time PCR was performed using a SYBR Green real-time PCR Master Mix  on a 7500 Real-Time PCR System . Primer sequences are listed in κB/P65, RIP, GAPDH, and beta-actin that were diluted at the indicated concentration. Then, the membranes were washed three times with TBST  and incubated with secondary antibodies  for 1.5 h at room temperature. After washing for 3 × 10 min with TBST, target bands were developed using ECL  and exposed on a film . The intensity of the protein bands was measured using Quantity One version 4.6.2 Image software .For whole cell extraction, cells and tissue samples were lysed in radioimmune precipitation assay buffer  supplemented with protease and phosphatase inhibitor. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% nonfat dry milk, membranes were incubated overnight at 4°C with primary antibodies against VCAM-1, E-selectin, ERK, P-ERK, JNK1/2, p-JNK1/2, TNF-R1, TRAF2, NF-κB/P65, and beta-actin primary antibodies were purchased from Abcam . All other primary antibodies were purchased from Cell Signaling Technology .The anti-GAPDH primary antibody was purchased from Bioworld . The anti-p-JNK1/2, TRAF2, NF-μm thickness were fixed in 4% paraformaldehyde for 15 minutes and then rinsed with Tris-buffered saline (TBS). After being blocked with 5% goat serum in TBS for 1 h at 37°C, the sections were then incubated with primary antibodies of NF-κB and incubated overnight at 4°C. After washing thrice with TBS, the slides were then incubated with a secondary antibody for 1 h at 37°C. Sections were then washed with TBS and incubated with the DAPI to highlight cell nuclei. Slides were visualized under a fluorescent microscope .Frozen tissue sections of 8 P < 0.05 was considered statistically significant.Data are expressed as the mean ± SEM. Differences between mean values were analyzed by one-way analysis of variance. Dunnett's post hoc test was used for multiple group comparisons. Flow cytometry determination of lentivirus transfection efficiency was 90% . PCR resα. The effect of TNF-α on JNK/MAPK and ERK/MAPK phosphorylation was assessed at 15 min. Besides, cells were preincubated with or without AT-Lipoxin A4 for 1 h before stimulation with TNF-α for various periods: 15 min for the measurement of NF-κB/p65 phosphorylation; 1 h for RIP, TRAF2, and TRADD translocation; and 6 h for the measurement of protein expression of VCAM-1, E-selectin, RIP, and TRAF2. To investigate whether the inhibition of inflammation by AT-Lipoxin A4 was involved in the regulation of RIP and TRAF2, we examined the expressions of RIP and TRAF2 of HPMECs using western blot assay. We found that the expression of RIP and TRAF2 was increased significantly following TNF-α stimulation compared with the control (P < 0.01) in HPMECs, whereas AT-Lipoxin A4 downregulated RIP and TRAF2 expression in TNF-α-stimulated HPMECs ((-/-) HPMECs) were pretreated with AT-Lipoxin A4 (50 ng/mL) for 1 h and then stimulated with TNF-α (50 ng/mL). The expression of RIP and TRAF2 was examined by western blot assay. And we found AT-Lipoxin A4 downregulated RIP and TRAF2 expression in TNF-α-stimulated TNFR2(-/-) HPMECs . Before each experiment, cells were grown in RPMI-1640 supplemented with 1% FBS for 12 h. Then, cells were treated with 50 ng/mL AT-Lipoxin A4 or vehicle (0.035% ethanol) for 1 h before the addition of 50 ng/mL TNF-d HPMECs . In addi) HPMECs .α induced p38 MAPK activation [α in HPMECs using western blot assay. A 15 min treatment of TNF-α was determined as previous studies have shown that TNF-α causes MAPK phosphorylation within 15 min in human endothelial cells [α induced a marked increase in ERK and JNK phosphorylation in HPMECs and these effects of TNF-α were inhibited by AT-Lipoxin A4 in HPMECs (α-induced TNFR2(-/-) HPMECs using western blot analysis. We found that TNF-α induced a marked increase in JNK and ERK phosphorylation in TNFR2(-/-) HPMECs. AT-Lipoxin A4 reduced TNF-α-induced phosphorylation of JNK and ERK in TNFR2(-/-) HPMECs  HPMECs .α-induced expression of VCAM-1 and E-selectin on the surface of the HPMECs were evaluated by western blot assay. Exposure of cells to TNF-α for 6 h induced significantly increased expressions of VCAM-1 and E-selectin. Pretreatment with AT-Lipoxin A4 significantly inhibited TNF-α-induced expressions of VCAM-1 and E-selectin in a dose-dependent manner . Histopathological changes in lung tissues were scored as described by Mayer et al. [Our previous studies found that AT-Lipoxin A4 downregulation of ICAM-1 expression in TNF-a stimulated HPMECs . The eff Figures . Attenua Figures . The pat Figures . The Sch Figures . Histopar et al. . Signifiα exerts its biological functions via interaction with its' cognate membrane receptors, namely, TNF-R1 and TNF-R2. TNF-α binds to TNF-R1, subsequently docked by adaptor proteins TRADD, TRAF2, and RIP and then triggers a series of intracellular events that ultimately activates proinflammatory signal transduction pathways [It is well known that TNF-pathways . TherefoκB is a transcriptional factor that is essential for the gene expression of inflammatory mediators in AP-associated ALI. Our previous study showed that LXA4 suppressed TNF-α-induced phosphorylation and translocation of NF-κB/p65 in HPMECs. In this experiment, we found that AT-Lipoxin A4 markedly reduced the NF-κB/p65 level in both the nucleus and cytoplasm in TNF-α-stimulated TNF-R2(-/-) HPMECs  HPMECs, while this effect was significantly inhibited by AT-Lipoxin A4  HPMECs.NF- Figures . In addi Figures . Thus, AκB at 12 h and 24 h. However, treatment with AT-Lipoxin A4 significantly inhibited NF-κB nuclear translocation at both time points . AT-Lipoxin A4 treatment resulted in a significant reduction of NF-κB compared to the SAP group   HPMECs. Therefore, we suspect the mechanism of AT-Lipoxin A4 protects the SAP-associated lung injury by affect TNF-α/TNF-R1 and downstream-related proteins.TNFR1 signaling depends on the ligand-induced formation of intracellular signaling complexes consisting of TRADD, TRAF2, RIP, and FADD, each exerting a different and specific function to activate the signaling , 25, 26.α and IL-6 and alleviates pancreatic and lung tissue pathological damage. In vitro, we found that AT-Lipoxin A4 markedly suppressed the protein expression of VCAM-1 and E-selectin in TNF-α-exposed HPMECs and TNF-R2(-/-) HPMECs. Taken together, we provided evidence that AT-Lipoxin A4 ameliorates SAP and SAP-associated lung injury. The outcomes of the study indicate that AT-Lipoxin A4 can break this waterfall-like vicious chain-reaction process, inhibit SIRS, and prevent this process so as to treat the critically ill.High concentrations of proinflammatory cytokines cause the injury of alveolar endothelial cells . In our α generation if any of the three MAPK pathways is inhibited [α-induced human pulmonary microvascular endothelial cells and the MAPK inhibitor can inhibit the expression of cytokines and adhesion molecules [MAPK is a serine/threonine protein kinase formed by a variety of isoenzymes, including extracellular signal-regulated kinase (ERK), P38, and JNK. It was shown that it is sufficient to prevent LPS-induced TNF-nhibited . In addinhibited . Studiesolecules , 31. MAP(-/-) HPMECs. The results showed that AT-Lipoxin A4 inhibited TNF-α-induced JNK MAPK and ERK MAPK phosphorylation in HPMECs and TNF-R2(-/-) HPMECs, which have shown that MAPK activation is attenuated in the presence of AT-Lipoxin A4. Considering our previous experimental results in vitro verifying the anti-inflammatory effect of AT-Lipoxin A4, these results suggest that the anti-inflammatory effect of AT-Lipoxin A4 may depend on the regulation of the JNK MAPK and ERK MAPK signaling pathway. Further, our previous results indicated that LXA4 inhibits p38 MAPK activation in HPMECs induced by TNF-α and that LXA4 alone did not significantly change it in HPMECs [To further investigate the mechanism responsible for the anti-inflammatory effect of AT-Lipoxin A4, we examined the effect of AT-Lipoxin A4 on JNK MAPK and ERK MAPK phosphorylation in HPMECs and TNF-R2n HPMECs . Therefoα/TNF-R1 and downstream signals such as TRADD, TRAF2, and RIP, which results in the suppression of the NF-κB pathway and MAPK pathway activity and in turn inhibits the generation of an inflammatory mediator. Downregulation of TNF-α/TNF-R1 signals by AT-Lipoxin A4 may be a significant mechanism of attenuation in SAP-associated lung injury.Taken together, our results clarified that AT-Lipoxin A4 can suppress the activation of TNF-