Carbon Chain Length Determines Inhibitory Potency of Perfluoroalkyl Sulfonic Acids on Human Placental 3ß-Hydroxysteroid Dehydrogenase 1: Screening, Structure-Activity Relationship, and In Silico Analysis.

Objective: This study aimed to compare 9 perfluoroalkyl sulfonic acids (PFSA) with carbon chain lengths (C4-C12) to inhibit human placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), aromatase, and rat 3ß-HSD4 activities. Methods: Human and rat placental 3ß-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS-MS, and human aromatase activity was determined by radioimmunoassay. Results: PFSA inhibited human 3ß-HSD1 structure-dependently in the order: perfluorooctanesulfonic acid (PFOS, half-maximum inhibitory concentration, IC 50: 9.03 ± 4.83 µmol/L) > perfluorodecanesulfonic acid (PFDS, 42.52 ± 8.99 µmol/L) > perfluoroheptanesulfonic acid (PFHpS, 112.6 ± 29.39 µmol/L) > perfluorobutanesulfonic acid (PFBS) = perfluoropentanesulfonic acid (PFPS) = perfluorohexanesulfonic acid (PFHxS) = perfluorododecanesulfonic acid (PFDoS) (ineffective at 100 µmol/L). 6:2FTS (1H, 1H, 2H, 2H-perfluorooctanesulfonic acid) and 8:2FTS (1H, 1H, 2H, 2H-perfluorodecanesulfonic acid) did not inhibit human 3ß-HSD1. PFOS and PFHpS are mixed inhibitors, whereas PFDS is a competitive inhibitor. Moreover, 1-10 µmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells. Docking analysis revealed that PFSA binds to the steroid-binding site of human 3ß-HSD1 in a carbon chain length-dependent manner. All 100 µmol/L PFSA solutions did not affect rat 3ß-HSD4 and human placental aromatase activity. Conclusion: Carbon chain length determines inhibitory potency of PFSA on human placental 3ß-HSD1 in a V-shaped transition at PFOS (C8), with inhibitory potency of PFOS > PFDS > PFHpS > PFBS = PFPS = PFHxS = PFDoS = 6:2FTS = 8:2FTS.

Overactivation of mitogen-activated protein kinase and suppression of mitofusin-2 expression are two independent events in high mobility group box 1 protein-mediated T cell immune dysfunction.

High mobility group box 1 protein (HMGB1), a critical proinflammatory cytokine, has recently been identified to be an immunostimulatory signal involved in sepsis-related immune dysfunction when released extracellularly, but the potential mechanism involved remains elusive. Here, we showed that the treatment with HMGB1 in vitro inhibited T lymphocyte immune response and expression of mitofusin-2 (Mfn-2; a member of the mitofusin family) in a dose- and time-dependent manner. Upregulation of Mfn-2 expression attenuated the suppressive effect of HMGB1 on T cell immune function. The phosphorylation of both extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) was markedly upregulated by treating with high amount of HMGB1, while pretreatment with ERK1/2 and p38 MAPK-specific inhibitors (U0126 and SB203580) could attenuate suppression of T cell immune function and nuclear factor of activated T cell (NFAT) activation induced by HMGB1, respectively. HMGB1-induced activity of ERK1/2 and p38 was not fully inhibited in the presence of U0126 or SB203580. Interestingly, overexpression of Mfn-2 had no marked effect on HMGB1-mediated activation of MAPK, but could attenuate the suppressive effect of HMGB1 on the activity of NFAT. Thus, the mechanisms involved in HMGB1-induced T cell immune dysfunction in vitro at least partly include suppression of Mfn-2 expression, overactivation of ERK1/2, p38 MAPK, and intervention of NFAT activation, while the protective effect of Mfn-2 on T cell immune dysfunction induced by HMGB1 is dependent on other signaling pathway associated with NFAT, but not MAPK. Taken together, we conclude that overactivation of MAPK and suppression of Mfn-2 expression are two independent events in HMGB1-mediated T cell immune dysfunction.

Burn injury induces gelsolin expression and cleavage in the brain of mice.

Gelsolin is an actin filament-severing and capping protein, affecting cellular motility, adhesiveness and apoptosis. Whether it is expressed in the brain of burned mice has not yet been characterized. Mice were subjected to a 15% total body surface area scald injury. Neuropathology was examined by hematoxylin and eosin staining. Cerebral gelsolin mRNA, distribution and cleavage were demonstrated by quantitative polymerase chain reaction (QPCR), immunohistochemistry and Western blot, respectively. Cysteinyl aspartate-specific protease (caspase)-3-positive cells and activity were also measured. Burn injury could induce pathological alterations in the brain including leukocyte infiltration, necrosis, microabscess and gliosis. Compared with sham-injured mice, gelsolin mRNA was up-regulated at 8h post-burn (pb) in a transient manner in the cortex and striatum of burned mice, while it remained at higher levels in the hippocampus up to 72 hpb. Of interest, gelsolin was further cleaved into 42 and 48 kDa (kilo Dalton) fragments as illustrated in the hippocampus at 24 hpb, and was widely expressed in the brain by activated monocyte/macrophages, astrocytes and damaged neurons. In the meantime, caspase-3-positive cells were noted in the striatum of burned mice and its activity peaked at 24 hpb. To clarify inflammation-induced gelsolin expression and cleavage in the brain, rat pheochromocytoma cells were exposed to lipopolysaccharide to show increased gelsolin expression and caspase-3-dependent cleavage. The results suggest that burn-induced cerebral gelsolin expression would be involved in the activation of both the monocytes and astroglial cells, thereby playing a crucial role in the subsequent inflammation-induced neural apoptosis following burn injury.

Curcumin inhibits suppressive capacity of naturally occurring CD4+CD25+ regulatory T cells in mice in vitro.

Accumulating evidence has demonstrated that naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) are critical for maintenance of immunological tolerance and have been shown to be important in regulating the immune responses in many diseases. Curcumin, a phytochemical obtained from the rhizome of the plant Curcuma longa, has achieved the potential therapeutic interest to numerous immune-related disorders. However, the effect and mechanism of curcumin on Tregs remain largely elusive. In the present study, curcumin inhibition of the suppressive activity of CD4(+)CD25(+) regulatory T cells appears to be dependent on three categories: inhibiting cell-cell contact by down-regulation of CTLA-4, suppressing inhibitory cytokine secretion and decreasing the ability to consume IL-2 and/or suppress IL-2 production. In addition, Foxp3 expression was also reduced on Tregs after curcumin stimulation. Moreover, we found that nuclear translocation of p65 and c-Rel, which is critical for Foxp3 and CD25 expressions, was markedly decreased in Tregs with curcumin stimulation. Based on the role of curcumin in the suppressive activity of Tregs, it may be feasible to use curcumin as an immunotherapy for Treg-related diseases, such as tumors and sepsis.

[The extracellular role of high mobility group box-1 protein in regulation of immune response].

High mobility group box-1 protein (HMGB1), a highly conserved nuclear DNA-binding protein, is involved in maintenance of nucleosome structure and regulation of gene replication, transcription and translation. Recently, there is accumulating evidence that HMGB1 can be passively or actively released from the nucleus to the extracellular milieu and act as a late proinflammatory cytokine that mediates development of inflammatory diseases, including sepsis. In addition, HMGB1 can also act as an "alarm signal" regulating immune response of host. In this article, we summarized the structure, secretion and receptor signaling pathways of HMGB1. Furthermore, the role and potential mechanism underlying HMGB1 in regulation of immune function were also discussed.

Expression of high mobility group protein B1 in the lungs of rats with sepsis.

BACKGROUND: Vibrio vulnificus inside the body could activate the NF-κB signaling pathway and initiate the inflammatory cascade. The lung is one of the earliest organs affected by sepsis associated with acute lung injury. High mobility group protein B1 (HMGB1) is an important late-acting pro-inflammatory cytokine involving in the pathophysiology of sepsis. It is also involved in the injury process in the lung, liver and intestine. There has been no report on the involvement of HMGB1 in Vibrio vulnificus sepsis-induced lung injury. METHODS: Sixty rats were randomly divided into a normal control group (group A, n=10) and a Vibrio vulnificus sepsis group (group B, n=50). Sepsis was induced in the rats by subcutaneous injection of Vibrio vulnificus (concentration 6×10(8) cfu/mL, volume 0.1 mL/100g)) into the left lower limbs. The rats in group B were sacrificed separately 1, 6, 12, 24, and 48 hours after the infection. Their lungs were stored as specimens, lung water content was measured, and lung pathology was observed under a light microscope. The expressions of the HMGB1 gene and protein in the lungs were detected by RT-PCR and Western blot. Data were analyzed with one-way analysis of variance (ANOVA) and the LSD method for pair-wise comparison between the two groups. P<0.05 was considered statistically significant. RESULTS: Compared to group A (0.652±0.177), HMGB1 mRNA expression in the lungs of group B was significantly higher at 0 hour (1.161±0.358, P=0.013), 24 hours (1.679±0.235, P=0.000), and 48 hours (1.258±0.274, P=0.004) (P<0.05), and peaked at 24 hours. Compared to group A (0.594±0.190), HMGB1 protein expression at 6 hours (1.408±0.567, P=0.026) after infection was significantly increased (P<0. 05), and peaked at 24 hours (2.415±1.064, P=0.000) after infection. Compared to group A (0.699±0.054), lung water content was significantly increased at 6 hours (0.759±0.030, P=0.001),12 hours (0.767±0.023, P=0.000), 24 hours (0.771±0.043, P=0.000) and 48 hours (0.789±0.137, P=0.000) after infection (P<0.05). Compared to group A, pathological changes at 12 hours in group B indicate marked pulmonary vascular congestion, interstitial edema and inflammatory infiltration. Alveolar cavity collapse and boundaries of the alveolar septum could not be clearly identified. CONCLUSION: Vibrio vulnificus sepsis can lead to injury in rat lungs, and increased HMGB1 expression in lung tissue may be one of the mechanisms for injury from Vibrio vulnificus sepsis.