The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.
Background: Bronchiolitis obliterans (BO), first mentioned in 1901, is a severe and rare chronic lung disease in children. BO has various etiologies and the most common in children is post-infectious BO (PIBO). High resolution CT (HRCT) is an often-used image tool for the diagnosis of BO, and pulmonary scintigraphy is an alternative tool that can functionally evaluate BO. Recently, dual-energy computed tomography (DECT) have also been applied to BO for its accuracy and safety. Here we described the characteristics of HRCT, pulmonary scintigraphy, DECT, and the clinical profiles of patients with PIBO. Methods: This is a retrospective and descriptive study. Data were collected from patients diagnosed with PIBO from 2014 to 2019 in the Pediatric Cardiopulmonary Outpatient Clinics of Kaohsiung Medical University Hospital. The diagnosis was based on clinical, chest X-ray, and HRCT findings. Clinical profile, radiological characteristics, and images of pulmonary scintigraphy were documented. Results: Eight children (4 boys and 4 girls) were diagnosed with PIBO at a mean age of 25.8 months (range 15 to 41 months). Two of our patients developed pulmonary hypertension. The most common HRCT finding is mosaic pattern, where match ventilation/perfusion (V/Q) defects is a general feature in pulmonary scintigraphy. DECT pulmonary blood vasculature images revealed various degrees of decreased perfusion and is compatible with the decreased perfusion on pulmonary scintigraphy. Conclusion: The therapeutic strategy of PIBO is still lacking of standardization. HRCT and V/Q scans are important image tools in diagnosis and follow-up of BO. DECT may be used in BO patients as it has no additional radiation exposure and add value on functional information of HRCT.
Objective: NXY-059 is a novel free-radical trapping neuroprotectant. Digoxin treatment is common in acute ischaemic stroke, the intended patient population for NXY‑059. Since both digoxin and NXY‑059 are eliminated primarily renally, with a substantial contribution by active renal secretion, and because digoxin has a narrow therapeutic window, this open, randomised, crossover, two-period study investigated whether NXY‑059 affects the pharmacokinetics (PK) of digoxin.Research design and methods: Twenty-two healthy subjects received 0.5 mg oral digoxin 2 h after the start of 60‑h intravenous infusions of NXY‑059 and placebo separated by a 14-day washout. Blood and urine were collected for 60 h. Digoxin concentrations were measured by a novel liquid chromatography–mass spectrometry assay.Main outcome measures: The ratio of the geometric mean (90% confidence interval) between NXY‑059 and placebo for the digoxin area under the concentration-versus-time curve was 0.91 (0.83–0.99) and was within the predefined range for no interaction (0.80–1.25). No safety concerns were raised in the study. No serious adverse events were recorded. The most common adverse event was headache with similar frequencies in the two treatments.Conclusions: NXY‑059 had no clinically significant effect on the PK of digoxin.
Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.
Eisenmenger syndrome (ES) refers to congenital heart diseases (CHD) with reversal flow associated with increased pulmonary pressure and irreversible pulmonary vascular remodeling. Previous reports showed limited therapeutic strategies in ES. In this study, 5 ES patients (2 males and 3 females), who had been followed regularly at our institution from 2010 to 2019, were retrospectively reviewed. We adopted an add-on combination of sildenafil, bosentan, and iloprost and collected the clinical characteristics and outcomes as well as findings of echocardiography, computed tomography, pulmonary perfusion-ventilation scans, positron emission tomography, and biomarkers. The age of diagnosis in these ES patients ranged from 23 to 54 years (38.2 ± 11.1 years; mean ± standard deviation), and they were followed for 7 to 17 years. Their mean pulmonary arterial pressure and pulmonary vascular resistance index were 56.4 ± 11.3 mmHg and 24.7 ± 8.5 WU.m2, respectively. Intrapulmonary arterial thrombosis was found in 4 patients, ischemic stroke was noted in 2 patients, and increased glucose uptake of the right ventricle was observed in 4 patients. No patient mortality was seen within 5 years of follow-up. Subsequently, 2 patients died of right ventricular failure, 1 died of sepsis related to brain abscess, and another died of sudden death. The life span of these patients was 44-62 years. Although these patients showed longer survival, the beneficial data on specific-target pharmacologic interventions in ES is still preliminary. Thus, larger trials are warranted, and the study of cardiac remodeling in ES from various CHD should be explored.
Human follicle dermal papilla cells (HFDPCs) are essential for the induction and maintenance of hair growth 1. Interventions that block the senescence of dermal papilla may effectively expand cell numbers for implantation. However, in vitro culture of HFDPCs has many difficulties in the proliferation and senescence, particularly the lower proliferative capacity 2. Cellular senescence is caused by a variety of stress and leads to growth arrest. Senescence is associated with declining cellular nicotinamide adenine dinucleotide (NAD+) levels, whereas activation of the NAD+ pathway can extend cellular replicative lifespan 3. AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis and was reported to inhibit mTORC1 4 and activate NAD+-dependent enzymes 3, 5 by increasing cellular NAD+ levels to promote proliferation and prevent senescence. Recently, we reported that adenine can act as a true AMPK activator following conversion to AMP during purine salvage 6. Proteomic analysis revealed that adenine has a similar effect on NIH-3T3 cells to another AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). In this study, we investigated that adenine delays senescence in cultural HFDPC. We investigated whether adenine supplementation could promote HFDPC proliferation in vitro, thereby preventing senescence. Experimental procedures are described in Appendix S1. Cultured HFDPCs purchased from PromoCell were subcultured for five passages and then cultured in the presence or absence of adenine. Cells were counted and replated at 3-day intervals (Fig. 1a). In the absence of adenine, the cell proliferation ceased after passage 14 (P14), but adenine supplementation expanded proliferation during consecutive cultures. We also detected senescent cells by measuring β-galactosidase activity under bright-field microscopy (Fig. 1b). No β-galactosidase activity was detected in P5 HFDPCs; however, the activity was apparent at P13. Notably, β-galactosidase activity was lower in cells treated with adenine (P13 and P16, Fig. 1c), indicating fewer senescent cells in adenine-treated cultures. To investigate the effect of adenine on HFDPC culture, we used cDNA array to analyse gene expression at P10 and P12. As indicted in the heat map presented in Fig. S1a, the expression of a total of 3605 genes was upregulated by over twofold, in which cell cycle and NAD metabolism were the most relevant pathways (Fig. S1b). Using quantitative PCR, we also confirmed that adenine supplementation prevented the decreases in gene expression associated with proliferation and telomere maintenance, including CCNB1, CCNB2, CCNE1, ASPM, PKB, SPAG5, CDC25C, PCNA and TERT (Fig. S1c). Several AMPK activators are available today. Adenine can be converted to AMP by adenosyl phosphoribosyl transferase (APRTase) 7 and it subsequently binds and activates AMPK 6. Another AMPK activator, AICAR, binds to AMPK as ZMP. We compared the effect of these two indirect AMPK activators on HFDPCs. To investigate the downstream effectors of AMPK that mediate the antisenescence effect of adenine, we used Western blot to assess the activation of AMPK and its downstream targets. HFDPCs (P5-P12) were cultured under different concentrations of adenine or AICAR (0, 60, 200, 600 μm) and cell lysates from P12 were assessed using Western blot. Adenine induced Thr172 phosphorylation of AMPK in dose-dependent manner, but less potently than equal molar concentrations of AICAR (Fig. 2a). Besides, AICAR-induced AMPK phosphorylation increased with the duration of incubation; however, adenine-induced AMPK phosphorylation did not (Fig. S2). The mTOR signalling pathway is inhibited by AMPK activation, and the inhibition of mTORC1 has been reported to promote cellular senescence 8. However, in this study, adenine did not increase the phosphorylation of raptor nor did it decrease the cellular levels of p-p70 S6 kinase and p-4E-BP1 in HFDPCs (Fig. 2b). Furthermore, the same concentrations of adenine did not inhibit mTOR signalling. This observation is not surprising because at lower levels AICAR also did not inhibit mTOR signalling. Another target of AMPK is known to modulate cellular senescence through activation of NAD+-dependent enzymes such as Sirt1 3. We also observed that adenine increased the intracellular NAD+ levels (Fig. 2c). In this study, we observed that adenine significantly stimulated the proliferation and inhibited the senescence of cultured HFDPCs. Microarray analysis also confirmed that adenine prevents the decreases in gene expression associated with senescence. Our results indicate, for the first time, that adenine activated AMPK in HFDPCs. However, adenine-induced AMPK activation was not sufficient to promote the inhibition of mTOR. Nevertheless, adenine supplementation increased cellular NAD+ levels and promoted the NAD+-dependent enzymes activity. In conclusion, our study demonstrate that adenine could exert proliferation and antisenescence effects possibly through activating AMPK and increasing cellular NAD+ levels. Future application of adenine in aiding HFDPC proliferations in vitro may thus contribute to the implantation in therapeutic use. The authors thank Ms. Shu-Ching Yang for secretarial work, Dr. Chun-Fang Huang for editing the manuscript, Mr. Jen-Chie Lee and Mr. Li-Ming Chen for their help in experiments. YF Cheng designed the study and performed the research, GH Young analysed the data and wrote the manuscript, TM Chiu, JT Lin, PR Huang, CY Kuo, YJ Liang, PK Chen, SF Chen and CY Cheng contributed essential reagents and HM Chen supervised the study. The authors have declared no conflict of interests. 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Objective: NXY-059 is a novel, free-radical trapping, neuroprotectant that reduces infarct size and preserves brain function in animal models of acute ischaemic stroke. This study evaluated the safety, tolerability and pharmacokinetics of NXY-059 in the unbound steady-state plasma concentration (Cuss) exposure range of 50-300 μmol/L in healthy young and elderly subjects.Research design and methods: The primary objective of this two-centre, randomised, double-blind, placebo-controlled, dose-escalating study was to investigate the safety and tolerability, including renal function parameters and vasoirritative effects, of 8‐h and 72‐h intravenous infusions of NXY‐059 in healthy young (20–45 years) and elderly (55–75 years) male and female subjects. The secondary objective of the study was to evaluate the pharmacokinetics of 8‐h and 72‐h intravenous infusions of NXY‐059 in these subjects, using blood and urine samples taken during and after NXY‐059 infusion as well as the doses administered.Results: Of the 104 healthy volunteers who participated in the study, 72 were young and 32 were elderly. The type and incidence of adverse events in NXY‐059 and placebo subjects were similar, although headache was more common in the NXY‐059 group. Renal function was not altered in either group. Thrombophlebitis was reported in two elderly subjects: one receiving NXY-059 and one receiving placebo. A proportional relationship between AUC and dose for the 8‐h and 72‐h infusions was observed, and clearance did not change with dose.Conclusions: NXY-059 was well tolerated at all plasma concentrations tested in both the young and elderly subjects, and no safety concerns were raised. Linear pharmacokinetics were observed following 8‐h and 72‐h infusions of NXY‐059 at doses resulting in an average Cuss in the 52–317 µmol/L range.
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