11 research outputs found
Canagliflozin Regulates Ferroptosis, Potentially via Activating AMPK/PGC-1α/Nrf2 Signaling in HFpEF Rats
Aims: Sodium-glucose cotransporter-2 (SGLT2) inhibitors have been found to ameliorate major adverse cardiovascular events in patients with heart failure with preserved ejection fraction (HFpEF), but the exact mechanism is unknown. Ferroptosis is a form of programmed necrosis. Herein, we verified that canagliflozin (CANA) ameliorates heart function in HFpEF rats, partly by regulating ferroptosis, which may be activated by AMPK/PGC-1α/Nrf2 signaling.Methods: An HFpEF model was established and subjected to CANA treatment. Blood pressure was monitored, and echocardiography was performed at the 12th week. Pathological examination was performed, and expression of ferroptosis-associated proteins and AMPK/PGC-1α/Nrf2 signaling related proteins was detected.Results: CANA had an antihypertensive effect and increased E/A ratios in HFpEF rats. Myocardial pathology was ameliorated, on the basis of decreased cross-sectional area and intercellular fibrosis. Acyl-CoA synthetase long-chain family member 4 (ACSL4) expression increased, whereas ferritin heavy chain 1 (FTH1) expression decreased in HFpEF rats, which showed iron overload. CANA reversed changes in ACSL4 and FTH1, and decreased iron accumulation, but did not alter glutathione peroxidase 4 (GPX4) expression. The expression of AMPK/PGC-1α/Nrf2 signaling related proteins and heme oxygenase 1 (HO-1) in the HFpEF group decreased but was reverted after CANA treatment.Conclusions: CANA regulates ferroptosis, potentially via activating AMPK/PGC-1α/Nrf2 signaling in HFpEF rats
Evidence That the P\u3csub\u3ei\u3c/sub\u3e Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle
Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein–protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle
α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al
Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated
Canagliflozin Alleviates Atherosclerosis Progression through Inflammation, Oxidative Stress, and Autophagy in Western Diet-fed ApoE −/− Mice
Purpose: This study was aimed at investigating the effect of canagliflozin (Cana) on atherosclerosis and further exploring its potential mechanism. Methods: ApoE −/− mice were fed a Western diet (WD) and randomly divided into a WD group and WD+Cana group. After 15 weeks of canagliflozin treatment, serum levels of fasting insulin and inflammatory cytokines were determined with ELISA kits. HE, Oil Red O, and Masson staining were used to estimate the extent of atherosclerosis. Immunohistochemistry, immunofluorescence, ROS staining, and RT-PCR were used to further investigate Cana’s potential mechanism. Results: Histological analysis indicated that Cana restrained atherosclerotic plaque development. Furthermore, Cana decreased the percentage of F4/80 positive cells, and the areal density of ROS and relative fluorescence intensity of P62, but enhanced the relative fluorescence intensity of LC3 in the aortic root. Analysis of factors associated with the inflammatory response mediated by AP-1, oxidative stress mediated through the ROS/Nrf2 pathway, and autophagy in the aorta indicated elevated mRNA levels of F4/80, MCP-1, VCAM-1, AP-1, ROS, NOX4, P62, NLRP3, and IL-1β, but diminished mRNA levels of Nrf2, GST, eNOS, and LC3, in the WD+Cana group. Conclusion: Canagliflozin may attenuate atherosclerosis by decreasing the inflammatory response mediated by AP-1, alleviating oxidative stress through the ROS/Nrf2 pathway, and enhancing autophagy in WD-fed ApoE −/− mice
Caffeic Acid Reduces Cutaneous Tumor Necrosis Factor Alpha (TNF-α), IL-6 and IL-1β Levels and Ameliorates Skin Edema in Acute and Chronic Model of Cutaneous Inflammation in Mice
Contribution of glutaredoxin-1 to S-glutathionylation of endothelial nitric oxide synthase for mesenteric nitric oxide generation in experimental necrotizing enterocolitis
Evidence That the P<sub>i</sub> Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle
Nitrogenase
reduction of dinitrogen (N<sub>2</sub>) to ammonia
(NH<sub>3</sub>) involves a sequence of events that occur upon the
transient association of the reduced Fe protein containing two ATP
molecules with the MoFe protein that includes electron transfer, ATP
hydrolysis, P<sub>i</sub> release, and dissociation of the oxidized,
ADP-containing Fe protein from the reduced MoFe protein. Numerous
kinetic studies using the nonphysiological electron donor dithionite
have suggested that the rate-limiting step in this reaction cycle
is the dissociation of the Fe protein from the MoFe protein. Here,
we have established the rate constants for each of the key steps in
the catalytic cycle using the physiological reductant flavodoxin protein
in its hydroquinone state. The findings indicate that with this reductant,
the rate-limiting step in the reaction cycle is not protein–protein
dissociation or reduction of the oxidized Fe protein, but rather events
associated with the P<sub>i</sub> release step. Further, it is demonstrated
that (i) Fe protein transfers only one electron to MoFe protein in
each Fe protein cycle coupled with hydrolysis of two ATP molecules,
(ii) the oxidized Fe protein is not reduced when bound to MoFe protein,
and (iii) the Fe protein interacts with flavodoxin using the same
binding interface that is used with the MoFe protein. These findings
allow a revision of the rate-limiting step in the nitrogenase Fe protein
cycle