Hydrogen sulfide antagonizes formaldehyde-induced ferroptosis via preventing ferritinophagy by upregulation of GDF11 in HT22 cells.

Formaldehyde (FA) has neurotoxic characteristics and causes neurodegenerative disease. Our previous study demonstrated the neuroprotective effects of hydrogen sulfide (H2S) on FA-induced neurotoxicity in HT22 cells. Emerging evidence have supported that ferroptosis is involved in FA-induced neurotoxicity. To understand the mechanism of the protection of H2S against FA-induced neurotoxicity, this study explored the regulatory effect of H2S on FA-induced ferroptosis and the underlying mechanisms. The researcher found that H2S (100, 200, and 400 µM, 30 min) reverses the ferroptosis induced by FA (100 µM, 24 h) in HT22 cells (a cell line of mouse hippocampal neurons), including decreases in free iron, reactive oxygen species (ROS), 4-hydroxy-2-trans-nominal (4-HNE), and malondialdehyde (MDA) contents, as well as an increase in glutathione (GSH) content. H2S (100, 200, and 400 µM, 30 min) also inhibited ferritinaphagy in FA-exposed HT22 cells, as evidenced by the downregulation of the ferritinophagy receptor nuclear receptor coactivator 4 (NCOA4) and microtubule-associated protein 1 light chain-3B (LC3B) as well as the upregulation of the main iron storage protein ferritin heavy chain 1 (FTH1) and p62. H2S (100, 200, and 400 µM, 30 min) also up-regulated the expression of growth differentiation factor-11 (GDF11) in FA-exposed HT22 cells. Furthermore, knockdown of GDF11 in HT22 cells cancelled the beneficial effects of H2S in FA-induced ferroptosis and ferritinaphagy. These data indicated that the protective mechanism underlying H2S-prevented neurotoxicity of FA is involved in alleviating FA-induced ferroptosis via inhibiting ferritinaphagy by upregulation of GDF11.

Pathogen distribution and bacterial resistance in children with severe pneumonia: A single-center retrospective study.

ABSTRACT: To examine the etiological distribution of pathogens in pediatric patients with severe pneumonia and analyze the drug resistance of major pathogen species.Nasopharyngeal secretion specimens were collected for bacterial culture from pediatric patients admitted to the Xiamen children's hospital who were diagnosed with severe pneumonia from January 2016 to December 2019. Pathogen species were detected by quantitative polymerase chain reaction, direct immunofluorescence, and bacterial culture and we examined the drug susceptibility of the bacterial pathogens.At least 1 species of the pathogen was detected in 576 of 734 patients and a total of 444 bacterial samples were isolated, of which 284 were gram-negative and 160 were gram-positive. The most frequently detected bacteria were Haemophilus influenzae, Streptococcus pneumonia, Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli. In addition, we isolated 186 viral samples, of which the majority were respiratory syncytial virus (n = 90) and adenovirus (n = 70) as well as 142 Mycoplasma pneumonia samples.Gram-negative bacteria are dominant among the pathogens causing severe pneumonia in pediatric patients and the major pathogen species are resistant to a variety of antibiotics. Appropriate antibiotic use has an important role in preventing the emergence of resistant strains.

Hydrogen Sulfide Inhibits High Glucose-Induced Neuronal Senescence by Improving Autophagic Flux via Up-regulation of SIRT1.

Hyperglycemia, a key characteristic and risk factor for diabetes mellitus (DM), causes neuronal senescence. Hydrogen sulfide (H2S) is a novel neuroprotectant. The present work was to investigate the potential effect of H2S on hyperglycemia-induced neuronal senescence and the underlying mechanisms. We found that NaHS, a donor of H2S, inhibited high glucose (HG)-induced cellular senescence in HT22 cells (an immortalized mouse hippocampal cell line), as evidenced by a decrease in the number of senescence associated-ß-galactosidase (SA-ß-gal) positive cells, increase in the growth of cells, and down-regulations of senescence mark proteins, p16INK4a and p21CIP1. NaHS improved the autophagic flux, which is judged by a decrease in the amount of intracellular autophagosome as well as up-regulations of LC3II/I and P62 in HG-exposed HT22 cells. Furthermore, blocked autophagic flux by chloroquine (CQ) significantly abolished NaHS-exerted improvement in the autophagic flux and suppression in the cellular senescence of GH-exposed HT22 cells, which indicated that H2S antagonizes HG-induced neuronal senescence by promoting autophagic flux. We also found that NaHS up-regulated the expression of silent mating type information regulation 2 homolog 1 (SIRT1), an important anti-aging protein, in HG-exposed HT22 cells. Furthermore, inhibition of SIRT1 by sirtinol reversed the protection of H2S against HG-induced autophagic flux blockade and cellular senescence in HT22 cells. These data indicated that H2S protects HT22 cells against HG-induced neuronal senescence by improving autophagic flux via up-regulation of SIRT1, suggesting H2S as a potential treatment strategy for hyperglycemia-induced neuronal senescence and neurotoxicity.

Genistein Protects H9c2 Cardiomyocytes against Chemical Hypoxia-Induced Injury via Inhibition of Apoptosis.

BACKGROUND/AIMS: Hypoxia can induce cell injury in cardiomyocytes and further lead to cardiovascular diseases. Genistein (Gen), the predominant isoflavone found in soy products, has shown protective effects on cardiovascular system. The aim of the present study was to investigate the cardioprotective effect of Gen against chemical hypoxia-induced injury. METHODS: Cobalt chloride (CoCl2) was administrated to trigger chemical hypoxia in H9c2 cardiomyocytes. Cell proliferation was detected by using MTT assay. The expression level of hypoxia-related proteins (hypoxia-inducible factor [HIF]-1α and Notch-1) and apoptosis-related proteins (B cell lymphoma [Bcl]-2, Bax, and caspase-3) were evaluated by Western blot analysis. RESULTS: In response to hypoxia, cell viability was reduced dramatically, whereas the expression of HIF-1α was upregulated. Hypoxia also induced cardiomyocytes apoptosis by reducing the ratio of Bcl-2/Bax and increasing expression of caspase-3. Interestingly, Gen attenuated CoCl2-induced cell death and suppressed HIF-1α expression, as well as upregulated the expression of Notch-1. Furthermore, Gen could antagonize CoCl2-induced apoptosis through upregulating Bcl-2/Bax ratio and inhibiting caspase-3 expression. CONCLUSIONS: Gen prevents chemical hypoxia-induced cell apoptosis through inhibition of the mitochondrial apoptotic pathway, exerting protective effects on H9c2 cardiomyocytes.

BDNF/TrkB Pathway Mediates the Antidepressant-Like Role of H2S in CUMS-Exposed Rats by Inhibition of Hippocampal ER Stress.

Our previous works have shown that hydrogen sulfide (H2S) significantly attenuates chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and hippocampal endoplasmic reticulum (ER) stress. Brain-derived neurotrophic factor (BDNF) generates an antidepressant-like effect by its receptor tyrosine protein kinase B (TrkB). We have previously found that H2S upregulates the expressions of BDNF and p-TrkB in the hippocampus of CUMS-exposed rats. Therefore, the present work was to explore whether BDNF/TrkB pathway mediates the antidepressant-like role of H2S by blocking hippocampal ER stress. We found that treatment with K252a (an inhibitor of BDNF/TrkB pathway) significantly increased the immobility time in the forced swim test and tail suspension test and increased the latency to feed in the novelty-suppressed feeding test in the rats cotreated with sodium hydrosulfide (NaHS, a donor of H2S) and CUMS. Similarly, K252a reversed the protective effect of NaHS against CUMS-induced hippocampal ER stress, as evidenced by increases in the levels of ER stress-related proteins, glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12. Taken together, our results suggest that BDNF/TrkB pathway plays an important mediatory role in the antidepressant-like action of H2S in CUMS-exposed rats, which is by suppression of hippocampal ER stress. These data provide a novel mechanism underlying the protection of H2S against CUMS-induced depressive-like behaviors.

Fabrication and characterization of a novel self-assembling micelle based on chitosan cross-linked pectin-doxorubicin conjugates macromolecular pro-drug for targeted cancer therapy.

Cancer is one of the leading causes of morbidity and mortality worldwide. Doxorubicin is one of the most effective anticancer drugs approved by FDA. However, like all the other anticancer drugs, the efficacy of DOX is associated with high systemic toxicity to healthy tissues. In this study, chitosan cross-linked pectin-doxorubicin conjugates macromolecular pro-drug (CS-PDC-M) was prepared to enhance the therapeutic effects on liver cancer. CS-PDC-M was characterized in terms of size, size distribution, zeta potential, scanning electron microscope (SEM) and drug loading content. The CS-PDC-M achieved prolonged releasing ability was demonstrated by the in vitro drug release and in vitro cellular uptake assay. Biocompatibility of CS-PDC-M was screened by hemolysis activity examination, BSA adsorption test and cell viability evaluation in endothelial cells and LO2 cells. The CS-PDC-M achieved significantly high antitumor efficiency and targeting efficiency, which was demonstrated by the in vitro MTT assay and cellular targeting assay toward HepG2 cells, MCF-7 cells and A549 cells. The in vivo antitumor efficacy of CS-PDC-M was studied in athymic BALB/c nude mice bearing HepG2 cell xenografts. The organ damage assays of CS-PDC-M was studied in SD rats. Compared with that of free DOX and PDC-M, the CS-PDC-M exhibited higher antitumor efficacy and lower toxicity, implying that CS-PDC-M is a highly promising drug delivery system for hepatocellular carcinoma treatment.

Inhibition of ALDH2 protects PC12 cells against formaldehyde-induced cytotoxicity: involving the protection of hydrogen sulphide.

Formaldehyde (FA), a common environmental contaminant, has toxic effects on the central nervous system (CNS). We have previously found that hydrogen sulphide (H2 S), the third endogenous gaseous mediator, protects neuron against the toxicity of FA. However, the underlying mechanism is poor. Aldehyde-dehydrogenase-2 (ALDH2) plays a major role in detoxification of reactive aldehyde in a range of organs and cell types. Therefore, we speculated that H2 S antagonizes FA-induced neurotoxicity by modulating ALDH2. In the present study, we found that the exposure of PC12 cells to FA causes increase in ALDH2 expression and activity. Daidzin, an inhibitor of ALDH2, significantly antagonizes FA-exerted cytotoxicity and oxidative stress including the accumulation of intracellular reactive oxygen species (ROS), 4-hydroxy-2-trans-nonenal (4-HNE), and malondialdehyde (MDA), in PC12 cells. We also showed that daidzin markedly attenuated FA-induced apoptosis in PC12 cells. Furthermore, we found that H2 S reverses FA-elicited upregulation of ALDH2 in PC12 cells. Our results demonstrated the involvement of downregulation of ALDH2 in the protection of H2 S against FA neurotoxicity.

Adsorption kinetics, isotherms and thermodynamics of atrazine on surface oxidized multiwalled carbon nanotubes.

The adsorption kinetics, isotherms and thermodynamic of atrazine on multiwalled carbon nanotubes (MWCNTs) containing 0.85%, 2.16%, and 7.07% oxygen was studied. Kinetic analyses were performed using pseudo-first-order, pseudo-second-order and intraparticle diffusion models. The regression results showed that the pseudo-second-order law fit the adsorption kinetics. The calculated thermodynamic parameters indicated that adsorption of atrazine on MWCNTs was spontaneous and exothermic. Standard free energy (DeltaG(0)) became less negative when the oxygen content of MWCNTs increased from 0.85% to 7.07% which is consistent with the low adsorption affinity of MWCNTs for atrazine.

Heat shock protein 90 acts as a molecular chaperone in late-phase activation of extracellular signal-regulated kinase 1/2 stimulated by oxidative stress in vascular smooth muscle cells.

AIM: To investigate whether cytosolic heat shock protein 90 (HSP90) acts as a molecular chaperone on the activated extracellular signal-regulated kinase 1/2 (ERK1/2) and cell proliferation stimulated by reactive oxygen species (ROS) in rat vascular smooth muscle cells (VSMC). METHODS: VSMC were exposed to 1 micromol/L LY83583 (6-anilinoquinoline-5,8-quinolinedione, producer of ROS) for 120 min in the presence or absence of 5 micromol/L geldanamycin, a specific inhibitor of HSP90. Then the total, soluble, and insoluble proteins of the cells were collected. HSP90, ERK1/2, and phosphor-ERK1/2 in the cell lysate were measured by Western blotting. The interaction of HSP90 and phosphor-ERK1/2 was analyzed by immunoprecipitation assay, and the nuclear phosphor-ERK1/2 was measured by Western blotting and immunofluorescence. Cell proliferation was tested by cell counting and 3-(4,5-dimethylthiazol-2-y1)-3,5-di-phenyltetrazolium bromide (MTT). RESULTS: The cytosolic HSP90 of VSMC was upregulated by LY83583 in a time-dependent manner with the peak at 120 min, which is consistent with the late peak of phosphor-ERK1/2. Immunoprecipitation and Western blotting analyses showed that LY83583 increased the interaction of HSP90 with phosphor-ERK1/2, the phosphor-ERK1/2 level, and the soluble phosphor-ERK1/2 level by 1.8-, 2.5-, and 2.9-fold, respectively. In contrast, the insoluble phosphor-ERK1/2 of VSMC was decreased. Interestingly, LY83583 treatment promoted the nuclear phosphor-ERK1/2 by 7.6-fold as confirmed by Western blotting and immunofluorescence assays. Furthermore, cell counting and the MTT assay showed that LY83583 stimulated VSMC proliferation with the increased expression of HSP90 and levels of soluble and nuclear phosphor-ERK1/2. Pretreatment of geldanamycin antagonized the effect of LY83583. CONCLUSION: HSP90 could mediate the oxidative stress-stimulated, late-phase activation of ERK1/2 and VSMC proliferation by promoting the ERK1/2 phosphorylation, the association of itself with phosphor-ERK1/2, and the solubility and nuclear translocation of phosphor-ERK1/2.

Phytoestrogen genistein supplementation increases eNOS and decreases caveolin-1 expression in ovariectomized rat hearts.

This study examined whether genistein influences the production of nitric oxide (NO) and expression of endothelial nitric oxide synthase (eNOS) and the modulators of eNOS activity in ovariectomized (OVX) rat hearts. Female mature Sprague-Dawley rats were subjected to bilateral ovariectomy, OVX rats were randomly divided into four groups: 17beta-estradiol (0.1 mg/kg, s.c. daily) was used as the positive control; low dose of genistein (0.5 mg/kg, s.c. daily); high dose of genistein (5.0 mg/kg, s.c. daily) and model. Sham operations as controls, the treatment lasted 6 weeks. Blood pressure, heart rate, plasma estradiol, heart and uterine weights were measured. Nitrite production in the myocardium was determined by nitrate reductase method. Protein level of eNOS, caveolin-1 and calmodulin was determined by Western blot. The results showed that nitrite production and eNOS protein in homogenized ventricular tissue was attenuated by approximately 53% and 67% in OVX rats compared with those in sham rats, respectively. Genistein increased nitrite production in rat heart in a dose-dependent manner, genistein at the dose of 5 mg/kg.d(-1) resumed nitrite production to a level similar to that in sham operated rats. Administration of genistein also increased eNOS protein expression in OVX rats myocardium with a concomitant decrease in the expression of caveolin-1, an endogenous eNOS inhibitory protein. Another eNOS stimulatory protein, calmodulin, was unchanged in these treatments. These effects were also observed in rats treated with 17beta-estradiol. Genistein at the dose of 5.0 mg/kg.d(-1) augmented uterine weight but this side effect in reproductive system was less than that of 17beta-estradiol. These results suggest that genistein supplementation and estrogen replacement therapy directly increase eNOS functional activity and NO production in the hearts of the OVX rats, but genistein has less side effects on the reproductive system than 17beta-estradiol.

Identification of peptides inhibiting adhesion of monocytes to the injured vascular endothelial cells through phage-displaying screening.

Using oxidized low-density lipoprotein (LDL)-injured vascular endothelial cells (ECs) as target cells, peptides specifically binding to the injured ECs were screened from a phage-displaying peptide library by using the whole-cell screening technique after three cycles of the adsorption-elution-amplification procedure. Positive phage clones were identified by ELISA, and the inserted amino acid sequences in the displaying peptides were deduced from confirmation with DNA sequencing. The adhesion rate of ECs to monocytes was evaluated by cell counting. The activity of endothelial nitric oxide synthase (eNOS), and the expression levels of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1) were determined by Western blotting. Six positive clones specifically binding to injured ECV304 endothelial cells were selected from fourteen clones. Interestingly, four phages had peptides with tandem leucine, and two of these even shared an identical sequence. Functional analysis demonstrated that the YCPRYVRRKLENELLVL peptide shared by two clones inhibited the expression of ICAM-1, increased nitric oxide concentration in the culture media, and upregulated the expression of caveolin-1 and eNOS. As a result, the adhesion rate of monocytes to ECV304 cells was significantly reduced by 12.1%. These data suggest that the anti-adhesion effect of these novel peptides is related to the regulation of the caveolin-1/nitric oxide signal transduction pathway, and could be of use in potential therapeutic agents against certain cardiovascular diseases initiated by vascular endothelial cell damage.

Onychin inhibits proliferation of vascular smooth muscle cells by regulating cell cycle.

AIM: To investigate the effects of onychin on the proliferation of cultured rat artery vascular smooth muscle cells (VSMCs) in the presence of 10% new-born calf serum (NCS). METHODS: Rat VSMCs were incubated with onychin 150 micromol/L or genistein 10 micromol/L in the presence of 10% NCS for 24 h. The proliferation of VSMCs was measured by cell counting and MTS/PMS colorimetric assays. Cell cycle progression was evaluated by flow cytometry. Retinoblastoma (Rb) phosphorylation, and expression of cyclin D1 and cyclin E were measured by Western blot assays. The tyrosine phosphorylation of ERK1/2 was examined by immunoprecipitation techniques using anti-phospho-tyrosine antibodies. RESULTS: The proliferation of VSMCs was accelerated significantly in the presence of 10% NCS. Onychin reduced the metabolic rate of MTS and the cell number of VSMCs in the presence of 10% NCS in a dose-dependent manner. Flow cytometry analysis revealed that the G1-phase fraction ratio in the onychin group was higher than that in the 10% NCS group (85.2% vs 70.0%, P<0.01), while the S-phase fraction ratio in the onychin group was lower than that in 10% NCS group (4.3% vs 16.4%, P<0.01). Western blot analysis showed that onychin inhibited Rb phosphorylation and reduced the expression of cyclin D1 and cyclin E. The effects of onychin on proliferation, the cell cycle and the expression of cyclins in VSMCs were similar to those of genistein, an inhibitor of tyrosine kinase. Furthermore immunoprecipitation studies showed that both onychin and genistein markedly inhibited the tyrosine phosphorylation of ERK1/2 induced by 10% NCS. CONCLUSION: Onychin inhibits the proliferation of VSMCs through G1 phase cell cycle arrest by decreasing the tyrosine phosphorylation of ERK1/2, and the expression of cyclin D1 and cyclin E, and sequentially inhibiting Rb phosphorylation.

Probucol inhibits oxidized-low density lipoprotein-induced adhesion of monocytes to endothelial cells in vitro.

AIM: To investigate the mechanism by which probucol (PBC) affected adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). METHODS: Effects of PBC on expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, a nd E-selectin in human umbilical vein endothelial cells were examined. Moreover, the inhibitory effect of PBC were compared with that of monoclonal antibodies (mAbs) to ICAM-1, VCAM-1, P-selectin, and E-select in on adhesion induced by oxidized-low density lipoprotein(Ox-LDL). RESULTS: PBC at 10 to 80 micromol/L inhibited Ox-LD L-induced adhesion index from 16.7 % to 7.0 % (P < 0.01) and Ox-LDL-induced expression of ICAM-1 (75 %) and P-selectin (72 %). mAbs to ICAM -1 or P-selectin, when used alone, could only slightly reduce the adhesion of monocyte to HUVEC. When both monoclonal antibodies were used in combination, the adhesion was markedly inhibited from 16.7 % to 11.3 % (P < 0.01), but the effect was still weaker than that of PBC (average 9.3 %). CONCLUSION: PBC exerts its inhibitory effect on the adhesion of monocyte to HUVEC by inhibiting the expression of ICAM-1 and P-selectin.