Selenium protects against Pb-induced renal oxidative injury in weaning rats and human renal tubular epithelial cells through activating NRF2.

BACKGROUND: Lead (Pb) poisoning posing a crucial health risk, especially among children, causing devastating damage not only to brain development, but also to kidney function. Thus, an urgent need persists to identify highly effective, safe, and low-toxicity drugs for the treatment of Pb poisoning. The present study focused on exploring the protective effects of Se on Pb-induced nephrotoxicity in weaning rats and human renal tubular epithelial cells, and investigated the possible mechanisms. METHODS: Forty weaning rats were randomly divided into four groups in vivo: control, Pb-exposed, Pb+Se and Se. Serum creatinine (Cr), urea nitrogen (BUN) and hematoxylin and eosin (H&E) staining were performed to evaluate renal function. The activities of antioxidant enzymes in the kidney tissue were determined. In vitro experiments were performed using human renal tubular epithelial cells (HK-2 cells). The cytotoxicity of Pb and Se was detected by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Inverted fluorescence microscope was used to investigate cell morphological changes and the fluorescence intensity of reactive oxygen species (ROS). The oxidative stress parameters were measured by a multi-detection reader. Nuclear factor-erythroid-2-related factor (NRF2) signaling pathways were measured by Western blot and reverse transcription polymerase chain reaction (RT-PCR) in HK-2 cells. RESULTS: We found that Se alleviated Pb-induced kidney injury by relieving oxidative stress and reducing the inflammatory index. Se significantly increased the activity of the antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), whereas it decreased the excessive release of malondialdehyde (MDA) in the kidneys of weaning rats and HK-2 cells. Additionally, Se enhanced the antioxidant defense systems via activating the NRF2 transcription factor, thereby promoting the to downstream expression of heme oxygenase 1. Furthermore, genes encoding glutamate-cysteine ligase synthetase catalytic (GCLC), glutamate-cysteine ligase synthetase modifier (GCLM) and NADPH quinone oxidoreductase 1 (NQO1), downstream targets of NRF2, formed a positive feedback loop with NRF2 during oxidative stress responses. The MTT assay results revealed a significant decrease in cell viability with Se treatment, and the cytoprotective role of Se was blocked upon knockdown of NRF2 by small interfering RNA (siRNA). MDA activity results also showed that NRF2 knockdown inhibited the NRF2-dependent transcriptional activity of Se. CONCLUSIONS: Our findings demonstrate that Se ameliorated Pb-induced nephrotoxicity by reducing oxidative stress both in vivo and in vitro. The molecular mechanism underlying Se's action in Pb-induced kidney injury is related to the activation of the NRF2 transcription factor and the activity of antioxidant enzymes, ultimately suppressing ROS accumulation.

Complete genetic characterization of carbapenem-resistant Acinetobacter johnsonii, co-producing NDM-1, OXA-58, and PER-1 in a patient source.

The emergence of carbapenemase-producing Acinetobacter spp. has been widely reported and become a global threat. However, carbapenem-resistant A. johnsonii strains are relatively rare and without comprehensive genetic structure analysis, especially for isolates collected from human specimen. Here, one A. johnsonii AYTCM strain, co-producing NDM-1, OXA-58, and PER-1 enzymes, was isolated from sputum in China in 2018. Antimicrobial susceptibility testing showed that it was resistant to meropenem, imipenem, ceftazidime, ciprofloxacin, and cefoperazone/sulbactam. Whole-genome sequencing and bioinformatic analysis revealed that it possessed 11 plasmids. bla OXA-58 and bla PER-1 genes were located in the pAYTCM-1 plasmid. Especially, a complex class 1 integron consisted of a 5' conserved segment (5' CS) and 3' CS, which was found to carry sul1, arr-3, qnrVC6, and bla PER-1 cassettes. Moreover, the bla NDM-1 gene was located in 41,087 conjugative plasmids and was quite stable even after 70 passages under antibiotics-free conditions. In addition, six prophage regions were identified. Tracking of closely related plasmids in the public database showed that pAYTCM-1 was similar to pXBB1-9, pOXA23_010062, pOXA58_010030, and pAcsw19-2 plasmids, which were collected from the strains of sewage in China. Concerning the pAYTCM-3 plasmids, results showed that strains were collected from different sources and their hosts were isolated from various countries, such as China, USA, Japan, Brazil, and Mexico, suggesting that a wide spread occurred all over the world. In conclusion, early surveillance is warranted to avoid the extensive spread of this high-risk clone in the healthcare setting.

Genome sequence of a tigecycline-resistant Acinetobacter seifertii recovered in human bloodstream infection in China.

OBJECTIVES: The phylogenetic characteristics of Acinetobacter seifertii clinical strain are not well-studied. Here, we reported one tigecycline-resistant ST1612Pasteur A. seifertii isolated from bloodstream infections (BSI) in China. METHODS: Antimicrobial susceptibility tests were conducted via broth microdilution. Whole-genome sequencing (WGS) was performed and annotation was conducted using rapid annotations subsystems technology (RAST) server. Multilocus sequence typing (MLST), capsular polysaccharide (KL), and lipoolygosaccharide (OCL) were analysed using PubMLST and Kaptive. Resistance genes, virulence factors, and comparative genomics analysis were performed. Cloning, mutations of efflux pump-related genes, and expression level were further investigated. RESULTS: The draft genome sequence of A. seifertii ASTCM strain is made up of 109 contigs with a total length of 4,074,640 bp. Based on the RAST results, 3923 genes that belonged to 310 subsystems were annotated. Acinetobacter seifertii ASTCM was ST1612Pasteur with KL26 and OCL4, respectively. It was resistant to gentamicin and tigecycline. ASTCM harboured tet(39), sul2, and msr(E)-mph(E), and one amino acid mutation in Tet(39) (T175A) was further identified. Nevertheless, the signal mutation failed to contribute to susceptibility change of tigecycline. Of note, several amino acid substitutions were identified in AdeRS, AdeN, AdeL, and Trm, which could lead to overexpression of adeB, adeG, and adeJ efflux pump genes and further possibly lead to tigecycline resistance. Phylogenetic analysis showed that a huge diversity was observed among A. seifertii strains based on 27-52,193 SNPs difference. CONCLUSION: In summary, we reported a tigecycline-resistant ST1612Pasteur A. seifertii in China. Early detection is recommended to prevent their further spread in clinical settings.

Emergence of IS26-mediated pLVPK-like virulence and NDM-1 conjugative fusion plasmid in hypervirulent carbapenem-resistant Klebsiella pneumoniae.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has been widely reported and poses a global threat. However, the comprehensive genetic structure of ST11-KL64 hv-CRKP and the possible evolutionary mechanisms from a genetic structure perspective of this high-risk clone remain unclear. Here, a blaKPC-2-blaNDM-1-positive ST11-KL64 hv-CRKP isolate was obtained from a human bloodstream infection (BSI). Whole-genome sequencing and bioinformatics analyses revealed that it contained a fusion plasmid, pKPTCM2-1. pKPTCM2-1 is a conjugative plasmid composed of an oriT-positive pLVPK-like virulence plasmid and a type IV secretion system-produced blaNDM-1-bearing IncX3 plasmid mediated by IS26-based co-integration. This progress generated 8-bp target site duplications (TGAAAACC) on both sides. The fusion plasmid possessed self-transferability and could be transferred to blaKPC-2-harboring ST11-KL64 CRKP to form the ST11-KL64 hv-CRKP clone. The pLVPK-like-positive ST11-KL64 strain exhibited virulence levels similar to those of the typical hypervirulent K. pneumoniae NTUH-2044. The mutation, Tet(A) (A276S), which was believed to lead to tigecycline resistance was observed. Overall, this high-risk clone has emerged as a tremendous threat in fatal BSIs and thus, targeted surveillance is an urgent need to contain the hv-CRKP clones.

Whole genome sequencing of OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae in human bloodstream infection co-harboring chromosomal ISEcp1-based bla CTX-M-15 and one rmpA2-associated virulence plasmid.

Objectives: To characterize one OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae (CRKP) co-harboring chromosomal bla CTX-M-15 and one rmpA2-associated virulence plasmid. Methods: Minimum inhibitory concentrations (MICs) were measured via broth microdilution method. Conjugation, chemical transformation, string test and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing (WGS) was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants were identified using ABRicate program with ResFinder database. Insertion sequences (ISs) were identified using ISfinder. Bacterial virulence factors were identified using virulence factor database (VFDB). Wzi, capsular polysaccharide (KL) and lipoolygosaccharide (OCL) were analyzed using Kleborate with Kaptive. Phylogenetic analysis of 109 ST15 K. pneumoniae strains was performed using core genome multilocus sequence typing (cgMLST) on the Ridom SeqSphere+ server. MLST, replicons type, SNP strategies and another cgMLST analysis for 45 OXA-232-producing K. pneumoniae strains were further conducted using BacWGSTdb server. Results: K. pneumoniae KPTCM strain belongs to ST15 with wzi93, KL112 and O1. It possessed a multidrug-resistant (MDR) profile and was resistant to carbapenems (meropenem and ertapenem), ciprofloxacin and amikacin. Virulence assays demonstrated KPTCM strain possesses a low virulence phenotype. WGS revealed it contained one circular chromosome and nine plasmids. The carbapenemase-encoding gene bla OXA-232 was located in a 6141-bp ColKP3-type non-conjugative plasmid and flanked by ΔISEcp1 and ΔlysR-ΔereA. Interestingly, bla CTX-M-15 was located in the chromosome mediated by ISEcp1-based transposon Tn2012. Importantly, it harbored a rmpA2-associated pLVPK-like virulence plasmid with iutA-iucABCD gene cluster and one IS26-mediated MDR fusion plasmid according to 8-bp (AGCTGCAC or GGCCTTTG) target site duplications (TSD). Based on the cgMLST and SNP analysis, data showed OXA-232-producing ST15 K. pneumoniae isolates were mainly isolated from China and have evolved in recent years. Conclusions: Early detection of CRKP strains carrying chromosomal bla CTX-M-15, OXA-232 carbapenemase and pLVPK-like virulence plasmid is recommended to avoid the extensive spread of this high-risk clone.

Eupalinolide O Induces Apoptosis in Human Triple-Negative Breast Cancer Cells via Modulating ROS Generation and Akt/p38 MAPK Signaling Pathway.

Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited therapeutic options. Eupalinolide O (EO) was reported to inhibit tumor growth. This study is aimed at exploring the role of EO on TNBC both in vivo and in vitro. Methods. In in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay were conducted to measure the impact of EO on TNBC cell growth at different concentrations and time points. Flow cytometry was conducted to evaluate cell apoptosis. Mitochondrial membrane potential (MMP) loss, caspase-3 activity, and reactive oxygen species (ROS) generation were assessed. The expressions of apoptosis-related mRNAs and Akt/p38 MAPK signaling pathway-related proteins were measured. In in vivo experiments, by injecting TNBC cells into the nude mice to induce xenograft tumor, mice were treated with EO for 20 days. Then, in vivo bioluminescence imaging system was utilized to monitor the growth and distribution of TNBC cells. Tumor volume and weight were also recorded. Hematoxylin-eosin (HE) staining and ELISA assay were applied to observe tumor tissue morphology and ROS levels. Furthermore, western blotting was conducted to observe the expression of apoptosis-related proteins and Akt/p38 MAPK signaling pathway-associated proteins. Results: EO inhibited the cell viability and proliferation of TNBC cells but not normal epithelial cells. Furthermore, EO induced apoptosis, decreased MMP, and elevated caspase-3 activity and ROS content in TNBC cells. Meanwhile, the expression of apoptosis-related mRNAs and Akt/p38 MAPK pathway-related proteins was regulated by EO treatment. Besides, in vivo experiments demonstrated EO not only suppressed tumor growth, Ki67 expression, ROS generation, and Akt phosphorylation but also upregulated caspase-3 expression and p-38 phosphorylation. Conclusion: EO may induce cell apoptosis in TNBC via regulating ROS generation and Akt/p38 MAPK pathway, indicating EO may be a candidate drug for TNBC.

Emergence of uncommon KL38-OCL6-ST220 carbapenem-resistant Acinetobacter pittii strain, co-producing chromosomal NDM-1 and OXA-820 carbapenemases.

Objective: To characterize one KL38-OCL6-ST220 carbapenem-resistant Acinetobacter pittii strain, co-producing chromosomal NDM-1 and OXA-820 carbapenemases. Methods: A. pittii TCM strain was isolated from a bloodstream infection (BSI). Antimicrobial susceptibility tests were conducted via disc diffusion and broth microdilution. Stability experiments of bla NDM-1 and bla OXA-820 carbapenemase genes were further performed. Whole-genome sequencing (WGS) was performed on the Illumina and Oxford Nanopore platforms. Multilocus sequence typing (MLST) was analyzed based on the Pasteur and Oxford schemes. Resistance genes, virulence factors, and insertion sequences (ISs) were identified with ABRicate based on ResFinder 4.0, virulence factor database (VFDB), and ISfinder. Capsular polysaccharide (KL), lipooligosaccharide outer core (OCL), and plasmid reconstruction were tested using Kaptive and PLACNETw. PHASTER was used to predict prophage regions. A comparative genomics analysis of all ST220 A. pittii strains from the public database was carried out. Point mutations, average nucleotide identity (ANI), DNA-DNA hybridization (DDH) distances, and pan-genome analysis were performed. Results: A. pittii TCM was ST220Pas and ST1818Oxf with KL38 and OCL6, respectively. It was resistant to imipenem, meropenem, and ciprofloxacin but still susceptible to amikacin, colistin, and tigecycline. WGS revealed that A. pittii TCM contained one circular chromosome and four plasmids. The Tn125 composite transposon, including bla NDM-1, was located in the chromosome with 3-bp target site duplications (TSDs). Many virulence factors and the bla OXA-820 carbapenemase gene were also identified. The stability assays revealed that bla NDM-1 and bla OXA-820 were stabilized by passage in an antibiotic-free medium. Moreover, 12 prophage regions were identified in the chromosome. Phylogenetic analysis showed that there are 11 ST220 A. pittii strains, and one collected from Anhui, China was closely related. All ST220 A. pittii strains presented high ANI and DDH values; they ranged from 99.85% to 100% for ANI and from 97.4% to 99.9% for DDH. Pan-genome analysis revealed 3,200 core genes, 0 soft core genes, 1,571 shell genes, and 933 cloud genes among the 11 ST220 A. pittii strains. Conclusions: The coexistence of chromosomal NDM-1 and OXA-820 carbapenemases in A. pittii presents a huge challenge in healthcare settings. Increased surveillance of this species in hospital and community settings is urgently needed.

Antioxidant and Anti-Inflammatory Activities of Six Flavonoids from Smilax glabra Roxb.

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, 1H NMR and 13C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (-)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1ß, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (-)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS+ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1ß, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra.

Protective Effects of Smilax glabra Roxb. Against Lead-Induced Renal Oxidative Stress, Inflammation and Apoptosis in Weaning Rats and HEK-293 Cells.

Lead (Pb) is an important environmental pollutant. Oxidative stress and the inflammatory response have been postulated as mechanisms involved in lead-induced renal damage. Smilax glabra Roxb. has been used for treatment of heavy-metal poisoning in China for 500 years. We investigated S. glabra flavonoids extract (SGF) could attenuate lead acetate-induced nephrotoxicity in weaning rats and human embryonic kidney (HEK)-293 cells, and investigated the possible mechanisms. Compared with Pb exposed group of weaning rats, SGF could significantly promote lead excretion in the blood and kidney, and increase the content of the renal-function indicators blood urea nitrogen, serum uric acid, and serum creatinine. SGF could improve the glomerular filtration rate (GFR) and histologic changes in the kidneys of weaning rats exposed to Pb. SGF could also reduce lead-induced cytotoxicity, improve DNA damage-induced apoptosis and cleaved caspase-3-mediated apoptosis in HEK-293 cells stimulated with Pb. SGF significantly increased the activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase, and decreased excessive release of reactive oxygen species (ROS) and malondialdehyde in the kidneys of the weaning rats and in HEK-293 cells. The antioxidant mechanism of SGF related to activation of the Kelch-like ECH-associated protein 1/nuclear-factor-E2-related factor 2/hemeoxygenase-1(Keap1/Nrf2/HO-1) pathway. SGF could inhibit secretion of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α induced by Pb in vivo and in vitro. The anti-inflammatory mechanism of SGF related to inhibition of ROS and pro-inflammatory cytokines triggered the nuclear factor-kappa B (NF-κB) pathway through blockade of inhibitors of I-κB degradation, phosphorylation of NF-κB p65, and nuclear translocation of p65. Our findings indicate that SGF could be a natural antioxidant and anti-inflammatory agent for treating lead-induced nephrotoxicity.

[Effect of ethanolic extract of Polygonum cuspidatum on acute gouty arthritis in mice through NLRP3/ASC/caspase-1 axis].

The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1ß, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1ß, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1ß, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.