Nrf2-dependent hepatoprotective effect of ellagic acid in titanium dioxide nanoparticles-induced liver injury.

BACKGROUND AND AIMS: Previous studies suggest that titanium dioxide nanoparticles (TiO2 NPs) induce liver injury, possibly due to oxidative stress and inflammation. Ellagic acid (EA) is a dietary polyphenol extracted from natural sources and possesses antioxidant and anti-inflammatory properties. Nonetheless, the efficacy of EA in mitigating liver injury induced by TiO2 NPs remains to be elucidated. METHODS: Primary hepatocytes and L02 cells were cultured with 45 µM EA and 10 µg/ml TiO2 NPs. Mice were orally administered TiO2 NPs (150 mg kg-1) and EA (25/50/100 mg kg-1) for eight weeks. sulforaphane (SFN) as a positive control to evaluate the inhibitory effect of EA on TiO2 NP-induced liver injury (SFN 10 mg kg-1). RNA sequencing (RNA-seq) was employed to elucidate the mechanisms underlying oxidative stress, inflammation, and liver fibrosis. RESULTS: We assessed the impact of EA on cytotoxicity, oxidative stress, inflammation, and fibrosis in both cells and mice exposed to TiO2 NPs for an extended period. Our findings indicated that EA had a protective effect on TiO2 NP-exposed hepatocytes, reducing cytotoxicity, oxidative stress, and inflammation. Furthermore, EA treatment markedly reduced serum aminotransferase levels in mice exposed to TiO2 NPs. Furthermore, EA treatment notably reduced hepatic stress response, inflammation, and fibrosis in mice. The treatment of EA demonstrates non-inferiority compared to SFN. The protective effects of EA were attributed to the upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2), EA promoted the translocation and phosphorylation of Nrf2, as indicated by the finding that Nfe2l2 shRNA and inhibition of Nrf2 by ML385 reversed the EA-induced hepatoprotective effects in TiO2 NP-exposed hepatocytes and mice. CONCLUSION: EA significantly mitigated liver injury induced by TiO2 NPs. Importantly, we identified that the nuclear translocation and phosphorylation of Nrf2 are the primary mechanisms through which EA alleviates liver injury resulting from exposure to TiO2 NPs. As a natural activator of Nrf2, EA emerges as a promising therapeutic candidate for treating TiO2 NPs-induced liver injury, further enhancing our understanding of its potential as a hepatoprotective agent and its underlying molecular mechanisms.

Emergence of a clinical Klebsiella pneumoniae harboring an acrAB-tolC in chromosome and carrying the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 in a plasmid.

Objective: The emergence of clinical Klebsiella pneumoniae strains harboring acrAB-tolC genes in the chromosome, along with the presence of two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 genes on a plasmid, has presented a significant clinical challenge. Methods: In order to study the detailed genetic features of K. pneumoniae strain SC35, both the bacterial chromosome and plasmids were sequenced using Illumina and nanopore platforms. Furthermore, bioinformatics methods were employed to analyze the mobile genetic elements associated with antibiotic resistance genes. Results: K. pneumoniae strain SC35 was found to possess a class A beta-lactamase and demonstrated resistance to all tested antibiotics. This resistance was attributed to the presence of efflux pump genes, specifically acrAB-tolC, on the SC35 chromosome. Additionally, the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65, as well as bla TEM-1 with rmtB, which shared overlapping structures with mobile genetic elements as In413, Tn3, and TnAs3. Through plasmid transfer assays, it was determined that the SC35 plasmid p1 could be successfully transferred with an average conjugation frequency of 6.85 × 10-4. Conclusion: The structure of the SC35 plasmid p1 appears to have evolved in correlation with other plasmids such as pKPC2_130119, pDD01754-2, and F4_plasmid pA. The infectious strain SC35 exhibits no susceptibility to tested antibioticst, thus effective measures should be taken to prevent the spread and epidemic of this strain.

Vitamin D Receptor Activation Reduces Hepatic Inflammation via Enhancing Macrophage Autophagy in Cholestatic Mice.

Macrophage autophagy dysfunction aggravates liver injury by activating inflammasomes, which can cleave pro-IL-1ß to its active, secreted form. We investigated whether the vitamin D/vitamin D receptor (VDR) axis could up-regulate macrophage autophagy function to inhibit the activation of inflammasome-dependent IL-1ß during cholestasis. Paricalcitol (PAL; VDR agonist) was intraperitoneally injected into bile duct-ligated mice for 5 days. Up-regulation of VDR expression by PAL reduced liver injury by reducing the oxidative stress-induced inflammatory reaction in macrophages. Moreover, PAL inhibited inflammasome-dependent IL-1ß generation. Mechanistically, the knockdown of VDR increased IL-1ß generation, whereas VDR overexpression exerted the opposite effect following tert-butyl hydroperoxide treatment. The inflammasome antagonist glyburide, the caspase-1-specific inhibitor YVAD, and the reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) blocked the increase in Vdr shRNA-induced IL-1ß production. Interestingly, up-regulation of VDR also enhanced macrophage autophagy. Autophagy reduction impaired the up-regulation of VDR-inhibited macrophage inflammasome-generated IL-1ß, whereas autophagy induction showed a synergistic effect with VDR overexpression through ROS-p38 mitogen-activated protein kinase (MAPK) pathway. This result was confirmed by p38 MAPK inhibitor, MAPK activator, and ROS inhibitor NAC. Collectively, PAL triggered macrophage autophagy by suppressing activation of the ROS-p38 MAPK pathway, which, in turn, suppressed inflammasome-generated cleaved, active forms of IL-1ß, eventually leading to reduced inflammation. Thus, triggering the VDR may be a potential target for the anti-inflammatory treatment of cholestatic liver disease.

Occurrence and characterization of plasmids carrying tmexCD1-toprJ1, blaDHA-1, and blaCTX-M-127, in clinical Klebsiella pneumoniae strains.

Objective: Today, the emergence of Klebsiella pneumoniae with the tmexCD1-toprJ1 gene cassette in patients has presented a significant clinical challenge. Methods: To present the detailed genetic features of the tmexCD1-toprJ1 gene cassette of K. pneumoniae strain F4_plasmid pA, the whole bacterial genome was sequenced by Illumina and nanopore platforms, and mobile genetic elements related to antibiotic resistance genes were analyzed with a series of bioinformatics methods. Results: K. pneumoniae strain F4 was determined to be a class A+C beta-lactamase, and was resistant to routinely used antibiotics, especially tigecycline, because of the oqxAB gene localized on the F4_chromosome and tmexCD1-toprJ1 on F4_plasmid A. After plasmid transfer assays, the F4_plasmid pA or F4_plasmid pB could be recovered with an average conjugation frequencies of 3.42×10-4 or 4.19×10-4. F4_plasmid pA carried tmexCD1-toprJ1 and bla DHA-1 accompanied by genetic intermixing of TnAs1, Tn5393, TnAs3, and In641, while F4_plasmid pB, bearing bla CTX-M-174, had structural overlap of TnAs3 and In641. Conclusions: We suggested that plasmids carrying tmexCD1- toprJ1 might be strongly related to IS26-integrated loop intermediates. This study showed that due to the structural evolution of F4 and related strains, their resistances were so strong that effective antibiotics were virtually unavailable, therefore their spread and prevalence should be strictly controlled.

Characterization of the two tandem repeats for the KPC-2 core structures on a plasmid from hospital-derived Klebsiella pneumoniae.

Today, Klebsiella pneumoniae strains are sophisticatedly associated with the transmission of KPC, and ST11 clones carrying KPC-2 are an important target for anti-infective clinical therapy, posing a very high threat to patients. To present the detailed genetic features of two KPC-2 core structures of F94_plasmid pA, the whole genome of K. pneumoniae strain F94 was sequenced by nanopore and illumina platform, and mobile genetic elements associated with antibiotic-resistance genes were analyzed with a series of bioinformatics methods. K. pneumoniae strain F94, identified as a class A carbapenemase-resistant Enterobacteriaceae, was resistant to most tested antibiotics, especially to low-levels of ceftazidime/avibactam (avibactam ≤ 4 mg/L), owing to overexpression of the two KPC-2 in F94_plasmid pA. However, strain F94 was sensitive to high-levels of ceftazidime/avibactam (avibactam ≥ 8 mg/L), which correlated with further inhibition of ceftazidime hydrolysis by the KPC-2 enzyme due to the multiplication of avibactam. Collinearity analysis indicated that multi-drug resistance (MDR) regions of plasmids with the tandam repeats of two or more KPC-2 core structures share highly similar structures. This study characterized the MDR region of the F94_ plasmid pA as homologous to plasmids pKPC2_090050, pKPC2_090374, plasmid unnamed 2, pC2414-2-KPC, pKPC2-020037, pBS1014-KPC2, pKPC-J5501, and pKPC2-020002, which contained the tandem repeats of one, two, or more KPC-2 core structures, providing insight into the evolution of multidrug resistance in K. pneumoniae. An alternative theoretical basis for exploring the tandem repeats of two or more KPC-2 core structures was developed by analyzing and constructing the homologous sequence of F94_ plasmid pA.

Vitamin D Receptor Activation Targets ROS-Mediated Crosstalk Between Autophagy and Apoptosis in Hepatocytes in Cholestasic Mice.

BACKGROUND & AIMS: Observational epidemiologic studies have associated vitamin D deficiency with cholestasis. We reported previously that activation of the vitamin D/vitamin D receptor (VDR) axis in cholangiocytes mitigates cholestatic liver injury by remodeling the damaged bile duct. However, the function of VDR in hepatocytes during cholestasis remains unclear. METHODS: Paricalcitol (VDR agonist, 200 ng/kg) was injected intraperitoneally into bile duct-ligated mice every other day for 5 days. Primary hepatocytes and HepG2 hepatoma cells were transfected with Vdr short hairpin RNA, control short hairpin RNA, Vdr plasmid, control vector, Atg5 small interfering RNA (siRNA), and control siRNA. Liver histology, cell proliferation, and autophagy were evaluated. RESULTS: Treatment with the VDR agonist paricalcitol improved liver injury in bile duct-ligated mice by up-regulating VDR expression in hepatocytes, which in turn reduced hepatocyte apoptosis by inhibiting reactive oxygen species (ROS) generation via suppressing the Ras-related C3 botulinum toxin substrate 1/reduced nicotinamide adenine dinucleotide phosphate oxidase 1 pathway. Mechanistically, upon exposure to an ROS-inducing compound, Vdr siRNA contributed to apoptosis, whereas the Vdr overexpression caused resistance to apoptosis. Interestingly, up-regulated VDR expression also increased the generation of autophagosomes and macroautophagic/autophagic flux, which was the underlying mechanism for reduced apoptosis following VDR activation. Autophagy depletion impaired the positive effects of VDR overexpression, whereas autophagy induction was synergystic with VDR overexpression. Importantly, up-regulation of VDR promoted autophagy activation by suppressing the activation of the extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (p38MAPK) pathway. Thus, a p38MAPK inhibitor abrogated the Vdr siRNA-induced decrease in autophagy and the Vdr siRNA-induced increase in apoptosis. In contrast, a Mitogen-activated protein kinase kinase (MEK)/ERK activator prevented the enhancement of autophagy and decreased apoptosis following Vdr overexpression. Moreover, the ROS inhibitor N-acetylcystein (NAC) blocked Vdr siRNA-enhanced activation of the ERK/p38MAPK pathway. CONCLUSIONS: VDR activation mitigated liver cholestatic injury by reducing autophagy-dependent hepatocyte apoptosis and suppressing the activation of the ROS-dependent ERK/p38MAPK pathway. Thus, VDR activation may be a potential target for the treatment of cholestatic liver disease.

Characterization of Klebsiella pneumoniae carrying the blaNDM-1 gene in IncX3 plasmids and the rare In1765 in an IncFIB-IncHI1B plasmid.

Background: Today, the blaNDM gene is widely distributed on several plasmids from a variety of Gram-negative bacteria, primarily in transposons and gene cassettes within their multidrug-resistant (MDR) regions. This has led to the global dissemination of the blaNDM gene. Methods: The determination of class A beta-lactamase, class B and D carbapenemases was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). Antimicrobial susceptibility testing was performed using both the BioMerieux VITEK2 system and antibiotic paper diffusion methods. Plasmid transfer was then evaluated by conjugation experiments and plasmid electroporation assays. To comprehensively analyze the complete genome of K. pneumoniae strain F11 and to investigate the presence of mobile genetic elements associated with antibiotic resistance and virulence genes, Nanopore and Illumina sequencing platforms were used, and bioinformatics methods were applied to analyze the obtained data. Results: Our findings revealed that K. pneumoniae strain F11 carried class A beta-lactamase and classes B+D carbapenemases, and exhibited resistance to commonly used antibiotics, particularly tigecycline and ceftazidime/avibactam, due to the presence of relevant resistance genes. Plasmid transfer assays demonstrated successful recovery of plasmids pA_F11 and pB_F11, with average conjugation frequencies of 2.91×10-4 and 1.56×10-4, respectively. However, plasmids pC_F11 and pD_F11 failed in both conjugation and electroporation experiments. The MDR region of plasmid pA_F11 contained rare In1765, TnAs2, and TnAs3 elements. The MDR2 region of plasmid pB_F11 functioned as a mobile genetic "island" and lacked the blaNDM-1 gene, serving as a "bridge" connecting the early composite structure of bleMBL and blaNDM-1 to the recent composite structure. Additionally, the MDR1 region of plasmid pB_F11 comprised In27, TnAs1, TnAs3, and Tn2; and plasmid pC_F11 harbored the recent composite structure of bleMBL and blaNDM-1 within Tn3000 which partially contained partial Tn125. Conclusion: This study demonstrated that complex combinations of transposons and integron overlaps, along with the synergistic effects of different drug resistance and virulence genes, led to a lack of effective therapeutic agents for strain F11, therefore its dissemination and prevalence should be strictly controlled.

Ferroportin-dependent ferroptosis induced by ellagic acid retards liver fibrosis by impairing the SNARE complexes formation.

Chronic liver injury causing liver fibrosis is a major cause of morbidity and mortality worldwide. Targeting the suppression of hepatic stellate cell (HSC) activation is recognized as an effective strategy for the treatment of liver fibrosis. Ellagic acid (EA), a natural polyphenol product isolated from fruits and vegetables, possesses many biological functions. Here, EA exerts its antifibrotic activity by inducing ferroptotic cell death of activated HSCs, which is accompanied by redox-active iron accumulation, lipid peroxidation, and GSH depletion in CCl4 mice and human LX-2 cells. The specific ferroptosis inhibitor ferrostatin-1 prevented EA-induced ferroptotic cell death. Mechanistically, EA impairs the formation of vesicle-associated membrane protein 2 (VAMP2)/syntaxin 4 and VAMP2/synaptosome-associated protein 23 complexes by suppressing VAMP2 expression by enhancing its degradation in a proteasome-dependent pathway. This leads to the impairment of ferroportin (FPN, an iron exporter) translocation and intracellular iron extrusion. Interestingly, VAMP2 overexpression inhibits the role of EA in blocking FPN translocation and increasing intracellular ferritin content (an iron storage marker). In contrast, VAMP2 knockdown shows a synergistic effect on EA-mediated ferroptotic events in both HSCs. Additionally, HSC-specific overexpression of VAMP2 impaired EA-induced HSC ferroptosis in mouse liver fibrosis, and HSC-specific VAMP2 knockdown increased the inhibitory effect of EA on fibrosis. Taken together, our data suggest that the natural product EA exerts its antifibrotic effects by inducing FPN-dependent ferroptosis of HSCs by disrupting the formation of SNARE complexes, and EA will hopefully serve as a prospective compound for liver fibrosis treatment.

Yes-associated protein regulates the hepatoprotective effect of vitamin D receptor activation through promoting adaptive bile duct remodeling in cholestatic mice.

Mounting clinical evidence has revealed that the vitamin D receptor (VDR) is associated with cholestatic liver injury, although the functions of VDR in this condition remain largely unexplored. Here, we investigated the effects of VDR activation on bile duct ligation (BDL) mice, and the underlying mechanisms were further investigated. A low-calcemic VDR agonist, paricalcitol (PAL, 200 ng/kg), was intraperitoneally injected into BDL mice every other day for 5 days or 28 days. Liver histology, liver function indicators, cholangiocyte proliferation, fibrosis scores, and inflammation were evaluated. Mice treated with PAL were rescued from the decreased survival rate induced by BDL and liver damage was reduced. Mechanistically, PAL promoted cholangiocyte proliferation, which was likely conducive to proliferating bile duct maturation and increased branching of bile ducts. PAL treatment also increased the expression of Yes-associated protein (YAP) and its target protein epithelial cell adhesion molecule (EpCam) and decreased the level of inactive cytoplasmic phosphorylated YAP. YAP knockdown abrogated PAL-induced primary bile duct epithelial cell proliferation, confirmed with YAP inhibitor administration. In addition, BDL-induced liver fibrosis and inflammatory cell infiltration were reduced by PAL treatment at both day 5 and day 28 post-BDL. In conclusion, VDR activation mitigates cholestatic liver injury by promoting adaptive bile duct remodeling through cholangiocytic YAP upregulation. Because PAL is an approved clinical drug, it may be useful for treatment of cholestatic liver disease. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Paricalcitol inhibits oxidative stress-induced cell senescence of the bile duct epithelium dependent on modulating Sirt1 pathway in cholestatic mice.

BACKGROUND: Clinical studies indicate that vitamin D receptor (VDR) expression is reduced in primary biliary cirrhosis patient livers. However, the mechanism by which activated VDR effect cholestatic liver injury remains unclear. METHODS: Mice were injected intraperitoneally with the VDR agonist paricalcitol or a vehicle 3 days prior to bile duct ligation (BDL) and for 5 or 28 days after surgery. The analyses of liver morphology and necrotic areas were based on H&E staining. Serum biochemical indicators of liver damage were analyzed by commercial kits. The mechanisms of paricalcitol on cholestatic liver injury were determined by Western blot analysis. RESULTS: Paricalcitol ameliorated the BDL-induced liver damage in mice. Paricalcitol increased the proliferation of BECs to promote the repair of the bile duct. Paricalcitol also reduced the BDL-induced oxidative stress level in the mice. Mechanistic analysis revealed that paricalcitol decreased the number of SA-ß-gal-positive cells and downregulated the expression of p53, p21 and p16 proteins which was associated with reducing oxidative stress. Additionally, paricalcitol exerted the inhibitory effect of cell senescence was through reducing DNA damage and promoting DNA repair. Interesting, we found that paricalcitol prevented the downregulation of oxidative stress-induced Sirt1 expression in the BDL mice and t-BHP-induced BECs models. Moreover, paricalcitol suppressed cell senescence through a Sirt1-dependent pathway. These results were confirmed by antioxidant ALCAR and the Sirt1 inhibitor EX-527. CONCLUSION: Paricalcitol alleviated cholestatic liver injury through promoting the repair of damaged bile ducts and reducing oxidative stress-induced cell senescence of the bile duct via modulating Sirt1 pathway.