Dual role of Nrf2/HO-1 pathway in Z-ligustilide-induced ferroptosis against AML cells.

BACKGROUND: The scarcity of drugs targeting AML cells poses a significant challenge in AML management. Z-Ligustilide (Z-LIG), a phthalide compound, shows promising pharmacological potential as a candidate for AML therapy. However, its precise selective mechanism remains unclear. PURPOSE: In order to assess the selective inducement effects of Z-LIG on ferroptosis in AML cells and explore the possible involvement of the Nrf2/HO-1 pathway in the regulation of ferroptosis. METHODS: Through in vitro cell proliferation and in vivo tumor growth tests, the evaluation of Z-LIG's anticancer activity was conducted. Ferroptosis was determined by the measurement of ROS and lipid peroxide levels using flow cytometry, as well as the observation of mitochondrial morphology. To analyze the iron-related factors, western blot analysis was employed. The up-regulation of the Nrf2/HO-1 axis was confirmed through various experimental techniques, including CRISPR/Cas9 gene knockout, fluorescent probe staining, and flow cytometry. The efficacy of Z-LIG in inducing ferroptosis was further validated in a xenograft nude mouse model. RESULTS: Our study revealed that Z-LIG specifically triggered lipid peroxidation-driven cell death in AML cells. Z-LIG downregulated the total protein and nuclear entrance levels of IRP2, resulting in upregulation of FTH1 and downregulation of TFR1. Z-LIG significantly increased the susceptibility to ferroptosis by upregulating ACSL4 levels and simultaneously suppressing the activity of GPX4. Notably, the Nrf2/HO-1 pathway displayed a twofold impact in the ferroptosis induced by Z-LIG. Mild activation suppressed ferroptosis, while excessive activation promoted it, mainly driven by ROS-induced labile iron pool (LIP) accumulation in AML cells, which was not observed in normal human cells. Additionally, Nrf2 knockout and HO-1 knockdown reversed iron imbalance and mitochondrial damage induced by Z-LIG in HL-60 cells. Z-LIG effectively inhibited the growth of AML xenografts in mice, and Nrf2 knockout partially weakened its antitumor effect by inhibiting ferroptosis. CONCLUSION: Our study presents biological proof indicating that the selective initiation of ferroptosis in leukemia cells is credited to the excessive activation of the Nrf2/HO-1 pathway triggered by Z-LIG.

Z-ligustilide preferentially caused mitochondrial dysfunction in AML HL-60 cells by activating nuclear receptors NUR77 and NOR1.

BACKGROUND: Nuclear receptors NUR77 and NOR1 were identified as critical targets in acute myeloid leukemia (AML) therapy. Previously, we showed that Z-ligustilide (Z-LIG) selectively targeted AML by restoring NUR77 and NOR1. However, its downstream mechanisms are yet to be elucidated. METHODS: SRB staining assay was used to measure cell viability. Cell apoptosis, mitochondrial membrane potential and mitochondrial reactive oxygen species were analyzed using flow cytometry. The potential targets of Z-LIG in AML HL-60 cells were evaluated by RNA sequencing. Changes in RNA levels were measured using quantitative RT-qPCR and western blot analysis was used to detect the expression of proteins. RESULTS: Z-LIG preferentially induced mitochondrial dysfunction in HL-60 cells compared with 293T cells. Furthermore, RNA sequencing revealed that mitochondrial transcription and translation might be potential Z-LIG targets inhibiting HL-60 cells. NUR77/NOR1 overexpression significantly reduced the mitochondrial ATP and mitochondrial membrane potential and increased mitochondrial reactive oxygen species in HL-60 cells but not in 293T cells. Moreover, Z-LIG induced mitochondrial dysfunction by restoring NUR77 and NOR1 in HL-60 cells. Compared with HL-60 cells, the apoptosis-inducing activities of NUR77/NOR1 and Z-LIG were significantly reduced in HL-60 ρ0 cells depleted in mitochondrial DNA (mt-DNA). Moreover, NUR77/NOR1 and Z-LIG downregulated mitochondrial transcription and translation related proteins in HL-60 cells. Notably, Z-LIG remarkably reduced mitochondrial ATP in primary AML cells and showed anti-AML activity in mouse models of human AML. CONCLUSIONS: Collectively, our findings suggested that Z-LIG selectively induces mitochondrial dysfunction in AML HL-60 cells by restoring NUR77 and NOR1, a process associated with interference in mtDNA transcription.

Neutralization sensitivity, fusogenicity, and infectivity of Omicron subvariants.

BACKGROUND: The emergence of SARS-CoV-2 Omicron subvariants has raised questions regarding resistance to immunity by natural infection or immunization. We examined the sensitivity of Delta and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) to neutralizing antibodies from BBIBP-CorV-vaccinated and BBIBP-CorV- or ZF2001-boosted individuals, as well as individuals with Delta and BA.1 breakthrough infections, and determined their fusogenicity and infectivity. METHODS: In this cross-sectional study, serum samples from two doses of BBIBP-CorV-vaccinated individuals 1 (n = 36), 3 (n = 36), and 7 (n = 37) months after the second dose; BBIBP-CorV- (n = 25) or ZF2001-boosted (n = 30) individuals; and fully vaccinated individuals with Delta (n = 30) or BA.1 (n = 26) infection were collected. The serum-neutralizing reactivity and potency of bebtelovimab were assessed against D614G, Delta, and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) through a pseudovirus neutralization assay. The fusogenicity and infectivity of D614G, Delta, and Omicron subvariants were determined by cell-cell fusion assay and pseudovirus infection assay, respectively. RESULTS: Omicron subvariants markedly escaped vaccine-elicited neutralizing antibodies after two doses of BBIBP-CorV with comparable efficiency. A third dose vaccination of BBIBP-CorV or ZF2001 increased neutralizing antibody titers and breadth against Delta and three Omicron subvariants. Delta and BA.1 breakthrough infections induced comparable neutralizing antibody titers against D614G and Delta variants, whereas BA.1 breakthrough infections elicited a stronger and broader antibody response against three Omicron subvariants than Delta breakthrough infections. BA.2.12.1 and BA.4/5 are more resistant to immunity induced by breakthrough infections. Bebtelovimab had no significant loss of potency against the Delta and Omicron subvariants. Cell culture experiments showed Omicron subvariants to be less fusogenic and have higher infectivity than D614G and Delta with comparable efficiency. CONCLUSIONS: These findings have important public health implications and highlight the importance of repeated exposure to SARS-CoV-2 antigens to broaden the neutralizing antibody response against Omicron subvariants.

Retroperitoneal tumor finally diagnosed as a bronchogenic cyst: A case report and review of literature.

BACKGROUND: Retroperitoneal bronchogenic cyst (RBC) is an extremely rare developmental abnormality. Most are benign tumors but malignant transformation is possible. Because of their anatomical position, RBCs are easily misdiagnosed as adrenal or pancreatic solid tumors on radiological evaluation. Here, we report a case of RBC, review the literature, and summarize some important features. CASE SUMMARY: A 49-year-old woman was incidentally found to have a retroperitoneal tumor during a physical examination. Enhanced computed tomography and laboratory evaluations, including routine blood examination, blood biochemistry, 24-h urine 17 ketones, 17 hydroxyls, adrenocortical hormone, serum potassium concentration, serum amylase, lipase, and epithelial tumor markers, revealed a moderate density, 54 mm × 40 mm mass with a clear boundary near the left adrenal gland. The were no abnormalities in the blood and urine values. Because the patient had a history of hypertension and the location of the mass was adjacent to the adrenal gland, it was initially diagnosed as a left adrenal tumor and was resected by retroperitoneal laparoscopy. However, the pathological examination after surgery confirmed it to be a bronchogenic cyst. CONCLUSION: Retroperitoneal laparoscopic surgery can be prioritized for symptomatic RBC patients. Conservative treatment is feasible for selected patients.

Persistence of Antibody and Cellular Immune Responses in Coronavirus Disease 2019 Patients Over Nine Months After Infection.

BACKGROUND: The duration of humoral and T and B cell response after the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unclear. METHODS: We performed a cross-sectional study to assess the virus-specific antibody and memory T and B cell responses in coronavirus disease 2019 (COVID-19) patients up to 343 days after infection. Neutralizing antibodies and antibodies against the receptor-binding domain, spike, and nucleoprotein of SARS-CoV-2 were measured. Virus-specific memory T and B cell responses were analyzed. RESULTS: We enrolled 59 patients with COVID-19, including 38 moderate, 16 mild, and 5 asymptomatic patients; 31 (52.5%) were men and 28 (47.5%) were women. The median age was 41 years (interquartile range, 30-55). The median day from symptom onset to enrollment was 317 days (range 257 to 343 days). We found that approximately 90% of patients still have detectable immunoglobulin (Ig)G antibodies against spike and nucleocapsid proteins and neutralizing antibodies against pseudovirus, whereas ~60% of patients had detectable IgG antibodies against receptor-binding domain and surrogate virus-neutralizing antibodies. The SARS-CoV-2-specific IgG+ memory B cell and interferon-γ-secreting T cell responses were detectable in more than 70% of patients. CONCLUSIONS: Severe acute respiratory syndrome coronavirus 2-specific immune memory response persists in most patients approximately 1 year after infection, which provides a promising sign for prevention from reinfection and vaccination strategy.

Effects of irrigation fluid temperature during flexible ureteroscopic holmium laser lithotripsy on postoperative fever and shivering: a randomized controlled trial.

BACKGROUND: Flexible ureteroscopic holmium laser lithotripsy is used to treat urinary tract calculi, but postoperative complications include shivering, fever and infection. To investigate the effects of irrigation fluid temperature on postoperative complications. METHODS: This randomized controlled trial included 120 consecutive patients undergoing flexible ureteroscopic holmium laser lithotripsy at the Urology Department, Suining Central Hospital, Sichuan, China between January 2017 and July 2019. Patients were randomized 1:1:1 into three groups (17 °C, 27 °C or 37 °C). Primary outcome was fever incidence (body temperature > 37.5 °C) within 48 h after surgery. Secondary outcomes included shivering incidence during recovery from anesthesia, white blood cell count (WBC), serum procalcitonin (PCT) and incidence of suspected infection (temperature > 38.5 °C and PCT > 0.5 µg/L). RESULTS: There were 108 patients, (17 °C group, n = 36; 27 °C group, n = 35; 37 °C group, n = 37), received flexible ureteroscopic holmium laser lithotripsy and analyzed. Age, gender distribution, body mass index, ASA grade, stone burden, preoperative creatinine, preoperative core temperature and irrigation fluid volume did not differ significantly between groups. 17 °C, 27 °C and 37 °C groups exhibited significant differences in the incidences of postoperative fever (38.9% vs. 17.1% vs. 13.5%) and shivering (22.2% vs. 5.7% vs. 2.7%) (p < 0.05 for all pairwise comparisons). There was no significant difference of WBC, PCT and incidence of suspected infection in 37 °C or 27 °C group compared with 17 °C group. One case each of flash pulmonary edema and bleeding occurred in 37 °C group. CONCLUSION: Warming the irrigation fluid can reduce the incidence of postoperative fever and shivering, but further studies are needed to determine the optimal temperature. Trial registration The trial was registered at the Chinese Clinical Trials Registry and allocated as ChiCTR2000031683. The trial was registered on 07/04/2020 and this was a retrospective registration.

[Application of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe in cellular magnetic resonance imaging].

OBJECTIVE: To prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI). METHODS: We prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI. RESULTS: Atomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05). CONCLUSIONS: We prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.

[Reversion of multidrug resistance of hepatocellular carcinoma by antisense oligonucleotides and ultrasonic microbubble intensifier transfection combined with ultrasound irradiation].

OBJECTIVE: To study whether antisense oligonucleotides and ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is an effective and directional way in reversing multidrug resistance (MDR) in tumors. METHODS: Mdr1, mrp, and lrp genes antisense oligonucleotides on the ultrasound microbubble intensifier were transfected for the human HepG2/ADM cell lines and then the cells were radiated with low intensity ultrasound. The effects of the reversion of carcinoma cells' MDR and the reduction of their malignancy and growth capability in vitro and in vivo were assessed using RT-PCR, Western blot and MTT. RESULTS: The treatment restrained the multiplication of the human HepG2/AMD cell lines. The levels of their mRNA and protein of cells' mdr1 and mrp genes dropped significantly. Growth of the subcutaneous transplanted tumors in the nude mice decreased. CONCLUSIONS: Transfection of MDR genes antisense oligonucleotides on the ultrasonic microbubble intensifier combined with low intensity ultrasound radiation may serve as a new treatment method for hepatocellular carcinoma.