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BACKGROUND: HucMSCs had shown promising efficacy in treating childhood diseases, but oxidative stress induced by the poor microenvironment at the site of damage resulted in low cell survival after transplantation, thus preventing the cells from maximizing therapeutic efficacy. Therefore, this study aimed to investigate the role and mechanism of keap1 in oxidative stress injury of human umbilical cord mesenchymal stem cells (hucMSCs), and to provide theoretical support for improving the efficacy of stem cell therapy. METHODS: The hucMSCs were treated with hypoxic low-sugar-free serum (GSDH) to mimic the damaged site microenvironment after implantation. Adenoviral overexpression of keap1 gene of hucMSCs was performed in vitro, and cell proliferation ability was detected by CCK8 assay, crystal violet staining assay, and cell cycle assay. Cellular redox level was assessed by Amplex Red, MDA, and GSH/GSSG kit. Mitochondrial morphology was evaluated by mitotracker Red staining. ATP production was estimated by ATP detection kit. The mRNA and protein expression levels were tested by western blotting and RT-qPCR. RESULTS: GSDH treatment substantially upregulated keap1 expression. Subsequently, we found that overexpression of keap1 notably inhibited cell proliferation and caused cells to stagnate in G1 phase. At the same time, overexpression of keap1 induced the production of large amounts of H2O2 and the accumulation of MDA, but suppressed the GSH/GSSG ratio and the expression of antioxidant proteins NQO1 and SOD1, which caused oxidative stress damage. Overexpression of keap1 induced cells to produce a large number of dysfunctional mitochondria resulting in reduced ATP production. Moreover, Overexpression of keap1 significantly decreased the IKKß protein level, while upregulating IkB mRNA levels and downregulating P50 mRNA levels. CONCLUSIONS: Overexpression of keap1 may induce oxidative stress injury in hucMSCs by down-regulating IKKß expression and inhibiting NF-κB pathway activation. This implies the importance of keap1 in hucMSCs and it may be a potential gene for genetic modification of hucMSCs.
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Peróxido de Hidrogênio , Células-Tronco Mesenquimais , Criança , Humanos , Trifosfato de Adenosina , Dissulfeto de Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Quinase I-kappa B/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , RNA Mensageiro/metabolismo , Cordão UmbilicalRESUMO
BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are seed cells that can be used for alternative treatment of myocardial damage. However, their immaturity limits their clinical application. Mitochondrial development accompanies cardiomyocyte maturation, and PINK1 plays an important role in the regulation of mitochondrial quality. However, the role and mechanism of PINK1 in cardiomyocyte development remain unclear. METHODS: We used proteomic and phosphoproteomic to identify protein and phosphosite changes in hiPSC-CMs deficient in PINK1. Bioinformatics analysis was performed to identify the potential biological functions and regulatory mechanisms of these differentially expressed proteins and validate potential downstream mechanisms. RESULTS: Deletion of PINK1 resulted in mitochondrial structural breakdown and dysfunction, accompanied by disordered myofibrils arrangement. hiPSC-CMs deficient in PINK1 exhibited significantly decreased expression of mitochondrial ATP synthesis proteins and inhibition of the oxidative phosphorylation pathway. In contrast, the expression of proteins related to cardiac pathology was increased, and the phosphoproteins involved in cytoskeleton construction were significantly altered. Mechanistically, PINK1 deletion damaged the mitochondrial cristae of hiPSC-CMs and reduced the efficiency of mitochondrial respiratory chain assembly. CONCLUSION: The significantly differentially expressed proteins identified in this study highlight the important role of PINK1 in regulating mitochondrial quality in hiPSC-CMs. PINK1-mediated mitochondrial respiratory chain assembly is the basis for mitochondrial function. Whereas the cytoskeleton may be adaptively altered in response to mitochondrial dysfunction caused by PINK1 deletion, inadequate energy supply hinders myocardial development. These findings facilitate the exploration of the mechanism of PINK1 in cardiomyocyte development and guide efforts to promote the maturation of hiPSC-CMs.
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Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Proteômica , Mitocôndrias , Proteínas Mitocondriais , Proteínas Quinases/genéticaRESUMO
OBJECTIVE: Anastomotic Leakage (AL) is one of the most common complications after resection of rectal cancer. Recognition of the incidence and risk factors related to AL is important. This study aimed develops a model that can predict anastomotic leakage after anterior rectal resection. METHODS: Data from 188 patients undergoing anterior resection of rectal cancer were collected for retrospective analysis. Patients were randomly divided in the development set and validation set at a 1:1 ratio. We first included age, sex, preoperative chemoradiotherapy, tumor size, degree of tumor differentiation, stage, TNM stage, lymph vascular invasion, distance, anastomotic method, diabetes, intraoperative time, intraoperative bleeding and smoking as candidates for variable selection with a LASSO method. A ROC curve was constructed with the validation set to assess the accuracy of the prediction model. RESULTS: AL occurred in 20 of 188 patients (10.6%). Preoperative chemoradiotherapy (p=0.04), medium degree of tumor differentiation (p=0.04), anastomotic method (p<0.01), intraoperative bleeding≥400ml (p<0.01), smoking (p<0.01), diabetes (p<0.01) were significantly related to AL. The area under the ROC curve of the prediction model is 0.952. CONCLUSIONS: This study developed a model that can predict anastomotic leakage after anterior rectal resection, which may aid the selection of preventive ileostomy and postoperative management.
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BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.
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Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Vitamina K 3/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mitochondrial dysfunction is considered one of the major pathogenic mechanisms of sepsis-induced cardiomyopathy (SIC). Pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of mitochondrial metabolism, is essential for maintaining mitochondrial function. However, its specific role in SIC remains unclear. To investigate this, we established an in vitro model of septic cardiomyopathy using lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Our study revealed a significant increase in PDK4 expression in LPS-treated H9C2 cardiomyocytes. Inhibiting PDK4 with dichloroacetic acid (DCA) improved cell survival, reduced intracellular lipid accumulation and calcium overload, and restored mitochondrial structure and respiratory capacity while decreasing lactate accumulation. Similarly, Oxamate, a lactate dehydrogenase inhibitor, exhibited similar effects to DCA in LPS-treated H9C2 cardiomyocytes. To further validate whether PDK4 causes cardiomyocyte and mitochondrial damage in SIC by promoting lactate production, we upregulated PDK4 expression using PDK4-overexpressing lentivirus in H9C2 cardiomyocytes. This resulted in elevated lactate levels, impaired mitochondrial structure, and reduced mitochondrial respiratory capacity. However, inhibiting lactate production reversed the mitochondrial dysfunction caused by PDK4 upregulation. In conclusion, our study highlights the pathogenic role of PDK4 in LPS-induced cardiomyocyte and mitochondrial damage by promoting lactate production. Therefore, targeting PDK4 and its downstream product lactate may serve as promising therapeutic approaches for treating SIC.
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Ácido Láctico , Lipopolissacarídeos , Miócitos Cardíacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Lipopolissacarídeos/toxicidade , Animais , Ratos , Ácido Láctico/metabolismo , Linhagem Celular , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/antagonistas & inibidores , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Proteínas QuinasesRESUMO
The enhancement of the heat transfer assisted by ultrasound is considered to be an interesting and highly efficient cooling technology, but the investigation and application of ultrasound in minichannel heat sinks to strengthen the flow boiling heat transfer are very limited. Herein, a novel installation of ultrasound transducers in the flow direction of a minichannel heat sink is designed to experimentally study the characteristics of heat transfer in flow boiling and the influence of operation parameters (e.g., heat flux, mass flux rate) and ultrasound parameters (e.g., frequency, power) on the flow boiling heat transfer in a minichannel heat sink with and without ultrasound field. Bubble motion and flow pattern in the minichannel are analyzed by high-speed flow visualization, revealing that the ultrasound field induces more bubbles at the same observation position and a forward shift of the onset of nucleation boiling along the flow direction, as ultrasonic cavitation produces a large number of bubbles. Moreover, bubbles hitting the channel wall on the left and right sides are found, and the motion speed of the bubbles is increased by 31.9% under the ultrasound field. Our results demonstrate that the heat transfer coefficient obtained under the ultrasound field is 53.9% higher than in the absence of the ultrasound field under the same conditions, and the enhancement ratio is decreased in the high heat flux region due to the change of the flow regime with increasing heat flux. This study provides a theoretical basis for the application of an ultrasound field in minichannel heat sinks for the enhancement of flow boiling heat transfer.
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Ultrasound is considered to be an effective active heat transfer enhancement method, which is widely used in various fields. But there is no clear understanding of flow boiling heat transfer characteristics in micro/mini-channels under ultrasonic field since the studies related are limited up to now. In this paper, a novel minichannel heat exchanger with two ultrasonic transducers inside the inlet and outlet plenum respectively is designed to experimentally investigate the impacts of ultrasound on flow boiling heat transfer enhancement in a minichannel heat sink. Flow visualization analyses reveal that ultrasound can promote rapid bubble motion, bubble detachment from heating wall surface and thereby new bubble generation, and decrease the length of confined bubble. Furthermore, the flow boiling experiments are initiated employing working fluid R141b at different ultrasonic parameters (e.g., frequency, power, angle of radiation) and heat flux under three types of ultrasound excitations: no ultrasound (NU), single inlet ultrasound (IU), inlet and outlet ultrasound (IOU). The results indicate that ultrasound has obvious augmentation effects on flow boiling heat transfer even though the intensification effects will be limited with the heat flux increases. The higher ultrasonic power, the lower ultrasonic frequency and the higher ultrasonic radiation angle, the better intensification efficiency. The maximum enhancement ratio of have in the saturated boiling section reaches 1.88 at 50 W, 23 kHz and 45° under the experimental conditions. This study will be beneficial for future applications of ultrasound on flow boiling heat transfer in micro/mini-channels.
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The objective of the study was to compare the therapeutic outcomes between the ultrasonography-guided (USG) and the fluoroscopy-guided (FG) mini-percutaneous nephrolithotomy (MPCNL) for the treatment of large or complex upper urinary tract stones (S.T.O.N.E. scores 5-11) in patients with autosomal dominant polycystic kidney disease (ADPKD). 45 ADPKD patients who were suffering from large or complex upper urinary tract stones (S.T.O.N.E. scores 5-11, mean score 7.6) in 45 renal units were accrued into this retrospective study. They were treated by either USG (20 patients) or FG (25 patients) MPCNL in our center. The treatment results as well as the complications according to the modified Clavien system were assessed and compared. The FG MPCNL group had a higher success rate in accessing the targeted calyces than the USG MPCNL group (96 vs. 70 %, p = 0.048). There was no significant difference observed between the two groups with respect to the operative time, the mean hemoglobin drop, and the stone free rate. The overall operative complications and the perioperative blood transfusion rates were significantly higher in the USG than the FG MPCNL groups, 71.4 vs. 29.2 %, p = 0.011, and 35.7 vs. 4.2 %, p = 0.018, respectively. There was no significant difference between these two groups in terms of major complications (Clavien score 3a-4a) (p = 0.542). In our center, the FG MPCNL was a superior modality to the USG MPCNL in the treatment of large or complex kidney stones in the ADPKD patients. It resulted in higher successful calyceal punctures and less operative complications.
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Nefrolitotomia Percutânea/efeitos adversos , Nefrolitotomia Percutânea/métodos , Rim Policístico Autossômico Dominante/cirurgia , Complicações Pós-Operatórias/epidemiologia , Urolitíase/cirurgia , Adulto , Transfusão de Sangue/estatística & dados numéricos , Feminino , Fluoroscopia , Hemoglobinas/análise , Humanos , Cálices Renais/cirurgia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Minimamente Invasivos , Nefrostomia Percutânea/efeitos adversos , Nefrostomia Percutânea/métodos , Duração da Cirurgia , Assistência Perioperatória/métodos , Rim Policístico Autossômico Dominante/complicações , Rim Policístico Autossômico Dominante/diagnóstico por imagem , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Ultrassonografia de Intervenção , Urolitíase/complicações , Urolitíase/diagnóstico por imagemRESUMO
BACKGROUND: Drug resistance and disease progression are still the major obstacles in the treatment of chronic myeloid leukemia (CML). Increasing researches have demonstrated that autophagy becomes activated when cancer cells are subjected to chemotherapy, which is involved in the development of drug resistance. Therefore, combining chemotherapy with inhibition of autophagy serves as a new strategy in cancer treatment. Tigecycline is an antibiotic that has received attention as an anti-cancer agent due to its inhibitory effect on mitochondrial translation. However, whether combination of tigecycline with inhibition of autophagy could overcome drug resistance in CML remains unclear. METHODS: We analyzed the biological and metabolic effect of tigecycline on CML primary cells and cell lines to investigate whether tigecycline could regulate autophagy in CML cells and whether coupling autophagy inhibition with treatment using tigecycline could affect the viabilities of drug-sensitive and drug-resistant CML cells. RESULTS: Tigecycline inhibited the viabilities of CML primary cells and cell lines, including those that were drug-resistant. This occurred via the inhibition of mitochondrial biogenesis and the perturbation of cell metabolism, which resulted in apoptosis. Moreover, tigecycline induced autophagy by downregulating the PI3K-AKT-mTOR pathway. Additionally, combining tigecycline use with autophagy inhibition further promoted the anti-leukemic activity of tigecycline. We also observed that the anti-leukemic effect of tigecycline is selective. This is because the drug targeted leukemic cells but not normal cells, which is because of the differences in the mitochondrial biogenesis and metabolic characterization between the two cell types. CONCLUSIONS: Combining tigecycline use with autophagy inhibition is a promising approach for overcoming drug resistance in CML treatment.
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Proteínas Relacionadas à Autofagia/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Minociclina/análogos & derivados , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Minociclina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , TigeciclinaRESUMO
Palladium-graphene (Pd-GR) nanocomposite was acted as modifier for construction of the modified electrode with direct electrochemistry of hemoglobin (Hb) realized. By using Nafion as the immobilization film, Hb was fixed tightly on Pd-GR nanocomposite modified carbon ionic liquid electrode. Electrochemical behaviors of Hb modified electrode were checked by cyclic voltammetry and a pair of redox peaks originated from direct electron transfer of Hb was appeared. The Hb modified electrode had excellent electrocatalytic activity to the reduction of trichloroacetic acid and sodium nitrite in the concentration range from 0.6 to 13.0mmol·L-1 and from 0.04 to 0.5 mmol·L-1. Therefore Pd-GR nanocomposite was proven to be a good candidate for the fabrication of third-generation electrochemical biosensor.
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Hemoglobinas/química , Técnicas Biossensoriais , Catálise , Técnicas Eletroquímicas , Eletrodos , Grafite , Peróxido de Hidrogênio , Líquidos Iônicos , PaládioRESUMO
OBJECTIVE: To investigate the feasibility and relibility of rapidly and accurately acquiring the informations of gene mutations in MPN patients by using self-designed custom MPN mutation-related multipe-PCR primer kit and next generation Ion Torrent PGM sequencing platform. METHODS: The bone marrow samples of 10 MPN patients with JAK2V617F and/or CALR+, Ph- confirmed by sanger sequencing method were collected and were re-detected by using next generation Ion Torrent PGM sequencing method, then the consistence of results of above-mentioned 2 kinds of detection methods was compared. RESULTS: In terms of JAK2V617F, MPL and CALR mutations, the results of Ion Torrent PGM sequencing were complete consistent with results of Sanger sequencing, except 52 bp deletion of CALR gene, which conld not be detected by next generation Ion Torrent PGM sequencing method in all bone marrow samples. CONCLUSION: The detection of multiple gene mutations in MPN patients by Ion Torrent PGM sequencing platform is feasible and can meet the needs of clinical testing. This method can complete detection of all 23 mutetions within 1-2 days, moreover, possesses advantages of high sensitivity, specificity, rapidity, high throughput and low cost.
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Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Transtornos Mieloproliferativos/genética , Humanos , Deleção de SequênciaRESUMO
Tyrosine kinase inhibitors (TKIs), including imatinib, dasatinib and nilotinib, are effective forms of therapy for various types of solid cancers and Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia. A number of TKIs have been known to have strong effects on T cells, particularly cluster of differentiation (CD) 4+CD25+ T cells, also known as regulatory T cells (Tregs). There is currently a deficit in the available clinical data regarding this area of study. In the present study, a total of 108 peripheral blood samples were collected from patients with chronic myeloid leukemia (CML) at diagnosis (n=31), and at 3 and 6 months following treatment with TKI [imatinib (n=12), dasatinib (n=11) and nilotinib groups (n=8)] and healthy controls (n=15). Peripheral blood mononuclear cells were collected from the patients prior to and following TKI treatment. The subtype and number of T lymphocytes in patients and healthy donors were analyzed using flow cytometry. Additionally, flow cytometry and ELISA were used to detect the proliferation and suppression of Tregs. Expression of cytokines and other molecules [forkhead box P3 (FOXP3), glucocorticoid-induced tumor necrosis factor receptor (GITR) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)] were also analyzed at 3 and 6 months following treatment with TKIs. It was indicated that, at diagnosis, a similar number of lymphocytes were detected in patients and control. However, following treatment with a TKI, the number of total T cells, Tregs, CD4+ T and CD8+ T cells decreased to various degrees in patients. Furthermore, the decrease in the number of Tregs was more significant with time. Although treatment with imatinib, dasatinib and nilotinib demonstrated similar inhibitory effects on the quantity of Tregs in vivo, the TKIs exhibited differential effects on the function of Tregs in vitro. Proliferation, suppression and expression of cytokines [interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-ß] and molecules (FOXP3, GITR and CTLA-4) decreased significantly in treatment groups with imatinib and dasatinib. The decrease was not significant in the nilotinib treatment group. Imatinib and dasatinib may exert more marked inhibitory roles compared with nilotinib on regulating the number and function of Tregs. These results suggest that personalized treatment and follow-up of CML patients during TKI treatment, particularly for those who received post-transplant TKI treatment may be beneficial.
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Ketamine has been reported to impair human cognitive function as a recreational drug of abuse. However, chronic effects of ketamine on central nervous system need to be further explored. We set out to establish chronic ketamine addiction models by giving mice a three or six month course of daily intraperitoneal injections of ketamine, then examined whether long-term ketamine administration induced cognition deficits and changed hippocampal post-synaptic protein expression in adult mice. Behavior tests results showed that mice exhibited dose- and time-dependent learning and memory deficits after long-term ketamine administration. Western blot results showed levels of GluA1, p-S845 and p-S831 proteins demonstrated significant decline with ketamine 60 mg/kg until six months administration paradigm. But levels of p-S845 and p-S831 proteins exhibited obvious increase with ketamine 60 mg/kg three months administration paradigm. NR1 protein levels significantly decrease with ketamine 60 mg/kg three and six months administration paradigm. Our results indicate that reduced expression levels and decreased phosphorylation levels of hippocampal post-synaptic membrane GluA1- containing AMPA receptors maybe involved in cognition impairment after long-term ketamine administration. These findings provide further evidence for the cognitive damage of chronic ketamine addiction as a recreational drug.