OBJECTIVE To investigate the role of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in regulating ferroptosis,malignant biological behavior,and cisplatin resistance in laryngeal squamous cell carcinoma(LSCC)under hypoxic conditions.METHODS Human laryngeal cancer Hep-2 and TU212 cell lines were cultured in vitro under hypoxic(1%O2)and normoxic(21%O2)conditions without serum.Hep-2 cells were divided into five experimental groups:DMSO control group,Erastin treatment group,Hypoxia+Erastin group,Hypoxia+Erastin+NC-GAPDH group,and Hypoxia+Erastin+Silenced GAPDH group,to study the cellular response under various experimental conditions.Quantitative polymerase chain reaction(qPCR)and Western blot(WB)techniques were employed to analyze GAPDH mRNA and protein expression levels in each cell line.Cell Counting Kit-8(CCK-8),colony formation,and Transwell invasion assays were used to assess the vitality,proliferation,and invasion capabilities of each group of laryngeal cancer cells.Additionally,CCK-8 assays were used to determine cell resistance to various concentrations of cisplatin(5,15,20 μg/ml).Kit methods were used to evaluate intracellular malondialdehyde(MDA)and glutathione(GSH)levels.WB was employed to detect the expression levels of glutathione peroxidase 4(GPX4)and 4-hydroxynonenal(4-HNE)proteins.Fe2+probes and reactive oxygen species(ROS)fluorescence probes were used to measure intracellular Fe2+content and ROS levels.Transmission electron microscopy was utilized to observe mitochondrial morphology changes to assess ferroptosis occurrence.RESULTS Compared to normoxic conditions,hypoxia significantly increased the expression of GAPDH mRNA and protein in Hep-2 and Tu212 cells(P<0.05).Under hypoxic conditions,the number of cell colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment were significantly higher than those under normoxic conditions(P<0.05).When GAPDH was silenced,these hypoxia-induced enhancements were reversed.Under normoxic conditions,compared to the DMSO control group,Erastin treatment significantly reduced the number of colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment(P<0.05).However,under hypoxic conditions,the inhibitory effect of Erastin was weakened.Additionally,in the hypoxia+Erastin+si-GAPDH group,the number of colonies,migration capacity,and cell viability at 15 and 20 μg/ml cisplatin treatment were significantly lower than those in the hypoxia+Erastin+NC-GAPDH group(P<0.05).CONCLUSION This study reveals that silencing GAPDH under hypoxic conditions can reverse the inhibition of ferroptosis in LSCC cells,thereby suppressing their malignant biological behavior and cisplatin resistance.

Background/Aims: Despite the high efficacy of direct-acting antivirals (DAAs), approximately 1–3% of hepatitis C virus (HCV) patients fail to achieve a sustained virological response. We conducted a nationwide study to investigate risk factors associated with DAA treatment failure. Machine-learning algorithms have been applied to discriminate subjects who may fail to respond to DAA therapy. Methods: We analyzed the Taiwan HCV Registry Program database to explore predictors of DAA failure in HCV patients. Fifty-five host and virological features were assessed using multivariate logistic regression, decision tree, random forest, eXtreme Gradient Boosting (XGBoost), and artificial neural network. The primary outcome was undetectable HCV RNA at 12 weeks after the end of treatment. Results: The training (n=23,955) and validation (n=10,346) datasets had similar baseline demographics, with an overall DAA failure rate of 1.6% (n=538). Multivariate logistic regression analysis revealed that liver cirrhosis, hepatocellular carcinoma, poor DAA adherence, and higher hemoglobin A1c were significantly associated with virological failure. XGBoost outperformed the other algorithms and logistic regression models, with an area under the receiver operating characteristic curve of 1.000 in the training dataset and 0.803 in the validation dataset. The top five predictors of treatment failure were HCV RNA, body mass index, α-fetoprotein, platelets, and FIB-4 index. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of the XGBoost model (cutoff value=0.5) were 99.5%, 69.7%, 99.9%, 97.4%, and 99.5%, respectively, for the entire dataset. Conclusions Machine learning algorithms effectively provide risk stratification for DAA failure and additional information on the factors associated with DAA failure.

AIM: To observe changes in fundus microcirculation of myopic adolescents after wearing orthokeratology by applying optical coherence tomography angiography(OCTA).METHODS: Prospective study. A total of 40 cases(40 eyes)of adolescents with low to moderate myopia who chose orthokeratology to correct visual acuity at our hospital from April 2021 to June 2022 were collected. The uncorrected distant visual acuity and axial length were evaluated at 1, 3 and 6mo before and after wearing orthokeratology, respectively. Furthermore, the changes in superficial vessel density(SVD), deep vessel density(DVD), central retinal thickness(CRT), foveal avascular zone area(FAZ-A), foveal avascular zone perimeter(FAZ-P), retinal nerve fiber layer(RNFL)thickness and radial peripapillary capillaries density(RPCD)were observed by applying OCTA.RESULTS: The uncorrected distant visual acuity was significantly improved at 1, 3 and 6mo after wearing orthokeratology(P<0.001). There was no statistically significant difference in axial length before and after wearing orthokeratology(P>0.05). Moreover, there were significant differences in both SVD of fovea quadrant and DVD of fovea and lower quadrant(P<0.01), but there were no differences in CRT, FAZ-A and FAZ-P, RNFL thickness and RPCD(P>0.05).CONCLUSION: Wearing orthokeratology can significantly improve visual acuity and increase local retinal vessel density in the macula in adolescents with low to moderate myopia.

Objective To investigate the effects of paclitaxel and doxorubicin on the immune microenvironment of breast cancer in mice. Methods The CTR-DB database, a database for analysis of gene expression profiles and drug resistance characteristics related to tumor drug response, was used to analyze the effect of chemotherapeutic drugs on the immune microenvironment of breast cancer. Mouse models with breast cancer were established by in situ injection with 4T1 cells, a triple-negative breast cancer (TNBC) cells. Then they were treated with doxorubicin and paclitaxel, respectively. The sizes of tumor were recorded and analyzed by growth curve. The number of different types of immune cells was analyzed using flow cytometry. The expressions of Ki67, S100 calcium binding protein A9 (S100A9) and matrix metalloproteinase 9 (MMP9) were detected by immunohistochemistry. The cell cycles of 4T1 cells in paclitaxel group and doxorubicin group were analyzed by flow cytometry. Results The results of CTR_Microarray_75 analysis showed that the immune scores, and the number of cytotoxic lymphocytes, B lineages, CD8⁺ T cells, dendritic cells (DCs), monocytic lineages and natural killer (NK) cells in chemotherapy-sensitive breast cancer were higher than those in chemotherapy-insensitive breast cancer. Through growth curve analysis in mice with breast cancer, we found that both paclitaxel and doxorubicin could inhibit the increase of the tumor sizes, and the paclitaxel showed a higher inhibitory effect. The results of cytometry displayed that both paclitaxel and doxorubicin could restrain the expression of Ki67 and increase the number of breast cancer cells in G2/M phase, and in the paclitaxel group, the expression of Ki67 was lower and the number of breast cancer cells in G2/M phase was larger. Paclitaxel and doxorubicin enhanced the infiltration of CD45⁺ immune cells but decreased the infiltration of neutrophils. Additionally, paclitaxel promoted the infiltration of CD3⁺CD4⁺ T helper cells, CD3⁺CD8⁺ cytotoxic T cells and CD45⁺CD19⁺B cells, while doxorubicin increased the infiltration of CD4⁺CD25⁺ regulatory T cells (Tregs). The results of immunohistochemistry displayed that the paclitaxel significantly inhibited the expression of S100A9, while the doxorubicin significantly restrained the expression of MMP9. Conclusion Paclitaxel and doxorubicin can effectively inhibit the growth of breast cancer cells and change immune microenvironment of TNBC by regulating the different patterns of cell infiltration and the expression of different extracellular matrix components.
Animals ; Mice ; Humans ; Paclitaxel/pharmacology* ; Matrix Metalloproteinase 9 ; Triple Negative Breast Neoplasms/drug therapy* ; CD8-Positive T-Lymphocytes ; Ki-67 Antigen ; Doxorubicin/pharmacology* ; Calgranulin B ; Tumor Microenvironment

Objective:To investigate the expressions of Nod-like receptor protein 3 (NLRP3), cysteine-aspartic acid protease 1 (Caspase-1), and Gasdermin D (GSDMD) in nasopharyngeal carcinoma (NPC), and their relationships with the recurrence and metastasis of NPC.Methods:A retrospective study was conducted on 421 patients diagnosed with NPC between December 2014 and January 2020. The expressions of NLRP3, Caspase-1, and GSDMD in pathological specimens were examined with immunohistochemistry and multiplex immunofluorescence staining. Univariate and multivariate Cox regression analyses were applied to identify the factors influencing NPC recurrence and metastasis. In vitro experiments with NPC cell line HNE-2 were used to explore the functional mechanisms of NLRP3, Caspase-1, and GSDMD.Results:Multivariate Cox analysis revealed that tumor staging of Ⅲ-Ⅳ( HRrecurrence=2.74, 95% CIrecurrence: 1.61-4.65; HRmetastasis=1.90, 95% CImetastasis: 1.04-3.49) and pre-treatment plasma EBV-DNA levels≥1 500 copies/ml ( HRrecurrence=1.91, 95% CIrecurrence: 1.13-3.23; HRmetastasis=2.07, 95% CImetastasis: 1.23-3.50)were independent risk factors for NPC recurrence and metastasis, while positive expression of NLRP3( HRrecurrence=0.17, 95% CIrecurrence: 0.08-0.35; HRmetastasis=0.30, 95% CImetastasis: 0.15-0.59), Caspase-1( HRrecurrence=0.32, 95% CIrecurrence: 0.18-0.59; HRmetastasis=0.43, 95% CImetastasis: 0.25-0.76), and GSDMD( HRrecurrence=0.48, 95% CIrecurrence: 0.25-0.91; HRmetastasis=0.96, 95% CImetastasis: 0.53-1.74) served as independent protective factors. Age ( HR=1.02, 95% CI: 1.01-1.04) and intensity-modulated radiotherapy ( HR=0.51, 95% CI: 0.30-0.88) were independent factors for NPC recurrence, whereas chemotherapy ( HR=0.50, 95% CI: 0.29-0.88) acted as an independent protective factor for NPC metastasis (all P<0.05). NPC patients with positive expressions of the three proteins had higher locoregional recurrence-free survival, distant metastasis-free survival, and overall survival compared to those with negative expressions (all P<0.05). In vitro experiments revealed that the overexpression of NLRP3 activated the NLRP3/Caspase-1/GSDMD signaling pathway, as evidenced by Western Blot analysis. Enzyme-linked immunosorbent assay and scanning electron microscopy demonstrated that overexpression of NLRP3 promoted pyroptosis in HNE-2 cells. Cellular functional assays further confirmed that overexpression of NLRP3 significantly inhibited the proliferation, invasion, and migration of HNE-2 cells. Conclusion:Positive expressions of NLRP3, Caspase-1, and GSDMD serves as independent protective factors for recurrence and metastasis of NPC, potentially by promoting cell pyroptosis and thus inhibiting NPC cell proliferation, invasion, and migration.

OBJECTIVE: To observe the clinical efficacy of acupoint injection combined with Vitalstim electrical stimulation for post-stroke dysphagia. METHODS: A total of 98 patients with dysphagia after first stroke were randomized into an acupoint injection group (35 cases, 2 cases dropped off), an electrical stimulation group (31 cases, 3 cases dropped off) and a combination group (32 cases, 3 cases dropped off). Injection of mecobalamin into Tunyan point, Vitalstim electrical stimulation and the combination of injection of mecobalamin into Tunyan point and Vitalstim electrical stimulation were applied respectively in the 3 groups, once a day, 10 times as one course, 2 courses were required. Before and after treatment, the tongue muscle thickness and video fluoroscopic swallowing study (VFSS) score were observed in the 3 groups. RESULTS: After treatment, the tongue muscle thickness was decreased (P<0.05), the VFSS scores were increased (P<0.05) compared with before treatment in the 3 groups, and the variation of tongue muscle thickness and VFSS score in the combination group was greater than the acupoint injection group and the electrical stimulation group (P<0.05). CONCLUSION Both acupoint injection of mecobalamin and Vitalstim electrical stimulation have therapeutic effect on dysphagia after stroke, and the two have synergistic effect.
Acupuncture Points ; Acupuncture Therapy ; Deglutition ; Deglutition Disorders/therapy* ; Electric Stimulation ; Humans ; Treatment Outcome

OBJECTIVE: To study the regulatory effect of Cheng's Juanbi Decoction (JBT) on autophagy in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and role of PI3K/Akt/mTOR signaling axis in the mechanism mediating this effect. METHODS: CCK8 assay was used to determine the optimal concentration and treatment time of JBT for inhibiting the viability of RA- FLS. The effect of freeze-dried powder of JBT, RAPA, or both on morphology of the autophagosomes in RA-FLS was observed under transmission electron microscope, and the changes in the number of autophagosomes and autolysosomes were observed with autophagy double-labeled adenovirus experiment. RT-qPCR and Western blotting were used to detect the expression levels of the related indicators. RESULTS: The results of CCK8 assay showed that treatment with 0.5 mg/mL JBT for 12 h produced the optimal effect for inhibiting RA-FLS viability. Observation with transmission electron microscope and the results of the autophagy double-labeled adenovirus experiment both showed the presence of a small number of autophagosomes in control RA-FLS group, and treatment with JBT significantly increased the number of autophagosomes and lowered the number of autophagolysosomes in the cells. Compared with the control cells and the cells treated with JBT or RAPA alone, the cells treated with both JBT and RAPA showed significantly decreased mRNA levels of PI3K, Akt and mTOR (P < 0.01) but without significant changes in their protein expressions (P > 0.05); the combined treatment significantly inhibited the protein expressions of p-PI3K, p-Akt, p-mTOR, and P62 (P < 0.05) and upregulated the protein expressions of Beclin-1 and LC3B (P < 0.05) in the cells. CONCLUSION JBT can inhibit the survival rate of RA-FLS and increase the level of autophagy possibly through a mechanism that down-regulates PI3K/Akt/mTOR signaling pathway.
Humans ; Phosphatidylinositol 3-Kinases ; Autophagy ; Fibroblasts ; Synoviocytes ; Arthritis, Rheumatoid ; Adenoviridae ; TOR Serine-Threonine Kinases

Objective:To analyze the clinical characteristics and prognosis of patients with AIDS-related lymphoma (ARL).Methods:The clinical data of 84 patients with ARL admitted in the First Affiliated Hospital Zhejiang University School of Medicine from March 2013 to October 2021 were retrospectively reviewed, and the influencing factors for prognosis of patients were analyzed by Cox proportional risk regression model.Results:Non-Hodgkin’s lymphoma accounted for 95.2% (80/84) of all cases; 52.3% (44/84) of the patients presented with B symptoms of lymphoma such as fever, night sweats, and weight loss, and 84.5% (71/84) of them were classified as stage Ⅲ~Ⅳ by Ann Arbor staging; 58.0% (47/81) patients had CD4 + T cell count ≤200/μL at baseline; 30 (35.7%) patients had pulmonary infection, 11 (13.1%) patients had digestive tract infection, and 21.4% (18/84) patients had sepsis. The 1-year survival rate of 84 patients was 70.2% (59/84). Multivariate Cox survival analysis showed that International prognostic index (IPI) score >3 ( HR=5.094, 95% CI 1.877-13.824, P=0.001) was an independent risk factors and rituximab treatment ( HR=0.354, 95% CI 0.152-0.823, P=0.016) was an independent protective factor for the prognosis of patients with ARL. Conclusions:The clinical manifestations of ARL are diverse, and it is often diagnosed in the late stage of the disease with adverse prognosis. IPI score and Rituximab treatment are key prognostic factors in patients with ARL.

Objective:To evaluate the effect of general practice service based on network platform in health management of patients with multiple chronic conditions (MCC).Methods:Based on network platform, the general practitioners provided health management service for 626 MCC patients in a petrochemical company in Panjin city of Liaoning province from June 2015 to June 2016. The individualized health management plan included health counseling, diet and exercise guidance, health information delivery, health file establishment, and rational medication guidance. After intervention the patient activeness, quality of life, clinical variables were evaluated.Results:Ninety participants dropped out of study (14.38%, 90/626), the questionnaire survey was conducted in rest 536 participants. After 1-year intervention, the scores of physical functioning, role-physical, general health, vitality, social functioning, role-emotional, mental health, and total scores in SF-36 were significantly increased ( F=12.350, 13.215, 10.515, 29.114, 42.972, 10.951, 89.783 and 33.311, respectively, all P<0.05); the values of BMI, waist, systolic and diastolic blood pressure, total cholesterol, triglycerides, low density lipoprotein cholesterol, fasting blood glucose were significantly decreased( F= 30.039, 33.849, 7.152, 27.453, 59.938, 214.270, 73.488 and 62.014, respectively, all P<0.05); the values of high density lipoprotein cholesterol were markedly increased ( F=16.491, P<0.01); the overall scores of activation were increased ( F=9.471, P<0.01), the level of activation was changed (χ 2=16.183, P<0.05); but there was no change in body pain ( F=1.486, P=0.227). There was a weak correlation between patient activation and the general health ( r=0.099, P=0.013), vitality ( r=0.101, P=0.011), mental health ( r=0.099, P=0.013). Conclusion:General practice service based on network platform is feasible and effective for patients with multiple chronic conditions, after the implementation, the activation and the health outcomes are improved significantly.

OBJECTIVEOsteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation.

METHODS3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.

RESULTSTRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.

CONCLUSIONOur results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.

14-3-3 Proteins ; genetics ; metabolism ; Cell Proliferation ; genetics ; Humans ; Osteosarcoma ; genetics ; Ribonucleoproteins ; genetics ; metabolism ; Tumor Cells, Cultured