OBJECTIVES: To investigate the role of ferroptosis in diquat-induced acute kidney injury (AKI) and its molecular mechanisms. METHODS: Transgenic zebrafish models with Tg (Eco.Tshb:EGFP) labeling of the renal tubules and Tg (lyz:dsRed2) labeling of the neutrophils were both divided into control group, gentamicin (positive control) group, diquat poisoning group, ferroptosis inhibitor group. The indicators of kidney injury, inflammatory response, and ferroptosis were examined in the zebrafish, and the changes in expressions of voltage-dependent anion-selective channel protein 1 (VDAC1) and mitochondrial ferritin (FTMT) were detected using Western blotting. RESULTS: AKI induced by diquat exhibited a significant dose-effect relationship, and the severity of injury was proportional to the exposure concentration. Diquat also caused marked oxidative stress and inflammatory responses in the zebrafish models. Rhodamine metabolism assay and HE staining revealed significantly declined glomerular filtration function of the zebrafish as diquat exposure concentration increased. Immunofluorescence staining highlighted significant changes in the expressions of ferroptosis markers GPX4 and FTH1 in zebrafish renal tissues following diquat exposure. In diquat-exposed zebrafish, treatment with ferrostatin-1, a ferroptosis inhibitor, obviously upregulated GPX4 and downregulated FTH1 expressions and improved the metabolic rate of glucan labeled with rhodamine B. Diquat exposure significantly upregulated the expression of VDAC1 and FTMT in zebrafish, and the application of ferrostatin-1 and VBIT-12 (a VDAC1 inhibitor) both caused pronounced downregulation of FTMT expression. CONCLUSIONS Ferroptosis is a critical mechanism underlying diquat-induced AKI, in which VDAC1 and FTMT play important regulatory roles, suggesting their potential as therapeutic target for AKI caused by diquat.
Animals ; Zebrafish ; Ferroptosis/drug effects* ; Acute Kidney Injury/chemically induced* ; Diquat/toxicity* ; Animals, Genetically Modified ; Voltage-Dependent Anion Channel 1/metabolism* ; Ferritins/metabolism* ; Oxidative Stress

Depression (Dep) is one of the most common concomitant symptoms of Parkinson's disease (PD), but there is a lack of detailed pathologic evidence for the occurrence of PD-Dep. Currently, the management of symptoms from both conditions using conventional pharmacological interventions remains a formidable task. In this study, we found impaired activation of extracellular signal-related kinase (ERK), reduced levels of transcription and translation, and decreased expression of brain-derived neurotrophic factor (BDNF) in the medial prefrontal cortex (mPFC) of PD-Dep rats. We demonstrated that the abnormal phosphorylation of α-synuclein (pS129) induced tropomyosin-related kinase receptor type B (TrkB) retention at the neuronal cell membrane, leading to BDNF/TrkB signaling dysfunction. We chose SEW2871 as an ameliorator to upregulate ERK phosphorylation. The results showed that PD-Dep rats exhibited improvement in behavioral manifestations of PD and depression. In addition, a reduction in pS129 was accompanied by a restoration of the function of the BDNF/ERK signaling loop in the mPFC of PD-Dep rats.
Animals ; Brain-Derived Neurotrophic Factor/metabolism* ; alpha-Synuclein/metabolism* ; Male ; Prefrontal Cortex/drug effects* ; Rats, Sprague-Dawley ; Depression/metabolism* ; MAP Kinase Signaling System/drug effects* ; Rats ; Parkinson Disease/metabolism* ; Receptor, trkB/metabolism* ; Phosphorylation ; Disease Models, Animal ; Signal Transduction

Objective:To observe the therapeutic effects of electroacupuncture(EA)stimulation on chronic pain and associated negative emotions,and to investigate the activity changes of glutamatergic neurons in the primary motor cortex(M1)following EA stimulation.Methods:Male C57BL/6J mice were randomly divided into four groups:sham surgery(Sham),pure electroacupuncture stimulation(EA),nerve injury(SNI)and electroacupuncture treatment of nerve injury(SNI+EA).Thirty days after the SNI model establishment,EA stimulation was administered bilaterally to the Zusanli(ST36)acupoints in mice.von Frey filaments and Hargreaves heat sensitivity testing were used to detect mechanical and thermal hyperalgesia.The elevated plus maze test was conducted to assess the impact on anxiety-like behaviors in mice.Dual immunofluorescence staining was employed to observe changes in c-Fos expression in M1 gluta-matergic neurons.Results:As compared to Sham group,SNI-treated mice exhibited significant mechanical and thermal hyperalgesia in bilateral hindpaws at 30 days post-modeling(P＜0.01)and displayed obvious anxiety-like behaviors(P＜0.01).The SNI+EA group showed significant relief in pain and anxiety-like behaviors(P＜0.01).c-Fos expression in M1 glutamatergic neurons was significantly decreased in SNI mice.Conversely,after electroacupuncture(EA)treatment,compared to the SNI group,M1 glutamatergic neurons in the SNI+EA group showed significantly in-creased c-Fos expression(P＜0.01).Conclusion:Electroacupuncture significantly alleviated chronic pain and associ-ated anxiety-like behaviors induced by SNI,which might involve the activation of glutamatergic neurons in the M1 in this process.

Objective:To observe the therapeutic effects of electroacupuncture(EA)stimulation on chronic pain and associated negative emotions,and to investigate the activity changes of glutamatergic neurons in the primary motor cortex(M1)following EA stimulation.Methods:Male C57BL/6J mice were randomly divided into four groups:sham surgery(Sham),pure electroacupuncture stimulation(EA),nerve injury(SNI)and electroacupuncture treatment of nerve injury(SNI+EA).Thirty days after the SNI model establishment,EA stimulation was administered bilaterally to the Zusanli(ST36)acupoints in mice.von Frey filaments and Hargreaves heat sensitivity testing were used to detect mechanical and thermal hyperalgesia.The elevated plus maze test was conducted to assess the impact on anxiety-like behaviors in mice.Dual immunofluorescence staining was employed to observe changes in c-Fos expression in M1 gluta-matergic neurons.Results:As compared to Sham group,SNI-treated mice exhibited significant mechanical and thermal hyperalgesia in bilateral hindpaws at 30 days post-modeling(P＜0.01)and displayed obvious anxiety-like behaviors(P＜0.01).The SNI+EA group showed significant relief in pain and anxiety-like behaviors(P＜0.01).c-Fos expression in M1 glutamatergic neurons was significantly decreased in SNI mice.Conversely,after electroacupuncture(EA)treatment,compared to the SNI group,M1 glutamatergic neurons in the SNI+EA group showed significantly in-creased c-Fos expression(P＜0.01).Conclusion:Electroacupuncture significantly alleviated chronic pain and associ-ated anxiety-like behaviors induced by SNI,which might involve the activation of glutamatergic neurons in the M1 in this process.

Bone Transplantation/adverse effects* ; Transplantation, Autologous/adverse effects*

Objective:To explore genes related to costimulatory molecule related to the prognosis of bladder cancer,and to construct and evaluate prognosis model based on costimulatory molecule-based signature(CMS).Methods:Gene expression matrix and clinical information of bladder cancer patients were downloaded from TCGA database and GEO database(GSE31684),and costimulatory molecule-related genes were retrieved from the literature.The univariate and multivariate Cox analysis were used to screened prognostic-related genes and constructed prognostic model.Forecast accuracy of model was verified in TCGA training group,TCGA validation data group and GEO group by Kaplan-Meier survival analysis and receiver operating characteristic curve(ROC).Considering risk score and clinical characteristics,we constructed a nomogram and evaluated its performance by consistency analysis and ROC.CIBERSORT algorithm was used to analyze immune cell composition of tumor microenvironment infiltration,and gene set enrichment analysis(GSEA)was performed to explore the potential mechanism.Results:Four prognostic-related CMSs were found:TNFRSF14,CD276,ICOS and TMIGD2,of which three were included in the risk score construction.Multivariate Cox regression results showed that the risk score based on CMS was an independent prognostic factor for bladder cancer patients.Consistency analysis and ROC results showed that the nomogram had ideal prognosis prediction accuracy.Immune infiltration analysis showed that the high risk group was likely to be in immunosuppressive state.GSEA results suggested that genes in high risk group were enriched in extracel-lular matrix(ECM)receptors interaction,cell cycle and other pathways.Conclusion:TNFRSF14,CD276 and ICOS may be potential prognostic biomarkers for bladder cancer patients.CMS-based risk score and nomogram could contribute to early prognosis and choice of personalized treatment.

AIM:To investigate the impact of aquaporin 4(AQP4)expression inhibition on autophagy and apoptosis in a mouse model of cerebral ischemia-reperfusion(I/R)injury,and to elucidate its underlying mechanism.METHODS:Cerebral I/R injury was induced in mice via transient middle cerebral artery occlusion(tMCAO).Totally 60 mice were randomly divided into sham group,I/R group,AQP4 inhibition group,and 3-methyladenine(3-MA)group,with 15 mice in each group.Among them,the mice in sham and I/R groups received intraperitoneal injections of normal saline,while those in AQP4 inhibition group and 3-MA group received intraperitoneal injections of AER-271(2 mg·kg-1·d-1)and AER-271+3-MA(2 mg·kg-1·d-1)for 3 d,respectively,once per day.Longa score was adopted to assess the neu-rological function,and to record changes in body weight.Cerebral infarction volume and histopathological alterations were evaluated using hematoxylin-eosin staining.Western blot analysis was performed to determine the levels of AQP4,LC3-Ⅱ,P62 and cleaved caspase-3,while the LC3-Ⅱ,P62,cleaved caspase-3 and NeuN(neuronal marker)colocalization and expression assessment were conducted with immunofluorescence.RESULTS:The mice in I/R and AQP4 inhibition groups exhibited extensive cerebral infarction,cerebral edema,and elevated Longa scores.However,in comparision to I/R group,the mice in AQP4 inhibition group showed significantly reduced cerebral infarct volume,cerebral edema vol-ume,and Longa score(P<0.05).Additionally,in contrast to sham group,the mice in I/R group displayed increased ex-pression of AQP4,LC3-Ⅱ and cleaved caspase-3(P<0.01),accompanied by decreased body weight and P62 expression(P<0.05 or P<0.01).Furthermore,compared with I/R group,the mice in both AQP4 inhibition group and 3-MA group demonstrated a decrease in the expression levels of AQP4,LC3-Ⅱ and cleaved caspase-3(P<0.05 or P<0.01),along with increased body weight and P62 expression(P<0.05 or P<0.01).Nonetheless,no significant differences were ob-served between AQP4 inhibition group and 3-MA group regarding Longa score,cerebral infarct volume,body weight,and the expression of AQP4,LC3-Ⅱ,cleaved caspase-3 and P62.CONCLUSION:Inhibition of AQP4 expression signifi-cantly reduces cerebral infarction area and nerve injury severity in tMCAO mice.Moreover,AQP4 expression inhibition decelerates autophagy and apoptosis after cerebral infarction,with the additional autophagy inhibitor showing no notable impact on the protective effect of AQP4 inhibition.

Objective:To explore the causal relationship between human immune cells and hypertrophic scar (HS) using two-sample bidirectional Mendelian randomization (MR) method.Methods:This study was based on two-sample MR method, and the datasets of 731 immune cells and HS were obtained from the genome-wide association study (GWAS) catalog database and Finngen database, respectively. A significance threshold was established to discern single nucleotide polymorphism (SNP) significantly correlated with immune cells or HS, thereby eliminating the impact of weak instrumental variable bias. The inverse variance weighted (IVW) method (meanwhile, the Benjamini-Hochberg (BH) procedure of false discovery rate (FDR) to adjust P values) was used for preliminary detection of the causal relationship between immune cells and HS and screen the immune cells that had a significant causal relationship with HS. Further, the causal relationship between the selected immune cells and HS was detected through five two-sample MR methods: IVW method, weighted median method, simple mode method, weighted mode method, and MR-Egger method, and the scatter plot was drawn. SNPs conformed to the hypothesis were subjected to Cochran Q test for heterogeneity assessment, MR-Egger regression coupled with MR-PRESSO to eliminate horizontal pleiotropic effects, and a leave-one-out analysis was also conducted to determine if significant results were driven by individual SNP. Finally, the IVW method contained in the two-sample MR analysis was utilized to inversely examine the causal relationship between HS and immune cells. Results:The number of SNPs in 731 immune cells reaching the significance threshold varied from 7 to 1 786, while in HS, 119 SNPs met the significance threshold, with the F values of all SNPs being greater than 10, suggesting a low likelihood of bias from weak instrumental variables. The IVW method revealed that 60 types of immune cells potentially had a causal relationship with HS (with all P values <0.05), and after adjustment using the BH method, only CD45RA and CD39 positive regulatory T cell (Treg) maintained a potentially strong causal relationship with HS ( PFDR<0.05). The IVW method (with odds ratio of 1.16 and 95% confidence interval of 1.08-1.24, P<0.05, PFDR<0.05), weighted median method (with odds ratio of 1.16 and 95% confidence interval of 1.05-1.28, P<0.05), weighted mode method (with odds ratio of 1.14 and 95% confidence interval of 1.02-1.27, P<0.05), and MR-Egger method (with odds ratio of 1.18 and 95% confidence interval of 1.07-1.30, P<0.05) of scatter plot all suggested a causal relationship between the 14 SNPs of CD45RA and CD39 positive Treg and risk of HS, only simple mode method of scatter plot suggested a not obvious relationship between the 14 SNPs of CD45RA and CD39 positive Treg and risk of HS ( P>0.05). Cochran Q test indicated no heterogeneity in the causal relationship between CD45RA on CD39 positive Treg and HS ( P>0.05). MR-Egger regression and MR-PRESSO analyses showed that there was no horizontal pleiotropy in the significant causal relationship between CD45RA and CD39 positive Treg and HS ( P>0.05). Leave-one-out analysis confirmed that the significant causal relationship between CD45RA and CD39 positive Treg and HS remained stable after sequentially removing individual SNP. Reverse two-sample MR analysis showed that HS had no potential causal relationship with any of the 731 types of immune cells ( P>0.05). Conclusions:From the perspective of genetics, it is revealed that immune cells CD45RA and CD39 positive Treg may increase the risk of HS.

Background and objective Current studies suggest that for early-stage lung cancers with a component of ground-glass opacity measuring ≤2 cm,sublobar resection is suitable if it ensures adequate margins.However,lobectomy may be necessary for some cases to achieve this.The aim of this study was to explore the impact of size and depth on surgical techniques for wedge resection,segmentectomy,and lobectomy in early-stage lung cancer ≤2 cm,and to determine methods for ensuring a safe resection margin during sublobar resections.Methods Clinical data from 385 patients with early-stage lung can-cer ≤2 cm,who underwent lung resection in 2022,were subject to a retrospective analysis,covering three types of procedures:wedge resection,segmentectomy and lobectomy.The depth indicator as the OA value,which is the shortest distance from the inner edge of a pulmonary nodule to the opening of the corresponding bronchus,and the AB value,which is the distance from the inner edge of the nodule to the pleura,were measured.For cases undergoing lobectomy and segmentectomy,three-dimensional computed tomography bronchography and angiography(3D-CTBA)was performed to statistically determine the number of subsegments required for segmentectomy.The cutting margin width for wedge resection and segmentectomy was recorded,as well as the specific subsegments and their quantities removed during lung segmentectomy were documented.Results In wedge resection,segmentectomy,and lobectomy,the sizes of pulmonary nodules were(1.08±0.29)cm,(1.31±0.34)cm and(1.50±0.35)cm,respectively,while the depth of the nodules(OA values)was 6.05(5.26,6.85)cm,4.43(3.27,5.43)cm and 3.04(1.80,4.18)cm for each procedure,showing a progressive increasing trend(P＜0.001).The median resec-tion margin width obtained from segmentectomy was 2.50(1.50,3.00)cm,significantly greater than the 1.50(1.15,2.00)cm from wedge resection(P＜0.001).In wedge resections,cases where AB value＞2 cm demonstrated a higher proportion of cases with resection margins less than 2 cm compared to those with margins greater than 2 cm(29.03％vs 12.90％,P=0.019).When utilizing the size of the nodule as the criterion for resection margin,the instances with AB value＞2 cm continued to show a higher proportion in the ratio of margin distance to tumor size less than 1(37.50％vs 17.39％,P=0.009).The median number of subsegments for segmentectomy was three,whereas lobectomy cases requiring segmentectomy involved five subsegments(P＜0.001).Conclusion The selection of the surgical approach for lung resection is influenced by both the size and depth of pulmonary nodules.This study first confirms that larger portions of lung tissue must be removed for nodules that are deeper and larger to achieve a safe margin.A distance of ≤2 cm from the inner edge of the pulmonary nodule to the nearest pleura may be the ideal indication for performing wedge resection.

Objective:To investigate the effect of keloid fibroblasts on the polarization and expression of inflammatory factors of M0 macrophages and possible mechanisms, and provide theoretical basis for new targets for keloid therapy.Methods:Keloids, normal skin tissues and paraffin specimens from patients undergoing plastic surgery in the First Affiliated Hospital of Sun Yat-sen University from November 2020 to September 2021 were collected, and fibroblasts of keloids and normal skins were isolated and co-cultured with M0 cells formed form THP-1 by phorbol ester (PMA)-stimulation to detect the expression of macrophage polarization markers and cytokines. Besides, keloid fibroblasts were treated with exogenous tumor necrosis factor-α(TNF-α) to detect its effect on the proliferation and extracellular matrix expression.Results:Macrophages were dominated by CD163 + (M2) in keloid tissues. Moreover, M0 cells expressed more TNF-α when co-cultured with keloid fibroblasts, compared with those with normal skin fibroblasts, in which, the positive staining rates of TNF-α were 19.32% and 29.52% respectively by flow cytometry. Furthermore, the proliferation was promoted and the expression of extracellular matrix proteins (COL3A1 and FN1)and Vimentin were upregulated in keloid fibroblasts under TNF-α stimulation. However, there was no significant difference in the expression of polarization surface markers CD86 and CD163 in macrophages, when co-cultured with keloid fibroblasts or normal skin fibroblasts. Conclusions:Keloid fibroblasts promote the expression of TNF-α in macrophages, which in turn promotes the proliferation and extracellular matrix secretion of keloid fibroblasts.