Objective: To investigate the role and mechanism of ATP6V1B2 in the inhibiton of lipid deposition by promoting lysosomal acidification in lipotoxinexposed hepatocytes. Methods: Hepatic steatosis model mice were constructed by feeding male C57BL/6 mice with a highfat diet for 14 weeks. Lipotoxic injury was induced in hepatocyte lines L02 and HepG2 using a mixture of oleic acid and palmitic acid. The expression of ATP6V1B2 gene in L02 cells was knocked down or overexpressed by siRNA and plasmid transfection. The mRNA and protein expressions of ATP6V1B2 were detected by qRT-PCR and Western blotting, respectively. Hepatocyte lipid deposition was detected by oil red O staining and Nile red staining. Lysosomal activity was detected by lyso-tracker in lipotoxic hepatocytes. Lysosomal membrane integrity was detected by acridine orange staining. The JASPAR database was used to predict transcription factors regulating ATP6V1B2, and the regulatory effect of transcription factors on ATP6V1B2 expression was verified. Results: Compared with the normal control group, ATP6V1B2 expression was decreased in hepatic tissues of high-fat diet mice and lipotoxic hepatocytes (P<0.05). ATP6V1B2 knockdown exacerbated hepatocyte lipid deposition, inhibited hepatocyte lysosomal acidification, and increased hepatocyte lysosomal membrane permeability. Overexpression of ATP6V1B2 alleviated cellular lipid deposition. Transcription factor SP1 could transcriptionally regulate ATP6V1B2 expression. Compared with the control group, silencing SP1 upregulates the mRNA and protein expression of ATP6V1B2 in L02 cells (P<0.01). Conclusion: ATP6V1B2 may inhibit hepatocyte lipid deposition by promoting hepatocyte lysosomal acidification.

Objective: To investigate the therapeutic effect and mechanism of Clitoria ternatea flower (CF) on nonalcoholic fatty liver disease (NAFLD) in mice induced by a highfat diet (HFD). Methods: Twentyfour healthy ICR mice were randomly divided into four groups: normal control group (NC group), NAFLD model group (NAFLD group), highdose CF group (CFH group, 200 mg/kg), and lowdose CF group (CFL group, 100 mg/kg), with 6 mice in each group. The NC group was fed a normal diet, while the NAFLD, CFH, and CFL groups were fed a 60% highfat diet to induce NAFLD. Additionally, the CFH and CFL groups received corresponding doses of CF by gavage, and the NC and NAFLD groups received an equal volume of normal saline (06 mL) by gavage daily for 12 weeks. Serum levels of hepatic enzymes, blood lipids, and oxidative stress factors were detected, combined with untargeted metabolomics analysis. Fecal microbiota composition was analyzed via 16S rRNA gene sequencing, and the contents of three major shortchain fatty acids (acetate, propionate, and butyrate) were determined. Meanwhile, hepatic inflammationrelated indicators were examined by quantitative realtime polymerase chain reaction (qRTPCR). Results: Compared with the NAFLD group, the CFL and CFH groups showed significant reductions in serum levels of alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), and lowdensity lipoprotein cholesterol (LDLC) (all P<005). In the liver, the mRNA expressions of proinflammatory cytokines IL1β and TNFα were decreased, while those of antiinflammatory cytokines IL4 and IL10 were increased (all P<001). Additionally, serum superoxide dismutase (SOD) activity was markedly enhanced, whereas malondialdehyde (MDA) content was significantly decreased (all P<001). 16S rRNA sequencing results indicated that compared with the NAFLD group, the CF groups had increased abundances of multiple beneficial gut bacteria including Prevotella and Lactobacillus, along with elevated concentrations of acetate, propionate, and butyrate in feces. Serum untargeted metabolomics analysis revealed that compared with the NAFLD group, the CF groups had higher levels of various antiinflammatory and antioxidative stress metabolites, which were mainly involved in glycerophospholipid metabolism, pyrimidine metabolism, and other pathways. Conclusion: CF can alleviate liver damage in NAFLD mice by improving lipid levels and hepatic inflammation, regulating gut microbiota balance and serum metabolic pathways, showing potential therapeutic effects for NAFLD.

Objective: To explore the disrupting effects of low-dose methyl-2-benzimidazole carbamate (CBZ) on glycolipid metabolism in mice during pubertal development period and its mechanism. Methods: A developmental (4-week-old) and adult (8-week-old) ICR mouse model with low-dose CBZ exposure was established. The mice were randomly divided into a blank control group (administered with corn oil) and different dose CBZ exposure groups (0.3, 3, 30 and 60 mg/kg ), and were given intragastric administration for 28 days. Commercial kits were used to detect the serum levels of total cholesterol (TC), total triglyceride (TG), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as the activity of glucose-6-phosphate dehydrogenase (G6PD) in the liver. Fasting blood glucose (FBG) was measured by a blood glucose meter. Body weight was monitored by weighing. The expression levels of genes related to glycolipid metabolism and endoplasmic reticulum stress pathway were detected by quantitative realtime PCR. Results: Compared with the blank control group, CBZ exposure significantly increased the serum lipid levels and various blood biochemical indexes in developing and adult mice (P<0.05 or P<0.01), and only significantly increased the FBG level and decreased the body weightof adult mice (P<0.05). CBZ exposure upregulated the expression of lipid synthesis and transport genes such as CD36 and SREBP-1, down-regulated the expression of lipid metabolism genes such as PPARα and HNF4α, as well as gluconeogenesis and glycogen synthesis genes such as G6PC, PEPCK and GSK3β in developing and adult mice(P<0.05). Meanwhile, CBZ up-regulated the expression of GRP78, PERK, CHOP, Bax, Caspase3 and other genes (P<0.05), down-regulated the expression of ATF4 and Bcl-2 genes (P<0.05), and activated the endoplasmic reticulum stress and apoptotic pathways. Conclusion: CBZ exerts endocrine and metabolic toxicity to developing mice, which can interfere with glycolipid metabolism and cause metabolic dysfunction by affecting the expression of glycolipid metabolism-related genes and activating the endoplasmic reticulum stress signaling pathway.

Objective: To investigate the correlation between the characteristic parameters of brown adipose tissue (BAT) and coronary artery calcification in 18F-FDG PET/CT images. Methods: A total of 2 399 patients who underwent 18F-FDG PET/CT examination at the Affiliated Hospital of Jiangsu University from December 2013 to April 2024 were collected. After 1∶1 propensity score matching (PSM), the patients were divided into BAT positive group (n=171) and BAT negative group (n=171) according to whether BAT was positive. Differences in general clinical data and the incidence of coronary artery calcification between the two groups were analyzed. Differences in BAT imaging characteristic parameters among six anatomical sites (the neck, supraclavicular fossa, axilla, mediastinum, paravertebral region, and abdomen) in patients of the BAT-positive group were analyzed, and the site with the highest BAT activity (the paravertebral region) was screened out. Furthermore, differences in paravertebral BAT imaging characteristic parameters among groups with different numbers of coronary artery calcification-involved branches and different severities of coronary artery calcification were analyzed. Results: After PSM, the incidence of coronary artery calcification and the coronary artery calcium score in the BAT positive group were significantly lower than those in the BAT negative group. In the BAT positive group, the SUVmax of paravertebral BAT was the highest among the six anatomical sites, and the difference was statistically significant compared with the SUVmax of abdominal and axillary BAT (all P<0.001). The SUVmean of paravertebral BAT significantly differed from that of axillary BAT (P<0.001), but showed no significant differences with other anatomical sites (all P>0.05). Both the volume and surface area of paravertebral BAT demonstrated statistically significant differences compared to abdominal and axillary BAT (all P<0.001). As both the number of coronary artery branches involved and the severity of calcification increased, all characteristic parameters of paravertebral BAT progressively decreased (all P<0.01). Conclusion: The presence of BAT is associated with a lower risk of coronary artery calcification. Paravertebral BAT may have a potential inhibitory effect on the occurrence and development of coronary artery calcification.

Objective: To investigate the expression levels of Rho GTPase-activating protein 12 (ARHGAP12) within tertiary lymphoid structures (TLS) in gastric cancer and its impact on tumor immune response. Methods: ARHGAP12 mRNA expression differences between gastric cancer (n=414) and normal tissues (n=211) were analyzed using TCGA databases. Western blotting was employed to validate the protein expression levels of ARHGAP12 in 12 pairs of gastric cancer and adjacent tissue samples. Immunofluorescence was used to detect ARHGAP12 protein expression in TLS and tumor-draining lymph nodes (TDLN) in gastric tissue pathological sections. Hallmark and KEGG pathway enrichment analyses were conducted to assess the correlation between ARHGAP12 mRNA expression and the activation of immune-related pathways. Immunohistochemical staining was performed to examine the protein expression levels of ARHGAP12 in pathological sections of tumor tissues from 28 gastric cancer patients, and clinical data were analyzed to explore the correlation between cytokine expression levels and ARHGAP12 immunohistochemical staining scores. KM plotter database was used to analyze the relationship between ARHGAP12 mRNA expression and prognosis of patients with gastric cancer. Results: TCGA database analysis showed that ARHGAP12 mRNA expression in gastric cancer tissues was significantly higher than that in adjacent tissues (P<0.001). Western blotting results indicated that there was no significant difference in ARHGAP12 protein expression levels between the gastric adenocarcinoma and adjacent tissue samples (P>0.05). Multiplex immunofluorescence results demonstrated higher expression of ARHGAP12 protein in gastric cancer cells, TLS, and TDLN regions (P<0.05). Hallmark and KEGG pathway enrichment analysis suggested that ARHGAP12 mRNA expression is involved in the activation of interferon response pathways and antigen presentation processes. Furthermore, high ARHGAP12 immunohistochemical staining score was positively correlated with preoperative serum IFN-γ levels in patients with gastric cancer (P<0.05). KM plotter database analysis showed that high expression of ARHGAP12 mRNA in gastric cancer tissues is positively correlated with longer overall survival period (P<0.05). Conclusion: ARHGAP12 is highly expressed in gastric cancer TLS, and it is positively correlated with the activation of interferon-γ-related anti-tumor immune pathways.

Objective: To investigate the therapeutic effects and underlying mechanisms of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles (HucMSC-sEVs) on renal injury in diabetic kidney disease (DKD) rats. Methods: HucMSCs were isolated, cultured, and used to extract HucMSC-sEVs from their culture supernatant. A DKD rat model was induced by a high-fat diet combined with STZ injection. After successful modeling validation, rats were randomized into the DKD group and the HucMSC-sEVs group, with normal rats as controls. At 8 weeks, HucMSC-sEVs were injected via the tail vein. After 24 weeks, kidney tissues were collected. Renal histopathology was assessed by HE staining, while Masson staining was employed to evaluate collagen deposition. The expression levels of necroptosis markers (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL) were quantified using qRT-PCR, Western blotting, and immunohistochemical staining. NRK-52E cells were treated with high glucose and HucMSC-sEVs. Transmission electron microscopy was used to observe necrotic phenomena in NRK-52E cells, and the expression levels of necroptosis marker proteins such as p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL were detected by Western blotting and qRT-PCR. Results: Compared with the control group, the glomerular basement membrane in the DKD group rats was significantly thickened, and the interstitium exhibited fibrotic characterization, with a marked increase in the expression of necroptosis marker proteins. After treatment with HucMSC-sEVs, the pathological damage and fibrosis degree of the rat kidney tissues were alleviated, and the expression of necroptosis execution proteins was reduced. In vitro experiments revealed that high glucose-treated NRK-52E cells exhibited necrotic features including mitochondrial cristae fragmentation, endoplasmic reticulum dilation, and elevated expression of necroptosis markers (p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL). Treatment with HucMSC-sEVs significantly attenuated these cellular alterations and reduced the expression levels of necroptosis-related proteins. Conclusion: HucMSC-sEVs can improve renal function and delay the progression of DKD in rats, and the mechanism may be attributed to the inhibition of necroptosis of renal tubular epithelial cells.

Objective: To explore the effect of crocin on the migration and invasion ability of anaplastic thyroid carcinoma (ATC) cells and its molecular mechanism of regulating epithelial-mesenchymal transition (EMT) via the Smad-dependent signaling pathway. Methods: ATC cells were treated with different concentrations of crocin. Cell proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry, respectively. The effects of crocin on ATC cell migration and invasion were detected by Transwell assays. Western blotting analysis was used to examine the expression of EMT markers (E-cadherin, N-cadherin, vimentin, fibronectin) and Smad signaling pathway-related proteins. A subcutaneous tumor model was established using BHT-101 cells to evaluate the inhibitory effect of crocin on tumor invasiveness. Immunohistochemistry and Western blotting were performed to detect the expression changes of MMP-2, MMP-9, N-cadherin and vimentin in tumor tissues. Results: At concentrations below 40 μmol/L, crocin treatment significantly reduced cell invasion and migration compared with the untreated group (P<0.01), accompanied by increased E-cadherin expression and decreased N-cadherin, vimentin and fibronectin expression. The inhibitory effect of crocin on Smad2/3 phosphorylation was alleviated after exogenous TGF-β intervention. In vivo experiments showed that crocin notably downregulated the expression of MMP-2, MMP-9, N-cadherin and vimentin in BHT-101 subcutaneous xenograft tumors. Conclusion: Crocin can inhibit the invasion and EMT of ATC cells by modulating the Smad-dependent signaling pathway.

Objective: To investigate the role of the long noncoding RNA (lncRNA) NONRATT021203.2 in the progression of cancer-induced bone pain (CIBP) and its potential molecular mechanisms. Methods: A CIBP model was constructed in Sprague-Dawley (SD) rats by intratibial inoculation of Walker 256 breast cancer cells. Sixteen adult female SD mice weighing 180-200 g were randomly divided into control group and CIBP group, with 8 rats in each group, and injected into tibiae with 10 μL normal saline and 10 μL Walker 256 breast cancer cell suspension (1×10⁸ cells/mL), respectively. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of the modeled lower limb of rats were detected by behavioral analysis. The expression levels of lncRNA NONRATT021203.2 and miRNA-138-5p in DRG were measured by using quantitative real-time PCR (qRT-PCR), and the localization of miRNA-138-5p in DRG tissue was examined by fluorescence in situ hybridization (FISH). Seven days after modeling, 10 rats with CIBP were selected and divided into CIBP+siRNA group (n=5) and CIBP+siNC group (n=5), lncRNA NONRATT021203.2-siRNA and siNC were injected intrathecally, respectively. The relative expression levels of lncRNA NONRATT021203.2 and miRNA-138-5p in the two groups were detected by qRT-PCR. Additionally, seven days after modeling, 16 rats with CIBP were divided into CIBP+mimics (n=8) and CIBP+NC group (n=8), miRNA-138-5p mimics and negative control oligonucleotide were injected intrathecally, respectively. PWT and PWL were detected by behavioral test. Finally, the binding sites of lncRNA NONRATT021203.2 and miRNA-138-5p were analysed by using miRand and miRwalk3.0 software. Results: Compared with control group, CIBP group showed significantly reduced PWT and PWL (P<0.05), markedly increased expression of lncRNA NONRATT021203.2 (P<0.01), and significantly decreased expression of miRNA-138-5p (P<0.001). FISH results showed that miRNA-138-5p was localized in neuronal cytoplasm. Compared with CIBP+siNC group, CIBP+siRNA group exhibited significantly reduced lncRNA NONRATT021203.2 expression (P<0.05) and increased miRNA-138-5p expression (P<0.05). Compared with CIBP+NC group, PWT was significantly increased in CIBP+miRNA-138-5p mimics group (P<0.01). Bioinformatics analysis revealed the presence of a potential binding site between lncRNA NONRATT021203.2 and miRNA-138-5p. Conclusion: LncRNA NONRATT021203.2 could reduce the intracellular free miRNA-138-5p level by competitively binding to miRNA-138-5p, thereby promoting the occurrence and development of bone cancer pain.

Objective: To investigate the association between the polymorphisms of meteorinlike protein (METRNL) gene rs4986080 and type 2 diabetes mellitus (T2DM). Methods: A total of 467 subjects were selected, including 315 T2DM patients and 152 individuals with normal glucose tolerance (NGT). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect the polymorphisms of METRNL gene rs4986080 in all subjects, then analyzed the association between these polymorphisms and T2DM. Results: The constituent ratio of GG genotype and the frequency of G allele at rs4986080 locus of METRNL gene was higher in the T2DM group compared to the NGT group (P<0.05). Compared to individuals with GG genotype at the METRNL gene rs4986080 locus, those with AA+AG genotypes showed significant lower waisttohip ratio, fasting plasma glucose (FPG), 2hour postprandial glucose (2hPG), glycated hemoglobin (HbA1c), and homeostasis model assessment of insulin resistance (HOMA-IR) (P<0.05). Logistic regression analysis showed that A allele at rs4986080 locus of METRNL gene was probably a protective factor for T2DM. Conclusion: The polymorphisms of METRNL gene rs4986080 may be associated with the occurrence of T2DM, and A allele may be a protective factor for T2DM.

Objective: To explore the feasibility of preoperative prediction of perineural invasion (PNI) and lymphovascular invasion (LVI) in advanced gastric cancer using dual-energy computed tomography (DECT) venous phase imaging features and spectral parameters of a hypotonic water-filled stomach, along with clinical laboratory information. Methods: A retrospective analysis was conducted on 161 cases of advanced gastric cancer that underwent DECT imaging within one week before surgery, and the cases were randomly divided into training sets and test sets in a 7∶3 ratio. Based on postoperative pathological assessment, 115 cases demonstrated LVI and/or PNI positivity, whereas 46 cases were negative for both. A predictive model for LVI/PNI was developed using venous phase imaging of a hypotonic water-filled stomach, DECT parameters ［including the slope of the spectral Hounsfeld unit curve (between 40 keV and 100 keV), normalized iodine concentration (NIC), and effective atomic number］, and clinical laboratory data (inflammatory and tumor markers). The predictive performance of the model was evaluated using the area under the ROC curve (AUC), and its clinical utility was assessed using decision curve analysis. Results: The AUC values of the radiomics model (Rad-score) in the training sets and test sets were 0.776 (95%CI: 0.653-0.821) and 0.781（95%CI：0.582~0.847）, respectively. The independent predictors for the DECT parametric model was NIC, with AUC values of 0.729（95%CI：0.615~0.790 in the training sets and 0.771（95%CI：0.604~0.864）in the test sets. For the clinical information predictive model, the independent predictor was lymphocyte percentage, with AUC values of 0.693 (95%CI: 0.638-0.805) in the training sets and 0.502 (95%CI: 0.352-0.648) in the test sets. The combined model integrating the Rad-score, DECT parameters, and clinical information had independent predictors including Rad-score, NIC, and lymphocyte percentage. The AUC values for this combined model were 0.880 (95%CI: 0.701-0.859) in the training sets and 0830 (95%CI: 0.602-0.857) in the test sets, demonstrating superior performance compared to the radiomics model, DECT parametric model, and clinical model. The DeLong test showed that the AUC of the combined model was significantly higher than that of the radiomics model, DECT parametric model, and clinical information model in the training sets (Z=1.979, P=0.048; Z=3.199, P=0.001; Z=3.053, P=0.001). In the test sets, the AUC of the combined model was also significantly higher than that of the clinical information model (Z=2.417, P=0.015). Decision curve analysis revealed that when the risk threshold ranges from 0.15 to 0.96, adopting the combined model for treatment guidance yielded a higher clinical net benefit rate. Conclusion: The integrated model, incorporating radiomics, NIC, and lymphocyte percentage, serves as a comprehensive predictive model for assessing lymphovascular invasion and perineural invasion status in advanced gastric cancer.

Objective: To investigate the median effective dose (ED50) and 95% effective dose (ED95) of ciprofol for combined procedural sedation during spinal anesthesia in patients with different ages. Methods: A total of 67 patients scheduled for lower limb orthopedic surgery under subarachnoid block from December 2023 to March 2024 were enrolled and divided into three groups by age: the youth group (18-40 years old, 22 cases), the middleaged group (41-64 years old, 24 cases), and the elderly group (65-80 years old, 21 cases). After completion of spinal anesthesia, the Dixon up-and-down sequential method was used to administer ciprofol for procedural sedation, with an initial dose of 0.2 mg/kg and a dose gradient of 0.05 mg/kg between consecutive patients. Two minutes after administration, a modified observer′s assessment of alertness/sedation scale (MOAA/S) score ≤3 and a bispectral index (BIS) <85 were defined as satisfactory sedation. The dose for subsequent patients was adjusted, and the study was terminated after MOAA/S score ≥7 cross inflection points appeared. The Probit regression analysis was used to calculate ED50 and ED95 in each group. Vital signs, MOAA/S scores, BIS values, and the incidence of adverse reactions were recorded. Results: The ED50 and ED95 of the youth group were 0.263 mg/kg (95%CI: 0.232-0.300 mg/kg) and 0.318 mg/kg (95%CI: 0.288-0.509 mg/kg), respectively; those of the middle-aged group were 0.208 mg/kg (95%CI: 0.178-0.238 mg/kg) and 0.264 mg/kg (95%CI: 0.235-0.417 mg/kg); those of the elderly group were 0.178 mg/kg (95%CI: 0.130-0.217 mg/kg) and 0244 mg/kg (95%CI: 0.209-0.526 mg/kg). There were no statistically significant differences in vital signs, MOAA/S scores, BIS values, or the incidence of adverse reactions among the three groups during procedural sedation (all P>0.05). Conclusion: The effective doses of ciprofol for combined procedural sedation during spinal anesthesia depend on patients, age groups, and ED50 and ED95 decrease with increasing age.