[摘 要] 目的：探究重楼皂苷Ⅱ（PPⅡ）抑制甲状腺癌（TC）恶性生物学行为的分子机制。方法：常规培养甲状腺癌细胞TPC1，实验分为sh-NC、sh-可溶性半乳糖凝集素3（sh-LGALS3）、OE-NC、OE-LGALS3和 OE-LGALS3 + PPⅡ组，用转染试剂将应用质粒转染至各组TPC1细胞中。qPCR法检测TPC1细胞中LGALS3 mRNA的表达，WB法检测各组TPC1细胞中LGALS3、PI3K/AKT信号通路相关蛋白的表达，CCK-8法、Transwell实验、划痕愈合实验和流式细胞术分别检测各组TPC1细胞增殖、迁移和侵袭能力，以及细胞凋亡情况。结果：PPⅡ抑制TPC1细胞的增殖、迁移和侵袭，并诱导其凋亡（均P < 0.000 1）。数据库数据分析显示LGALS3在甲状腺癌组织中高表达（P < 0.001）且是PPⅡ的靶基因。LGALS1在TPC1细胞中呈高表达（P < 0.000 1），敲减LGALS3抑制TPC1细胞的恶性生物学行为，并促进其凋亡（均P < 0.000 1），PPⅡ通过抑制LGALS3 mRNA和蛋白的表达（P < 0.01或P < 0.001）从而抑制TPC1细胞的恶性生物学行为（P < 0.01或P < 0.000 1），PPⅡ抑制LGALS3表达抑制PI3K/AKT信号通路的激活水平（P <0.001或P <0.000 1），LGALS3通过PI3K/AKT信号通路促进TPC1细胞的恶性生物行为（P < 0.000 1）。结论：PPⅡ通过抑制TPC1细胞中LGALS3的表达，缓解PI3K/AKT信号通路的过度激活从而发挥抑癌作用。

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

BACKGROUND:Studies have shown that upregulation of heme oxygenase-1 expression enhances cellular antioxidant and anti-apoptotic abilities.However,the effects of upregulating heme oxygenase-1 expression on the proliferation and differentiation of neural stem cells under oxidative stress conditions remain unclear.OBJECTIVE:To investigate the influence of heme oxygenase-1 overexpression on the survival and differentiation capacity of neural stem cells under oxidative stress conditions.METHODS:(1)Mouse primary neural stem cells were isolated and cultured from newborn Balb/c mice.Immunofluorescence was used to detect the neural stem cell marker Nestin.(2)Lentivirus was used to infect neural stem cells to induce heme oxygenase-1 overexpression.Flow cytometry was used to assess green fluorescent protein fluorescence.Western blot assay was performed to detect the expression levels of heme oxygenase-1.(3)H2O2 was added to the lentivirus-infected neural stem cell culture medium to simulate the oxidative stress microenvironment after spinal cord injury.Effects of heme oxygenase-1 overexpression on neural stem cell proliferation and apoptosis levels were analyzed using cell proliferation assay kits,cell apoptosis assay kits,and TUNEL staining kits.(4)The levels of lipid oxidation markers malondialdehyde,catalase,superoxide dismutase,and glutathione peroxidase were detected using assay kits.(5)Flow cytometry was used to measure intracellular reactive oxygen species levels,and neural stem cell differentiation into astrocytes and neurons.(6)The effect of heme oxygenase-1 overexpression on neuronal axon growth during neural stem cell differentiation was observed under optical and fluorescence microscopes.RESULTS AND CONCLUSION:(1)Mouse neural stem cells exhibited stable morphology,good growth status,and high expression of Nestin as detected by immunofluorescence.(2)Western blot analysis showed that the overexpression of heme oxygenase-1 in the overexpression group was significantly higher than that in the empty carrier control group.Flow cytometry was used to detect the expression of green fluorescent protein in the neural stem cells of the heme oxygenase-1 overexpression group and empty vector control group.(3)Overexpression of heme oxygenase-1 maintained the proliferative activity of neural stem cells and significantly reduced the number of apoptotic cells under oxidative stress conditions.(4)Overexpression of heme oxygenase-1 inhibited lipid peroxidation of neural stem cells under oxidative stress microenvironment,enhanced the expression of enzymes related to maintaining the oxidative-reductive balance,and significantly reduced intracellular reactive oxygen species levels.(5)Overexpression of heme oxygenase-1 promoted the differentiation of neural stem cells into neurons and reduced differentiation into astrocytes.(6)The heme oxygenase-1 overexpression group exhibited longer axons,and more intercellular connections.The above results indicate that overexpression of heme oxygenase-1 can alleviate oxidative damage of H2O2-induced neural stem cells,reduce neural stem cell apoptosis,promote proliferation,and facilitate differentiation of neural stem cells into neurons.

BACKGROUND:Formononetin demonstrates potent anti-inflammatory and antioxidant abilities.However,its protective effect on nucleus pulposus mesenchymal stem cells is not yet clear.OBJECTIVE:To investigate the effect and mechanism of formononetin on nucleus pulposus mesenchymal stem cells under an inflammatory microenvironment.METHODS:(1)Primary nucleus pulposus mesenchymal stem cells were isolated from the intervertebral discs of SD rats,and flow cytometry was performed to identify the surface markers of mesenchymal stem cells.(2)The CCK-8 assay was employed to evaluate the impact of lipopolysaccharide and formononetin on the proliferation viability of nucleus pulposus mesenchymal stem cells,aiming to determine the appropriate concentration of formononetin for subsequent cell treatments.(3)An inflammatory microenvironment was simulated by adding 5 μg/mL lipopolysaccharide to the DMEM/F-12 culture medium,and nucleus pulposus mesenchymal stem cells were treated with different concentrations of formononetin for 24 hours.Levels of inflammation markers were detected using western blot assay,real-time quantitative PCR,and immunofluorescence.Western blot assay was conducted to measure the protein levels of the nuclear factor kappa B signaling pathway.RESULTS AND CONCLUSION:(1)The nucleus pulposus mesenchymal stem cells cultured in adherent wall were shuttle-shaped with good growth status.The results of flow cytometry showed that the surface markers of mesenchymal stem cells were positive for CD29,CD44,and CD90,and the surface markers of hematopoietic stem cells were negative for CD34 and CD45.(2)The treatment with formononetin at 12.5,25,50,100,and 200 μmol/L concentrations for 24 hours had no significant proliferation inhibitory effect on nucleus pulposus mesenchymal stem cells.Compared with the lipopolysaccharide group,the cell viability of nucleus pulposus mesenchymal stem cells treated with 12.5,25,and 50 μmol/L formononetin for 24 hours was significantly increased,so formononetin at 12.5,25,and 50 μmol/L concentrations was subsequently selected as the low,medium,and high concentrations for treating nucleus pulposus mesenchymal stem cells.(3)Compared with the lipopolysaccharide group,the protein and mRNA expressions of matrix metalloproteinase-3,matrix metalloproteinase-13,and tumor necrosis factor-α in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.(4)Compared with the lipopolysaccharide group,the expressions of phosphorylated nuclear factor kappa B and phosphorylated nuclear factor kappa B inhibitor protein in nucleus pulposus mesenchymal stem cells in the low,medium,and high concentrations of formononetin groups were significantly decreased(P＜0.05)in a dose-dependent manner.The above results suggest that formononetin may attenuate lipopolysaccharide-induced inflammation in rat nucleus pulposus mesenchymal stem cells by inhibiting the activation of the nuclear factor kappa B signaling pathway.

AIM:To investigate the therapeutic effects and potential mechanism of pachymic acid(PA)on li-popolysaccharide(LPS)-induced acute kidney injury(AKI)in mice.METHODS:(1)Genes related to AKI were screened using the DAVID database.Core genes were identified by intersecting related genes and analyzed using Cyto-scape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed through the DAVID database for the cross-targets.Molecular docking and activity assays were conducted on the primary core targets.(2)A total of 100 C57BL/6J mice were randomly divided into five groups:normal control(NC),model(LPS),solvent control(LPS+DMSO),and treatment groups(LPS+PA-10 and LPS+PA-20),with 20 mice in each group.The LPS-AKI model was established by intraperitoneal injection of 18 mg/kg LPS.The treatment groups received 10 mg/kg and 20 mg/kg PA,respectively,and the solvent control group was administered an equivalent dose of DMSO.Mice were euthanized 24 h after injection.Serum was collected for biochemical analysis,and Western blot was used to detect neutro-phil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),caspase-3,cleaved caspase-3,interleu-kin-1β(IL-1β),and monocyte chemoattractant protein-1(MCP-1)protein expression.RT-qPCR was employed to detect inflammatory factor mRNA levels.Molecular docking was used to simulate the optimal binding site of PA to caspase-3.En-zyme activity assays were performed to assess caspase protein activity,and renal lesions were observed via hematoxylin and eosin(HE)staining.Apoptosis was detected by TUNEL staining.RESULTS:(1)Thirty-one potential targets of PA against AKI were identified through network pharmacology.GO and KEGG enrichment analyses indicated that these tar-gets were primarily involved in immune response,inflammatory processes,apoptosis and survival,angiogenesis and hemo-dynamics,oxidative stress,and endoplasmic reticulum stress.Key targets included CASP3(caspase-3),PTGS2,BCL2,CCL2,and CYP219.(2)PA treatment improved renal function and reduced tubular epithelial injury.It significantly de-creased NGAL,KIM-1,and cleaved caspase-3 protein levels,as well as inflammatory factors TNF-α,IL-1β,and MCP-1 mRNA and protein expression.PA also reduced apoptosis of renal tubular epithelial cells.Enzyme activity assays and mo-lecular docking revealed that PA exerted its anti-apoptotic effect by directly binding to caspase-3,thereby inhibiting its ac-tivation by caspase-8.CONCLUSION:PA demonstrated a therapeutic effect in LPS-AKI,potentially through the inhibi-tion of inflammatory factor synthesis and release,as well as the inhibition of caspase-3 activation by caspase-8,reducing apoptosis in renal tubular epithelial cells.

Objective To investigate the correlation of solute carrier protein 17 family member 1 (SLC17A1) gene single nucleotide polymorphisms (SNPs) and susceptibility to hyperuricemia (HUA) in automotive manufacturing workers. Methods A total of 192 Han male workers diagnosed with HUA were selected as the case group, 192 Han male workers without HUA from the same enterprises were selected as the control group. These workers were determined by the matching factor of age, total length of service, and body mass index by the 1∶1 case-control study method. Peripheral venous blood from the workers was collected for DNA extraction. Two SNPs of SLC17A1 were genotyped by MassArray system. Results The gene frequency distributions of SLC17A1 rs2096386 and rs1183201 of workers in the control group were in consistent with the Hardy-Weinberg equilibrium test (both P>0.05). The allele frequency distribution of rs2096386, and the genotype and allele frequency distribution of rs1183201 were significantly different between workers in the two groups (all P<0.05). There was no significant difference in genotype frequency distribution of rs2096386 between workers in the two groups (P>0.05). The results of conditional logistic regression analysis showed that workers with G allele at rs2096386 increased the risk of HUA [odds ratio (OR)=1.43, 95% confidence interval (CI)=1.01-2.04], workers with T allele at rs1183201 increased the risk of HUA (OR=2.03, 95%CI =1.29-3.19), after adjusting for confounding factors such as serum creatinine, blood urea nitrogen, alanin aminotransferase and aspartate aminotransferase. While workers with TA and TA+AA genotypes at rs1183201 had a lower risk of HUA than those with TT genotype (OR=0.51, 95%CI =0.30-0.85; OR=0.50, 95%CI =0.30-0.83), workers with TA genotype at rs1183201 had a lower risk of HUA than those with TT+AA genotype (OR=0.53, 95%CI =0.32-0.88). Conclusion The polymorphisms at rs2096386 and rs1183201 of SLC17A1 gene may be correlated with HUA susceptibility among automobile manufacturing workers in Guangzhou City.

Objective:To explore the application effectiveness of the large language model DeepSeek in the cultivation of clinical thinking skills for medical laboratory technology students.Methods:A non-randomized controlled study was conducted. In the 2024-2025 academic year, two classes of second-year medical laboratory technology students from Hubei University of Chinese Medicine were selected and divided into a DeepSeek-assisted teaching group (Class A, n=53) and a traditional teaching control group (Class B, n=53), totaling 106 students. Both groups followed a 20-week problem-based learning (PBL) framework with identical teaching content, instructors, and class hours. Class A utilized DeepSeek via the"Learning Pass AI"platform for case diagnosis reasoning, prompt construction training, test plan formulation, and result analysis, while Class B received traditional PBL instruction. Paired t-tests were used to compare pre-and post-teaching scores in clinical thinking skills, AI interaction literacy, and prompt construction in Class A. Independent samples t-tests and chi-square ( χ2) tests were used to evaluate differences in case reasoning scores, etiology analysis accuracy, and teaching satisfaction between groups. Structured questionnaires supplemented the evaluation of model-assisted teaching processes. Results:The comparison of pre-and post-teaching scores in Class A showed that post-teaching scores significantly improved in clinical thinking skills[(4.02±0.45) points vs. (3.09±0.50) points, t=2.23)] and AI interaction literacy [(4.62±0.41) points vs. (3.27±0.54) points, t=2.18]. Compared to Class B, Class A demonstrated superior performance in case reasoning scores [(81.1±3.8) points vs.(74.3±4.2) points, t=8.97], etiology analysis accuracy [94.3% (50/53) vs. 81.1% (43/53), χ2=4.29], and teaching satisfaction [(95.6±3.2)points vs. (82.6±4.8) points, t=11.86] ( P<0.05). The results of questionnaires indicated that during model application, the prompt construction improved in logic [(2.85±0.58) points to (4.25±0.50) points, t=14.23, P<0.01] and innovation [(2.60±0.53) points to (4.05±0.46) points, t=11.57, P<0.05], but question clarity (77.4%, 41/53) and medical terminology accuracy (43.4%, 23/53) remained primary shortcomings. Conclusion:Integrating large language models into AI-teacher collaborative learning pathways can effectively promote students′ autonomous inquiry and clinical reasoning skills, thereby enhancing medical laboratory technology students′ clinical thinking skills.

AIM:To investigate the therapeutic effects and potential mechanism of pachymic acid(PA)on li-popolysaccharide(LPS)-induced acute kidney injury(AKI)in mice.METHODS:(1)Genes related to AKI were screened using the DAVID database.Core genes were identified by intersecting related genes and analyzed using Cyto-scape software.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses were performed through the DAVID database for the cross-targets.Molecular docking and activity assays were conducted on the primary core targets.(2)A total of 100 C57BL/6J mice were randomly divided into five groups:normal control(NC),model(LPS),solvent control(LPS+DMSO),and treatment groups(LPS+PA-10 and LPS+PA-20),with 20 mice in each group.The LPS-AKI model was established by intraperitoneal injection of 18 mg/kg LPS.The treatment groups received 10 mg/kg and 20 mg/kg PA,respectively,and the solvent control group was administered an equivalent dose of DMSO.Mice were euthanized 24 h after injection.Serum was collected for biochemical analysis,and Western blot was used to detect neutro-phil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),caspase-3,cleaved caspase-3,interleu-kin-1β(IL-1β),and monocyte chemoattractant protein-1(MCP-1)protein expression.RT-qPCR was employed to detect inflammatory factor mRNA levels.Molecular docking was used to simulate the optimal binding site of PA to caspase-3.En-zyme activity assays were performed to assess caspase protein activity,and renal lesions were observed via hematoxylin and eosin(HE)staining.Apoptosis was detected by TUNEL staining.RESULTS:(1)Thirty-one potential targets of PA against AKI were identified through network pharmacology.GO and KEGG enrichment analyses indicated that these tar-gets were primarily involved in immune response,inflammatory processes,apoptosis and survival,angiogenesis and hemo-dynamics,oxidative stress,and endoplasmic reticulum stress.Key targets included CASP3(caspase-3),PTGS2,BCL2,CCL2,and CYP219.(2)PA treatment improved renal function and reduced tubular epithelial injury.It significantly de-creased NGAL,KIM-1,and cleaved caspase-3 protein levels,as well as inflammatory factors TNF-α,IL-1β,and MCP-1 mRNA and protein expression.PA also reduced apoptosis of renal tubular epithelial cells.Enzyme activity assays and mo-lecular docking revealed that PA exerted its anti-apoptotic effect by directly binding to caspase-3,thereby inhibiting its ac-tivation by caspase-8.CONCLUSION:PA demonstrated a therapeutic effect in LPS-AKI,potentially through the inhibi-tion of inflammatory factor synthesis and release,as well as the inhibition of caspase-3 activation by caspase-8,reducing apoptosis in renal tubular epithelial cells.

Objective: To investigate the survival rate of female breast cancer patients in Panyu District, Guangzhou City, so as to provide the basis for improving the prognosis of breast cancer patients. Methods: Basic information including age, clinical stage and surgical treatment of female breast cancer patients registered in Panyu District and diagnosed in 2017 were collected through the Guangzhou Municipal Cancer Registration and Reporting Management System, and were followed up until December 31, 2022. The survival rate was calculated using the life table. Factors affecting survival time among female breast cancer patients were analyzed using a multivariable Cox proportional hazard regression model. Results: A total of 227 female breast cancer patients were reported in Panyu District in 2017, and had a median age of 51 (interquartile range, 17) years. There were 43 cases (18.94%) in stage Ⅰ, 55 cases (24.23%) in stage Ⅱ, 63 cases (27.75%) in stage Ⅲ, 27 cases (11.89%) in stage Ⅳ, and 39 cases (17.18%) with unknown staging. Surgical treatment was performed in 204 cases (89.87%), and chemotherapy was administered in 73 cases (32.16%). By December 31, 2022, there were 40 deaths and 14 patients lost to follow-up. The one-year, three-year and five-year survival rates were 96.44%, 87.45% and 82.87%, respectively. Multivariable Cox proportional hazard regression analysis showed that older age (HR=1.023, 95%CI: 1.002-1.046), clinical stage Ⅲ (HR=10.050, 95%CI: 1.324-76.270) or IV (HR=42.663, 95%CI: 5.588-325.742) were associated with a higher risk of mortality in female breast cancer patients, while surgical treatment (HR=0.278, 95%CI: 0.130-0.598) was associated with a lower risk of mortality. Conclusions The five-year survival rate of female breast cancer patients in Panyu District was 82.87%. Age, clinical stage and surgical treatment were the main influencing factors for the survival time of female breast cancer patients.

Objective:To investigate the expression of m6A methyltransferase-like protein 3(METTL3)in human osteosarcoma(OS)tissue and the effect of METTL3 on the proliferation and invasion of MG-63 cells.Methods:Quantitative PCR and Western blotting were used to measure the expression level of METTL3 in 5 pairs of human OS tissue samples and investigate the effect of METTL3 on the downstream long non-coding RNA(lncRNA)small nucleolar RNA host gene 5(SNHG5)and the Wnt/β-catenin signaling path-way;methylated RNA immunoprecipitation was used to observe the effect of METTL3 knockdown on the m6A level of lncRNA SNHG5;CCK-8 assay and Transwell assay were used to measure cell proliferation and invasion;Western blotting was used to measure the change in the activity of the Wnt/β-catenin signaling pathway.Results:Compared with the paracancerous normal tissue,human OS tis-sue showed significant increases in the mRNA and protein expression levels of METTL3(P=0.034 and 0.002),and knockdown of METTL3 reduced the m6A level and mRNA expression level of lncRNA SNHG5(P=0.027 and 0.002).Functionally,knockdown of METTL3 inhibited the proliferation and invasion of MG-63 cells(P=0.014 and 0.001)and the protein expression levels of Wnt1,Wnt3a,Wnt10a,β-catenin,and C-myc(all P<0.01),while overexpression of SNHG5 promoted cell proliferation(P=0.027)and inva-sion(P=0.006),activated the Wnt/β-catenin signaling pathway(P<0.01),and reversed the antitumor effect induced by METTL3 knockdown(P<0.01).Conclusion:METTL3-mediated m6A modification of lncRNA SNHG5 promotes the proliferation and invasion of MG-63 cells by activating the Wnt/β-catenin signaling.