To investigate the mechanism of dihydroartemisinin （DHA） against influenza A virus （IAV） in human non-small cell lung cancer cells （A549） and normal human lung epithelial cells （BEAS-2B）.
Methods
The CC50 values of DHA on A549 and BEAS- 2B cells was calculated after treatment with different concentrations of DHA for 24 hours. A549 cells were infected with H1N1 for 3 hours， then treated with different concentrations of DHA （0.1， 1， and 10 μmol/ L）for 24 hours， and cell viability was measured using the CCK-8 assay. The inhibitory effect of DHA on H1N1 replication was detected by plaque assay. Metabolomic was employed to analyze the differential metabolites induced by DHA following H1N1 infection， and the KEGG pathway enrichment analysis was conducted to map the pathways associated with these differential metabolites.The contents of reduced glutathione （GSH） and oxidized glutathione （GSSG）， intracellular reactive oxygen species （ROS）， and mitochondrial function were measured using commercial kits. The expression of RIG1， MAVS， p-TBK1， and p-IRF3 proteins was detected by Western blot. The mRNA expression and secretion levels of IFN-α and IFN-β were measured by real-time quantitative PCR（RT-qPCR） and enzyme-linked immunosorbent assay （ELISA）， respectively. The comparison of means between two groups was performed using an independent samples t-test， the comparison of means among multiple groups was conducted using one-way ANOVA， and pairwise comparisons of means among multiple groups were tested using the Tukey's test method.
Results
The CC50 values of DHA in A549 and BEAS-2B cells was 18.26 μmol/ L and 28.61 μmol/L， respectively. At a concentration of 1 μmol/L， DHA enhanced cell viability and reduced plaque formation. DHA inhibited the metabolism of glutamate， glycine， and ornithine induced by influenza virus infection by regulating glutathione metabolism， and preventing GSH depletion as well as the formation of GSSG and ROS，thereby alleviating mitochondrial damage. Furthermore， DHA treatment increased the expression of RIG1 and MAVS proteins， promoting the aggregation of MAVS to mitochondria， and activating the phosphorylation of TBK1 and IRF3 proteins， while enhanced both mRNA expression and protein secretion of IFN-α and IFN-β.
Conclusion
DHA exerts its antiviral effects against influenza virus by regulating intracellular glutathione homeostasis， reducing ROS formation， alleviating mitochondrial damage， and enhancing the innate immune response.
To investigate the effect and regulatory mechanism of chromosome structure maintenance protein 4 （SMC4） on hypoxemic human pulmonary artery smooth muscle（hPASMC）pyroptosis.
Method
hPASMCs were used as experimental subjects， and the cells were randomly divided into normoxic group， hypoxic group， hypoxic + NC group， and hypoxic + si-SMC4 group. Except for the normoxic group， which was cultured under 37 ℃ and 5% CO2 conditions，the hypoxic group and hypoxic + si-SMC4 group were cultured for 24 hours under 3%O2 and 5%CO2 restrictions to establish a hypoxic model. Western blot was used to detect the expression levels of SMC4 and apoptosis related proteins such as interleukin-1β， GSDMD， GSDMD-N， and NOD like receptor protein 3 （NLRP3）. Lactate dehydrogenase （LDH） release assay was used to detect the release of LDH release in cell culture medium. Cell immunofluorescence DAPI/PI double staining to was used to detect the proportion of pyroptosis cells. Rats were divided into normoxic group and 10% O2 low oxygen group. Blood pressure monitoring and HE staining were used to determine the success of the animal model. Tissue immunofluorescence was used to detect SMC4 tissue localization，and Western blot was used to detect differential expression of SMC4 in various tissues and organs.Independent sample t-test was used for comparison between the two groups； Multiple groups were analyzed using one- way ANOVA， and pairwise comparisons were processed using SNK-q test.
Result
Compared with the normoxic group， the expression level of SMC4 protein was increased in the lung tissue of rats in the hypoxic group （0.86 ± 0.06 vs 0.75 ± 0.05， P ＜ 0.05）， and SMC4 was localized in the smooth muscle layer of pulmonary vessels. Compared with the normoxic group， the hypoxic group showed increased pulmonary artery thickness [（30.33 ± 3.49 ） vs （13.52 ± 2.34） μm］，NEMO （0.77 ± 0.14 vs 0.31 ± 0.09）， NLRP3 （1.04 ± 0.15 vs 0.54 ± 0.22）， GSDMD （1.34 ± 0.32 vs 0.86 ± 0.34）， GSDMD-N （0.90 ± 0.11 vs 0.45 ± 0.02）， IL-1β （3.15 ± 0.56 vs 0.53 ± 0.06）expression， percentage of LDH release （71.69 ± 10.36 vs 19.94 ± 3.54）， and PI positive cells （4.81 ±0.64 vs 2.18 ± 0.31）， P ＜ 0.05. The low oxygen+si-SMC4 group can reverse the above effects.
Conclusion
SMC4 is upregulated in hypoxic pulmonary hypertension， activates the expression of cell apoptosis related proteins， and increases the release of LDH from hPASMCs， and increasing the proportion of PI positive cells. Knocking down SMC4 may reverse these changes， which may be related to NEMO regulation.
To evaluate the therapeutic effects of human umbilical cord mesenchymal stem cell-derived exosomes （hucMSC-Exo） in inhibiting the activation of hepatic stellate cells （HSCs） and improving liver fibrosis in vivo and in vitro.
Methods
The exosomes were isolated and purified by ultracentrifugation and characterized by transmission electron microscopy（TEM）， nanoparticle size analysis， and Western blot. For in vivo experiments， 40 male C57BL/6J mice were divided into 4 groups： normal control group， model group， exosome treatment group，and colchicine treatment group （n = 10）. The hepatic fibrosis model was built by intragastric administration of CCl4-olive oil （1 ： 3）， 2 times/week for 8 weeks. After 4 weeks of modeling，intervention treatments were carried out： exosomes were proceeded by intravenous injection administration （300μg/100g body weight）， and colchicine was given by intragastric administration（0.01mg/100g body weight）. At the end of 8 weeks， levels of ALT， AST， IV-C， LN， PC-III， and HAase in serum were measured using biochemical assays and ELISA methods. Liver histopathological changes were assessed with H&E and Masson staining， and α-smooth muscle actin （α-SMA） and Collagen-Ⅰ expression in liver tissue were detected using immunohistochemistry. For in vitro experiments， the human HSC cell line LX-2 was treated with TGF-β1 and hucMSC- Exo， and named blank control， TGF-β1 induction group， TGF-β1 induction+hucMSC-Exo group. Immunofluorescence was used to analyze the expression of α-SMA， and Western blot was performed to detect the expression levels of α-SMA and Collagen-Ⅰ proteins. One-way analysis of variance was used for comparison between multiple groups， and LSD-t test was used for pairwise comparison between groups.
Results
Compared to the normal control group， the levels of ALT， AST， Collagen-Ⅳ， LN，PC-Ⅲ， and HAase were increased （all P ＜ 0.01）. Compared to the model group， the levels of ALT[（3.17 ± 0.70） vs （ 6.34 ± 0.37） U/ L］， AST [（2.91 ± 0.60） vs （7.11 ± 0.41）U/ L］， Collagen-Ⅳ[（116.38 ± 4.96）vs （143.79 ± 6.71） μg/ L］， LN [（762.96 ± 120.45） vs （950.86 ± 29.77） μg/ L］，PC- Ⅲ[（14.88 ± 0.83）vs （18.39 ± 2.57） μg/ L］and HAase [（109.92 ± 11.32）vs （187.58 ±10.42） ng/L］ were decreased significantly in the hucMSC-Exo treatment group （all P ＜ 0.01）.Immunohistochemical results of liver tissues showed that the level of the expression of α-SMA and Collagen-Ⅰ was increased （all P ＜ 0.01）. Compared to the model group， the expression of α-SMA（ 337.05 ± 39.13 vs 2 681.83 ± 312.63）， and Collagen-Ⅰ expression （479.32 ± 86.25 vs 1 022.57 ±102.13）were decreased in liver tissues in hucMSC-Exo intervention group （all P ＜ 0.01）. The Western blot data of LX-2 cell lysate indicated that hucMSC- Exo inhibited the expression of α-SMA and Collagen-Ⅰ in LX-2 cells compared to the model group [（0.87 ± 0.09 vs 1.01 ± 0.34） and（0.46 ± 0.07 vs 0.75 ± 0.22）］（all P ＜ 0.05）.
Conclusion
Early stage transplantation of hucMSCExo ameliorates hepatic fibrosis induced by CCl4 in rats， which may be induced by suppressing HSCs activation and the synthesis and secretion of collagen.
To explore whether zinc finger E-box binding homeobox 1 （ZEB1）promotes the proliferation， migration and invasion of prostate cancer cells by regulating the Wnt/β-catenin signaling pathway.
Methods
siZEB1 and Wnt3a overexpression plasmids were transfected into PC-3 and LNCaP prostate cancer cells， and named control group， the siZEB1 group， and the siZEB1+OE-Wnt3a group， respectively. Real-time fluorescence quantitative polymerase chain reaction （RT-qPCR） was used to detect the mRNA expression levels of ZEB1， Wnt3a， β-catenin，c-Myc， Cyclin D1， E-cadherin， N-cadherin in human normal prostate epithelial cell RWPE- 1 and PC-3， LNCaP prostate cancer cells. Western blot was used to detect the protein contents of Wnt3a，β-catenin， c-Myc， Cyclin D1， E-cadherin and N-cadherin in PC-3 as well as LNCaP prostate cancer cells. Cell counting kit-8 （CCK-8） assay and plate clone assay were used to detect the proliferation changes of prostate cancer cells after different treatments. Scratch assay and Transwell assay were used to detect the invasion and migration ability of prostate cancer cells after different treatments.Independent sample t-test was used for comparison between two groups， one- way ANOVA was used for comparison among multiple groups， and LSD-t test was used for pairwise comparison between groups.
Results
Compared with RWPE1 cells， the expression level of ZEB1 mRNA in prostate cancer cells PC-3 and LNCaP were upregulated [（1.99 ± 0.11）， （1.96 ± 0.13） vs （1.00 ± 0.05）］（both P ＜ 0.05）. Compared with the control group， the viability， the colony numbers [PC-3 cells：（21.33 ± 2.05） vs （48.67 ± 3.68） cells， LNCaP cells： （21.67 ± 2.87） vs （47.33 ± 2.05） cells］， the migration rate [PC-3 cells： （30.67 ± 2.49）% vs （48.33 ± 2.49）%， LNCaP cells： （28.33 ± 3.40） %vs （43.00 ± 2.94）%］， and the number of invaded cells [PC-3 cells： （43.00 ± 3.56） vs （64.00 ± 3.74）cells， and LNCaP cells： （42.67 ± 4.50） vs （61.33 ± 2.62）cells］ were decreased in cells transfected with siZEB1 （all P ＜ 0.05）， while the apoptosis rate [PC- 3 cells： （9.63 ± 0.66）% vs （1.25 ± 0.34）%，LNCaP cells： （9.34 ± 0.72）% vs （1.54 ± 0.42）%］ were increased （all P ＜ 0.001）. Compared with the control group， the expression levels of protein and mRNA of Wnt3a， c-Myc， Cyclin D1， N-cadherin andthe expression of β-catenin protein was down-regulated， while the expression levels of of E-cadherin protein and mRNA in PC-3 and LNCaP cells after transfected with siZEB1 （all P ＜ 0.05），which could be partially restored after co-transfection of siZEB1 and OE- Wnt3a. After co-transfection of siZEB1 and OE-Wnt3a， the viability， the colony number [PC-3 cells： （35.67 ± 2.05） vs （18.33 ±2.87） cells； LNCaP cells： （31.33 ± 2.52） vs （15.00 ± 3.61） cells］， the migration rate [PC- 3 cells：（50.67 ± 3.30）% vs （43.67 ± 3.68）%； LNCaP cells： （53.67 ± 3.21）% vs （41.33 ± 5.50）%］（all P ＜ 0.05）， the invaded cell number [PC-3 cells： （35.67 ± 1.24） vs （24.00 ± 2.45） cells； LNCaP cells： （34.33 ± 4.04） vs （22.00 ± 3.00） cells］ （all P ＜ 0.05） were increased， and the apoptosis rate was decreased [PC-3 cells： （6.74 ± 0.73）% vs （9.75 ± 1.08）% ； LNCaP cells： （8.30 ± 0.47）%vs （11.50 ± 0.56）%］ （all P ＜ 0.05） in PC-3 and LNCaP cells compared with siZEB1 group.
Conclusion
ZEB1 plays a significant role in the proliferation， migration， invasion， and apoptosis of prostate cancer cells by regulating the Wnt/β-catenin signaling pathway. Knockdown of ZEB1 can significantly inhibit the malignant phenotype of prostate cancer cells， while overexpression of Wnt3a can partially reverse this inhibitory effect.
To investigate the effects and mechanisms of atorvastatin on macrophage polarization in high-fat diet-induced metabolic-associated fatty liver disease （MAFLD）in New Zealand rabbits.
Methods
Single-cell datasets from healthy individuals and MAFLD patients were downloaded from the GEO database. Fifteen male New Zealand rabbits were divided into normal control group （NCG）， high-fat diet model group （MCG）， and high-fat diet +atorvastatin treatment group （PCG）. The rabbits in NCG were fed with standard diet， while those in other groups were fed with high-fat diet. Atorvastatin was administered at a dose of 4 mg/kg/day.After 8 weeks， Oil Red O staining was used to assess the differences in hepatic lipid deposition among groups. Western blot was performed to measure the protein expression levels of Toll-like receptor 4/ nuclear factor kappa B （TLR4/NF-κB） in liver tissues. Flow cytometry was used to determine the proportions of macrophage subtypes in peripheral blood. ANOVA-test was used for comparisons among multiple groups， and the Bonferroni method was used for pairwise comparisons between groups.
Results
Single-cell data analysis revealed that TLR4 is expressed across all macrophage subtypes in MAFLD patients and associated with M2 macrophage polarization. Compared to the NCG group， N the hepatic lipid depositionand the expression levels of TLR4 （0.68 ± 0.08 vs 0.37 ± 0.06），NF-κB proteins （1.81 ± 0.14 vs 1.27 ± 0.13） were significantly elevated in the MCG group after 8 weeks of high-fat diet feeding （P ＜ 0.001）， which could be restored after atorvastatin treatment（TLR4 protein 0.58 ± 0.08 vs 0.68 ± 0.08， NF-κB protein expression 1.60 ± 0.14 vs 1.81 ± 0.14） .Compared with MCG group， theproportion of M2 macrophages （0.42 ± 0.05）% vs （2.22 ± 0.16）%in peripheral blood was increased in PCG group （P ＜ 0.001）.
Conclusion
Peripheral blood macrophage subtypes are associated with MAFLD progression. Atorvastatin reduces hepatic lipid deposition in high-fat diet-induced New Zealand rabbits by modulating the TLR4/NF-κB signaling pathway and regulating M1/M2 macrophage polarization.
Based on bibliometric methods， a visualized analysis of research related to ferroptosis in colorectal cancer was conducted to explore the research hotspots and development trends in this field from 2017 to 2024.
Methods
Relevant literature published from January 1， 2017 to December 31， 2024 in the Web of Science Core Collection， China National Knowledge Infrastructure （CNKI）， Wanfang， and VIP databases were retrieved. The publication volume， countries， institutions， journals， and keywords were visualized by CiteSpace 6.4.R1 and Microsoft Excel 2016. Co-occurrence and clustering analysis of keywords were also performed.
Results
A total of 739 articles were included， with 598 in English and 141 in Chinese. The analysis results showed that the number of papers published from 2017 to 2019 was relatively low， but the number has increased since 2020. Globally， China is the country with the most papers published，Fudan University is the institution with the highest number of papers published， and Frontiers in Oncology is the journal that publishes the most such studies. Co-occurrence and clustering analysis of keywords identified four core research themes： the molecular mechanisms and metabolic regulation of ferroptosis， the expression and signaling pathway regulation of ferroptosis-related genes， ferroptosis and tumor immune regulation， and clinical therapeutic strategies and interventions based on ferroptosis. Keyword emergence analysis showed that ferroptosis regulation mechanism and metabolic regulation are continuous research hotspots in this field. In recent years， the research focus has gradually shifted to clinical applications， with particular attention paid to the research on endoplasmic reticulum stress， stem cell technology， photodynamic therapy， and inflammatory response.
Conclusion
The research on ferroptosis in colorectal cancer is in a rapid development stage， the exploration of the mechanism of ferroptosis in colorectal cancer and its clinical application are current research hotspots and emerging research directions.
