ObjectiveTo investigate the effect and mechanism of transplantation of human umbilical cord mesenchymal stem cell （hUC-MSC） with overexpression of the Numb gene in the treatment of cholestatic liver fibrosis （CLF）. MethodsThe technique of lentiviral transfection was used to induce the overexpression of the Numb gene in hUC-MSC （hUC-MSC^Numb-OE）， and hUC-MSC transfected with empty vector （hUC-MSC^OE-EV） was used as negative control. Bile duct ligation （BDL） was performed to establish a rat model of CLF， and then the rats were randomly divided into BDL group， hUC-MSC group， hUC-MSC^OE-EV group， and hUC-MSC^Numb-OE group， while a sham-operation group was also established. The rats in the intervention groups were given a single splenic injection of the corresponding cells after BDL， and samples were collected at the end of week 4. Related indicators were measured， including serum biochemistry， liver histopathology， the content of hydroxyproline （Hyp） in the liver， hepatic stellate cell activation， ductular reaction， liver regeneration， and the expression levels of key molecules in the Numb-p53 signaling axis. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the BDL group， the hUC-MSC group and the hUC-MSC^OE-EV group had significant reductions in the levels of serum biochemical parameters （aspartate aminotransferase， gamma-glutamyl transpeptidase， total bile acid， total bilirubin， and direct bilirubin）， liver fibrosis markers （the content of Hyp and the expression levels of alpha-smooth muscle actin， tumor necrosis factor-α， and transforming growth factor-beta 1）， and ductular reaction markers （the expression levels of CK7 and CK19） （all P <0.05）， and compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significantly greater improvements in the above indicators （all P <0.05）. In addition， compared with the hUC-MSC^OE-EV group， the hUC-MSC^Numb-OE group had significant improvements in the expression levels of liver regeneration-related markers （albumin and hepatocyte nuclear factor 4α） and the molecules associated with the Numb-p53 signaling axis （Numb， pNumb， Mdm2， and p53） （all P <0.05）. ConclusionOverexpression of the Numb gene can enhance the therapeutic effect of hUC-MSC on CLF， possibly by activating the Numb-PTB^L-p53-HNF4α axis， promoting the hepatic differentiation of hUC-MSCs and subsequently enhancing liver regeneration.

ObjectiveBased on the theory of "spleen governing transportation and transformation"， this study investigates the efficacy of Atractylodis Macrocephalae Rhizoma processed with Aurantii Fructus Immaturus juice（AMR-AFI） in improving slow-transit constipation（STC）， as well as the synergistic regulatory mechanism involving the microbiota-metabolism axis， thereby elucidating the scientific basis of its processing theory. MethodsAnimals were randomly divided into the control group， model group， positive drug（mosapride） group（3 mg·kg^-1）， and low-， medium-， and high-dose groups of AMR-AFI（3.9， 7.8， 15.6 g·kg^-1）. Except for the control group， the remaining five groups were induced with STC using loperamide hydrochloride. Following modeling， interventions were administered. All groups received continuous administration for 15 d， during which fecal samples， colon tissue， and serum were collected. Constipation improvement was assessed by measuring fecal moisture content and small intestinal propulsion rate， histological morphology of colonic tissue was observed via hematoxylin-eosin（HE） staining， and the levels of interleukin（IL）-6， tumor necrosis factor（TNF）-α， and IL-2 in serum were detected using enzyme-linked immunosorbent assay（ELISA）. Furthermore， the microbial community structure in mouse feces was analyzed by 16S rRNA sequencing， while transcriptomic sequencing was employed to screen differentially expressed genes in colonic tissue， followed by gene ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analyses. Finally， Spearman correlation analysis was conducted to explore the association between differential microbiota and differential genes. ResultsCompared with the control group， the intestinal propulsion rate and fecal moisture content in the model group were significantly decreased（P<0.01）， while serum levels of IL-6， TNF-α， and IL-2 were significantly elevated（P<0.01）. HE staining showed damage and shedding of colonic mucosal epithelial cells， along with a reduction in goblet cells in the model group. In comparison with the model group， all treatment groups improved the pathological state of the colonic mucosa to varying degrees and reduced serum levels of IL-6， TNF-α， and IL-2（P<0.01）. Among these， the high-dose group of AMR-AFI significantly increased the intestinal propulsion rate and fecal moisture content of rats（P<0.05， P<0.01）. Further transcriptomic analysis revealed that a total of 104 differentially expressed genes were identified from comparisons between the model group and the control group， as well as between the model group and the high-dose group of AMR-AFI. These genes were mainly enriched in pathways closely related to STC pathogenesis， such as arachidonic acid metabolism and aldosterone-regulated sodium reabsorption. 16S rRNA sequencing results indicated that AMR-AFI reversed the structural imbalance of the gut microbiota in model mice， increased species richness， downregulated the relative abundance of pro-inflammatory bacteria such as Parasutterella， and enriched beneficial and butyrate-producing bacteria， including Lachnospiraceae_NK4A136_group， Ruminococcaceae， and Lachnospiraceae. Spearman correlation analysis further showed that the beneficial bacteria enriched in the AMR-AFI group were negatively correlated with genes involved in the arachidonic acid metabolic pathway and positively correlated with genes in the aldosterone-regulated sodium reabsorption pathway. In contrast， pro-inflammatory bacteria in the model group exhibited the opposite correlation trends. ConclusionAMR-AFI can effectively exert synergistic therapeutic effects on STC by regulating intestinal microbiota， arachidonic acid-mediated inflammatory metabolism， and aldosterone-regulated water-salt balance pathways.

ObjectiveBased on the theory of "spleen governing transportation and transformation"， this study investigates the efficacy of Atractylodis Macrocephalae Rhizoma processed with Aurantii Fructus Immaturus juice（AMR-AFI） in improving slow-transit constipation（STC）， as well as the synergistic regulatory mechanism involving the microbiota-metabolism axis， thereby elucidating the scientific basis of its processing theory. MethodsAnimals were randomly divided into the control group， model group， positive drug（mosapride） group（3 mg·kg^-1）， and low-， medium-， and high-dose groups of AMR-AFI（3.9， 7.8， 15.6 g·kg^-1）. Except for the control group， the remaining five groups were induced with STC using loperamide hydrochloride. Following modeling， interventions were administered. All groups received continuous administration for 15 d， during which fecal samples， colon tissue， and serum were collected. Constipation improvement was assessed by measuring fecal moisture content and small intestinal propulsion rate， histological morphology of colonic tissue was observed via hematoxylin-eosin（HE） staining， and the levels of interleukin（IL）-6， tumor necrosis factor（TNF）-α， and IL-2 in serum were detected using enzyme-linked immunosorbent assay（ELISA）. Furthermore， the microbial community structure in mouse feces was analyzed by 16S rRNA sequencing， while transcriptomic sequencing was employed to screen differentially expressed genes in colonic tissue， followed by gene ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analyses. Finally， Spearman correlation analysis was conducted to explore the association between differential microbiota and differential genes. ResultsCompared with the control group， the intestinal propulsion rate and fecal moisture content in the model group were significantly decreased（P<0.01）， while serum levels of IL-6， TNF-α， and IL-2 were significantly elevated（P<0.01）. HE staining showed damage and shedding of colonic mucosal epithelial cells， along with a reduction in goblet cells in the model group. In comparison with the model group， all treatment groups improved the pathological state of the colonic mucosa to varying degrees and reduced serum levels of IL-6， TNF-α， and IL-2（P<0.01）. Among these， the high-dose group of AMR-AFI significantly increased the intestinal propulsion rate and fecal moisture content of rats（P<0.05， P<0.01）. Further transcriptomic analysis revealed that a total of 104 differentially expressed genes were identified from comparisons between the model group and the control group， as well as between the model group and the high-dose group of AMR-AFI. These genes were mainly enriched in pathways closely related to STC pathogenesis， such as arachidonic acid metabolism and aldosterone-regulated sodium reabsorption. 16S rRNA sequencing results indicated that AMR-AFI reversed the structural imbalance of the gut microbiota in model mice， increased species richness， downregulated the relative abundance of pro-inflammatory bacteria such as Parasutterella， and enriched beneficial and butyrate-producing bacteria， including Lachnospiraceae_NK4A136_group， Ruminococcaceae， and Lachnospiraceae. Spearman correlation analysis further showed that the beneficial bacteria enriched in the AMR-AFI group were negatively correlated with genes involved in the arachidonic acid metabolic pathway and positively correlated with genes in the aldosterone-regulated sodium reabsorption pathway. In contrast， pro-inflammatory bacteria in the model group exhibited the opposite correlation trends. ConclusionAMR-AFI can effectively exert synergistic therapeutic effects on STC by regulating intestinal microbiota， arachidonic acid-mediated inflammatory metabolism， and aldosterone-regulated water-salt balance pathways.

ObjectiveTo observe the effect of M1 bone marrow-derived macrophages （M1-BMDM） with Wnt5a knockdown on liver fibrosis and regeneration in a rat model of liver cirrhosis， and to investigate its gain-of-function effect compared with unmodified M1-BMDM. MethodsPrimary bone marrow-derived macrophages were isolated from rats and were polarized to M1 phenotype to construct M1-BMDM^Wnt5a-KD cells. A rat model of liver cirrhosis induced by CCl₄/2-AAF was established， and at the end of week 8， rats were randomly divided into model group， M1-BMDM group， M1-BMDM Wnt5a-knockdown empty vector group （M1-BMDM^KD-EV group）， and M1-BMDM Wnt5a-knockdown group （M1-BMDM^Wnt5a-KD group）， with 6 rats in each group. On the first day of week 9， the rats in each group were given a single injection of the corresponding cells via the caudal vein， along with an intraperitoneal injection of a CCR2 inhibitor. Six rats without any treatment were used as normal control group. Samples were collected at the end of week 12 to assess liver histopathology， serum liver function parameters， hepatic stellate cell activation， and the expression levels of mature hepatocyte markers. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the model group， all cell treatment groups had significant alleviation of liver inflammatory response and significant reductions in the activities of alanine aminotransferase and aspartate aminotransferase （AST） in serum （all P<0.01）， and the M1-BMDM^Wnt5a-KD group had a significantly lower serum level of AST than the M1-BMDM group （P<0.05）. The semi-quantitative analysis based on immunohistochemical staining showed that compared with the model group， all cell treatment groups had a significant reduction in the percentage of CD68-positive area （all P<0.05）， and compared with the M1-BMDM^KD-EV group， the M1-BMDM^Wnt5a-KD group had a significant reduction in the percentage of CD68-positive area and a significant increase in the percentage of CD163-positive area （both P<0.05）. Compared with the model group， all cell treatment groups had significant reductions in the mRNA expression levels of CD68 and tumor necrosis factor-α （all P<0.05） and the protein expression level of CD68 （all P<0.01）； compared with the M1-BMDM^KD-EV group， the M1-BMDM^Wnt5a-KD group had significant increases in the protein and mRNA expression levels of CD163 （both P<0.05）， significant reductions in the protein and mRNA expression levels of CD68 （both P<0.05）， and a significant reduction in the protein expression level of tumor necrosis factor-α （P<0.01）. Sirius Red collagen staining and alpha-smooth muscle actin （α-SMA） immunohistochemical staining showed that compared with the model group， all cell treatment groups had significant alleviation of liver collagen deposition and α-SMA-positive area， with the most significant changes in the M1-BMDM^Wnt5a-KD group， and compared with the M1-BMDM^KD-EV group， the M1-BMDM^Wnt5a-KD group had significantly smaller Sirius Red-positive area and α-SMA-positive area and a significantly lower content of hydroxyproline in liver tissue （all P<0.05）. Compared with the M1-BMDM^KD-EV group， the M1-BMDM^Wnt5a-KD group had significant reductions in the protein and mRNA expression levels of α-SMA and the mRNA expression level of COL-I and TGF-β （all P<0.05）. Compared with the model group， all cell treatment groups had a significant increase in the protein expression level of HNF-4α in liver tissue （all P<0.05）， and the M1-BMDM^Wnt5a-KD group had significantly higher protein and mRNA expression levels of HNF-4α and hepatocyte specific antigen than the M1-BMDM^KD-EV group （both P<0.05）. The M1-BMDM^Wnt5a-KD group had a significantly higher serum level of albumin than the M1-BMDM^KD-EV group （P<0.01）. Immunofluorescence co-staining showed that compared with the model group， all cell treatment groups had a significant increase in the number of cells stained positive for HNF and HNF-4α and Ki67 （all P<0.01）， and the M1-BMDM^Wnt5a-KD group had a significantly higher number of such cells than the M1-BMDM^KD-EV group （P<0.05）. ConclusionInhibition of Wnt5a expression enhances the therapeutic effect of M1-BMDM on rats with liver cirrhosis induced by CCl₄/2-AAF， which provides new ideas for enhancing the anti-cirrhotic effect of M1-BMDM through genetic modification.

BACKGROUND:Steroid-induced femoral head necrosis is mostly caused by long-term and extensive use of hormones,but its specific pathogenesis is not yet clear and needs further study. OBJECTIVE:To screen out the differential miRNAs in osteoclast exosomes after the intervention of Panax notoginseng saponins,and on this basis,to further construct an osteogenic-related ferroptosis regulatory network to explore the potential mechanism and research direction of steroid-induced osteonecrosis of the femoral head. METHODS:MTT assay was used to detect the toxic effects of different concentrations of dexamethasone and different mass concentrations of Panax notoginseng saponins on Raw264.7 cell line.Tartrate resistant acid phosphatase staining and TUNEL assay were used to detect the effects of Panax notoginseng saponins on osteoclast inhibition and apoptosis.Exosomes were extracted from cultured osteoclasts with Panax notoginseng saponins intervention.Exosomes from different groups were sequenced to identify differentially expressed miRNAs.CytoScape 3.9.1 was used to construct and visualize the regulatory network between differentially expressed miRNAs and mRNAs.Candidate mRNAs were screened by GO analysis and KEGG analysis.Finally,the differential genes related to ferroptosis were screened out,and the regulatory network of ferroptosis-related genes was constructed. RESULTS AND CONCLUSION:(1)The concentration of dexamethasone(0.1 μmol/L)and Panax notoginseng saponins(1 736.85 μg/mL)suitable for intervention of Raw264.7 cells was determined by MTT assay.(2)Panax notoginseng saponins had an inhibitory effect on osteoclasts and could promote their apoptosis.(3)Totally 20 differentially expressed miRNAs were identified from osteoclast-derived exosome samples,and 11 differentially expressed miRNAs related to osteogenesis were predicted by target mRNAs.The regulatory networks of 4 up-regulated differentially expressed miRNAs corresponding to 155 down-regulated candidate mRNAs and 7 down-regulated differentially expressed miRNAs corresponding to 238 up-regulated candidate mRNAs were constructed.(4)Twenty-four genes related to ferroptosis were screened out from the differential genes.Finally,12 networks were constructed(miR-98-5p/PTGS2,miR-23b-3p/PTGS2,miR-425-5p/TFRC,miR-133a-3p/TFRC,miR-185-5p/TFRC,miR-23b-3p/NFE2L2,miR-23b-3p/LAMP2,miR-98-5p/LAMP2,miR-182-5p/LAMP2,miR-182-5p/TLR4,miR-23b-3p/ZFP36,and miR-182-5p/ZFP36).These results indicate that Panax notoginseng saponins may regulate osteoblast ferroptosis by regulating the expression of miRNAs derived from osteoclast exosomes,thus providing a new idea for the study of the mechanism of steroid-induced femoral head necrosis.

ObjectiveThe utilization of assisted reproductive technology to rescue genetically modified mouse strains with reproductive disorders provides a reference for improving techniques to preserve valuable experimental mouse strains. MethodsIn vitro fertilization-embryo transfer (IVF-ET) technology was performed on 28 strains of infertile male mice aged 9-18 months. Several indicators such as sperm density and sperm motility in infertile male mice were assessed to select the most viable sperm for IVF-ET experiments. Fertility rate, abnormal egg rate, and birth rate were recorded after the birth of the pups. An optimized ovarian transplantation procedure was applied to 12 strains of infertile female mice aged 8-18 months. 6-week-old female mice with the same genetic background were selected as recipients. One intact ovary was removed from each recipient mouse, and the contralateral oviduct was ligated. An ovary from a donor mouse was isolated and transplanted orthotopically into the side where the ovary had been removed in the recipient mouse. Twenty-one days post-surgery, recipient mice were co-housed with 8-week-old wild type male mice of the same genetic background for breeding. Data such as the pregnancy rate and live birth rate of the recipients were recorded after the birth of the pups. ResultsIVF-ET successfully rescued 28 mouse strains, with the oldest male mice being 18 months old. The success rate of the first round of IVF-ET experiments was 89.29% (25/28). The average fertility rate of IVF in infertile male mice was (51.01±14.97)%, the abnormal egg rate was (9.03±5.28)%, and the birth rate of offspring mice was (18.60±7.03)%. 39 out of 40 ovarian transplant recipient mice survived, with a pregnancy rate of 33.33% (13/39) for ovarian transplant recipients, and a live birth rate of 17.95% (7/39). Four mouse strains were successfully rescued using optimized ovarian transplantation technology, with the oldest female mice being 18 months old. 8 strains were not rescued as they failed to produce offspring that survived to sexual maturity. ConclusionIVF-ET is an effective approach for rescuing mice with reproductive disorders caused by different reasons, especially for those beyond the optimal breeding age. Ovarian transplantation technology can also be used as an alternative for aged female mice. But its success rate is relatively lower than that of IVF-ET, and carries a higher experimental risk.

Avian influenza H10N3 is a type of avian influenza virus that can occasionally infect humans and cause severe pneumonia and acute respiratory distress syndrome (ARDS). On December 25, 2024, a 23-year-old obese female patient with H10N3 avian influenza complicated with severe ARDS was admitted to the Fourth People's Hospital of Nanning. The patient was transferred to our department due to "fever, cough, and shortness of breath for 13 days". Physical examination revealed moist rales in bilateral lungs. Chest imaging showed large areas of ground-glass opacity and consolidation in both lungs. Based on the patient's medical history, clinical manifestations, and laboratory findings, she was diagnosed with human infection of H10N3 avian influenza, severe pneumonia, and severe ARDS. Supported by mechanical ventilation and extracorporeal membrane oxygenation (ECMO), daily monitoring of airway peak pressure, plateau pressure (Pplat), driving pressure (ΔP), and lung compliance was performed to guide the adjustment of tidal volume (VT) and positive end-expiratory pressure (PEEP) during invasive mechanical ventilation. Medications including anti-avian influenza virus agents, antibacterial drugs, and antifungals were administered. Eventually, the patient's condition improved gradually, and she was successfully weaned from ECMO. No ventilator-induced lung injury (VILI) or multiple organ dysfunction syndrome (MODS) related to ARDS occurred during ECMO support. However, during the final stage of ventilator weaning after the restoration of spontaneous breathing, a right pneumothorax occurred. Closed thoracic drainage was performed, after which the ventilator was successfully discontinued. The patient was successfully transferred out of the intensive care unit (ICU), recovered fully, and was discharged from the hospital. In the invasive mechanical ventilation management of patients infected with H10N3 avian influenza complicated by ARDS, monitoring airway peak pressure, Pplat, ΔP, and assessing pulmonary compliance may facilitate more standardized management of such ARDS patients and help reduce VILI.
Humans ; Female ; Influenza, Human/complications* ; Respiratory Distress Syndrome/complications* ; Respiration, Artificial/methods* ; Obesity/complications* ; Young Adult ; Extracorporeal Membrane Oxygenation ; Influenza A virus

OBJECTIVE: Hepatocellular carcinoma (HCC) is sensitive to ferroptosis, a new form of programmed cell death that occurs in most tumor types. However, the mechanism through which ferroptosis modulates HCC remains unclear. This study aimed to investigate the oncogenic role and prognostic value of FANCD2 and provide novel insights into the prognostic assessment and prediction of immunotherapy. METHODS: Using clinicopathological parameters and bioinformatic techniques, we comprehensively examined the expression of FANCD2 macroscopically and microcosmically. We conducted univariate and multivariate Cox regression analyses to identify the prognostic value of FANCD2 in HCC and elucidated the detailed molecular mechanisms underlying the involvement of FANCD2 in oncogenesis by promoting iron-related death. RESULTS: FANCD2 was significantly upregulated in digestive system cancers with abundant immune infiltration. As an independent risk factor for HCC, a high FANCD2 expression level was associated with poor clinical outcomes and response to immune checkpoint blockade. Gene set enrichment analysis revealed that FANCD2 was mainly involved in the cell cycle and CYP450 metabolism. CONCLUSION To the best of our knowledge, this is the first study to comprehensively elucidate the oncogenic role of FANCD2. FANCD2 has a tumor-promoting aspect in the digestive system and acts as an independent risk factor in HCC; hence, it has recognized value for predicting tumor aggressiveness and prognosis and may be a potential biomarker for poor responsiveness to immunotherapy.
Humans ; Carcinoma, Hepatocellular/diagnosis* ; Liver Neoplasms/diagnosis* ; Immunotherapy ; Fanconi Anemia Complementation Group D2 Protein/metabolism* ; Prognosis ; Male ; Female ; Middle Aged ; Biomarkers, Tumor/metabolism*