Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Type 2 diabetes mellitus(T2DM) is a prevalent metabolic and endocrine disorder. Long-term hyperglycemia can lead to severe chronic complications, imposing substantial economic burdens on both society and patients. Despite the availability of various hypoglycemic agents for clinical use, these agents often fail to meet the therapeutic needs of T2DM and its complications. Consequently, there is an urgent need for novel therapeutic strategies and drugs. Lithocarpus litseifolius(L. litseifolius), commonly referred to as "cordyceps on trees", has a long history of use in traditional medicine and can be applied in tea, sugar, and medicine. Research indicates that L. litseifolius extracts are rich in dihydrochalcones, including trilobatin, phloridzin, and phloretin, which exhibit a range of pharmacological activities, such as anti-inflammatory, antioxidant, hypoglycemic, hypolipidemic, hepatoprotective, and cardioprotective effects. These properties suggest potential applications in the treatment of T2DM and its complications. This review systematically compiled and organized the relevant literature from the past decade on dihydrochalcones(trilobatin, phloridzin, and phloretin) from L. litseifolius extracts. It highlighted recent research progress regarding their role in treating T2DM and its complications through mechanisms such as reducing insulin resistance, regulating glucose transport, improving glucose and lipid metabolism, modulating enzyme activity, regulating gut microbiota, and alleviating inflammation and oxidative damage. The purpose of this review is to provide a reference and basis for future research on the prevention and treatment of T2DM and its complications using dihydrochalcones(trilobatin, phloridzin, and phloretin) from L. litseifolius extracts.
Chalcones/chemistry* ; Diabetes Mellitus, Type 2/metabolism* ; Humans ; Animals ; Elaeocarpaceae/chemistry* ; Drugs, Chinese Herbal/therapeutic use* ; Hypoglycemic Agents/chemistry* ; Plant Extracts/chemistry*

This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*

OBJECTIVE: To explore the accuracy of human-computer interaction software in identifying and locating type C1 distal radius fractures. METHODS: Based on relevant inclusion and exclusion criteria, 14 cases of type C1 distal radius fractures between September 2023 and March 2024 were retrospectively analyzed, comprising 3 males and 11 females(aged from 27 to 82 years). The data were assigned randomized identifiers. A senior orthopedic physician reviewed the films and measured the ulnar deviation angle, radial height, palmar inclination angle, intra-articular step, and intra-articular gap for each case on the hospital's imaging system. Based on the reduction standard for distal radius fractures, cases were divided into reduction group and non-reduction group. Then, the data were sequentially imported into a human-computer interaction intelligent software, where a junior orthopedic physician analyzed the same radiological parameters, categorized cases, and measured fracture details. The categorization results from the software were consistent with manual classifications (6 reduction cases and 8 non-reduction cases). For non-reduction cases, the software performed further analyses, including bone segmentation and fracture recognition, generating 8 diagnostic reports containing fracture recognition information. For the 6 reduction cases, the senior and junior orthopedic physicians independently analyzed the data on the hospital's imaging system and the AI software, respectively. Bone segments requiring reduction were identified, verified by two senior physicians, and measured for displacement and rotation along the X (inward and outward), Z (front and back), and Y (up and down) axes. The AI software generated comprehensive diagnostic reports for these cases, which included all measurements and fracture recognition details. RESULTS: Both the manual and AI software methods consistently categorized the 14 cases into 6 reduction and 8 non-reduction groups, with identical data distributions. A paired sample t-test revealed no statistically significant differences (P>0.05) between the manual and software-based measurements for ulnar deviation angle, radial ulnar bone height, palmar inclination angle, intra-articular step, and joint space. In fracture recognition, the AI software correctly identified 10 C-type fractures and 4 B-type fractures. For the 6 reduction cases, a total of 24 bone fragments were analyzed across both methods. After verification, it was found that the bone fragments identified by the two methods were consistent. A paired sample t-tests revealed that the identified bone fragments and measured displacement and rotation angles along the X, Y, and Z axes were consistent between the two methods. No statistically significant differences(P>0.05) were found between manual and software measurements for these parameters. CONCLUSION Human-computer interaction software employing AI technology demonstrated comparable accuracy to manual measurement in identifying and locating type C1 distal radius fractures on CT imaging.
Humans ; Male ; Female ; Radius Fractures/surgery* ; Middle Aged ; Adult ; Aged ; Aged, 80 and over ; Tomography, X-Ray Computed/methods* ; Retrospective Studies ; Software ; Wrist Fractures

OBJECTIVE: To explore the functional rehabilitation of patients with rheumatoid arthritis (RA) after total knee arthroplasty (TKA). METHODS: A retrospective analysis was conducted on 101 patients who needed TKA due to rheumatoid arthritis (RA) involving both knees from January 2017 to December 2020, including 16 males and 85 females, aged from 41 to 65 years old with an average of (58.13±5.53) years old;body mass index (BMI) ranged from 16.88 to 33.33 kg·m^-2 with an average of (23.16±3.49) kg·m^-2;63 patients with grade 1, 29 patients with grade 2, and 9 patients with grade 3 according to classification of American Society of Anesthesiologists (ASA). According to the latest follow-up results at 12 months after operation, 82 patients returned to work and 19 patients did not return to work. Visual analogue scale(VAS) was used to evaluate the degree of pain relief before operation and 12 months after operation, and work, osteoarthritis and joint replacement questionnaire (WORQ) was used to evaluate knee joint activity status of all patients before and after operation, and the working ability index was used to evaluate working ability of all patients before operation and 12 months after operation. For the 82 patients who returned to work, the labor time stopped before operation and within 12 months after operation was compared, and the changes in labor grades, types of work and labor hours of patients before and after operation were recorded. For the 19 patients who did not return to work, the specific reasons for their non-return to work was analyzed;the postoperative satisfaction of patients was evaluated by using Likert satisfaction scale. All patients were followed up for at least 12 months. VAS was decreased from (6.49±0.59) before operation to (1.10±0.43) at 12 months after operation (P<0.05);for WORQ questionnaire survey, scores of walking, sitting posture, standing and stair climbing were increased from (1.07±0.35), (1.05±0.29), (1.06±0.34) and (1.14±0.42) before operation to (3.00±0.00), (2.87±0.33), (2.95±0.21) and (2.95±0.21) after operation, respectively, had statistically significant (P<0.05);the labor work index of all patients increased from 1.11±0.46 before operation to 2.99±0.10 at 12 months after operation, and the difference was statistically significant (P<0.05). Among the 82 patients who returned to work after operation, regarding the time of stopping labor, 81 patients stopped working within 3 months before operation, 1 patient stopped working for 4 to 6 months after operation, and the number of patients who stopped working was 81, 1, and 0 respectively. Forty patients returned to work within 3 months after operation, 4 to 6 months after operation for 29 patients, and 12 months after operation for 13 patients. 95.1% (78/82) of patients engaged in light labor before operation, and 85.4% (70/82) of patients engaged in moderate labor after operation. At 12 months after operation, the types of jobs and working hours available to all patients increased compared with those before operation. Among 19 patients who did not return to work after TKA, 7 patients had poor control of rheumatoid arthritis, 5 patients still felt pain, swelling and numbness on knee joint, 2 patients had retired, and 5 patients had other reasons. Eighty-six patients (85%) expressed great satisfaction with the postoperative working ability, 8 patients (8%) expressed satisfaction with the postoperative working ability, 6 patients (6%) expressed acceptance of postoperative working ability, and 1 patient (1%) expressed dissatisfaction with postoperative working ability. CONCLUSION TKA is an effective treatment option for patients with RA. After undergoing TKA, patients could significantly improve pain and functional activities of knee joint, and effectively enhance the quality of life and working ability. For patients whose rehabilitation labor capacity is not fully met, postoperative management and personalized rehabilitation treatment need to be strengthened to achieve the best rehabilitation effect.
Humans ; Female ; Male ; Arthroplasty, Replacement, Knee/rehabilitation* ; Arthritis, Rheumatoid/physiopathology* ; Middle Aged ; Aged ; Retrospective Studies ; Adult

PURPOSE: To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss. METHODS: This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA⁺, K⁺, and Ca²⁺ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance. RESULTS: The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA⁺ and K⁺ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca²⁺ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group. CONCLUSIONS Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals ; Perfusion/methods* ; Disease Models, Animal ; Brain Injuries/etiology* ; Swine ; Male ; Hypothermia, Induced/methods*

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

OBJECTIVE: To explore the expression and clinical significance of XPO1 in newly diagnosed adult diffuse large B-cell lymphoma (DLBCL), and further investigate its functional mechanism. METHODS: Immunohistochemical testing was conducted for XPO1 expression in 93 cases of DLBCL and 30 cases of reactive lymphoid hyperplasia. A risk model was construed to find survival related genes in DLBCL patients. Cell proliferation, apoptosis, and cell cycle assays were performed to explore the effect of XPO1 inhibitor (KPT-8602) and XPO1 knockdown. Differential expression gene (DEG) was examined based on the transcriptomes. RESULTS The expression of XPO1 in DLBCL patients was higher than that of the controls. Compared with XPO1 low-expression group, XPO1 high-expression group had a worse prognosis. The constructed risk model indicated that XPO1 and 14 genes in nucleocytoplasmic transport pathway (NTP) might be potential prediction marker of adverse outcome in DLBCL. Moreover, KPT-8602 as well as the XPO1 knockdown could inhibit cell proliferation, promote apoptosis, and induce cell cycle arrest in two DLBCL cell lines, Farage and SU-DHL-4. Based on the gene expression profiling in the datasets of DLBCL, patients were classified into XPO1 high and XPO1 low expression groups, and the MYBL1 was identified as the down-stream effector of XPO1. Inhibiting the function of XPO1 or reducing its expression can significantly decrease the expression of MYBL1 Conclusion: XPO1 is highly expressed in DLBCL, which is associated with poor prognosis. The oncogenic roles of the new XPO1/MYBL1 signaling are identified in DLBCL and XPO1 inhibitor may be a potential option for newly-diagnosed DLBCL patients.
Humans ; Lymphoma, Large B-Cell, Diffuse/pathology* ; Exportin 1 Protein ; Karyopherins/metabolism* ; Receptors, Cytoplasmic and Nuclear/metabolism* ; Cell Proliferation ; Apoptosis ; Prognosis ; Cell Line, Tumor ; Clinical Relevance