Objective To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

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.

The gut microbiota regulates intestinal nutrient absorption, participates in modulating host glucolipid metabolism, and contributes to ameliorating glucolipid metabolism disorder. Dysbiosis of the gut microbiota can compromise the integrity of the intestinal mucosal barrier, induce inflammatory responses, and exacerbate insulin resistance and abnormal lipid metabolism in the host. Dangua Humai Oral Liquid, a hospital-developed formulation for regulating glucolipid metabolism, has been granted a national invention patent and demonstrates significant clinical efficacy. This study aimed to investigate the effects of Dangua Humai Oral Liquid on gut microbiota and the intestinal mucosal barrier in a mouse model with glucolipid metabolism disorder. A glucolipid metabolism disorder model was established by feeding mice a high-glucose and high-fat diet. The mice were divided into a normal group, a model group, and a treatment group, with eight mice in each group. The treatment group received a daily gavage of Dangua Humai Oral Liquid(20 g·kg~(-1)), while the normal group and model group were given an equivalent volume of sterile water. After 15 weeks of intervention, glucolipid metabolism, intestinal mucosal barrier function, and inflammatory responses were evaluated. Metagenomics and untargeted metabolomics were employed to analyze changes in gut microbiota and associated metabolic pathways. Significant differences were observed between the indicators of the normal group and the model group. Compared with the model group, the treatment group exhibited marked improvements in glucolipid metabolism disorder, alleviated pathological damage in the liver and small intestine tissue, elevated expression of recombinant claudin 1(CLDN1), occluding(OCLN), and zonula occludens 1(ZO-1) in the small intestine tissue, and reduced serum levels of inflammatory factors lipopolysaccharides(LPS), lipopolysaccharide-binding protein(LBP), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α). At the phylum level, the relative abundance of Bacteroidota decreased, while that of Firmicutes increased. Lipid-related metabolic pathways were significantly altered. In conclusion, based on the successful establishment of the mouse model of glucolipid metabolism disorder, this study confirmed that Dangua Humai Oral Liquid effectively modulates gut microbiota and mucosal barrier function, reduces serum inflammatory factor levels, and regulates lipid-related metabolic pathways, thereby ameliorating glucolipid metabolism disorder.
Animals ; Gastrointestinal Microbiome/drug effects* ; Mice ; Intestinal Mucosa/microbiology* ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Mice, Inbred C57BL ; Humans ; Glycolipids/metabolism* ; Lipid Metabolism/drug effects* ; Administration, Oral ; Disease Models, Animal

OBJECTIVE: To compare the clinical efficacy of oblique lateral interbody fusion(OLIF) combined with lateral fixation and transforaminal lumbar interbody fusion(TLIF) in patients with lumbar spinal stenosis. METHODS: Totally 47 patients with lumbar stenosis from November 2018 to December 2021 were analyzed retrospectively and were divided into two groups according to the surgical methods. Among them, 21 cases underwent oblique lumbar interbody fusion supplemental anterolateral screw and rod instrumentation, including 5 males and 16 females, with a mean age of (68.19±6.13) years old ranging 55 to 74 years; the other 26 cases underwent posterior pedicle screw fixation and reduction were recorded, including 8 males and 18 females with a mean age of (65.35±7.64) years old ranging 54 to 78 years. Visual analogue scale(VAS) of pain was recorded to evaluate the degree of low back pain and lower extremity pain. The radiographic parameters were collected to evaluate the efficacy of two approaches including disc height, foraminal height, canal sagittal diameter and cross-sectional area. RESULTS: All operations were completed successfully. The wound healed normally and bone fusion was obtained in the last final follow up. No serious complication was occurred in both groups. One case of dural tear occurred in direct compression group. Four cases of left thigh weakness and pain were recorded in indirect decompression group. The average postoperative follow-up was(21.69±4.37)months in direct compression group, while(20.43±4.80)months in another group. There were no significant difference in bone density, body mass index(BMI), hospital stay, Cobb angel(P>0.05). The differences in intra-operative blood loss, operation time, disc height, foraminal height between two groups were statistically significant(P<0.05). The area and sagittal diameter of the spinal canal in the two groups were significantly improved after surgery(P<0.05). CONCLUSION Both two fusion methods have achieved good clinical results in the treatment of lumbar spinal stenosis, with the advantages of good stability, fast recovery and high fusion rate. Compared with TLIF, the advantage of OLIF has greater advantages in less bleeding and less trauma.
Humans ; Male ; Female ; Spinal Stenosis/surgery* ; Spinal Fusion/methods* ; Aged ; Middle Aged ; Lumbar Vertebrae/surgery* ; Retrospective Studies ; Treatment Outcome

Diabetic foot (DF) is one of the most serious chronic complications of diabetes. The incidence rate among global diabetes patients is as high as 15% to 25%, and about 50% of patients will develop contralateral foot ulcers within 5 years after the first unilateral ulcer. As a non-invasive prevention and control solution, the application progress of functional insoles is mainly reflected in the following aspects:(1) Material innovation. The application of new composite materials and smart materials has significantly enhanced the pressure reduction effect and comfort. (2) Structural optimization. The development of multi-layer design and local pressure reduction structure has achieved more precise pressure distribution regulation. (3) Manufacturing process. 3D printing and parametric design have enabled the personalized customization of functional insoles. (4) Intelligent monitoring. It integrates functions such as pressure sensing and temperature monitoring, achieving real-time monitoring and early warning of foot conditions. Clinical research has confirmed that personalized functional insoles could reduce the incidence of foot ulcers and shorten the healing time of ulcers. At present, the research hotspots mainly focus on the development of smart materials, the construction of multi-functional integration and remote monitoring systems. However, in-depth research is still needed in the aspects of biomechanical mechanisms, standardized evaluation systems and long-term efficacy assessment. The development of future functional insoles should focus on the coordinated advancement of "personalization-intelligence-standardization", with the aim of providing more effective solutions for the prevention and treatment of DF.
Humans ; Diabetic Foot/therapy* ; Foot Orthoses

OBJECTIVE: To explore the therapeutic effects and underlying mechanisms of Dendrobium officinale (Tiepi Shihu) extract (DOE) on insomnia. METHODS: Forty-two male Sprague-Dawley rats were randomly divided into 6 groups (n=7 per group): normal control, model control, melatonin (MT, 40 mg/kg), and 3-dose DOE (0.25, 0.50, and 1.00 g/kg) groups. Rats were raised in a strong-light (10,000 LUX) and -noise (>80 db) environment (12 h/d) for 16 weeks to induce insomnia, and from week 10 to week 16, MT and DOE were correspondingly administered to rats. The behavior tests including sodium pentobarbital-induced sleep experiment, sucrose preference test, and autonomous activity test were used to evaluate changes in sleep and emotions of rats. The metabolic-related indicators such as blood pressure, blood viscosity, blood glucose, and uric acid in rats were measured. The pathological changes in the cornu ammonis 1 (CA1) region of rat brain were evaluated using hematoxylin and eosin staining and Nissl staining. Additionally, the sleep-related factors gamma-aminobutyric acid (GABA), glutamate (GA), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) were measured using enzyme linked immunosorbent assay. Finally, we screened potential sleep-improving receptors of DOE using polymerase chain reaction (PCR) array and validated the results with quantitative PCR and immunohistochemistry. RESULTS: DOE significantly improved rats' sleep and mood, increased the sodium pentobarbital-induced sleep time and sucrose preference index, and reduced autonomic activity times (P<0.05 or P<0.01). DOE also had a good effect on metabolic abnormalities, significantly reducing triglyceride, blood glucose, blood pressure, and blood viscosity indicators (P<0.05 or P<0.01). DOE significantly increased the GABA content in hippocampus and reduced the GA/GABA ratio and IL-6 level (P<0.05 or P<0.01). In addition, DOE improved the pathological changes such as the disorder of cell arrangement in the hippocampus and the decrease of Nissel bodies. Seven differential genes were screened by PCR array, and the GABA_A receptors (Gabra5, Gabra6, Gabrq) were selected for verification. The results showed that DOE could up-regulate their expressions (P<0.05 or P<0.01). CONCLUSION DOE demonstrated remarkable potential for improving insomnia, which may be through regulating GABA_A receptors expressions and GA/GABA ratio.
Animals ; Dendrobium/chemistry* ; Rats, Sprague-Dawley ; Male ; Sleep Initiation and Maintenance Disorders/blood* ; Plant Extracts/therapeutic use* ; Receptors, GABA-A/metabolism* ; Noise/adverse effects* ; Light/adverse effects* ; gamma-Aminobutyric Acid/metabolism* ; Sleep/drug effects* ; Rats ; Receptors, GABA/metabolism*

Metformin has been demonstrated to attenuate hyperglycaemia by modulating the gut microbiota. However, the mechanisms through which the microbiome mediates metformin monotherapy failure (MMF) are unclear. Herein, in a prospective clinical cohort study of newly diagnosed type 2 diabetes mellitus (T2DM) patients treated with metformin monotherapy, metagenomic sequencing of faecal samples revealed that Phocaeicola vulgatus abundance was approximately 12 times higher in nonresponders than in responders. P. vulgatus rapidly hydrolysed taurine-conjugated bile acids, leading to ceramide accumulation and reversing the improvements in glucose intolerance conferred by metformin in high-fat diet-fed mice. Interestingly, C22:0 ceramide bound to mitochondrial fission factor to induce mitochondrial fragmentation and impair hepatic oxidative phosphorylation in P. vulgatus-colonized hyperglycaemic mice, which could be exacerbated by metformin. This work suggests that metformin may be unsuitable for P. vulgatus-rich T2DM patients and that clinicians should be aware of metformin toxicity to mitochondria. Suppressing P. vulgatus growth with cefaclor or improving mitochondrial function using adenosylcobalamin may represent simple, safe, effective therapeutic strategies for addressing MMF.

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Background and Aims:Surgical treatment is a cornerstone of breast cancer management;however,the physical trauma and psychological burden associated with surgery may adversely affect patients'quality of life(QoL).Based on data from a prospective cohort,this study was conducted to evaluate changes in postoperative QoL among breast cancer patients and to identify mediating factors influencing QoL,thereby providing evidence for perioperative rehabilitation strategies.Methods:Female breast cancer patients who underwent local surgical treatment at Suining Central Hospital between June 2024 and January 2025 were enrolled.The Constant-Murley Shoulder Score,EORTC QLQ-C30,and QLQ-BR23 questionnaires were used pre-and postoperatively to assess shoulder joint function,and QoL.Paired t-tests were used to analyze changes in scores before and after surgery.A mediation model was applied to explore the indirect pathways through which surgery impacts QoL.Additionally,subgroup analyses were conducted to compare QoL changes across different surgical modalities.Results:A total of 148 patients were included,with median age of 54(46-60)years old Postoperative psychological health significantly improved,while shoulder function and overall QoL scores markedly declined(all P<0.001).According to QLQ-C30,functional domains such as physical and role functioning worsened,while symptom burden increased.The QLQ-BR23 revealed significantly higher scores for breast and upper limb symptoms postoperatively(both P<0.001).Subgroup analyses showed that patients undergoing axillary lymph node dissection experienced greater declines in shoulder function and QoL compared to those receiving sentinel lymph node biopsy(both P<0.05).QoL deterioration was comparable between mastectomy and breast-conserving surgery,although the former was associated with more prominent pain and breast symptoms.Mediation analysis indicated that shoulder function,breast symptoms,and physical functioning served as mediators in the impact of surgery on QoL,while better psychological health exerted a protective effect.Conclusion:Surgical treatment significantly affects postoperative QoL in breast cancer patients,with local functional impairment and symptom exacerbation serving as key mediators.Different surgical approaches have varying impacts on QoL.Enhancing postoperative shoulder rehabilitation,symptom management,and psychological support may improve patients'QoL.