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.

OBJECTIVE: To investigate the effectiveness of free palmaris longus tendon graft reconstruction in the treatment of gouty tophus erosion lesions in flexor tendon of wrist and hand. METHODS: A retrospective analysis was conducted on 8 patients with gouty tophus erosion lesions in flexor tendon of wrist and hand who underwent free palmaris longus tendon graft reconstruction between June 2017 and December 2023. All patients were male, aged 22-65 years, with an average of 45.9 years. The duration of gout history ranged from 2 to 18 years, with an average of 8.8 years. The duration from the discovery of gouty tophus to operation ranged from 12 to 26 months, with an average of 17.6 months. The gouty tophus eroded the flexor pollicis longus tendon in 4 cases, with Verdan flexor tendon zones being Ⅰ-Ⅱ in 1 case and Ⅳ-Ⅴ in 3 cases. The flexor digitorum profundus tendons were affected in 2 cases for the index finger, 1 for the middle finger, and 1 for the ring finger, all located in zone Ⅳ-Ⅴ. The long axis of the gouty tophus ranged from 2.3 to 4.5 cm, with an average of 3.4 cm. All 8 patients presented with limited finger flexion and extension. Among them, 4 cases were accompanied by median nerve compression symptoms, and 1 case had associated bone and joint destruction in the hand. The total active motion (TAM) of the affected finger was (81.3±30.2)° before operation according to the hand function evaluation criteria for tendon repair by the Chinese Society of Hand Surgery of the Chinese Medical Association, and the functional evaluation was poor. The harvested palmaris longus tendon intraoperatively was 7-9 cm in length. RESULTS: Surgical incisions in all 8 patients healed by first intention, with no infections, graft non-union, or significant adhesion complications. All patients were followed up 8-25 months, with an average of 14.8 months. Numbness symptoms resolved in all 4 patients who presented with median nerve compression symptoms. Patients did not experience wrist pain or other discomfort, and function was not compromised. At last follow-up, according to the hand function evaluation criteria for tendon repair by the Chinese Society of Hand Surgery of the Chinese Medical Association, the TAM of 8 patients was (197.5±55.8)°, which significantly improved when compared with that before operation ( t=11.638, P<0.001); the hand function of 1 patient with gouty tophus in zone Ⅰ-Ⅱ flexor pollicis longus tendon was good, and the other 7 patients were excellent. CONCLUSION Free palmaris longus tendon graft reconstruction demonstrates good effectiveness in treating gouty tophus erosion lesions in flexor tendon of wrist and hand.
Humans ; Middle Aged ; Male ; Adult ; Tendons/surgery* ; Retrospective Studies ; Aged ; Gout/complications* ; Wrist/surgery* ; Plastic Surgery Procedures/methods* ; Hand/surgery* ; Treatment Outcome ; Young Adult

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*

Human NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavoenzyme expressed at high levels in multiple solid tumors, making it an attractive target for anticancer drugs. Bioactivatable drugs targeting NQO1, such as β-lapachone (β-lap), are currently in clinical trials for the treatment of cancer. β-Lap selectively kills NQO1-positive (NQO1⁺) cancer cells by inducing reactive oxygen species (ROS) via catalytic activation of NQO1. In this study, we demonstrated that cryptotanshinone (CTS), a naturally occurring compound, induces NQO1-dependent necrosis without affecting NQO1 activity. CTS selectively kills NQO1⁺ cancer cells by inducing NQO1-dependent necrosis. Interestingly, CTS directly binds to NQO1 but does not activate its catalytic activity. In addition, CTS enables activation of JNK1/2 and PARP, accumulation of iron and Ca²⁺, and depletion of ATP and NAD⁺. Furthermore, CTS selectively suppressed tumor growth in the NQO1⁺ xenograft models, which was reversed by NQO1 inhibitor and NQO1 shRNA. In conclusion, CTS induces NQO1-dependent necrosis via the JNK1/2/iron/PARP/NAD⁺/Ca²⁺ signaling pathway. This study demonstrates the non-enzymatic function of NQO1 in inducing cell death and provides new avenues for the design and development of NQO1-targeted anticancer drugs.

ObjectiveTo observe the clinical efficacy and safety of Shugan Bushen Yulin Decoction （疏肝补肾毓麟汤， SBYD） in the treatment of asthenospermia and infertility with liver constraint and kidney deficiency. MethodsA multicenter， randomized， controlled clinical study was conducted in three hospitals in central China. Totally 95 patients with asthenospermia and infertility were controlled. According to random number table， the patients were divided into treatment group （47 cases） and control group （48 cases）. The control group was given vitamin E soft capsules （100 mg per time， twice daily） orally， and the treatment group was given SBYD （one dose daily， 30 min after breakfast and dinner， about 200 ml each time） orally. The course of treatment was 12 weeks in both groups. After the treatment， the sperm concentration， percentage of forward motile sperm （PR）， and percentage of total sperm activity， that is PR + percentage of non-progressively motile sperm （NP） were compared between groups， and the clinical efficacy was judged. Traditional Chinese medicine （TCM） syndrome score （inlcuding 6 single symptom scores and total symptoms score） and 21-item depression， anxiety， and stress scale （DASS-21） scores （including depression， anxiety and stress scores） were compared between the two groups before and after treatment. The patients were followed up for 6 months， and the pregnancy status of spouse between groups was compared. The occurrence of adverse events and vital signs during the trial were recorded for safety assessment. ResultsTwo cases in the treatment group and three cases in the control group dropped out. Finally， 45 cases in each group were included in the statistical analysis. The total effective rate was 86.67% （39/45） in the treatment group， significantly higher than 73.74% （33/45） in the control group （P＜0.05）. After treatment， PR and PR + NP significantly increased in both groups， and were much higher in the treatment group than in the control group （P＜0.05）. The scores of lumbar and knee soreness， emotional disturbance， cold sperm， chest and rib-side and lesser abdomen distension and pain， frequent sighing， and the total TCM syndrome score in the treatment group decreased after treatment （P＜0.05）； and except for cold sperm， the scores of other symptoms mentioned above as well as the total TCM syndrome score in the treatment group were lower than those in the control group （P＜0.05）. The scores of depression， anxiety and stress of DASS-21 in the treatment group decreased after treatment， and were lower than those in the control group （P＜0.05）. After 6 months of follow-up， the spouses of 5 patients in the treatment group and 3 patients in the control group got pregnant （P＞0.05）. No adverse events occurred in both groups during the treatment， and their vital signs were stable. ConclusionSBYD can obviously improve sperm vitality and effectively improve the clinical symptoms， anxiety and depression， and is safe in treating asthenospermia and infertility with liver constraint and kidney deficiency.

To understand Helicobacter pylori's drug resistance,genetic diversity,and relationship with clinical diseases in the Guiyang and Qiannan minority areas of Guizhou Province,we collected samples through endoscopy,and isolated and cul-tured H.pylori.The drug resistance and genotype characteristics were determined.The differences in different regions and dis-ease types were compared,and the structural characteristics of H.pylori and mixed infections with different strains of H.py-lori in Qiannan Prefecture were analyzed.A difference in the composition ratio of EPYIA typing in the cagA variable region was observed between the two areas(P=0.012),and the composition ratio of the vacA genotype differed(P=0.000).A total of 94.6％(53/56)new sequences of H.pylori strains from two regions were obtained by MLST.The rate of infection by H.pylori mixed with different strains was 44.4％in Qiannan Pre-fecture,and no significant difference was observed in the com-position of H.pylori mixed infections among patients with dif-ferent clinical diseases(P=0.349).Differences in EPI YA typ-ing and the vacA genotype composition ratio in the cagA varia-ble region of H.pylori were observed between the Qiannan Prefecture and Guiyang City.

Objective:To explore the biomechanical stability of a novel anchor-loop internal fixation system in the treatment of acromioclavicular joint dislocation using cadaveric specimens.Methods:The acromioclavicular ligaments were severed in 12 complete shoulder joint specimens, in which the quasi-static non-destructive cycle experiment was performed until the coracoclavicular ligaments failed. The failure intensities of the coracoclavicular ligaments were recorded. Next, the 12 specimens were randomly divided into groups A, B, C and D ( n=3), in which 4 different internal fixation materials were used respectively to reduce and fix the acromioclavicular joint. Group A was subjected to 3.5 mm clavicular hook locking compression plate, group B to 5 mm soft tissue with wire anchor, group C to 10 mm Endobutton steel plate, and group D to the novel anchor-loop internal fixation system (5 mm soft tissue with wire anchor + 10 mm Endobutton steel plate). An X-ray machine was used to evaluate the reduction and internal fixation of the acromioclavicular joint. After the shoulder specimens were securely fastened by a homemade fixation jig to a 100 KN electronic universal mechanical testing machine, each experimental specimen was subjected to a destructive static tensile mechanic determination in the vertical direction at a loading speed of 100 mm/min. The load-displacement curves were recorded and drawn by a computer connected with the biomechanical testing machine. The failure strength and failure causes were recorded for each internal fixation. Results:The fracture strength of the coracoclavicular ligament in 12 cadaver specimens was (374.6±0.8) N. The mechanical load of internal fixation failure was (409.5±2.6) N in group A, (297.8±3.4) N in group B, (375.2±3.1) N in group C and (376.2±3.1) N in group D. The internal fixation failure was due to clavicular fracture in 2 specimens and to acromial fracture in 1 specimen in group A, to anchor protrusion in all the 3 specimens in group B, to coracoid base fracture in all the 3 specimens in group C, and to anchor protrusion in all the 3 specimens in group D. The mechanical loads of internal fixation failure were significantly different among the 4 experimental groups ( P<0.05). The mechanical load of internal fixation failure in group D was significantly different from that in groups A and B ( P<0.05). Conclusions:Our self-developed novel anchor-loop internal fixation system can effectively reposit the acromioclavicular joint to treat acromioclavicular joint dislocation, because it conforms to the biomechanical characteristics of the acromioclavicular joint, and is easy to handle. Therefore, its feasibility is high.

Objective This study investigated the impact of occupational mercury(Hg)exposure on human gene transcription and expression,and its potential biological mechanisms. Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation.Hg-exposed cell models and PTEN low-expression models were established in vitro using 293T cells.PTEN gene expression was assessed using qRT-PCR,and Western blotting was used to measure PTEN,AKT,and PI3K protein levels.IL-6 expression was determined by ELISA. Results Combined findings from gene expression microarray analysis,bioinformatics,and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group.In the Hg-exposed cell model(25 and 10 μmol/L),a significant decrease in PTEN expression was observed,accompanied by a significant increase in PI3K,AKT,and IL-6 expression.Similarly,a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K,AKT,and IL-6 levels. Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene,activates the PI3K/AKT regulatory pathway,and increases the expression of inflammatory factors,ultimately resulting in kidney inflammation.

Objective We aimed to investigate the effect of ferroptosis on Shenling Baizhu Powder,a compound prescription of Chinese herbal medicine,in improving testicular spermatogenic function in hyperuricemic mice with spermatogenic dysfunction. Methods Sixty BALB/c mice were randomly divided into normal group,model group,Shenling Baizhu Powder high-,medium-,and low-dose groups (20.14,10.07,5.04 g/kg,by gavage),and ferrostatin-1(Fer-1) group (0.8 mg/kg,by tail vein injection),with 10 mice each group. Except for the normal group,the other groups were intraperitoneally injected with potassium oxonate suspension[600mg/(kg·d)]for 7 days to establish the hyperuricemic model,and then the corresponding intervention was given for consecutive 14 days. Content of serum uric acid (UA),testicular Fe2+,reduced glutathione (GSH),malondialdehyde (MDA) and superoxide dismutase (SOD) activity were detected by biochemical method. Epididymal and testicular indices were measured. The spermatogenic function of testes was evaluated by eosin-hematoxylin staining. Sperm quality was detected by an automatic animal sperm analyzer. Prussian blue staining was used to detect iron deposition in testicular tissue. Immunohistochemistry was used to detect the related protein expressions of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4) in testicular tissue. Western blotting was used to detect the related protein expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1(HO-1)/GPX4 signaling pathway in testicular tissue. Results Compared with the normal group,the contents of serum UA,MDA,and Fe2+in the testis tissue of the model group were increased,the GSH content and SOD activity were decreased,the epididymal and testicular index,testicular spermatogenic function,sperm density and activity rate were decreased,and the iron deposition and ACSL4 protein expression in the testis tissue were increased. The expressions of kelch-like ECH-associated protein-1 (Keap1) and Nrf2 were increased. The expressions of nuclear Nrf2,HO-1,GPX4,and recombinant solute carrier family 7 member 11 (SLC7A11) protein were decreased (P<0.01). Compared with the model group,the above indexes in the Shenling Baizhu Powder groups and the Fer-1 group were improved to varying degrees (P<0.05,P<0.01). Conclusion Shenling Baizhu Powder can inhibit the ferroptosis of testicular cells through the Nrf2/HO-1/GPX4 signaling pathway,and improve the testicular spermatogenic function of mice with hyperuricemia spermatogenic dysfunction.