Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

ObjectiveTo decipher the mechanism by which Zuoguiwan （ZGW） treat hyperthyroidism in rats with kidney-Yin deficiency based on the dopamine receptor D4 （DRD4）/nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4） signaling pathway. MethodsThe rat model of kidney-Yin deficiency was induced by unilateral intramuscular injection of dexamethasone （0.35 mg·kg^-1）. After successful modeling， the rats were randomized into model， methimazole （positive control， 5 mg·kg^-1）， low-， medium-， and high-dose （1.85， 3.70， 7.40 g·kg^-1， respectively） ZGW， and normal control groups. After 21 days of continuous gavage， the behavioral indexes and body weight changes of rats were evaluated. The pathological changes of the renal tissue were observed by hematoxylin-eosin staining. The serum levels of thyroid hormones ［triiodothyronine （T₃）， thyroxine （T₄）， thyroid-stimulating hormone （TSH）］， renal function indexes ［serum creatine （Scr） and blood urea nitrogen （BUN）］， energy metabolism markers ［cyclic adenosine monophosphate （cAMP） and cyclic guanosine monophosphate （cGMP）］， and oxidative stress-related factors ［superoxide dismutase （SOD）， malondialdehyde （MDA）， and NADPH）］ were measured by enzyme-linked immunosorbent assay （ELISA）. Western blot was employed to analyze the expression of DRD4， NOX4， mitochondrial respiratory chain complex proteins ［NADH：ubiquinone oxidoreductase subunit S4 （NDUFS4） and cytochrome C oxidase subunit 4 （COX4）］， and inflammation-related protein ［tumor necrosis factor-alpha （TNF-α）， interleukin-6 （IL-6）， p38 mitogen-activated protein kinase （MAPK）］ pathway in the renal tissue. ResultsCompared with the normal group， the model group showed mental malaise， body weight decreases （P<0.01）， inflammatory cell infiltration in the renal tissue， a few residual parotid glands in the thyroid， elevations in serum levels of T₃， T₄， Scr， BUN， cAMP， cAMP/cGMP， MDA， and NADPH （P<0.01）， down-regulation in protein levels of TSH， SOD， and DRD4 （P<0.05， P<0.01）， and up-regulation in expression of NOX4， p-p38 MAPK/p38 MAPK， and inflammatory factors （P<0.01）. Compared with the model group， ZGW increased the body weight （P<0.05， P<0.01）， reduced the infiltration of renal interstitial inflammatory cells， restored the thyroid structure and follicle size， lowered the serum levels of T₃， T₄， Scr， BUN， cAMP， cAMP/cGMP， MDA and NADPH （P<0.05， P<0.01）， up-regulated the expression of TSH， SOD and DRD4 （P<0.05， P<0.01）， and down-regulated the expression of NOX4， p-p38 MAPK/p38 MAPK， and inflammatory factors （P<0.05， P<0.01）. Moreover， high-dose ZGW outperformed methimazole （P<0.05）. ConclusionBy activating DRD4， ZGW can inhibit the expression of NOX4 mediated by the p38 MAPK pathway， reduce oxidative stress and inflammatory response， thereby ameliorating the pathological state of hyperthyroidism due to kidney-Yin deficiency. This study provides new molecular mechanism support for the clinical application of ZGW.

This paper summarizes the clinical experience in the syndrome differentiation and treatment of rosacea using the method of venting heat and resolving stagnation. It is considered that the key pathogenesis of rosacea is the accumulation of heat with stagnation. Accordingly， the method of venting heat and resolving stagnation is proposed， which vents and disperses constraint heat by applying approaches such as dredging defensive qi， clearing qi， venting ying （营） level， and cooling blood， while eliminating stagnation and masses through regulating qi， resolving dampness， dispelling phlegm and removing stasis. In clinical practice， a core prescription for venting heat and resolving stagnation is formulated， with flexible modifications made according to the clinical characteristics of different rosacea subtypes， including erythematotelangiectatic， papulopustular， phymatous， and ocular types， thereby providing therapeutic insights for the treatment of rosacea with traditional Chinese medicine.

Transfusion technicians constitute a core workforce in safeguarding blood safety, and the quality of their standardized training directly affects the operational efficiency of the blood services. At present, training programs for transfusion technicians across China have been developed in a diversified manner. Positive progress has been achieved in integrated theoretical and practical training, on-the-job faculty development, and regional collaborative training models. However, persistent challenges remain, including fragmented training providers, inconsistent training standards, limited coverage, and a disconnect between training and workforce utilization. Based on a review of current standardized training practice, this article systematically analyzes the structural challenges confronting current standardized training, and further proposes a framework for building a comprehensive training system, covering the definition and core components of standardized training, targeted improvement strategies and key assessment indicators. The study aims to facilitate the transformation of training for blood transfusion technicians in blood services from a project-based approach to an institutionalized model, and from fragmented implementation to a systematic framework.

Mammalian male germline development depends on a specialized gonadal somatic microenvironment. However, itsin vitro reconstruction remains challenging. Major barriers include the generation of appropriate supporting-cell lineages, organized tissue architecture, and culture conditions that sustain germ-cell maturation. Yoshino et al. established a mouse pluripotent stem cell-derived testicular organoid model. This model reconstitutes the sex-determining region Y (Sry)-SRY-box transcription factor 9 (Sox9)‑mediated sex-determination program and generates testicular somatic cell-like cells (TesLCs). When assembled with primordial germ cell-like cells (PGCLCs), TesLCs formed seminiferous tubule-like structures and supported male germline progression toward prospermatogonia and germline stem cell-like cells (GSCLCs). After transplantation into infertile recipient testes, organoid-derived GSCLCs produced fertilization-competent sperm and generated fertile offspring. The major significance of this work extends beyond sperm production. It integrates sex determination, germ-cell fate transition, and testicular niche self-organization into an experimentally tractable organoid system. This commentary discusses the technical strategy, sex-determination mechanism, value of testicular niche reconstruction, and translational limitations of this study. It also highlights its implications for in vitro gametogenesis, male infertility modeling, and reproductive medicine.

Objective To systematically evaluate the similarities between six-gene-modified pig red blood cells and human red blood cells in multiple dimensions, including cell morphology, hematological parameters, microbial safety and serological compatibility, and to verify the feasibility of using six-gene-modified pig red blood cells as an alternative for blood transfusion in cases of traumatic hemorrhage. Methods Blood samples from O-type human donor, six-gene-modified O-type pig and wild-type O-type pig were collected. The pig gene-modified phenotype was identified by flow cytometry. The morphology was observed using field emission scanning electron microscopy and the diameter was measured. Hematological tests, including blood routine, osmotic fragility test, adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) content and partial pressure of oxygen at 50% hemoglobin saturation (P50), were conducted to assess the deformability of red blood cells. Real-time fluorescent quantitative polymerase chain reaction was used to screen for zoonotic viruses, and cross-matching was performed using column gel method, condensation amine method and saline tube method. Results The six-gene-modified pig red blood cells successfully knocked out the α-Gal, Neu5Gc and Sda antigens and expressed hCD55. Wild-type pig red blood cells expressed these three antigens but did not express human complement regulatory proteins. All three types of red blood cells presented a biconcave discoid morphology. The diameter of O-type human red blood cells was larger than that of the six-gene-modified porcine red blood cells and wild-type pig red blood cells (all P < 0.05). The average red blood cell volume and average red blood cell hemoglobin of O-type humans were higher than those of the six-gene-modified pig red blood cells, and the distribution width-variable coefficient of O-type human red blood cells was lower than that of the six-gene-modified pig red blood cells (all P < 0.05). There were no statistically significant differences in the permeability fragility, ATP content, 2,3-DPG content, P50 and red blood cell deformability indicators of the three types of red blood cells (all P > 0.05). The screening for zoonotic viruses in human and animal cells of the six-gene-modified pigs and wild-type pigs was negative. The cross-matching results showed that in the column gel method and condensation amine method, the primary and secondary sides of the six-gene-modified pig red blood cells did not agglutinate with human red blood cells, while the wild-type pig red blood cells agglutinated with human red blood cells in all cross-matching methods. Conclusions The six-gene-modified pig red blood cells are highly similar to human red blood cells in terms of morphology, key hematological parameters, energy metabolism, oxygen-carrying capacity, mechanical properties, microbial safety and serological compatibility, providing an experimental basis for the clinical transformation of xenotransfusion.

OBJECTIVE To investigate the therapeutic effect of Qingchang Huazhuo Formula(QCHZF)on mice with ulcerative colitis(UC)and the influences of fecal metabolites based on non-targeted metabolomics to investigate the mechanism of action of QCHZF in the treatment of UC.METHODS UC mice were induced by dextran sulfate sodium salt(DSS)and were administered with QCHZF.During the experiment,the body weight,stool characteristics and blood in stool were recorded daily,and the disease activity index(DAI)was calculated.At the end of the experiment,the length of colon was measured,colonic tissue damage in mice were ob-served by hematoxylin-eosin and alcian blue staining,mRNA expression levels of inflammatory factors,IL-6,IL-18 and IL-1β,and intestinal barrier factors,ZO-1 and Muc2,were detected in colon tissues via qPCR method,and protein expression level of intestinal barrier,Muc2,was detected with immunofluorescence.Fecal metabolite changes in mice were detected employing un-targeted metabo-lomics and analyzed by MetaboAnalyst 5.0 for metabolic pathway enrichment.RESULTS QCHZF significantly alleviated colitis symptoms,increased body weight,decreased DAI score,reversed colonic shortening,inhibited inflammatory factors expression,im-proved colonic tissue structure disorders,increased the number of goblet cells,and restored the intestinal barrier in UC mice,regulated 58 metabolites,mainly involving pathways of methionine and cysteine metabolism,purine metabolism,steroid hormone biosynthesis,vitamin B6 metabolism,tryptophan metabolism and primary bile acid pathways.CONCLUSION QCHZF can improve colitis symp-toms,repaire the intestinal barrier and modulate fecal metabolites and related metabolic pathways in UC mice.

BACKGROUND:Soft tissue damage induced by cobalt nanoparticles is currently the most noticeable complication in patients with artificial joint prostheses.Therefore,an effective therapeutic strategy is needed to limit the toxicity of cobalt nanoparticles.OBJECTIVE:To investigate the protective effect of a ferroptosis inhibitor on cobalt nanoparticles-induced cytotoxicity.METHODS:To evaluate the detoxification effect of ferroptosis inhibitor on mouse fibroblasts(Balb/3T3),Balb/3T3 cells were treated with cobalt nanoparticles and ferroptosis inhibitor for 24 hours.The cell viabilities were measured by cell viability assay.Based on the results of the cell viability assay,the concentrations of cobalt nanoparticles and deferiprone were determined.The experiment was divided into four groups:the cobalt nanoparticles group(400 μmol/L cobalt nanoparticles),the cobalt nanoparticles+deferiprone group(400 μmol/L cobalt nanoparticles and 25 μmol/L deferiprone),the deferiprone group(25 μmol/L deferiprone),and the control group.The expressions of glutathione peroxidase 4 and solute carrier family 7 member 11 protein were examined by western blot assay.RESULTS AND CONCLUSION:(1)The cell viability assay results showed that as the exposure time or the drug concentration increased,cell viability decreased further,indicating that the cytotoxic effect of cobalt nanoparticles was time-and dose-dependent.Additionally,after 24 hours of exposure,cobalt nanoparticles significantly reduced cell viability and glutathione levels compared with the control group(P＜0.05).At the same time,compared with the control group,there was an increase in reactive oxygen species production,intracellular iron levels,and the expression of inflammatory cytokines such as tumor necrosis factor α,interleukin-1β,and interleukin-6.After the addition of deferiprone,compared with the cobalt nanoparticles group,cell viability significantly improved,and reactive oxygen species production,intracellular iron levels,and the expression of inflammatory cytokines(tumor necrosis factor α,interleukin-1β,and interleukin-6)significantly decreased(P＜0.05).This demonstrated that deferiprone had a protective effect on cells exposed to cobalt nanoparticles.(2)Western blot assay results showed that cobalt nanoparticles reduced the expression of glutathione peroxidase 4 and solute carrier family 7 member 11 protein(P＜0.05),while deferiprone inhibited this effect(P＜0.05).(3)The above findings verify that cobalt nanoparticles are highly cytotoxic and ferroptosis inhibitor deferiprone has a detoxification effect on cytotoxicity induced by cobalt nanoparticles.Ferroptosis plays an important role in the process by which cobalt nanoparticles induce cytotoxicity.The inhibitory effect of ferroptosis inhibitors on the toxicity of cobalt nanoparticles may provide valuable insights for further research into the mechanisms of cobalt nanoparticle toxicity and potential detoxification strategies.

Objective To investigate the effect of local histopathological changes of thyroid nodules after ultrasound-guided fine needle aspiration biopsy(US-FNBA)on laparoscopic thyroid surgery.Methods The clinical data of 120 patients with thyroid malignant tumors admitted to Xiaogan Hospital of Wuhan University of Science and Technology from January 2020 to December 2022 were retrospectively analyzed,and according to whether received US-FNAB before surgery they were divided into the observation group(with US-FNAB)and the control group(without US-FNAB),with 60 cases in each group.The operation time,change of parathyroid hormone(ΔPTH)before and after surgery,change of serum Ca2+concentration(serum ΔCa2+)before and after surgery,drainage volume 24 hours after surgery and incidence of complications were compared between the two groups.According to fine needle aspiration-to-surgery(FTS),the patients in the observation group were further divided into the group A(FTS≤3 days)and the group B(FTS>3 days),and the effect of US-FNAB on laparoscopic thyroid surgery was explored combined with the pathological changes of the thyroid puncture capsule of the two groups.Results The operation time of the observation group was longer than that of the control group(P<0.05),and there was no significant difference in the drainage volume 24 hours after surgery,ΔPTH,serum ΔCa2+,or proportions of facial or limb numbness,hypoparathyroidism,recurrent laryngeal nerve integrity,and hypocalcemia between the two groups(P>0.05).There was no significant difference in the operation time,ΔPTH,serum ΔCa2+,drainage volume 24 hours after surgery,hypocalcemia,recurrent laryngeal nerve integrity or hypothyroidism between the group A and group B(P>0.05).The proportion of patients with facial or limb numbness after surgery in the group A was significantly lower than that in the group B(P<0.05).Conclusion Preoperative US-FNAB for thyroid nodules may prolong the operation time in patients undergoing laparoscopic thyroid surgery,and surgical treatment in early aspiration can help decrease this effect and reduce the incidences of postoperative hypocalcemia and hypothyroidism.

Background and Aims:Laparoscopic transcystic bile duct exploration(LTCBDE)has become the preferred method for treating secondary bile duct stones due to its advantages of minimal trauma,fast recovery,and low complication rates.However,challenges remain in the dilation of the cystic duct,the insertion of the choledochoscope,and the exploration of the common hepatic duct and intrahepatic bile ducts.This study was performed to explore the clinical application and effectiveness of the programmed modified LTCBDE in the treatment of gallbladder stones combined with common bile duct stones.Methods:A retrospective analysis was conducted on the clinical data of 248 patients who underwent programmed modified LTCBDE at the Affiliated Suzhou Ninth Hospital of Soochow University from January 2018 to January 2024.The surgical strategies and treatment outcomes were summarized.Data from 913 patients who underwent laparoscopic common bile duct exploration(LCBDE)during the same period were also collected to compare surgical outcomes and postoperative complications between the two groups.Results:Through programmed surgical steps,the innovative"diaphragm"incision technique,and improved bile duct probe application,244 patients(98.4%)successfully underwent LTCBDE,while 4 patients were converted to LCBDE due to failure to insert a 4.9 mm choledochoscope through the cystic duct.After operation,1 patient(0.4%)had residual bile duct stones,which were successfully removed through T-tube tract stone extraction(this patient was converted to LCBDE during the procedure).Additionally,1 case of bile leakage and 1 case of abdominal infection(each 0.4%)occurred,both of which resolved with conservative treatment.No cases of intra-abdominal bleeding,bile duct stenosis,or bile duct injury were reported.The average operative time in the programmed modified LTCBDE group was comparable to that of the LCBDE group(85.2 min vs.88.0 min,P=0.398),but the postoperative hospital stay was significantly shorter(6.2 d vs.8.3 d,P<0.001),and the incidence of complications was lower(1.6%vs.4.7%,P=0.044).Conclusion:The programmed modified LTCBDE is a standardized,safe,and effective procedure with a low complication rate.It is worthy of further clinical promotion and application.