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.

AIM:To investigate the predictive value of aqueous humor stromal cell-derived factor-1(SDF-1)and epithelial neutrophil-activating peptide-78(ENA78)for visual impairment following vitrectomy in patients with diabetic retinopathy(DR).METHODS: Patients with DR who underwent vitrectomy in the ophthalmology department of our hospital from March 2022 to March 2024 were enrolled as study group. According to the results of uncorrected visual acuity examination during the 1-year follow-up after surgery, they were further divided into visual impairment group and good visual acuity group. In addition, patients with cataracts who were treated in our hospital during the same period were recruited as control group. The levels of SDF-1 and ENA78 in aqueous humor were detected by enzyme-linked immunosorbent assay(ELISA). Pearson correlation analysis was used to evaluate the correlation of indicators. Multivariate Logistic regression analysis was performed to identify the influencing factors of postoperative visual impairment in DR patients. Relative risk analysis was conducted to assess the impact of different aqueous humor SDF-1 and ENA78 levels on postoperative visual impairment in DR patients. Receiver operating characteristic(ROC)curve analysis was applied to determine the predictive value of aqueous humor SDF-1 and ENA78 levels for postoperative visual impairment in DR patients.RESULTS: Totally 156 cases(156 eyes)were enrolled in the study group, including 87 males and 69 females, with a mean age of 60.65±8.91 years old; and 91 cases(91 eyes)were enrolled in the control group, including 53 males and 38 females, with a mean age of 58.72±8.43 years old. The levels of SDF-1 and ENA78 in aqueous humor of the study group were significantly higher than those of the control group(P<0.001). According to the results of uncorrected visual acuity during the 1-year follow-up after surgery, the study group was further divided into the visual impairment group(n=49)and the good visual acuity group(n=107), including 28 males and 21 females in the visual impairment group, with a mean age of 63.73±7.41 years, and 59 males and 48 females in the good visual acuity group, with a mean age of 59.24±8.47 years. The age and glycated hemoglobin(HbA1c)levels of the visual impairment group were higher than those of the good visual acuity group(all P<0.05). Compared with the good visual acuity group, the levels of SDF-1 and ENA78 in aqueous humor of the visual impairment group were significantly elevated(all P<0.05). A positive correlation was found in the study group between the levels of SDF-1, ENA78 in aqueous humor and HbA1c(r=0.314, 0.362, all P<0.05). Elevated levels of aqueous humor SDF-1(OR=2.936, 95% CI: 1.628-5.296)and ENA78(OR=3.092, 95% CI: 1.738-5.502)were identified as risk factors for postoperative visual impairment in DR patients(all P<0.05). High levels of SDF-1 and ENA78 increased the risk of postoperative visual impairment by 1.958-fold and 2.030-fold compared with the low-level groups, respectively; the area under the curve(AUC)of combined detection of aqueous humor SDF-1 and ENA78 for predicting postoperative visual impairment in DR patients was 0.936, which was superior to the predictive value of SDF-1 or ENA78 alone(Zcombination-SDF-1=2.850, Zcombination-ENA78=2.445, all P<0.05).CONCLUSION: Elevated levels of aqueous humor SDF-1 and ENA78 are risk factors for postoperative visual impairment in DR patients, and the combined detection of SDF-1 and ENA78 has high clinical application value for predicting postoperative visual impairment in DR patients.

AIM: To investigate the correlation of serum leukocyte-derived chemotaxin 2(LECT2)and complement C1q/tumor necrosis factor-related protein 5(CTRP5)with diabetic retinopathy(DR).METHODS:A single-center cross-sectional analysis was conducted on 138 patients with type 2 diabetes mellitus(T2DM)admitted to our hospital from April 2023 to April 2025. According to the diagnostic results, they were divided into a non-DR group(60 cases)and a DR group(78 cases). The DR group was further divided into a proliferative DR(PDR)group(29 cases)and a non-proliferative DR(NPDR)group(49 cases). Pearson correlation analysis was used to assess the correlation of LECT2 and CTRP5 with glucose-lipid metabolism indicators. Logistic regression analysis was conducted to identify the influencing factors for the occurrence of DR in T2DM patients. ROC curve analysis was performed to evaluate the diagnostic value of serum LECT2 and CTRP5 for DR in T2DM patients.RESULTS: A comparison of general patient data between the two groups showed no significant differences. The DR group had higher serum levels of LECT2, CTRP5, total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), and homeostatic model assessment of insulin resistance(HOMA-IR)than the non-DR group, while high-density lipoprotein cholesterol(HDL-C)was lower(all P<0.05). The serum levels of LECT2 and CTRP5 in the PDR group were higher than those in the NPDR group(all P<0.05). Serum LECT2 and CTRP5 positively correlated with TC, TG, LDL-C, and HOMA-IR, and negatively correlated with HDL-C(all P<0.001). Logistic results showed that duration of diabetes, TC, TG, LDL-C, HOMA-IR, LECT2, and CTRP5 were risk factors for DR occurrence in T2DM patients(all P<0.05). The ROC curve showed that the AUCs for serum LECT2, CTRP5 alone, and combined in diagnosing DR in T2DM patients were 0.830, 0.839, and 0.915, respectively. The AUC for the combined diagnosis was higher than that of LECT2 or CTRP5 alone according to the DeLong test(Z=2.818, 2.824, P=0.015, 0.012).CONCLUSION:Serum LECT2 and CTRP5 levels are closely related to the development of DR in T2DM patients, and joint detection has certain clinical value in diagnosing DR in T2DM patients.

Hepatocellular carcinoma (HCC) develops within a profoundly immunosuppressive tumor immune microenvironment (TIME), which limits the efficacy of immunotherapy. Polarization of tumor-associated macrophages (TAMs) toward a pro-tumorigenic M2 phenotype is a major driver of immune escape. Ferroptosis, an iron-dependent regulated cell death program, intersects with hepatic iron metabolism and immune regulation and thus offers promising points of therapeutic intervention. This review systematically elucidates the mechanistic role of TAM ferroptosis in HCC immune evasion and highlights a “bidirectional regulation” intervention strategy grounded in the Traditional Chinese medicine (TCM) principle of “fortifying healthy qi and eliminating pathogens” (Fuzheng Quxie). This strategy employs “eliminating pathogens” (Quxie) approaches to exploit the metabolic vulnerability of M2-like TAMs and precisely induce their ferroptosis. Moreover, it utilizes “fortifying healthy qi” (Fuzheng) approaches to protect M1-like TAMs and CD8⁺ T cells from oxidative damage. This parallel “induction-protection” paradigm demonstrates the unique advantages of TCM in systemically remodeling TIME through multitarget synergistic actions. Accordingly, precision regulation of TAM ferroptosis based on the Fuzheng Quxie theory represents a promising integrative Chinese-Western medicine strategy for overcoming current bottlenecks in HCC immunotherapy, although its clinical translational potential warrants further validation.

Objective To analyze the genomic characteristics of Legionella pneumophila (L. pneumophila) isolated from industrial circulating cooling water in workplace. Methods A total of 20 L. pneumophila strains isolated from industrial circulating cooling water collected from eight chemical enterprises were selected as the research subjects. Whole-genome sequencing was performed on the strains, followed by multilocus sequence typing (MLST), whole-genome single nucleotide polymorphism analysis, virulence factor profiling, and antibiotic resistance gene detection. Results The dominant MLST typing among the 20 L. pneumophila strains was ST2. Serogroup 1 (Lp1) strains exhibited high genetic diversity. Strains from different enterprises showed substantial genomic diversity, suggesting possible inter-enterprise dissemination. Each isolate harbored 175-295 virulence genes; among them, virulence genes were identified, and 139 genes were detected in all the 20 strains. These genes primarily included those related to the Dot/Icm type Ⅳ bacterial secretion system, flagellar synthesis and motility, and type Ⅳ pili. The number of virulence genes carried by L. pneumophila strains of different ST types differed (P<0.01), with unidentified sequence types harboring more virulence genes than other STs (P<0.01). A total of four resistance genes were predicted, including aminoglycoside phosphotransferase-encoding gene APH(9)-Ia, carbapenemase-encoding gene OXA-29, and efflux pump genes-encoding genes LpeA and LpeB, with detection rates of 100.00%, 100.00%, 10.00%, and 10.00%, respectively. These genes may confer resistance to cephalosporins, penicillins, and aminoglycosides, and all strains were multidrug resistance. Conclusion L. pneumophila isolated from industrial recirculating cooling water exhibits high genetic diversity, strong pathogenicity, and significant multidrug resistance potential.

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.

This article reports a case of a male patient with anti-N-methyl-D-aspartate receptor （NMDAR） encephalitis comorbid with early-onset Alzheimer's disease （AD）. The patient presented with depression， hallucinations and delusions syndrome， and cognitive impairment， and was admitted to a psychiatric hospital. Through multidisciplinary consultation， the diagnosis was confirmed based on positive biological and pathological biomarkers. The patient's symptoms improved after receiving a therapeutic regimen comprising high-dose glucocorticoid therapy and psychiatric-related treatments. Based on relevant current research findings domestically and internationally， this article reviews the pathogenesis and clinical manifestations of early-onset AD and anti-NMDAR encephalitis， in order to enhance the discrimination ability of psychiatric specialists for such patients and improve the diagnosis and treatment level. ［Funded by Medical Research Project of Chengdu City （number， 2024403）］

OBJECTIVE To investigate the ameliorative effects of Coptidis Rhizoma-Ophiopogonis Radix herb pair （HLMD） in rats with type 2 diabetes mellitus （T2DM） and its potential mechanisms.METHODS Male Wistar rats were randomly divided into a control group （6 rats） and a model induction group （30 rats）. The T2DM rat model was established by feeding a high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin. The successfully modeled rats were subsequently divided into the model group， the metformin group （200 mg/kg）， and the low-， medium-， and high-dose HLMD groups （100， 200 and 400 mg/kg， respectively）， with six rats in each group. Rats in each group were administered the corresponding drug solution or normal saline by oral gavage once daily for six consecutive weeks. Body weight， fasting blood glucose （FBG）， the area under the curve （AUC） of the oral glucose tolerance test， serum lipid profiles [total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C）]， and inflammatory cytokine [interleukin-6 （IL-6）， IL-1β， tumor necrosis factor-α （TNF-α）， and IL-10] levels were measured. Histopathological changes in liver and pancreatic tissues， as well as hepatic lipid accumulation， were evaluated. Changes in gut microbiota composition were analyzed in the control， model， and high-dose HLMD groups. Network pharmacology and molecular docking analyses were performed to identify potential therapeutic targets of HLMD against T2DM， followed by experimental validation.RESULTS Compared with the model group， the body weight generally recovered in rats in all HLMD dose groups， while FBG levels consistently decreased. Histopathological abnormalities， including disrupted pancreatic islet architecture， disorganized hepatocyte arrangement， hepatic cord necrosis， and lipid accumulation， were alleviated to varying degrees. AUC （except for the low-dose group）， levels of TC， TG， LDL-C， IL-6（except for the low-dose group）， IL-1β and TNF-α， Simpson and Shannon indices， as well as the relative abundances of Firmicutes， Proteobacteria， and Clostridium were significantly reduced； meanwhile， the levels of HDL-C（except for the low-dose group） and IL-10 （except for the low-dose group）， as well as the relative abundances of Bacteroidota and Ligilactobacillus were significantly increased （ P ＜0.05）. Network pharmacology and molecular docking analyses suggested that the active constituents of HLMD， including quercetin， adenosine， oleanolic acid， stigmasterol， and ophiopogonin D′， may exert antidiabetic effects by targeting key proteins such as protein kinase B1（Akt1）， TNF， IL-6， P53 and IL-1β. Experimental validation further demonstrated that HLMD significantly downregulated the protein expressions of P53 （except for the low-dose group）， IL-6 （except for the low-dose group）， IL-1β and TNF-α in the liver tissues of T2DM rats （ P ＜0.05）.CONCLUSIONS HLMD alleviates glucose and lipid metabolism disorders， attenuates inflammatory responses， and ameliorates hepatic and pancreatic tissue damage in T2DM rats. These beneficial effects may be associated with the modulation of gut microbiota composition and the downregulation of inflammation-related targets， including P53， IL-6， IL-1β and TNF-α.

Objective To investigate the effect of miR-206 on LPS-induced apoptosis and inflamma-tory response in H9c2 cardiomyocytes.Methods An inflammatory injury model was established in H9c2 cells with treatment of 10 μg/ml LPS.Then the cells were divided into control group,LPS group,LPS1 group(LPS+anti-negative control),LPS2 group(LPS+anti-miR-206),LPS3 group(LPS+miR-206 negative control),and LPS4 group(LPS+miR-206 mimic).The LPS1,LPS2,LPS3,and LPS4 groups(n=3)were transfected with anti-negative control,anti-miR-206 se-quence,miR-negative control,or miR-206 mimic sequence,using Lipofectamine 3000 reagent.CCK-8 and EdU assays were used to detect cell proliferation,Caspase-Glo 3/7 activity kit was em-ployed to measure Caspase-3/7 activity,and ELISA was utilized to test the secretion of inflamma-tory cytokines,TNF-α,IL-1β,and IL-6 in the cells.Results Compared with the control group,the LPS group had significantly reduced cell survival rate and EDU-positive cell rate,while elevated miR-206 expression,Bax/Bcl-2,activated Caspase-3,Caspase-3/7,TNF-α,IL-1β,IL-6,p-p38 and p-p65(P＜0.05,P＜0.01).The expression of miR-206,Bax/Bcl-2,activated Caspase-3,Caspase-3/7,TNF-α,IL-1β,IL-6,p-p38 and p-p65 were obviously reduced,while the cell survival rate and EDU-positive cell rate were notably increased in the LPS 2 group than the LPS group(P＜0.05).In the LPS 4 group,the activity of Caspase-3/7 was remarkably stronger than the LPS group(1586±58 vs 816±51,P＜0.05).Conclusion Suppression of miR-206 can effectively inhibit LPS-induced apoptosis and inflammatory response in H9c2 cardiomyocytes.

Objective:To analyze a novel intronic variant in the SPTB gene and explore its effect on SPTB mRNA splicing.Methods:Clinical data of a child diagnosed with hereditary spherocytosis (HS) and admitted to the First Affiliated Hospital of Xi′an Jiaotong University in February 2022 were analyzed retrospectively. Whole genome sequencing was used to identify disease-causing variantions and the results were validated with Sanger sequencing, mRNA sequencing was used to determine the SPTB gene′s mRNA expression level, and bioinformatics tools were used for splicing site prediction and analysis.Results:The proband is a 2-month-old Han male child, clinically presenting with anemia and jaundice. In the past, jaundice appeared early and was severe during the neonatal period, with significantly elevated indirect bilirubin (203.5 μmol/L), accompanied by moderate anemia. This family consisted of four generations, eight of whom suffered from splenomegaly, jaundice, and anemia. In their peripheral blood, the percentage of microglobular erythrocytes was between 5% and 10%. Under scanning electron microscopy analysis of the proband's father's peripheral red blood cells, about 6% exhibited a mouth-shaped morphology, about 4% were spherical, and about 3% were oval. Following the splenectomy, the father′s anemia and jaundice recovered to normal level. Whole genome sequencing analysis of the proband identified a heterozygous variant in the SPTB gene (NM_ 001355436.2 (SPTB):c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC), which was verified to be co-segregating with the disease in this family line by Sanger sequencing. The results of the SPTB gene mRNA expression level detection showed that the expression levels of the SPTB variant gene were statistically increased in the proband and affected family members (father, grandmother, cousin, second cousin, great-grandmother, great-aunt) (all P<0.05). The SPTB gene′s intron can undergo selective splicing, as demonstrated by analysis using the bioinformatics program ESE Finder. Additionally, predictions from the SpliceAI and SpliceTool software indicated that activation of a new covert splicing donor can result in a code-shift variantion that introduces an early termination codon and nonsense-mediated degradation of the mRNA, which prevents the synthesis of proteins. Conclusion:A new variantion site c.6022+4_6022+18delinsTGGCTCCTCCGTGAAGGGACAGTCCTGC was found in SPTB gene. This variantion was the pathogenic factor of HS. By affecting the splicing process, this variantion triggers the nonsense mediated mRNA degradation pathway, resulting in inactivation of gene function.