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.

OBJECTIVE:Elevated blood pressure increases the risk of cardiovascular diseases.Isometric exercise training has been shown to significantly reduce resting blood pressure,but the factors influencing its effectiveness remain unclear,and specific application guidelines are yet to be established.This study aims to evaluate the impact of isometric exercise training on resting blood pressure through meta-analysis,explore its moderating factors,and provide evidence-based recommendations based on its dose-response relationship.METHODS:Following the PRISMA guidelines,a systematic search was conducted in PubMed,Embase,Cochrane Library,Scopus,and Web of Science databases using keywords"Isometric exercise training,""Systolic blood pressure,"and"Diastolic blood pressure,"covering literature up to September 2024.Randomized controlled trials involving isometric exercise training and resting blood pressure were included.Three independent researchers performed literature screening and data extraction,assessing bias risk and quality grades using the Risk of Bias 2.0 tool and GRADE framework.Main effect pooling,publication bias assessment,subgroup,and regression analysis were conducted using R software(version 4.3.4).RESULTS:A total of 28 articles(comprising 32 randomized controlled trials)involving 977 participants were included.(1)Meta-analysis results indicated that isometric exercise training significantly reduced resting systolic blood pressure(MD=-8.01,95％CI=-9.22 to-6.80,P＜0.01,I2=18.20％,low evidence grade)and diastolic blood pressure(MD=-3.46,95％CI=-4.64 to-2.28,P＜0.01,I2=0％,moderate evidence grade)compared to no exercise.(2)Subgroup analysis results revealed significant influences of gender,health status,exercise modality,frequency,intensity,duration,sets per session,rest duration,and baseline blood pressure on the main effects for both systolic(P＜0.01)and diastolic blood pressure(P＜0.05).(3)Regression analysis results did not show any significant influencing factors,but body mass index(β=-4.11,P=0.091)showed a significant negative trend on the main effect for systolic blood pressure.(4)No significant publication bias was observed in the meta-analysis results(P＞0.05).CONCLUSION:(1)Isometric exercise training significantly lowers systolic(low evidence grade)and diastolic(moderate evidence grade)blood pressure with clinically meaningful thresholds.(2)Participant characteristics(gender,health status,baseline blood pressure,and body mass index)and isometric exercise training protocols(modality,frequency,intensity,duration,cycle,sets per session,and rest duration)influence its antihypertensive effects.(3)The article recommends the optimal blood pressure management prescription:three sessions per week,with four sets per session,each set lasting 2 minutes with a 2-minute rest,at an intensity of 95％HRpeak using isometric wall squat exercises;the intervention period can be adjusted around a 6-week node.Future high-quality research is urgently needed to further validate and support these conclusions.

Objectives: . This study aimed to investigate the regulatory functions of gamma-aminobutyric acid (GABA)ergic neural circuits from the inferior colliculus (IC) to the medial geniculate body (MGB) in salicylate-induced tinnitus. Methods: . Mice were treated with salicylate to induce tinnitus, and tinnitus-like behaviors were evaluated via gap prepulse inhibition of acoustic startle. Using combined viral tracing methodologies, we identified and mapped the pathways and connections from the IC to the MGB. Furthermore, we employed Gq-coupled human M3 designer receptors exclusively activated by designer drugs (DREADDs) and Gi-coupled human M4 DREADDs to achieve targeted excitation or suppression of GABAergic neurons in the IC and MGB. Following the administration of clozapine N-oxide, which binds to these receptors, we modulated these neural circuits to assess their impact on tinnitus severity in a mouse model. Results: . Our findings demonstrated that mice exposed to salicylate exhibited tinnitus-like behaviors. GABAergic neurons projecting retrogradely from the MGB to the IC were primarily concentrated in the external nucleus of the IC. After clozapine N-oxide administration, chemogenetic activation of IC-MGB GABAergic neurons aggravated salicylate-induced tinnitus. Additionally, activation of GABAergic neurons between the IC and MGB induced the perception of tinnitus even without salicylate. However, chemogenetic inhibition of the IC-MGB GABAergic circuit did not reverse salicylate-induced tinnitus. Conclusion . These findings suggest that activation of the IC-MGB GABAergic neural circuit may contribute to tinnitus generation through a mechanism distinct from that of salicylate-induced tinnitus. This study provides novel insights into the mechanisms underlying tinnitus.

OBJECTIVE:Elevated blood pressure increases the risk of cardiovascular diseases.Isometric exercise training has been shown to significantly reduce resting blood pressure,but the factors influencing its effectiveness remain unclear,and specific application guidelines are yet to be established.This study aims to evaluate the impact of isometric exercise training on resting blood pressure through meta-analysis,explore its moderating factors,and provide evidence-based recommendations based on its dose-response relationship.METHODS:Following the PRISMA guidelines,a systematic search was conducted in PubMed,Embase,Cochrane Library,Scopus,and Web of Science databases using keywords"Isometric exercise training,""Systolic blood pressure,"and"Diastolic blood pressure,"covering literature up to September 2024.Randomized controlled trials involving isometric exercise training and resting blood pressure were included.Three independent researchers performed literature screening and data extraction,assessing bias risk and quality grades using the Risk of Bias 2.0 tool and GRADE framework.Main effect pooling,publication bias assessment,subgroup,and regression analysis were conducted using R software(version 4.3.4).RESULTS:A total of 28 articles(comprising 32 randomized controlled trials)involving 977 participants were included.(1)Meta-analysis results indicated that isometric exercise training significantly reduced resting systolic blood pressure(MD=-8.01,95％CI=-9.22 to-6.80,P＜0.01,I2=18.20％,low evidence grade)and diastolic blood pressure(MD=-3.46,95％CI=-4.64 to-2.28,P＜0.01,I2=0％,moderate evidence grade)compared to no exercise.(2)Subgroup analysis results revealed significant influences of gender,health status,exercise modality,frequency,intensity,duration,sets per session,rest duration,and baseline blood pressure on the main effects for both systolic(P＜0.01)and diastolic blood pressure(P＜0.05).(3)Regression analysis results did not show any significant influencing factors,but body mass index(β=-4.11,P=0.091)showed a significant negative trend on the main effect for systolic blood pressure.(4)No significant publication bias was observed in the meta-analysis results(P＞0.05).CONCLUSION:(1)Isometric exercise training significantly lowers systolic(low evidence grade)and diastolic(moderate evidence grade)blood pressure with clinically meaningful thresholds.(2)Participant characteristics(gender,health status,baseline blood pressure,and body mass index)and isometric exercise training protocols(modality,frequency,intensity,duration,cycle,sets per session,and rest duration)influence its antihypertensive effects.(3)The article recommends the optimal blood pressure management prescription:three sessions per week,with four sets per session,each set lasting 2 minutes with a 2-minute rest,at an intensity of 95％HRpeak using isometric wall squat exercises;the intervention period can be adjusted around a 6-week node.Future high-quality research is urgently needed to further validate and support these conclusions.

Background: and Purpose Live cell-based assays (LCBA) are increasingly used for serological antibody detection due to their ability to preserve antigen conformation, offering moderately higher sensitivity than fixed cell-based assays. However, the clinical necessity of prioritizing LCBA for the detection of aquaporin-4 immunoglobulin G (AQP4-IgG) in neuromyelitis optica spectrum disorder (NMOSD) remains unclear, especially when compared to its established role in diagnosing myelin oligodendrocyte glycoprotein antibody-associated disease. Methods: We compared the performance of live cell-based assays using immunofluorescence (LCBA-IF) and fixed cell-based assays using immunofluorescence (FCBA-IF) in detecting AQP4-IgG in 90 cases of NMOSD meeting 2015 International Panel for Neuromyelitis Optica Diagnosis criteria, alongside 40 controls. Additionally, we further investigated the relationship between AQP4-IgG titers as measured by LCBA-IF and FCBA-IF and clinical parameters in NMOSD patients. Results: Results showed 96.9% agreement between LCBA-IF and FCBA-IF (Cohen’s κ=0.935), with a strong Spearman correlation (0.977, p<0.001). Both methods demonstrated 100% specificity, with LCBA-IF showing slightly higher sensitivity compared to FCBA-IF. Within LCBAIF-tested groups, statistically significant differences in annualized relapse rates were observed across all pairwise comparisons (low-titer vs. moderate-titer, low-titer vs. high-titer, and moderate-titer vs. high-titer; all p<0.050). However, this association reached statistical significance in some FCBA-IF-tested groups. Conclusions Overall, there is a strong concordance between LCBA-IF and FCBA-IF in detecting AQP4-IgG, where LCBA-IF shows slightly higher sensitivity. Furthermore, there is a potential link between elevated AQP4-IgG titers and an increased risk of relapse, and this correlation may appear more pronounced when using LCBA-IF.

Objective To investigate the association of serum total testosterone(TT),free testosterone(FT),sex hormone-binding globulin(SHBG),and estradiol(E_2)with all-cause mortality rate(AMR)in males and to evaluate their predictive value. Methods Based on data from the National Health and Nutrition Examination Survey(NHANES)cycles(1999—2004 and 2013—2016),the relationships between different sex hormones and AMR was analyzed with Cox proportional hazards regression models. The dose-response relationships between different sex hormones and male AMR were assessed with restricted cubic splines(RCS). The predictive performances of different sex hormones for male AMR were compared with receiver operating characteristic(ROC)curves and the area under the curve(AUC). The survival differences across varying sex hormone levels were evaluated with Kaplan-Meier survival curves and log-rank tests. Results This study involved 3632 adult male participants with a median follow-up of 6.5 years,during which 320 AMR occurred(8.8%). Cox univariate analysis indicated that TT,FT and SHBG were risk factors for AMR(P＜0.001). Cox multivariate analysis revealed that FT and SHBG were independent risk factors for AMR(FT:HR=0.90,95%CI:0.86-0.94; SHBG:HR=1.01,95%CI:1.01-1.02). RCS revealed a U-shaped nonlinear relationship between TT,FT and AMR(P＜0.001); SHBG showed a positive dose-response relationship with AMR(P＜0.001). The AUCs of FT predicting the 5-year,10-year,and 15-year AMR were 0.78(95%CI:0.73-0.82),0.80(95%CI:0.76-0.84),and 0.84(95%CI: 0.80-0.88),respectively,significantly outperforming other indicators. Subgroup analysis revealed that FT was negatively correlated with male AMR,but this association was modulated by age,race,smoking status and diabetes status(P_(interaction)＜0.05). Kaplan-Meier survival analysis showed that different levels of TT and FT(Q1-Q4)were significantly positively correlated with survival rate(P＜0.001),while different levels of SHBG were significantly negatively correlated with survival rate,with patients in the Q4 group having the highest risk of death(P＜0.001). Conclusion Based on US NHANES,studies have shown that the FT and SHBG are independent risk factors for male AMR,and FT demonstrates greater predictive value for male AMR than TT.

Objective To investigate the expression of six-transmembrane epithelial antigen of prostate 4 (STEAP4) in prostate cancer (PCa), explore its correlation with the infiltration of regulatory T cells (Tregs) and CD8+T cells within the tumor-infiltrating T lymphocyte population, and to predict the potential role of STEAP4 in promoting PCa progression. Methods The clinicopathological data and tumor tissue specimens were collected from 90 PCa patients undergoing radical prostatectomy at our hospital during Jan.2018 and Dec.2024.The protein expressions of STEAP4, forkhead box transcription factor P3 (FoxP3), and CD8+T cells in tumor tissues were detected with immunohistochemistry.The correlation between STEAP4 expression and the infiltration of Tregs (FoxP3 T cells), CD8+T cells and clinicopathological characteristics was analyzed.The relationship between STEAP4 expression and prognosis was evaluated with Kaplan-Meier survival curves. Results STEAP4 expression was significantly higher in PCa tissues than in adjacent benign tissues (P＜0.001).STEAP4 expression was significantly associated with serum total prostate-specific antigen (tPSA) level, seminal vesicle invasion, extraprostatic extension, International Society of Urological Pathology (ISUP) grading, and pathological T stage (all P＜0.05).The mean infiltration density of Tregs (FoxP3 T cells) in PCa tissues was significantly higher than that in adjacent benign tissues[ (11.09±5.05) cells/HPF vs. (3.92±1.59) cells/HPF, P＜0.001], while the mean infiltration density of CD8+T cells was significantly lower[ (6.07±2.90) cells/HPF vs. (27.23±6.75) cells/HPF, P＜0.001].In PCa tissues, STEAP4 expression showed a significant positive correlation with Treg (FoxP3 T cells) infiltration density (r_s=0.472, P＜0.001) and a significant negative correlation with CD8+T-cell infiltration density (r_s=-0.495, P＜0.001).Kaplan-Meier survival curves indicated that high STEAP4 expression group and low STEAP4 expression group in PCa patients was not correlated with overall survival (OS) or recurrence-free survival (P=0.562, 0.346). Conclusion STEAP4 is highly expressed in PCa tissues, and its expression is closely associated with tumor-infiltrating lymphocytes, particularly Tregs and CD8+T cells, suggesting that STEAP4 may cooperate with Tregs and CD8+T cells to regulate the tumor microenvironment, thereby promoting PCa progression.

Tumorigenesis， invasion， and metastasis occur in the context of a persistent inflammatory microenvironment， and a variety of inflammatory factors can lead to the development of various tumors. Guided by the thought of "preventive treatment of disease" in TCM and the concept of tertiary prevention in modern medicine， it is of great significance to effectively intervene in the inflammatory stage of the disease， interrupt disease progression， prevent the occurrence of malignant tumors， and reverse the process of "inflammation-to-cancer transformation". Sinisan， a commonly used prescription in the Treatise on Febrile Diseases， has been widely applied in the treatment of precancerous lesions of the digestive system， demonstrating considerable advantages. This article reviewed literature from the past 20 years， summarizing the application of Sinisan in precancerous lesions of the digestive system from three aspects： the exploration of its prescription-syndrome relationship， clinical application， and mechanistic study. It is found that basic syndrome indications of Sinisan include harmonizing the Earth element to promote spleen-stomach transportation and transformation， soothing the liver and nourishing the Wood element to restore the smooth flow of Qi， and regulating Yin and Yang to relieve stagnation within the system. In clinical application， Sinisan has shown significant efficacy in atrophic gastritis and precancerous conditions such as intestinal metaplasia， gastric ulcer， ulcerative colitis， esophagitis， and pancreatitis. Mechanistic studies have revealed that Sinisan can inhibit inflammatory factors and improve the inflammatory microenvironment， inhibit cell proliferation and regulate apoptosis， exhibit anti-angiogenic and antitumorigenic effects， modulate immune function， and exert antioxidant effects. These mechanisms can be achieved by regulating pathways such as nuclear factor erythroid 2-related factor 2/heme oxygenase-1 （Nrf2/HO-1）， farnesoid X receptor （FXR）/Nrf2， phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt）， Takeda G protein-coupled receptor 5/cyclic adenosine monophosphate/protein kinase A （TGR5/cAMP/PKA）， interleukin-4/signal transducer and activator of transcription 6 （IL-4/STAT6）， Janus kinase/signal transducer and activator of transcription （JAK/STAT）， RhoA/Rho-associated protein kinase （RhoA/ROCK）， and transforming growth factor-β/Smad proteins （TGF-β/Smads）， confirming Sinisan's role in reversing the inflammation-to-cancer transformation. The current research status of Sinisan in precancerous lesions of the digestive system was thoroughly examined through the above three aspects， along with the identification of limitations and areas for improvement in current research. The aim is to provide a basis and support for future in-depth research on Sinisan， promote the development of new integrated treatment models combining TCM and Western medicine for precancerous lesions， and aid in the research and development of drugs related to precancerous lesions.

Objective To investigate whether glaucocalyxin A can reduce inflammatory response in rats with trau-matic fracture.Methods The rats were randomly divided into control group,model group,low GLA(GLA-L),high GLA dose group(GLA-H)and GLA?H+YC?1(HIF?1α inhibitor)group.The level of TNF?α,IL?6,CTX?Ⅰ,CTX?Ⅱ,S?Ca,S?P,BGP and the activities of iNOS and ALP were detected by ELISA.The density and thickness of bone trabeculae were measured by computed tomography.Apoptosis of femur tissue was detected by TUNEL.The cartilage morphology was observed by saffranin O?fast green staining.Western blot was applied to de?tect the expression of HIF?1α,VEGF,CD31,BMP?2,and collagenⅠ proteins in femoral tissue.Results Compared with the control group,the cartilage morphology of rats in the model group was significantly damaged,the level of serum TNF?α,IL?6,CTX?Ⅰ,CTX?Ⅱ,iNOS activity and apoptosis rate of femur tissue were in?creased.Serum S?Ca,S?P,BGP levels,ALP activity,bone trabecular density and thickness,HIF?1α,VEGF,CD31,BMP?2,collagen Ⅰ protein expression levels in femur tissue were all decreased(P<0.05).The mor?phology of cartilage tissue in GLA?L and GLA?H groups was significantly improved,and the level of serum TNF?α,IL?6,CTX?Ⅰ,CTX?Ⅱ,iNOS activity and apoptosis rate of femur tissue were all significantly de?creased.Serum levels of S?Ca,S?P,BGP,ALP activity,bone trabecular density and thickness,HIF?1α,VEGF,CD31,BMP?2,collagen Ⅰ protein expression level in femur tissue were significantly increased(P<0.05).YC?1 was able to weaken the therapeutic effect of GLA on traumatic fracture rats(P<0.05).Conclusions GLA alleviates inflammation and cartilage damage in rats with traumatic fractures,promotes angio?genesis and improves femoral morphology,possibly by activating the HIF?1α/VEGF signaling pathway.

Objective:To investigate the effect of insulin-like growth factor 1 (IGF-1) on acute lung injury in septic mice and its underlying molecular mechanism.Methods:Twenty SPF male C57BL/6J mice aged 6-8 weeks were randomly (random number) divided into the sham-operated group, sham-operated + IGF-1 group, sepsis group and sepsis + IGF-1 group, with 5 mice in each group. IGF-1 [60 μg/(kg·d)] was injected via the tail vein for 3 consecutive days in the sham-operated + IGF-1 group and sepsis + IGF-1 group, and mice in the sham-operated group and sepsis group were injected with an equal volume of saline. The tissue of the upper lobe of the right lung was taken to calculate the wet-to-dry ratio, and the upper lobe of the left lung was subjected to HE staining to analyze pathological changes and evaluate lung injury. The levels of interleukin (IL)-6 and IL-1β in the bronchoalveolar lavage fluid (BALF) and serum of mice were detected by ELISA. The expression of p-PI3K, PI3K, p-AKT and AKT in lung tissues was determined via Western blotting. The quantitative data with a normal distribution and homogeneity of variance were compared between the two groups by two independent sample t test. Results:Lung volume was reduced in the sepsis group than in the sham-operated group, obvious surface congestion, dark red color, large bruises and hemorrhagic foci were observed under the pericardium, and the wet-to-dry ratio was significantly elevated ( P<0.05). Compared with the sepsis group, the sepsis + IGF-1 group had slightly increased lung volume, less congestion, darker red color, fewer bruises and hemorrhagic foci, and a lower wet-to-dry ratio ( P<0.05). There was no significant change in lung tissue morphology in the sham-operated + IGF-1 group compared with the sham-operated group. HE staining and lung histopathological scores showed that lung tissue was significantly damaged in the sepsis group than the sham-operated group ( P<0.001), and the pathological score of lung tissue was less damaged in the sepsis + IGF-1 group compared with the sepsis group ( P<0.01). The ELISA results demonstrated that the serum levels of IL-6 and IL-1β were markedly decreased in the sepsis + IGF-1 group than in the sepsis group [(26.22±1.60) pg/mL vs. (45.61±7.85) pg/mL, P<0.05; (87.99±11.80) pg/mL vs. (181.26±10.11) pg/mL, P<0.001]. Moreover, the IL-6 and IL-1β contents in the BALF of the sepsis + IGF-1 group were notably lower than those in the BALF of the sepsis group [(7.67±0.42) pg/mL vs. (20.25±0.43) pg/mL, P<0.001; (17.00±6.08) pg/mL vs. (108.61±5.18), pg/mL P <0.001]. Western blot analysis revealed that the expression of p-PI3K, PI3K, p-AKT and AKT in the lung tissues of mice in the sepsis+IGF-1 group were markedly lower than that in the sepsis group [(0.71±0.05) vs. (1.21±0.09), P<0.05; (0.57±0.08) vs. (1.24±0.22), P<0.01; (0.29±0.07) vs. (1.10±0.04), P<0.001; (0.65±0.17) vs. (1.19±0.07), P<0.01]. Conclusion:IGF-1 ameliorates sepsis-induced acute lung injury in mice, and its protective effect may be achieved by inhibiting the PI3K/AKT pathway.