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 To study the improvement effect and mechanism of Tripterygium wilfordii multiglucoside （TWM） on nephrotic syndrome in rats. METHODS The nephrotic syndrome model was established by intravenous injection of adriamycin via the tail vein. The modeling rats were randomly divided into the model group （distilled water）， prednisone group （10 mg/kg）， and TWM high- and low-dose groups （10 and 5 mg/kg， respectively）. Additionally， blank group （distilled water） without model induction was established. Each group consisted of 9 rats. Rats in each group were administered the corresponding drugs or distilled water by gavage， once a day， for 6 consecutive weeks. The histopathological morphology of kidney tissues in rats was observed； the levels of 24-hour urinary protein （24 h-UTP） and serum biochemical indicators [albumin （ALB）， blood urea nitrogen （BUN）， serum creatinine （SCr）， cholesterol （CHOL）， and triglyceride （TG）] in rats were determined； the levels of oxidative stress indicators [superoxide dismutase （SOD）， malondialdehyde （MDA）] in kidney tissue of rats were determined； expressions of hypoxia-inducible factor-1α （HIF-1α）/microRNA-155-5p （miR-155-5p）/nuclear factor erythriod 2- related factor 2 （Nrf2） signaling pathway-related mRNA and protein in the renal tissues of rats were detected. RESULTS Compared with the blank group， the rats in the model group exhibited disordered renal tissue structure， with a small amount of glomerular necrosis and edema of the renal tubular epithelial cells. 24 h-UTP， serum levels of SCr， BUN， CHOL and TG， MDA content， mRNA and protein expressions of HIF-1α and Keap1 as well as the expression of miR-155-5p in renal tissues were increased significantly （ P ＜0.05）. Serum level of ALB， SOD level in renal tissue as well as mRNA and protein expressions of Nrf2 were decreased significantly （ P ＜0.05）. Compared with the model group， TWM high-dose and low-dose groups exhibited significant improvements in renal injury， with notable reversals in the levels of the above quantitative indicators （ P ＜0.05）. CONCLUSIONS TWM can alleviate oxidative stress-induced damage and thereby improve nephrotic syndrome in rats by regulating the HIF-1α/miR-155-5p/Nrf2 signaling pathway.

ObjectiveTaking the cuproptosis/oxidative stress pathway as the entry point， this study investigated the effect and mechanism of Liangyi Paste on hepatic lipid deposition in naturally aged mice fed with a high-fat diet. MethodsAfter adaptive feeding， 80 ten-week-old male C57BL/6 mice were used. Thirty of them were randomly divided into three groups （10 mice per group）： The 12-month-old control group （12MCON）， the 15-month-old control group （15MCON）， and the 15-month-old group with a high-fat diet （15MHFD）. The 12MCON and 15MCON groups were continuously fed a standard diet， while the 15MHFD group started receiving a high-fat diet at 12 months of age. Tissue samples were collected at the corresponding time points for each group. The remaining 50 mice were randomly divided into five groups （10 mice per group）： the 20-month-old control group （20MCON）， the model group， and the low-， medium-， and high-dose Liangyi Paste groups （2.91 ， 5.82 ， 11.64 g·kg^-1·d^-1， respectively）. The 20MCON group was continuously fed a standard diet， while the other groups started receiving a high-fat diet at 15 months of age. At 18 months of age， the Liangyi Paste groups were administered the corresponding doses of Liangyi Paste by gavage， while the 20MCON and model groups were given an equal volume of saline by gavage. After 8 weeks of continuous gavage （when the mice reached 20 months of age）， tissue samples were collected. Hepatic TG levels were measured using assay kits； liver histology and lipid deposition were observed via hematoxylin-eosin （HE） and oil red O staining； reactive oxygen species （ROS） were detected by enzyme-linked immunosorbent assay （ELISA）； Cu²⁺， superoxide dismutase （SOD）， and malondialdehyde （MDA） levels were measured by colorimetry； mRNA and protein expression of genes related to cuproptosis and oxidative stress pathways were analyzed by Real-time polymerase chain reaction（Real-time PCR） and Wes automated protein expression system. ResultsCompared with 12MCON， the 15MCON group showed significantly increased hepatic TG， Cu²⁺， ROS， and MDA levels （P<0.01）， decreased SOD （P<0.01）， hepatocyte swelling， and disordered arrangement. The mRNA and protein levels of ferredoxin 1 （FDX1）， dihydrolipoamide S-acetyltransferase （DLAT）， heat shock protein 70 （HSP70）， dihydrolipoamide dehydrogenase （DLD）， pyruvate dehydrogenase E1 subunit-β （PDHB）， nuclear factor erythroid 2-related factor 2 （Nrf2）， and peroxisome proliferator-activated receptor γ （PPARγ） were significantly elevated （P<0.05， P<0.01）. Compared with 15MCON group， the 15MHFD and 20MCON groups exhibited further increases in TG， Cu²⁺， ROS， and MDA （P<0.01）， reduced SOD （P<0.01）， and aggravated hepatocyte swelling and disorder. There were increased lipid droplets with mild vacuolization in the 15MHFD group， and no significant lipid deposition was observed in the 20MCON group. FDX1， DLAT， HSP70， DLD， PDHB， Nrf2， and PPARγ mRNA and protein levels were significantly increased （P<0.05， P<0.01）. Compared with 20MCON group， the model group demonstrated markedly elevated TG， Cu²⁺， ROS， and MDA （P<0.01）， reduced SOD （P<0.01）， severe hepatic steatosis， and upregulated expression of FDX1， DLAT， HSP70， DLD， PDHB， Nrf2， and PPARγ mRNA and proteins （P<0.05， P<0.01）. All abnormalities were significantly reversed after Liangyi Paste treatment. ConclusionLiangyi paste can ameliorate hepatic lipid deposition in naturally aged mice with a high-fat diet by modulating the cuproptosis/oxidative stress pathway.

Tumors are the second leading cause of death worldwide. Exosomes are a type of extracellular vesicle secreted from multivesicular bodies, with particle sizes ranging from 40 to 160 nm. They regulate the tumor microenvironment, proliferation, and progression by transporting proteins, nucleic acids, and other biomolecules. Compared with other drug delivery systems, exosomes derived from different cells possess unique cellular tropism, enabling them to selectively target specific tissues and organs. This homing ability allows them to cross biological barriers that are otherwise difficult for conventional drug delivery systems to penetrate. Due to their biocompatibility and unique biological properties, exosomes can serve as drug delivery systems capable of loading various anti-tumor drugs. They can traverse biological barriers, evade immune responses, and specifically target tumor tissues, making them ideal carriers for anti-tumor therapeutics. This article systematically summarizes the methods for exosome isolation, including ultracentrifugation, ultrafiltration, size-exclusion chromatography (SEC), immunoaffinity capture, and microfluidics. However, these methods have certain limitations. A combination of multiple isolation techniques can improve isolation efficiency. For instance, combining ultrafiltration with SEC can achieve both high purity and high yield while reducing processing time. Exosome drug loading methods can be classified into post-loading and pre-loading approaches. Pre-loading is further categorized into active and passive loading. Active loading methods, including electroporation, sonication, extrusion, and freeze-thaw cycles, involve physical or chemical disruption of the exosome membrane to facilitate drug encapsulation. Passive loading relies on drug concentration gradients or hydrophobic interactions between drugs and exosomes for encapsulation. Pre-loading strategies also include genetic engineering and co-incubation methods. Additionally, we review approaches to enhance the targeting, retention, and permeability of exosomes. Genetic engineering and chemical modifications can improve their tumor-targeting capabilities. Magnetic fields can also be employed to promote the accumulation of exosomes at tumor sites. Retention time can be prolonged by inhibiting monocyte-mediated clearance or by combining exosomes with hydrogels. Engineered exosomes can also reshape the tumor microenvironment to enhance permeability. This review further discusses the current applications of exosomes in delivering various anti-tumor drugs. Specifically, exosomes can encapsulate chemotherapeutic agents such as paclitaxel to reduce side effects and increase drug concentration within tumor tissues. For instance, exosomes loaded with doxorubicin can mitigate cardiotoxicity and minimize adverse effects on healthy tissues. Furthermore, exosomes can encapsulate proteins to enhance protein stability and bioavailability or carry immunogenic cell death inducers for tumor vaccines. In addition to these applications, exosomes can deliver nucleic acids such as siRNA and miRNA to regulate gene expression, inhibit tumor proliferation, and suppress invasion. Beyond their therapeutic applications, exosomes also serve as tumor biomarkers for early cancer diagnosis. The detection of exosomal miRNA can improve the sensitivity and specificity of diagnosing prostate and pancreatic cancers. Despite their promising potential as drug delivery systems, challenges remain in the standardization and large-scale production of exosomes. This article explores the future development of engineered exosomes for targeted tumor therapy. Plant-derived exosomes hold potential due to their superior biocompatibility, lower toxicity, and abundant availability. Furthermore, the integration of exosomes with artificial intelligence may offer novel applications in diagnostics, therapeutics, and personalized medicine.

This study aims to investigate the effect of Chaijin Jieyu Anshen Formula on the neuroinflammation of rats with insomnia complicated with depression through the regulation of triggering receptor expressed on myeloid cells 2(TREM2)/complement protein C1q signaling pathway. Rats were randomly divided into a normal group, a model group, a positive drug group, as well as a high, medium, and low-dose groups of Chaijin Jieyu Anshen Formula, with 10 rats in each group. Except for the normal group, the other groups were injected with p-chlorophenylalanine and exposed to chronic unpredictable mild stress to establish the rat model of insomnia complicated with depression. The sucrose preference experiment, open field experiment, and water maze test were performed to evaluate the depression in rats. Enzyme-linked immunosorbent assay was employed to detect serum 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) levels. Hematoxylin and eosin staining and Nissl staining were used to observe the damage in nucleus accumbens neurons. Western blot and immunofluorescence were performed to detect TREM2, C1q, postsynaptic density 95(PSD-95), and synaptophysin 1(SYN1) expressions in rat nucleus accumbens, respectively. Golgi-Cox staining was utilized to observe the synaptic spine density of nucleus accumbens neurons. The results show that, compared with the model group, Chaijin Jieyu Anshen Formula can significantly increase the sucrose preference as well as the distance and number of voluntary activities, shorten the immobility time in forced swimming test and the successful incubation period of positioning navigation, and prolong the stay time of space exploration in the target quadrant test. The serum 5-HT, DA, and NE contents in the model group are significantly lower than those in the normal group, with the above contents significantly increased after the intervention of Chaijin Jieyu Anshen Formula. In addition, Chaijin Jieyu Anshen Formula can alleviate pathological damages such as swelling and loose arrangement of tissue cells in the nucleus accumbens, while increasing the Nissl body numbers. Chaijin Jieyu Anshen Formula can improve synaptic damage in the nucleus accumbens and increase the synaptic spine density. Compared to the normal group, the expression of C1q protein was significantly higher in the model group, while the expression of TREM2 protein was significantly lower. Compared to the model group, the intervention with Chaijin Jieyu Anshen Formula significantly downregulated the expression of C1q protein and significantly upregulated the expression of TREM2. Compared with the model group, the PSD-95 and SYN1 fluorescence intensity is significantly increased in the groups receiving different doses of Chaijin Jieyu Anshen Formula. In summary, Chaijin Jieyu Anshen Formula can reduce the C1q protein expression, relieve the TREM2 inhibition, and promote the synapse-related proteins PSD-95 and SNY1 expression. Chaijin Jieyu Anshen Formula improves synaptic injury of the nucleus accumbens neurons, thereby treating insomnia complicated with depression.
Animals ; Male ; Rats ; Nucleus Accumbens/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Depression/complications* ; Membrane Glycoproteins/genetics* ; Rats, Sprague-Dawley ; Sleep Initiation and Maintenance Disorders/complications* ; Neurons/metabolism* ; Receptors, Immunologic/genetics* ; Signal Transduction/drug effects* ; Synapses/metabolism*

PURPOSE: To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss. METHODS: This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA⁺, K⁺, and Ca²⁺ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance. RESULTS: The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA⁺ and K⁺ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca²⁺ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group. CONCLUSIONS Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals ; Perfusion/methods* ; Disease Models, Animal ; Brain Injuries/etiology* ; Swine ; Male ; Hypothermia, Induced/methods*

OBJECTIVES: To explore the mediating role of sleep duration in the relationship between depression symptoms and myopia among middle school students. METHODS: This study was a cross-sectional research conducted using a stratified cluster random sampling method. A total of 1 728 middle school students were selected from two junior high schools and two senior high schools in certain urban areas and farms of the Xinjiang Production and Construction Corps. Questionnaire surveys and vision tests were conducted among the students. Spearman analysis was used to analyze the correlation between depression symptoms, sleep duration, and myopia. The Bootstrap method was employed to investigate the mediating effect of sleep duration between depression symptoms and myopia. RESULTS: The prevalence of myopia in the overall population was 74.02% (1 279/1 728), with an average sleep duration of (7.6±1.0) hours. The rate of insufficient sleep was 83.62% (1 445/1 728), and the proportion of students exhibiting depression symptoms was 25.29% (437/1 728). Correlation analysis showed significant negative correlations between visual acuity in both eyes and sleep duration with depressive emotions as measured by the Center for Epidemiologic Studies Depression Scale (with correlation coefficients of -0.064, -0.084, and -0.199 respectively; P<0.01), as well as with somatic symptoms and activities (with correlation coefficients of -0.104, -0.124, and -0.233 respectively; P<0.01) and interpersonal relationships (with correlation coefficients of -0.052, -0.059, and -0.071 respectively; P<0.05). The correlation coefficients for left and right eye visual acuity and sleep duration were 0.206 and 0.211 respectively (P<0.001). Sleep duration exhibited a mediating effect between depression symptoms and myopia (indirect effect=0.056, 95%CI: 0.029-0.088), with the mediating effect value for females (indirect effect=0.066, 95%CI: 0.024-0.119) being higher than that for males (indirect effect=0.042, 95%CI: 0.011-0.081). CONCLUSIONS Sleep duration serves as a partial mediator between depression symptoms and myopia in middle school students.
Humans ; Myopia/etiology* ; Male ; Female ; Depression/physiopathology* ; Cross-Sectional Studies ; Sleep ; Adolescent ; Students ; Child ; Time Factors ; Sleep Duration

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

Debates persist regarding the efficacy and safety of azvudine, particularly its real-world outcomes. This study involved patients aged ≥60 years who were admitted to 25 hospitals in mainland China with confirmed SARS-CoV-2 infection between December 1, 2022, and February 28, 2023. Efficacy outcomes were all-cause mortality during hospitalization, the proportion of patients discharged with recovery, time to nucleic acid-negative conversion (T _NANC), time to symptom improvement (T _SI), and time of hospital stay (T _HS). Safety was also assessed. Among the 5884 participants identified, 1999 received azvudine, and 1999 matched controls were included after exclusion and propensity score matching. Azvudine recipients exhibited lower all-cause mortality compared with controls in the overall population (13.3% vs. 17.1%, RR, 0.78; 95% CI, 0.67-0.90; P = 0.001) and in the severe subgroup (25.7% vs. 33.7%; RR, 0.76; 95% CI, 0.66-0.88; P < 0.001). A higher proportion of patients discharged with recovery, and a shorter T _NANC were associated with azvudine recipients, especially in the severe subgroup. The incidence of adverse events in azvudine recipients was comparable to that in the control group (2.3% vs. 1.7%, P = 0.170). In conclusion, azvudine showed efficacy and safety in older patients hospitalized with COVID-19 during the SARS-CoV-2 omicron wave in China.

Iron overload is strongly associated with heart disease. Ferroptosis is a new form of regulated cell death indicated in cardiac ischemia-reperfusion (I/R) injury. However, the specific molecular mechanism of myocardial injury caused by iron overload in the heart is still unclear, and the involvement of ferroptosis in iron overload-induced myocardial injury is not fully understood. In this study, we observed that ferroptosis participated in developing of iron overload and I/R-induced cardiomyopathy. Mechanistically, we discovered that Parkin inhibited iron overload-induced ferroptosis in cardiomyocytes by promoting the ubiquitination of long-chain acyl-CoA synthetase 4 (ACSL4), a crucial protein involved in ferroptosis-related lipid metabolism pathways. Additionally, we identified p53 as a transcription factor that transcriptionally suppressed Parkin expression in iron-overloaded cardiomyocytes, thereby regulating iron overload-induced ferroptosis. In animal studies, cardiac-specific Parkin knockout mice (Myh6-CreER ^T2 /Parkin ^fl/fl ) fed a high-iron diet presented more severe myocardial damage, and the high iron levels exacerbated myocardial I/R injury. However, the ferroptosis inhibitor Fer-1 significantly suppressed iron overload-induced ferroptosis and myocardial I/R injury. Moreover, Parkin effectively protected against impaired mitochondrial function and prevented iron overload-induced mitochondrial lipid peroxidation. These findings unveil a novel regulatory pathway involving p53-Parkin-ACSL4 in heart disease by inhibiting of ferroptosis.