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.

OBJECTIVES: To investigate the effect of Haematococcus pluvialis (HP) on bleomycin (BLM)-induced pulmonary fibrosis in mice and on TGF-β1-induced human fetal lung fibroblasts (HFL1). METHODS: Thirty male C57BL/6 mice were randomly divided into control group, BLM-induced pulmonary fibrosis model group, low- and high-dose HP treatment groups (3 and 21 mg/kg, respectively), and 300 mg/kg pirfenidone (positive control) group. The effects of drug treatment for 21 days were assessed by examining respiratory function, lung histopathology, and expression of fibrosis markers in the lung tissues of the mouse models. In TGF-β1-induced HFL1 cell cultures, the effects of treatment with 120, 180 and 240 μg/mL HP or 1.85 μg/mL pirfenidone for 48 h on expression levels of fibrosis markers were evaluated. Transcriptome analysis was carried out using the control cells and cells treated with TGF-β1 and 240 μg/mL HP. RESULTS: HP obviously alleviated BLM-induced lung function damage and fibrotic changes in mice, evidenced by improved respiratory function, lung tissue morphology and structure, inflammatory infiltration, and collagen deposition and reduced expressions of fibrotic proteins. HP at the high dose produced similar effect to PFD. In TGF-β1-induced HFL1 cells, treatment with 240 μg/mL HP significantly reduced the mRNA and protein expression levels of α-SMA and FN. Transcriptome analysis revealed that multiple key genes and pathways mediated the protective effect of HP against pulmonary fibrosis. CONCLUSIONS HP alleviates pulmonary fibrosis in both the mouse model and cell model, possibly as the result of the synergistic effects of its multiple active components.
Animals ; Pulmonary Fibrosis/chemically induced* ; Bleomycin/adverse effects* ; Mice, Inbred C57BL ; Male ; Mice ; Fibroblasts/drug effects* ; Lung/pathology* ; Transforming Growth Factor beta1/pharmacology* ; Myofibroblasts/drug effects* ; Humans ; Pyridones

OBJECTIVE: To investigate the feasibility and prognostic implications of assessing volume status during early fluid resuscitation in septic patients based on estimated plasma volume status (ePVS). METHODS: A prospective study was conducted. Patients with sepsis admitted to intensive care unit (ICU) of the First Affiliated Hospital of Kunming Medical University from March to December in 2023 were enrolled. The general information and laboratory indicators at ICU admission were recorded, and ePVS, sequential organ failure assessment (SOFA) score, acute physiology and chronic health status evaluation II (APACHE II) score were calculated. The vital signs, arterial blood gas analysis and volume status related indicators before liquid resuscitation (T0h) and 3 hours (T3h) and 6 hours (T6h) of fluid resuscitation were recorded. The diameter and variability of the inferior vena cava (IVC) were measured by ultrasound, and ePVS, percentage change value of estimated plasma volume status (ΔePVS%), difference in central venous-to-arterial partial pressure of carbon dioxide (Pcv-aCO₂), and lactate clearance rate (LCR) were calculated. Patients were divided into sepsis group and septic shock group based on the diagnosis at ICU admission, and septic patients were subdivided into survival group and death group based on their 28-day survival status. The differences in clinical data between the groups were compared. The correlation between ePVS or ΔePVS% and volume status related indicators during early liquid resuscitation was analyzed by Spearman rank sum correlation test. The predictive value of each variable for 28-day survival in patients with sepsis was analyzed by receiver operator characteristic curve (ROC curve), and 28-day death risk factors were analyzed by Logistic regression method. RESULTS: Fifty-four septic patients were enrolled in the final analysis, including 17 with sepsis and 37 with septic shock; 34 survived at 28 days and 20 died, with a 28-day survival rate of 63.0%. Compared with the sepsis group, the septic shock group had a lower venous ePVS at ICU admission [dL/g: 4.96 (3.67, 7.15) vs. 7.55 (4.36, 10.07), P < 0.05]. Compared with the death group, the survival group had higher T6h arterial and venous ΔePVS%, and albumin [Alb; T6h arterial ΔePVS% (%): 11.57% (-1.82%, 31.35%) vs. 0.48% (-5.67%, 6.02%), T6h venous ΔePVS%: 9.62% (3.59%, 25.75%) vs. 1.52% (-9.65%, 7.72%), Alb (g/L): 27.57±4.15 vs. 23.77±6.97, all P < 0.05], lower SOFA score, APACHE II score, AST, T0h Lac, and T3h and T6h norepinephrine dosage [SOFA score: 9.00 (8.00, 10.00) vs. 11.50 (9.25, 14.50), APACHE II score: 18.00 (14.75, 21.25) vs. 25.50 (21.00, 30.00), AST (U/L): 34.09 (23.20, 56.64) vs. 79.24 (25.34, 196.59), T0h Lac (mmol/L): 1.75 (1.40, 2.93) vs. 3.25 (2.33, 5.30), norepinephrine dosage (mg): 0.98 (< 0.01, 3.10) vs. 4.60 (1.05, 8.55) at T3h, 1.82 (0.38, 5.30) vs. 8.20 (2.80, 17.73) at T6h, all P < 0.05]. While there were no significantly differences in other basic data and ePVS at all of the time points before and after resuscitation between the two groups. Correlation analysis showed that T6h venous ePVS was significantly positively correlated with T6h IVC variability in septic patients (r = 0.360, P < 0.05), T0h arterial ePVS was significantly negatively correlated with T3h and T6h liquid intake volume (r₁ = -0.367, r₂ = -0.280, both P < 0.05), and venous ePVS at ICU admission was significantly positively correlated with NT-proBNP at ICU admission (r = 0.409, P < 0.05). T6h venous ΔePVS% was significantly positively correlated with T3h liquid intake volume and T6h LCR (r₁ = 0.286, r₂ = 0.286, both P < 0.05), and significantly negatively correlated with T6h urine volume and T6h change value of Pcv-aCO₂ (ΔPcv-aCO₂; r₁ = -0.321, r₂ = -0.371, both P < 0.05). ROC curve analysis showed that the area under the ROC curve (AUC) of T6h venous ΔePVS% for predicting 28-day survival in septic patients was 0.726 [95% confidence interval (95%CI) was 0.578-0.875, P = 0.006], with a sensitivity of 82.4%, a specificity of 60.0%, and an optimal cut-off value of 3.09%. Binary multifactorial Logistic regression analysis showed that an increase in T6h venous ΔePVS% was a protective factor for 28-day death in patients with sepsis on early fluid resuscitation [odds ratio (OR) = 0.900, 95%CI was 0.834-0.972, P = 0.007]. CONCLUSIONS ePVS may have potential for assessing the volume status of septic patients during early fluid resuscitation. The ΔePVS% during early fluid resuscitation may help to identify septic patients with a poor prognosis.
Humans ; Prognosis ; Fluid Therapy ; Sepsis/physiopathology* ; Prospective Studies ; Plasma Volume ; Intensive Care Units ; Resuscitation ; Male ; Female ; Middle Aged ; Shock, Septic/therapy*

Objective To investigate the prevalence of common diseases among primary and secondary school students in Yichang City from 2019 to 2022, and to provide a scientific basis for formulating effective intervention measures in the future. Methods By random cluster sampling , 7 schools in urban areas and 5 schools in suburban counties were selected to screen common diseases such as myopia, dental caries, obesity and abnormal spinal curvature. Descriptive epidemiological methods were employed for statistical analysis. Results A total of 17 023 primary and secondary school students were screened from 2019 to 2022. The overall detection rate of common diseases from high to low was myopia (54.12%), caries (36.75%), overweight (15.17%), obesity (11.88%), malnutrition (5.80%), and abnormal spinal curvature (3.49%). The detection rates of myopia and abnormal curvature of the spine showed an increasing trend with years and school stages, while the detection rates of malnutrition and dental caries showed a decreasing trend with years and school stages. The detection rates of overweight and obesity showed no trend difference with years, and the detection rates of obesity showed a decreasing trend with school stages. The rates of myopia, overweight and obesity were higher in urban areas than those in suburban counties, and the rate of dental caries was higher in suburban counties than that in urban areas. The prevalence of overweight, obesity, and malnutrition in boys was higher than that in girls. The prevalence of myopia and dental caries in girls was higher than that in boys. The above differences were statistically significant (all P<0.05). Conclusion Myopia, dental caries, obesity, and abnormal curvature of the spine are the current focus of the prevention and treatment of common diseases in students. There are great differences between different regions, school stages, and genders. The ^“tripartite linkage^” of schools, families, and communities should be achieved with the joint efforts of the education and health departments to actively take targeted intervention measures to reduce the prevalence.

This study aims to explore the pharmacodynamic material basis of Jinbei Oral Liquid(JBOL) against idiopathic pulmonary fibrosis(IPF) based on serum pharmacochemistry and network pharmacology. The ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS) technology was employed to analyze and identify the components absorbed into rat blood after oral administration of JBOL. Combined with network pharmacology, the study explored the pharmacodynamic material basis and potential mechanism of JBOL against IPF through protein-protein interaction(PPI) network construction, "component-target-pathway" analysis, Gene Ontology(GO) functional enrichment, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. First, a total of 114 compounds were rapidly identified in JBOL extract according to the exact relative molecular mass, fragment ions, and other information of the compounds with the use of reference substances and a self-built compound database. Second, on this basis, 70 prototype components in blood were recognized by comparing blank serum with drug-containing serum samples, including 28 flavonoids, 25 organic acids, 4 saponins, 4 alkaloids, and 9 others. Finally, using these components absorbed into blood as candidates, the study obtained 212 potential targets of JBOL against IPF. The anti-IPF mechanism might involve the action of active ingredients such as glycyrrhetinic acid, cryptotanshinone, salvianolic acid B, and forsythoside A on core targets like AKT1, TNF, and ALB and thereby the regulation of multiple signaling pathways including PI3K/AKT, HIF-1, and TNF. In conclusion, JBOL exerts the anti-IPF effect through multiple components, targets, and pathways. The results would provide a reference for further study on pharmacodynamic material basis and pharmacological mechanism of JBOL.
Drugs, Chinese Herbal/pharmacokinetics* ; Animals ; Tandem Mass Spectrometry ; Network Pharmacology ; Rats ; Chromatography, High Pressure Liquid ; Rats, Sprague-Dawley ; Male ; Idiopathic Pulmonary Fibrosis/metabolism* ; Humans ; Administration, Oral ; Protein Interaction Maps/drug effects* ; Signal Transduction/drug effects*

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*

Researchers commonly use cyclization recombination enzyme/locus of X-over P1 (Cre/loxP) technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis. However, the shortcomings of these genetic tools include high costs, lengthy experimental periods, and limited accessibility for researchers. Therefore, exploring alternative gene silencing techniques is of great practical value. In this study, we employed adeno-associated virus (AAV) as a vector for gene silencing in Sertoli and Leydig cells. Our findings demonstrated that AAV serotypes 1, 8, and 9 exhibited high infection efficiency in both types of testis cells. Importantly, we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection. We achieved cell-specific knockouts of the steroidogenic acute regulatory ( Star ) and luteinizing hormone/human chorionic gonadotropin receptor (Lhcgr) genes in Leydig cells, but not in Sertoli cells, using AAV9-single guide RNA (sgRNA)-mediated gene editing in Rosa26-LSL-Cas9 mice. Knockdown of androgen receptor ( Ar ) gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpin RNA (shRNA)-mediated targeting. Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
Animals ; Male ; Leydig Cells/metabolism* ; Mice ; Dependovirus/genetics* ; Sertoli Cells/metabolism* ; Gene Silencing ; Genetic Vectors ; Testis/cytology*

This article reports the clinical features and gene mutation types of a large family with Nascimento form of syndromic X-linked intellectual developmental disorder (MRXSN), involving 9 individuals across 3 generations, and a literature review was conducted. In this family, 9 individuals had similar manifestations including mental retardation and unusual facies, and 4 of them had passed away. Genetic testing showed that the proband had the deletion of exons 2-3 of the UBE2A gene, which was inherited from the mother. Fluorescent quantitative polymerase chain reaction showed that the proband and his uncle had the deletion of exons 2-3 of the UBE2A gene; the proband's mother, grandmother, and great-aunt had a heterozygous deletion of exons 2-3 of the UBE2A gene; the proband's father, sister, and aunt had a normal copy number of exons 2-3 of the UBE2A gene. The 34 patients reported in the literature had diverse clinical phenotypes, and UBE2A gene mutations (22/34, 65%) and large fragment deletions (12/34, 35%) were the main mutation types. Moderate to severe mental retardation (34/34, 100%), speech and language impairment (33/34, 97%), and unusual facies (32/34, 94%) were the main clinical manifestations of MRXSN patients. The disease has obvious phenotypic heterogeneity, and early diagnosis facilitates optimal prenatal and postnatal management to improve reproductive outcomes.
Humans ; Male ; Ubiquitin-Conjugating Enzymes/genetics* ; Female ; X-Linked Intellectual Disability/genetics* ; Gene Deletion ; Child ; Pedigree ; Child, Preschool ; Adult

Objective:To explore the effects of Qingre Qudu Decoction for fumigation combined with three-gap drainage on wound healing and serum inflammatory factors in patients with acute perianal abscess.Methods:Randomized controlled trial was conducted. A total of 117 patients with acute perianal abscess in the hospital were enrolled as the observation objects between August 2022 and May 2024. According to random number table method, they were divided into observation group (59 cases) and control group (58 cases). Both groups received three-gap drainage therapy. On basis of three-gap drainage, control group was given potassium permanganate, while observation group was given Qingre Qudu Decoction for fumigation. All patients were treated for 14 d. The growth of granulation tissue and wound secretions before and after treatment was evaluated. VAS scale was used to evaluate the degree of incision pain, and Wexner score was used to assess incontinence; ELISA was used to detect serum activator A (ACTA), immunoturbidimetry was used to detect serum CRP, and radioimmunoassay was used to detect serum IL-6 levels. The occurrence of complications and abscess recurrence during treatment was recorded, and clinical efficacy was evaluated.Results:The total effective rate of the observation group was 96.61% (57/59), while that of the control group was 82.76% (48/58), with statistical significance ( χ2=6.10, P=0.014). After treatment, scores of granulation tissue growth and wound secretions in observation group, and scores of VAS and Wexner incontinence in observation group were lower than those in the control group ( t=9.66, 5.00, 7.98, 3.65, P<0.001), and wound healing time was shorter than that in control group ( t=8.41, P<0.001). After treatment, levels of serum ACTA, CRP and IL-6 in observation group were lower than those in control group ( t=15.30, 2.08, 19.34, P<0.01 or P<0.05). The incidence of postoperative complications in the observation group was 6.78% (4/59), while in the control group it was 27.59% (16/58), with statistical significance ( χ2=8.93, P=0.003). Conclusion:Qingre Qudu Decoction for fumigation combined with three-gap drainage can relieve postoperative incision pain, inhibit inflammatory response, accelerate the recovery of wound and promote the recovery of anal function and improve clinical efficacy.