ObjectiveThis study aimed to establish a monocrotaline （MCT）-induced pulmonary arterial hypertension （PAH） rat model to systematically evaluate the protective effect of Xiao Qinglongtang （XQLT） on right cardiac function in model rats and further elucidate the underlying regulatory mechanism. MethodsSixty male SD rats were randomly assigned to the normal group， model group， XQLT low-， medium-， and high-dose groups （XQLT-L/M/H）， and the beraprost sodium tablet group （BST）. Except for the normal group， rats in all other groups were given a single subcutaneous injection of MCT （60 mg·kg^-1） to induce PAH. Three weeks after injection， rats in the XQLT-L/M/H groups were administered XQLT intragastrically at 3.07， 6.14， 12.28 g·kg^-1·d^-1， respectively. Rats in the BST group received beraprost sodium at 12.6 μg·kg^-1·d^-1， and rats in the model group received an equal volume of saline. All treatments lasted for 3 weeks. Right ventricular systolic pressure （RVSP） was measured by right ventricular catheterization. Cardiac function was assessed by echocardiography. The right ventricle was weighed to calculate the right ventricular hypertrophy index （RVHI）. Hematoxylin-eosin （HE） staining， Masson staining， and transmission electron microscopy were used to observe myocardial morphology. Serum metabolomic changes were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. Data-independent acquisition （DIA） proteomics was used to detect differentially expressed （DE） proteins in the right ventricle， and Western blot was used to measure the expression of uncoupling protein 3 （UCP3）， phosphatidylinositol 3-kinase catalytic subunit p110α （PIK3CA）， L1 cell adhesion molecule （L1CAM）， and quinone oxidoreductase （CRYZ）. UPLC-MS/MS was used to analyze the chemical components of XQLT. ResultsCompared with the normal group， the model group showed significantly increased RVSP and RVHI （P<0.05）， along with pathological changes in myocardial morphology. Compared with the model group， all XQLT-treated groups exhibited reductions in RVSP and RVHI as well as significant improvements in cardiac function and myocardial morphology. Among the XQLT groups， XQLT-M showed the most pronounced effects （P<0.05）， comparable to the BST group. Serum metabolomics revealed 105 differential metabolites in the XQLT groups versus the model group ［variable importance in projection （VIP） >1， P<0.05］， including 58 upregulated and 47 downregulated metabolites. KEGG enrichment analysis indicated that XQLT intervention downregulated phenylalanine metabolism （P<0.01） and upregulated unsaturated fatty acid biosynthesis （P<0.05）. Proteomics analysis showed that 982 DE proteins were identified in the MCT groups versus the normal group， including 455 upregulated and 527 downregulated proteins （|fold change （FC）| >1.3， P<0.05）. Compared with the model group， 237 DE proteins were identified in the XQLT groups， including 124 upregulated and 113 downregulated proteins （|FC| >1.3， P<0.05）， with 57 overlapping DE proteins. KEGG enrichment suggested that XQLT mainly modulated pathways related to mineral absorption， ribosomal biogenesis， peroxisomes， glycolysis/gluconeogenesis， spliceosomes， and thyroid hormone signaling. Western blot analysis showed that， compared with the model group， XQLT increased the expression of UCP3， PIK3CA， and L1CAM， while decreasing the expression of CRYZ （P<0.05）. ConclusionXQLT exerts a protective effect on right heart function in MCT-induced PAH rats， and its mechanism is associated with maintaining myocardial homeostasis and alleviating right ventricular remodeling.

ObjectiveTo investigate the mechanism of Xiezhuo Jiedu prescription in the treatment of ulcerative colitis （UC） by inhibiting ferroptosis and alleviating intestinal mucosal injury based on the nuclear factor E₂ related factor 2/solute carrier family 7 member/glutathione peroxidase 4 （Nrf2/SLC7A11/GPX4） signaling pathway. MethodsA total of 60 male SD rats were divided into a normal group， a model group， high- and low-dose Xiezhuo Jiedu prescription groups （26.64 and 13.32 g·kg^-1， respectively）， a ferroptosis inhibitor group （Ferrostatin-1， 0.005 g·kg^-1）， and a mesalazine group （0.27 g·kg^-1）， with 10 rats in each group. A UC rat model was established by intrarectal administration of trinitrobenzene sulfonic acid （TNBS）-ethanol. The normal group and the model group were intragastrically administered normal saline. The other groups were given intragastric administration according to the corresponding dosage for 7 d. The general condition， disease activity index （DAI） score， colon length， and mucosal injury index （CDMI） score were observed in each group. The pathological changes of colon tissue in each group were observed by hematoxylin-eosin （HE） staining. The intestinal mucosa and mitochondrial morphology in each group were observed by transmission electron microscopy. The expression levels of Occludin， Claudin-1， mucin 2 （MUC2）， and E-cadherin in intestinal tissue were detected by immunofluorescence （IF）. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression levels of serum tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） in each group， and a lactic acid assay kit or ELISA was employed to detect the expression levels of reactive oxygen species （ROS）， ferrous ions （Fe²⁺）， glutathione （GSH）， malondialdehyde （MDA）， 4-hydroxynonenal （4-HNE）， diamine oxidase （DAO）， and D-lactate （D-LA）. Real-time quantitative polymerase chain reaction （Real-time PCR） was applied to detect the mRNA expression levels of Nrf2， SLC7A11， GPX4， Occludin， Claudin-1， MUC2， and E-cadherin in each group， and Western blot was adopted to detect the protein expression levels of Nrf2， p-Nrf2， SLC7A11， and GPX4 in each group. ResultsCompared with the normal group， rats in the model group exhibited listlessness， sluggish response， and mucopurulent and bloody stools. The model group also showed significantly increased DAI score， colon length， CDMI score， and expression levels of TNF-α， IL-6， ROS， Fe²⁺， MDA， 4-HNE， DAO， and D-LA （P<0.01）. In addition， it presented significantly decreased IF values of Occludin， Claudin-1， MUC2， and E-cadherin and mRNA and protein expression levels of IL-10， GSH， Nrf2， p-Nrf2， SLC7A11， and GPX4 （P<0.01）. There were different degrees of improvement in each administration group after treatment， and the improvement was the most significant in the high-dose Xiezhuo Jiedu prescription group （P<0.01）. ConclusionXiezhuo Jiedu prescription may alleviate intestinal mucosal injury by inhibiting ferroptosis of intestinal epithelial cells via regulating the Nrf2/SLC7A11/GPX4 signaling pathway， thereby exhibiting efficacy in the treatment of UC.

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.

BACKGROUND:The mesenchymal stem cell secretome contains bioactive substances,cytokines,and growth factors.Three-dimensional cell culture can regulate the secretion of these components,potentially enhancing the ability to promote injury repair.OBJECTIVE:To investigate the repair effect of three-dimensional cultured human umbilical cord mesenchymal stem cell secretome on skin injuries in mice.METHODS:Human umbilical cord mesenchymal stem cells were cultured in conventional two-dimensional culture dishes and 96-well U-bottom cell culture plates,from which their secretory components were subsequently collected.The expression of skin damage repair related secretory factors in umbilical cord mesenchymal stem cells was analyzed using RT-qPCR.The protein expression level of skin damage repair related factors in umbilical cord mesenchymal stem cell secretome was detected using enzyme-linked immunosorbent assay.The potential of human umbilical cord mesenchymal stem cell secretome to repair vascular injuries was evaluated using an immortalized human umbilical vein endothelial cell migration model.A mouse skin injury model was established,and the human umbilical cord mesenchymal stem cell secretome was injected subcutaneously.Repair effects on skin injury were assessed through wound healing rates and histopathological analysis.RESULTS AND CONCLUSION:(1)After three days of cultivation,human umbilical cord mesenchymal stem cells cultured in two dimensions exhibited a fibroblast-like,swirling growth pattern,whereas three-dimensional culture led to the formation of uniform microspheres.(2)Compared with two-dimensional culture,three-dimensional culture significantly increased the mRNA expression of transforming growth factor β and basic fibroblast growth factor in human umbilical cord mesenchymal stem cells.(3)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly enhanced the protein expression of vascular endothelial growth factor,interleukin-10,and granulocyte-macrophage colony-stimulating factor in the human umbilical cord mesenchymal stem cell secretome.(4)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly promoted the migration of immortalized human umbilical cord mesenchymal stem cells.(5)Compared with the untreated control group and the two-dimensional cultured human umbilical cord mesenchymal stem cell secretome,the three-dimensional cultured human umbilical cord mesenchymal stem cell secretome can significantly accelerate the skin wound healing rate and wound skin structure remodeling in mice.These results indicate that three-dimensional culture can enhance the expression of paracrine factors of human umbilical cord mesenchymal stem cells,and their secretome can significantly promote the repair of mouse skin damage.

BACKGROUND:The mesenchymal stem cell secretome contains bioactive substances,cytokines,and growth factors.Three-dimensional cell culture can regulate the secretion of these components,potentially enhancing the ability to promote injury repair.OBJECTIVE:To investigate the repair effect of three-dimensional cultured human umbilical cord mesenchymal stem cell secretome on skin injuries in mice.METHODS:Human umbilical cord mesenchymal stem cells were cultured in conventional two-dimensional culture dishes and 96-well U-bottom cell culture plates,from which their secretory components were subsequently collected.The expression of skin damage repair related secretory factors in umbilical cord mesenchymal stem cells was analyzed using RT-qPCR.The protein expression level of skin damage repair related factors in umbilical cord mesenchymal stem cell secretome was detected using enzyme-linked immunosorbent assay.The potential of human umbilical cord mesenchymal stem cell secretome to repair vascular injuries was evaluated using an immortalized human umbilical vein endothelial cell migration model.A mouse skin injury model was established,and the human umbilical cord mesenchymal stem cell secretome was injected subcutaneously.Repair effects on skin injury were assessed through wound healing rates and histopathological analysis.RESULTS AND CONCLUSION:(1)After three days of cultivation,human umbilical cord mesenchymal stem cells cultured in two dimensions exhibited a fibroblast-like,swirling growth pattern,whereas three-dimensional culture led to the formation of uniform microspheres.(2)Compared with two-dimensional culture,three-dimensional culture significantly increased the mRNA expression of transforming growth factor β and basic fibroblast growth factor in human umbilical cord mesenchymal stem cells.(3)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly enhanced the protein expression of vascular endothelial growth factor,interleukin-10,and granulocyte-macrophage colony-stimulating factor in the human umbilical cord mesenchymal stem cell secretome.(4)Compared with two-dimensional culture,three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly promoted the migration of immortalized human umbilical cord mesenchymal stem cells.(5)Compared with the untreated control group and the two-dimensional cultured human umbilical cord mesenchymal stem cell secretome,the three-dimensional cultured human umbilical cord mesenchymal stem cell secretome can significantly accelerate the skin wound healing rate and wound skin structure remodeling in mice.These results indicate that three-dimensional culture can enhance the expression of paracrine factors of human umbilical cord mesenchymal stem cells,and their secretome can significantly promote the repair of mouse skin damage.

ObjectiveTo investigate whether intestinal deficiency of lanosterol synthase （LSS）， a key enzyme in cholesterol synthesis， influences the progression of metabolic dysfunction-associated steatotic liver disease （MASLD） by regulating intestinal cholesterol absorption and immune response. MethodsLSS heterozygous knockout （LSS^+/-） mice and wild-type （WT） controls were generated using CRISPR/Cas9 technology and fed either a high-fat diet （HFD） or regular chow （CHOW）. The model was validated by genotyping. Hepatic steatosis was assessed by HE and oil red O staining. Immunohistochemistry was used to detect the localization and expression of NPC1L1 and CD36 proteins in the intestine. Western blot analysis was performed to measure JNK phosphorylation and TLR4 protein levels in intestinal tissues. Real-time quantitative polymerase chain reaction （qPCR） was employed to examine the mRNA expression of TLR4 and IL-6. ResultsLSS^+/- mice were successfully validated by genotyping and reduced intestinal LSS protein expression. HE and oil red O staining of liver sections showed that， compared with WT mice fed a CHOW diet， WT mice fed a HFD exhibited a marked increase in hepatic lipid vacuoles. In contrast， compared with HFD-fed WT mice， HFD-fed LSS^+/- mice displayed significantly attenuated hepatic lipid deposition and reduced serum ALT levels （P<0.05）. Immunohistochemical analysis revealed that， compared with WT mice， the expression of the cholesterol absorption protein NPC1L1 in the intestinal villi of LSS^+/- mice was downregulated under both CHOW and HFD conditions （P_HFD<0.001）. Conversely， the expression of the fatty acid transporter CD36 was upregulated in the intestines of LSS^+/- mice （P_CHOW<0.05， P_HFD<0.01）. Western blot analysis demonstrated that， compared with WT mice， TLR4 protein expression in the intestines of LSS^+/- mice significantly increased under both CHOW and HFD conditions （both P<0.05）. JNK phosphorylation level was significantly elevated in LSS^+/- mice under CHOW condition （both P<0.05）. Under HFD condition， total JNK protein expression increased， but its phosphorylation level showed no significant change. qPCR analysis showed that， compared with WT mice， the mRNA levels of TLR4 （P_CHOW<0.01， P_HFD<0.000 1） and IL-6 （P_CHOW<0.001， P_HFD<0.01） were significantly upregulated in the intestines of LSS^+/-mice. ConclusionLSS deficiency counteracts hepatic lipid deposition by orchestrating a synergistic reprogramming involving restricted intestinal cholesterol absorption， enhanced fatty acid utilization， and activation of immune pathways， suggesting intestinal LSS as a potential therapeutic target of MASLD.

ObjectiveTo investigate the effect of hypoxia on pemetrexed （PE）-mediated apoptosis in non-small cell lung cancer cells. MethodsA549 cells were cultured under 21% O₂ （normoxia） or 1% O₂ （hypoxia）， and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group， normoxic vehicle group， normoxic drug-treated group， hypoxic control group， hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1， Cleaved Caspase-3， E-Cadherin and N-Cadherin. ResultsTrypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group （P<0.001）. Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However， the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group （P<0.000 1）. Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group （P<0.05）， while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells （P<0.000 1）. Compared with the control group， Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia （P<0.05）， whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions， PE treatment downregulated the expression of E-Cadherin （P<0.000 1，P<0.001） and N-Cadherin （P<0.05，P<0.000 1）. In addition， the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group （all P<0.000 1）. ConclusionHypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.

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

Objective To identify the cuproptosis genes associated with prognosis and immune microenvironment in lung cancer with the use of bioinformatics.Methods The lung cancer dataset used in this study was obtained from the TCGA database.The cuproptosis-related genes(CRGs)were obtained from the previously reported literature.The R software Deseq2 package was used to identify the differentially expressed genes(DEGs)in lung cancer.The intersection of DEGs and CRGs was taken to obtain the differentially expressed CRGs(DE-CRGs).COX analysis and R software rms package were used to identify DE-CRGs associated with the prognosis of lung cancer.Estimate and Cibersort algorithms were applied to identify the correlation of DE-CRGs with the immune microenvironment.Results Compared to the normal tissues,a total of 5,269 DEGs were present in lung cancer tissues in the TCGA database,and there were 11 shared genes between DEGs and CRGs.The 11 DE-CRGs mainly regulated the energy metabolism,carbon metabolism and amino acid metabolism.In the DE-CRGs,LIPT1 was an independent risk factor for lung cancer,and the column chart constructed with the clinical features(age,TNM staging,and residual tumor)predicted the survival of lung cancer patients in a manner similar to their actual outcomes.LIPT1 showed a positive correlation with the infiltration of M1 and M2 type macrophages,activated natural killer cells cells and CD8+T cells,and showed a significant negative correlation with M0 type macrophages,activated mast cells,neutrophils and Treg cells(P<0.05).Conclusion LIPT1 may serve as a prognostic and immune microenvironment-associated cuproptosis gene,which is a novel biomarker for lung cancer therapy.

Objective : To explore the changes in behavior and spatial memory of C57BL/6J female mice of different ages (youth , middle-aged , and elderly) . Methods: C57BL/6J female mice were divided into female youth group (YG group) , female middle-aged group ( MG group) and female elderly group ( OG group) according to age. The Morris water maze test measured spatial memory ability , and the open field and elevated cross maze test observed activity level and anxiety level. Western blot was used to determine the protein expressions of CREB , CaMKⅡ(pan) and CaMKⅡ(p) in the hippocampus of the brain tissues of female mice in each group. Results: Compared with the YG group , the weight of the MG group and the OG group significantly increased (P < 0. 01 , P < 0. 001) . Compared with the OG group , the third quadrant escape latency and the number of crossings in the YG group and MG group were shortened , and the difference was not statistically significant. Compared with the OG group , there was a statistically significant difference in the exercise speed in the open field of the YG group (P < 0. 01) , there was no significant difference in the movement speed in the open field of the MG group , the number of entries into the central zone significantly increased in the MG group ( P < 0. 05 ) , and there was no significant difference in the number of entries in the YG group (P > 0. 05) . Compared with the OG group , the YG group had a statistically significant difference in the elevated cross maze (P < 0. 05) , the MG group had no statistically signif- icant difference in the elevated cross maze , and the number of closed arm entries in the YG group and MG group significantly increased (P < 0. 001 , P < 0. 01) . Compared with the YG group , the relative expression level of CaMKⅡ(pan) in the OG group was statistically significant ( P < 0. 05 ) , while the relative expression level of CaMKⅡ(pan) in the MG group was not statistically significant ( P > 0. 05) . Conclusion With the increase of age , the weight of C57BL/6J female mice gradually increased , the activity level and desire to explore gradually de- creased , the spatial memory ability also declined , and the anxiety level and anxiety-like behavior increased. This study helps to reveal the effect of age on the activity level and cognitive function of females , and provides a refer- ence for studying cognitive and memory decline in older females.