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.

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 role of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor-κB （NF-κB） signaling pathway in intestinal mucosal barrier damage in ulcerative colitis， as well as the intervention mechanism of Shaoyaotang. MethodsSixty SD rats were allocated into a blank group， a model group， a mesalazine （0.42 g·kg^-1） group， and low-， medium-， and high-dose （11.1， 22.2， 44.4 g·kg^-1， respectively） Shaoyaotang groups. A model of ulcerative colitis was induced by 2，4，6-trinitrobenzenesulfonic acid （TNBS）. After successful modeling， rats were administrated with corresponding agents via gavage for 7 days. Changes in colon length and colon weight were observed. Hematoxylin-eosin staining was performed to examine the pathological changes of the colon， and immunohistochemistry was employed to detect the expression of the inflammatory cytokine interleukin-8 （IL-8）， cyclooxygenase-2 （COX-2）， junction adhesion molecule-1 （JAM-1）， and claudin-1 in the colon. Western blot analysis was performed to determine the protein levels of TLR4， MyD88， and NF-κB in the colon. ResultsCompared with the blank group， the model group showed elevated DAI score （P<0.01）， reduced colon length and colon weight （P<0.01）， down-regulated protein levels of JAM-1 and claudin-1 （P<0.01）， and up-regulated protein levels of IL-8， COX-2， TLR4， MyD88， and NF-κB p65 （P<0.01） in the colon tissue. Compared with the model group， each treatment group showed decreased DAI score （P<0.05， P<0.01）， increased colon length and colon weight （P<0.05， P<0.01）， up-regulated protein levels of JAM-1 and claudin-1 （P<0.01）， and down-regulated protein levels of IL-8， COX-2， TLR4， MyD88， and NF-κB p65 （P<0.01） in the colon tissue. ConclusionShaoyaotang alleviates intestinal inflammation and intestinal mucosal damage to protect intestinal barrier integrity by regulating the TLR4/MyD88/NF-κB signaling pathway.

The bacterial CBASS(cyclic-oligonucleotide-based anti-phage signaling system) represents a novel innate immune defense mechanism mediated by cyclic nucleotides.The system employs cGAS/DncV-like nucleotidyltransferases (CD-NTases) to recognize exogenous nucleic acids, catalyzing the production of second messenger molecules such as cyclic GMP-AMP (cGAMP) to activate effector proteins (e.g., nucleases), thereby triggering "cell suicide" to combat phage infection. The molecular mechanisms and evolutionary features of CBASS not only uncover the diversity of bacterial immune defenses but also provide critical insights into the conservation of prokaryotic-eukaryotic immune pathways. Notably, the bacterial CBASS system exhibits profound homology with the eukaryotic cGAS-STING (Cyclic GMP-AMP Synthase—Stimulator of Interferon Genes) pathway across multiple dimensions, including the catalytic domains of CD-NTase/cGAS and cyclic nucleotide signaling mechanisms. This finding supports the hypothesis that eukaryotic innate immunity may have originated from prokaryotic horizontal gene transfer and highlights an evolutionary shift in defense strategies from bacterial "population lysis" to "individual inflammatory activation" in higher organisms.This review systematically synthesizes the functional architecture and mechanisms of the CBASS system, with a deep exploration of its evolutionary connections to the eukaryotic cGAS-STING pathway. The insights gained herein offer fresh perspectives for understanding the origin and evolution of the innate immune system, and for driving advancements in biotechnological research and development.

BACKGROUND:In recent years,numerous studies have shown that autophagy and mitophagy play an important role in the regulation of bone metabolism.Under non-physiological conditions,mitophagy breaks the balance of bone metabolism and triggers metabolism disorders,which affect osteoblasts,osteoclasts,osteocytes,chondrocytes,bone marrow mesenchymal stem cells,etc. OBJECTIVE:To summarize the mechanism of mitophagy in regulating bone metabolic diseases and its application in clinical treatment. METHODS:PubMed,Web of Science,CNKI,WanFang and VIP databases were searched by computer using the keywords of"mitophagy,bone metabolism,osteoblasts,osteoclasts,osteocytes,chondrocytes,bone marrow mesenchymal stem cells"in English and Chinese.The search time was from 2008 to 2023.According to the inclusion criteria,90 articles were finally included for review and analysis. RESULTS AND CONCLUSION:Mitophagy promotes the generation of osteoblasts through SIRT1,PINK1/Parkin,FOXO3 and PI3K signaling pathways,while inhibiting osteoclast function through PINK1/Parkin and SIRT1 signaling pathways.Mitophagy leads to bone loss by increasing calcium phosphate particles and tissue protein kinase K in bone tissue.Mitophagy improves the function of chondrocytes through PINK1/Parkin,PI3K/AKT/mTOR and AMPK signaling pathways.Modulation of mitophagy shows great potential in the treatment of bone diseases,but there are still some issues to be further explored,such as different stages of drug-activated mitophagy,and the regulatory mechanisms of different signaling pathways.

BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged

BACKGROUND:Foot position and seat height are important factors affecting "Sit-to-Stand",but most of the current research on "Sit-to-Stand" focuses on healthy people and Parkinson's disease patients. The kinematic and kinetic characteristics of the lower limbs of children with spastic cerebral palsy during the "Sit-to-Stand" task under different foot positions and seat heights are not known.OBJECTIVE:To investigate the effects of different foot positions and different seat height on lower limb kinematic and kinetic parameters during the "Sit-to-Stand" task in children with cerebral palsy. METHODS:Seven children with spastic cerebral palsy were selected as the research subjects. All subjects received the "Sit-to-Stand" test of six tasks,namely three seat heights (high,medium,and low stools) × two foot positions (front and back foot positions). The kinematic and dynamic data of children with cerebral palsy were collected under different foot positions and seat heights.RESULTS AND CONCLUSION:(1) The time characteristics results showed that the total time required for the children with cerebral palsy to perform the sit-to-stand transfer task was significantly smaller in the high stool condition compared to the low stool condition (P=0.046). (2) The kinetic results showed that at the moment of lifting,the knee flexion moment was significantly larger in the bipedal posterior condition than the bipedal anterior condition (P=0.049). The knee flexion moment was significantly smaller in the high stool condition compared to the medium stool condition (P<0.001). (3) It is concluded that raising the seat height and changing the foot position had an effect on the sit-to-stand transfer in children with spastic cerebral palsy. The children were able to perform the sit-to-stand maneuver with less motor compensation in the high-stool bipedal-rear position condition. Meanwhile,the high chair can be used as an aid to enhance the performance of sit-to-stand transfer in children with spastic cerebral palsy. The high stool bipedal hindfoot condition was the most effective in improving the sit-to-stand transfer in children with spastic cerebral palsy.

OBJECTIVE To explore the functional substance basis of Jinhong Tablet in the treatment of chronic superficial gastri-tis(CSG).METHODS Three different models were constructed to investigate the anti-inflammatory and antioxidant effects,func-tional material basis of Jinhong Tablet:inflammatory model in human gastric epithelial cells(GES-1)induced by lipopolysaccharide(LPS),LPS-induced inflammatory model in mouse gastric organoids,and ethanol-induced oxidative damage model in GES-1 cells.MTS assay was performed to detect cell proliferation activity;qPCR was applied to measure the relative mRNA expression of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),and interleukin-8(IL-8)in cells and gastric organoids;and the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and reactive oxygen species(ROS)in cells were detected.RESULTS Jinhong Tablet and 10 functional components significantly reduced the relative expression of inflammation-relat-ed genes TNF-α,IL-1β,IL-6,and IL-8 in LPS-induced GES-1 cells and gastric organoids,suggesting that these 10 components are the functional substance basis for the anti-inflammatory effects of Jin Hong Tablet.Jin Hong Tablet and 11 functional components markedly decreased the levels of MDA and ROS and increased the activity of SOD,indicating that these 11 components were the func-tional substance basis of the antioxidant effects of Jinhong Tablet.CONCLUSION Through in vitro cell and gastric organoid experi-ments,it has been preliminarily determined that allocryptopine,corydaline,dehydrocorydaline,palmatine hydrochloride,chlorogenic acid,costunolide,rutin,quercitrin,dehydrocostus lactone,tetrahydrocoptisine,isochlorogenic acid B,toosendanin,protopine,and quercetin are the functional material basis of Jinhong Tablet in treating CSG,accumulating scientific evidence for the enhancement of the quality standards of Jinhong Tablet.

Objective:To investigate the changes in hepatic phase II detoxification enzymes and their mechanism in metabolic associated steatohepatitis (MASH) induced by a methionine-choline-deficient (MCD) diet in mice.Methods:Ten C57BL/6J mice were randomly divided into two groups, with five mice in each group, and fed with a control diet (NCD group) and a methionine-choline-deficient diet (MCD group) for four consecutive weeks to establish the MASH model in mice. Mice body weight was recorded weekly. Mice peripheral blood and liver tissue samples were collected after four weeks. The liver histopathological changes were observed by hematoxylin-eosin staining and Sirius red staining in liver tissue. The levels of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglycerides were measured by an automatic biochemical analyzer. Triglyceride and total cholesterol were used to evaluate the lipid accumulation condition in the liver of mice with Oil red O staining. Real-time fluorescence quantitative PCR was used to detect the expression of liver inflammatory factors interleukin (IL)-1β and monocyte chemoattractant protein-1 (MCP-1) condition. Transcriptome sequencing and bioinformatics were used to analyze the changes in gene expression profiles in the liver of mice and screen differentially expressed genes. The expression conditions of phase Ⅱ detoxification enzymes glutathione S-transferase mu 4 (GSTM4), dihydronicotinamide riboside:quinone oxidoreductases (NQO-2), sulfotransferase 1β1 (SULT1β1), and uridine diphosphate glucuronosyltransferase 2 family, polypeptide A3(UGT2A3) were verified by real-time fluorescent quantitative PCR. Plasma malondialdehyde content, total antioxidant capacity (T-AOC), plasma and liver glutathione content were determined using commercial kits. The expression of nuclear factor E2-related factor 2 (Nrf2), GSTM4, and UGT1A6 was examined by Western blotting. The independent sample t-test was used for comparison between the groups. Results:The body weight of mice in the MCD group showed a gradual downward trend, while the body weight of mice in the NCD group did not change significantly following four weeks of different dietary feeding. The MCD group mice liver had yellow-white appearance with round edges. The liver/body mass index was significantly lower in the NCD group ( t=3.216, P<0.01). Hematoxylin-eosin staining showed that hepatocytes in the MCD group had an occurrence of fatty degeneration accompanied by inflammatory cell infiltration, with a higher NAFLD activity score (NAS) compared to the NCD group ( t=7.155, P<0.001). Sirius red staining showed that the the liver of the MCD group had mildly increased periportal fibers. Plasma biochemical tests indicated that plasma ALT, AST, and triglyceride levels were significantly higher in the MCD group than those in the NCD group ( t=8.920, P<0.001; t=6.696, P<0.001; t=3.904, P<0.01). Oil red O staining showed that a large number of lipid droplets accumulated in the liver tissue of the MCD group and were more severe than those in the NCD group ( t=7.405, P<0.001). The triglyceride content was significantly higher in the liver of the mice in the MCD group than that in the NCD group ( t=3.559, P<0.01), and the expression of inflammatory factors IL-1β and MCP-1 was significantly increased ( t=2.562 and 2.391, respectively, P<0.05). Transcriptome sequencing analysis showed that the expression profile of genes related to lipid metabolism was changed in the liver tissue of the mice in the MCD group. The expression of multiple phase Ⅱ detoxification enzymes was significantly downregulated. Real-time fluorescence quantitative PCR verification demonstrated that the expression of four phase Ⅱ detoxification enzymes GSTM4, NQO2, SUIL1β1, and UGT2A3 were significantly lower in the liver of the mice in the MCD group than those in the NCD group ( t=2.498, 3.570, 3.768, and 4.166, respectively, P<0.05). The detection kit showed that compared with the NCD group, the malondialdehyde content in the liver of mice in the MCD group increased ( t=3.601, P<0.01), while the plasma total glutathione ( t=11.93, P<0.001) and reduced glutathione levels were significantly reduced ( t=3.635, P<0.01). The total antioxidant capacity of the liver decreased ( t=2.872, P<0.05), and the total glutathione and reduced glutathione levels in the liver were significantly increased ( t=3.175 and 3.064, P<0.05). Western blotting showed that the expression of Nrf2, GSTM4, and UGT1A6 proteins was significantly lower in the MCD group than that in the NCD group ( t=3.385, 2.990, 2.168, P<0.05). Conclusions:The expressions of multiple phase Ⅱ detoxification enzymes and antioxidant capacity are reduced in the liver of MASH mice induced by the MCD diet, and its mechanism is related to the down-regulation of the expression of the upstream regulatory factor Nrf2 protein.