ObjectiveTo systematically compare the differential regulation of the maternal-fetal interface cell lineages and communication networks in the CBA/J×DBA/2 mouse model of recurrent pregnancy loss （RPL） by the two classic therapeutic methods-tonifying the kidney to stabilize the fetus and invigorating the spleen to stabilize the fetus （Shoutai Wan， Juyuan Jian）-of traditional Chinese medicine （TCM） at the single-cell resolution and clarify their modern scientific connotations. MethodsFemale non-pregnant CBA/J mice were caged with male BALB/c （blank group） and DBA/2 （modeling group） mice separately. Pregnant mice in the modeling group were randomly grouped as follows： high/low-dose Shoutai Wan， high/low-dose Juyuan Jian， model （RPL）， and positive control （dydrogesterone）， with 10 mice in each group. Starting from the day after the detection of the vaginal plug， mice were administrated with drugs or an equal volume of normal saline by gavage for 10 consecutive days. After the intervention， the following indicators were measured. ① Macroscopic evaluation： general conditions， uterine wet weight， embryo loss rate， four coagulation parameters ［prothrombin time （PT）， activated partial thromboplastin time （APTT）， fibrinogen （FIB）， and thrombin time （TT）］， and peripheral blood estradiol （E₂） and progesterone （Pg） levels. The decidua with embryos was stained with hematoxylin-eosin （HE） and evaluated by transmission electron microscopy （TEM）. The expression of B-cell lymphoma-2 （Bcl-2）， vascular endothelial growth factor （VEGF）， angiotensin Ⅱ （AngⅡ）， matrix metalloproteinase-2 （MMP-2）， interleukin-6 （IL-6）， leukemia inhibitory factor （LIF）， CXC chemokine ligand 12 （CXCL12）， and microtubule-associated protein 1 light chain 3 homolog （LC3）Ⅰ/Ⅱ was quantified by Western blot. ② Mechanism analysis at the single-cell level： The decidua with embryos from the blank， model， high-dose Shoutai Wan， and high-dose Juyuan Jian groups （6 mice per group， with 3 single-cell samples per group， totaling 24 mice） were analyzed by the BD Rhapsody™ platform， and the whole-cell atlas was drawn by uniform manifold approximation and projection （UMAP） dimensionality reduction clustering combined with the single-cell mouse cell atlas （scMCA）. The differentially expressed genes （DEGs） and cell interaction networks were analyzed via Gene Ontology （GO）， Kyoto Encyclopedia of Genes and Genomes （KEGG）， and CellChat， and the protein-protein interaction （PPI） map of subtype cells was constructed. The CytoTRACE pseudo-temporal analysis was performed to explore the developmental trajectories of core immune cells （natural killer cells， NK cells） from maternal and fetal sources. Results① Pathological and Western blot results indicated that compared with the blank group， the RPL group showed an increase in the embryo loss rate （P<0.01）， down-regulated expression of Bcl-2， LIF， MMP-2， and Vegf in the decidua with embryos （P<0.05）， up-regulated protein levels of CXCL-12， AngⅡ， and IL-6 （P<0.05）， blocked angiogenesis， apoptosis-inflammation imbalance， and coagulation dysfunction. Both prescriptions dose-dependently reduced the abortion rate and restored the angiogenesis-inflammation balance， and Shoutai pill showed superior performance in restoring the E₂ level to the Pg level （P<0.05）. ② Single-cell transcriptome analysis indicated that compared with the blank group， the RPL group showed differences in multiple key cell populations such as decidual cells， trophoblast cells， endothelial cells， erythroblasts， NK cells， and macrophages at the maternal-fetal interface. Immunity and angiogenesis were the key links in RPL. Compared with the RPL group， high-dose Shoutai Wan reversed the changes of NK cells in the embryonic layer （upregulating the mRNA levels of 17 genes and downregulating the mRNA levels of 29 genes） and macrophages （upregulating the mRNA levels of 117 genes and downregulating the mRNA levels of 53 genes） through the regulation of gene expression. High-dose Shoutai pill regulated the immune cells to affect unfolded proteins， cell adhesion， and programmed cell death， thereby promoting decidualization and angiogenesis and modulating embryo-membrane development. High-dose Juyuan Jian regulated the key subgroups of NK cells （up-regulating the mRNA levels of 9 genes and down-regulating the mRNA levels of 17 genes） and macrophages （up-regulating the mRNA levels of 110 genes and down-regulating the mRNA levels of 81 genes）， which affected decidual inflammation and apoptosis and intervened in glycolysis. ③ The pseudo-temporal analysis and communication network indicated that the communication frequency of the RPL group decreased. High-dose Shoutai Wan restored maternal-fetal tolerance through pathways such as NKG2D， CDH5， GDF， and FASLG. High-dose Juyuan Jian enhanced the IL-6/LIFR/JAK/signal transducer and activator of transcription 3 （STAT3） and desmosome/SEMA6/tumor necrosis factor-like weak inducer of apoptosis （TWEAK） signaling to improve endometrial receptivity. The RPL group showed an increased proportion of toxic dNK7， a decreased proportion of reparative dNK4， and blocked embryo fNK1. High-dose Shoutai Wan down-regulated dNK7 and up-regulated dNK4. High-dose Juyuan Jian inhibited the terminal differentiation of dNK7 and up-regulated LILRB1， thus restoring the balance of cytotoxicity and repair. ConclusionBoth the kidney-tonifying and spleen-invigorating methods are effective in treating RPL. NK and macrophages are the key immune cells in the interaction between the embryo and the membrane. The kidney-tonifying method （Shoutai Wan） has an advantage in regulating the phenotypes of unfolded protein， cell adhesion， and programmed cell death， and shows expression characteristics closer to the physiological state in the regulation of NKG2D and CDH5 signals. The spleen-invigorating method （Juyuan Jian） has an advantage in regulating epithelial-mesenchymal transition （EMT）， angiogenesis， and glycolysis and shows higher communication intensity in the IL-6 and LIFR pathways.

ObjectiveMultiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS); however, its underlying neurological pathogenic mechanisms remain incompletely understood. Endogenous formaldehyde (FA), a metabolic byproduct of methylation-demethylation cycles, has recently been implicated in neurotoxicity, oxidative damage, and cognitive impairment. This study aimed to investigate whether excessive FA contributes to myelin sheath demyelination in mice and to evaluate the protective effects and mechanisms of two FA-elimination strategies: sodium bisulfite (NaHSO₃), a classical FA scavenger, and polyethylene glycol-modified astaxanthin nanoparticles (PEG-ATX@NPs), a brain-targeted nano-antioxidant formulation. MethodsA chronic demyelination model was established by feeding female C57BL/6J mice a diet containing 0.2% cuprizone (CPZ) for four weeks, followed by a two-week intervention period. Eighty mice were randomly assigned to four groups: NS (normal saline), CPZ+NS, CPZ+NaHSO₃, and CPZ+PEG-ATX@NPs. Behavioral tests, including open-field, Y-maze, and pole-climbing assays, were conducted to assess locomotor activity, motor coordination, and working memory. FA levels in serum, corpus callosum, and spinal cord were measured using an Na-FA fluorescent probe and quantified via in vivo and ex vivo fluorescence imaging. Neuroinflammatory responses were evaluated by measuring TNF-α, IL-1β, and IL-6 levels using ELISA, while oxidative stress was assessed by reactive oxygen species (ROS) fluorescence intensity. Demyelination was examined via Luxol fast blue staining, and microglial activation was analyzed by Iba1 immunofluorescence. Correlation analyses were performed to explore relationships among FA levels, inflammatory cytokines, ROS intensity, and behavioral parameters. ResultsCompared with the NS group, mice in the CPZ+NS group exhibited significant weight loss, impaired motor coordination and memory, and markedly reduced myelin regeneration (P<0.05). FA levels and pro-inflammatory cytokines were significantly elevated in serum, corpus callosum, and spinal cord (P<0.05). FA-associated fluorescence in brain and spinal tissues, as well as ROS intensity across all tissues examined, also increased substantially (P<0.05). CPZ treatment induced pronounced microglial activation and severe demyelination in the corpus callosum (P<0.01). Both NaHSO₃ and PEG-ATX@NPs effectively reduced FA accumulation in the brain and spinal cord, attenuated demyelination, suppressed microglial activation, decreased inflammatory cytokine levels, and improved motor and cognitive performance. These results confirm that CPZ induced severe demyelination accompanied by oxidative stress, neuroinflammation, and abnormal FA accumulation. Following intervention with either NaHSO₃ or PEG-ATX@NPs, endogenous FA levels in the CNS were substantially reduced. Both treatments alleviated demyelination and significantly decreased the number of activated microglia. Levels of TNF-α, IL-1β, and IL-6 in serum, corpus callosum, and spinal cord were downregulated. Behavioral performance improved significantly, as evidenced by enhanced locomotor activity, better coordination, and improved memory function. These findings indicate that both FA-scavenging agents mitigate CPZ-induced biochemical and behavioral abnormalities. ConclusionThis study demonstrates that excessive endogenous FA is closely associated with cognitive impairment, inflammatory dysregulation, and demyelination in a CPZ-induced chronic demyelination mouse model. Clearing abnormally elevated FA effectively reduces neuroinflammation, suppresses microglial overactivation, decreases oxidative stress, and alleviates demyelination, ultimately improving motor and cognitive outcomes in mice. These results suggest that targeting endogenous FA represents a promising therapeutic strategy for MS and other demyelinating disorders. Further investigations are warranted to explore the long-term safety, dosage optimization, and molecular pathways involved in FA-mediated neurotoxicity.

ObjectiveMultiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS); however, its underlying neurological pathogenic mechanisms remain incompletely understood. Endogenous formaldehyde (FA), a metabolic byproduct of methylation-demethylation cycles, has recently been implicated in neurotoxicity, oxidative damage, and cognitive impairment. This study aimed to investigate whether excessive FA contributes to myelin sheath demyelination in mice and to evaluate the protective effects and mechanisms of two FA-elimination strategies: sodium bisulfite (NaHSO₃), a classical FA scavenger, and polyethylene glycol-modified astaxanthin nanoparticles (PEG-ATX@NPs), a brain-targeted nano-antioxidant formulation. MethodsA chronic demyelination model was established by feeding female C57BL/6J mice a diet containing 0.2% cuprizone (CPZ) for four weeks, followed by a two-week intervention period. Eighty mice were randomly assigned to four groups: NS (normal saline), CPZ+NS, CPZ+NaHSO₃, and CPZ+PEG-ATX@NPs. Behavioral tests, including open-field, Y-maze, and pole-climbing assays, were conducted to assess locomotor activity, motor coordination, and working memory. FA levels in serum, corpus callosum, and spinal cord were measured using an Na-FA fluorescent probe and quantified via in vivo and ex vivo fluorescence imaging. Neuroinflammatory responses were evaluated by measuring TNF-α, IL-1β, and IL-6 levels using ELISA, while oxidative stress was assessed by reactive oxygen species (ROS) fluorescence intensity. Demyelination was examined via Luxol fast blue staining, and microglial activation was analyzed by Iba1 immunofluorescence. Correlation analyses were performed to explore relationships among FA levels, inflammatory cytokines, ROS intensity, and behavioral parameters. ResultsCompared with the NS group, mice in the CPZ+NS group exhibited significant weight loss, impaired motor coordination and memory, and markedly reduced myelin regeneration (P<0.05). FA levels and pro-inflammatory cytokines were significantly elevated in serum, corpus callosum, and spinal cord (P<0.05). FA-associated fluorescence in brain and spinal tissues, as well as ROS intensity across all tissues examined, also increased substantially (P<0.05). CPZ treatment induced pronounced microglial activation and severe demyelination in the corpus callosum (P<0.01). Both NaHSO₃ and PEG-ATX@NPs effectively reduced FA accumulation in the brain and spinal cord, attenuated demyelination, suppressed microglial activation, decreased inflammatory cytokine levels, and improved motor and cognitive performance. These results confirm that CPZ induced severe demyelination accompanied by oxidative stress, neuroinflammation, and abnormal FA accumulation. Following intervention with either NaHSO₃ or PEG-ATX@NPs, endogenous FA levels in the CNS were substantially reduced. Both treatments alleviated demyelination and significantly decreased the number of activated microglia. Levels of TNF-α, IL-1β, and IL-6 in serum, corpus callosum, and spinal cord were downregulated. Behavioral performance improved significantly, as evidenced by enhanced locomotor activity, better coordination, and improved memory function. These findings indicate that both FA-scavenging agents mitigate CPZ-induced biochemical and behavioral abnormalities. ConclusionThis study demonstrates that excessive endogenous FA is closely associated with cognitive impairment, inflammatory dysregulation, and demyelination in a CPZ-induced chronic demyelination mouse model. Clearing abnormally elevated FA effectively reduces neuroinflammation, suppresses microglial overactivation, decreases oxidative stress, and alleviates demyelination, ultimately improving motor and cognitive outcomes in mice. These results suggest that targeting endogenous FA represents a promising therapeutic strategy for MS and other demyelinating disorders. Further investigations are warranted to explore the long-term safety, dosage optimization, and molecular pathways involved in FA-mediated neurotoxicity.

ObjectiveTo investigate whether Bushen Huoxue prescription improves embryo implantation through regulating exosomal miRNA to enhance maternal-fetal interface communication based on Bushen Huoxue therapy. MethodsIn the animal experiment， all the rats （except for the blank group） were administered hydroxyurea （450 mg·kg^-1） via gavage for 10 d， as well as epinephrine （0.3 mg·kg^-1） and mifepristone （5.5 mg·kg^-1） via subcutaneous injection for 7 d to establish an implantation disorder model of kidney deficiency and blood stasis type. The Bushen Huoxue prescription （BSHX） groups were administered the prescription at different doses （7.30 g·kg^-1 for the high-dose group， 3.65 g·kg^-1 for the medium-dose group， and 1.83 g·kg^-1 for the low-dose group） via gavage. The dydrogesterone group was administered the corresponding medicine （2.63 mg·kg^-1） via gavage. After intervention for 10 days， uterine histopathological changes were observed via hematoxylin-eosin （HE） staining. Mucin （MUC1）， forkhead box protein O1 （FoxO1）， and homeobox A10 （HoxA10） expression levels were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Cell experiment selected primary endometrial epithelial cells （EEC） and trophoblast cells （TC） as research subjects. Exosome-free medicated serum was prepared by ultracentrifugation and cultured in complete medium. Exosomes were isolated from cell supernatants by ultracentrifugation for cross-co-culture. After 48 h， migration and invasion abilities were assessed by scratch and Transwell assays. Sequencing was then performed on EEC-origin exosomal miRNA. ResultsThe model rats exhibited thin endometrium， along with reduced blood vessels， glandules， and pinopode numbers. BSHX improved endometrial morphology and increased pinopode numbers. MUC1， FoxO1， and HoxA10 expressions were downregulated in the model rats， while these parameters were upregulated after BSHX medium- and high-dose intervention. In the cell experiment， after exosome-free medicated serum intervention for 24 h， migration and invasion abilities were enhanced in the BSHX groups （P<0.01）. In EEC-origin exosomal miRNA sequencing， gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） analyses revealed enrichment in biological processes （gastrulation， neuronal differentiation， alongside cell development and regeneration）， involving the mitogen-activated protein kinase （MAPK）， FoxO1， Wnt， mammalian target of rapamycin （mTOR）， and tumor necrosis factor （TNF） signaling pathways. ConclusionBSHX promotes embryo implantation by improving endometrial receptivity via regulating exosomal miRNA. These findings provide potential targets for exosomal miRNA-based assisted reproductive strategies and a novel theoretical basis for infertility treatment by traditional Chinese medicine.

[Objective] To retrospectively analyse the serological characteristics of hemolytic transfusion reactions caused by Rh and Kidd antibodies, and to provide reference for safe, timely, and effective blood transfusion. [Methods] Two cases of patients with RhCcEe and Kidd blood type who experienced allogeneic transfusion at Dazhou Central Hospital were selected. A series of immunohematological tests were performed, including ABO, RhDCcEe and Kidd blood typing, unexpected antibody screening and identification, crossmatching, direct antiglobulin test, acid elution test, and capillary centrifugation to separate the patient's own red blood cells from donated red blood cells. [Results] Unexpected antibody screening, antibody identification, and direct antiglobulin test were positive in both patients. Case 1 had anti-Jk in the plasma, but no specific antibodies were found in the eluate. Case 2 had anti-c and E in the plasma, and anti-E was detected in the eluate. High-speed capillary centrifugation revealed corresponding antigen-positive erythrocytes at the distal end of the blood samples of both patients. [Conclusion] Case 1 received Kidd allogeneic red blood cells, and case 2 received RhCcEe allogeneic red blood cells, and both patients developed the corresponding unexpected antibodies, which led to the occurrence of immune haemolytic blood transfusion reaction.

Several types of arthritis share the common feature that the generation of inflammatory mediators leads to joint cartilage degradation. However, the shared mechanism is largely unknown. H2BK120ub1 was reportedly involved in various inflammatory diseases but its role in the shared mechanism in inflammatory joint conditions remains elusive. The present study demonstrated that levels of cartilage degradation, H2BK120ub1, and its regulator WW domain-containing adapter protein with coiled-coil (WAC) were increased in cartilage in human rheumatoid arthritis (RA) and osteoarthritis (OA) patients as well as in experimental RA and OA mice. By regulating H2BK120ub1 and H3K27me3, WAC regulated the secretion of inflammatory and cartilage-degrading factors. WAC influenced the level of H3K27me3 by regulating nuclear entry of the H3K27 demethylase KDM6B, and acted as a key factor of the crosstalk between H2BK120ub1 and H3K27me3. The cartilage-specific knockout of WAC demonstrated the ability to alleviate cartilage degradation in collagen-induced arthritis (CIA) and collagenase-induced osteoarthritis (CIOA) mice. Through molecular docking and dynamic simulation, doxercalciferol was found to inhibit WAC and the development of cartilage degradation in the CIA and CIOA models. Our study demonstrated that WAC is a key factor of cartilage degradation in arthritis, and targeting WAC by doxercalciferol could be a viable therapeutic strategy for treating cartilage destruction in several types of arthritis.

Notum, a negative feedback regulator of the Wnt signaling, has emerged as a promising target for treating glucocorticoid-induced osteoporosis (GIOP). This study showcases an efficient strategy for discovering the anti-Notum constituents from herbal medicines (HMs) as novel anti-GIOP agents. Firstly, a rapid-responding near-infrared fluorogenic substrate for Notum was rationally engineered for high-throughput identifying the anti-Notum HMs. The results showed that Bu-Gu-Zhi (BGZ), a known anti-osteoporosis herb, potently inhibited Notum in a competitive-inhibition manner. To uncover the key anti-Notum constituents in BGZ, an efficient strategy was adapted via integrating biochemical, phytochemical, computational, and pharmacological assays. Among all identified BGZ constituents, three furanocoumarins were validated as strong Notum inhibitors, while 5-methoxypsoralen (5-MP) showed the most potent anti-Notum activity and favorable safety profiles. Mechanistically, 5-MP acted as a competitive inhibitor of Notum via creating strong hydrophobic interactions with Trp128 and Phe268 in the catalytic cavity of Notum. Cellular assays showed that 5-MP remarkably promoted osteoblast differentiation and activated Wnt signaling in dexamethasone (DXMS)-challenged MC3T3-E1 osteoblasts. In dexamethasone-induced osteoporotic mice, 5-MP strongly elevated bone mineral density (BMD) and improved cancellous and cortical bone thickness. Collectively, this study constructs a high-efficient platform for discovering key anti-Notum constituents from HMs, while 5-MP emerges as a promising anti-GIOP agent.

Osteoarthritis (OA) causes chronic pain that significantly impairs quality of life, with current treatments often proving insufficient and accompanied by adverse effects. Recent research has identified the dorsal root ganglion (DRG) and its resident macrophages as crucial mediators of chronic OA pain through neuroinflammation driven by macrophage polarization. We present a novel injectable thermo-sensitive hydrogel system, KAF@PLEL, designed to deliver an anti-inflammatory peptide (KAF) specifically to the DRG. This biodegradable hydrogel enables sustained KAF release, promoting the reprogramming of DRG macrophages from pro-inflammatory to anti-inflammatory phenotypes. Through comprehensive in vitro and in vivo studies, we evaluated the hydrogel's biocompatibility, effects on macrophage polarization, and therapeutic efficacy in chronic OA pain management. The system demonstrated significant capabilities in preserving macrophage mitochondrial function, suppressing neuroinflammation, alleviating chronic OA pain, reducing cartilage degradation, and improving motor function in OA rat models. The sustained-release properties of KAF@PLEL enabled prolonged therapeutic effects while minimizing systemic exposure and side effects. These findings suggest that KAF@PLEL represents a promising therapeutic approach for improving outcomes in OA patients through targeted, sustained treatment.