ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， network pharmacology， molecular docking and cell experiments， the active ingredients， possible targets and molecular mechanisms of Baoxin granules（BXG） regulating ferroptosis in the treatment of heart failure（HF） were explored. MethodsBXG intestinal absorption fluid was prepared by everted gut sac and the chemical composition contained therein were identified by UPLC-Q-TOF-MS. According to the obtained components， the potential targets of BXG were predicted， and the HF-related targets and related genes of ferroptosis were retrieved at the same time， and the intersecting targets were obtained by Venn diagram. In addition， the protein-protein interaction（PPI） network and the component-target network were constructed， and the core components and core targets were obtained by topological analysis. Then Gene Ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis were performed on the core targets， and molecular docking validation of the key targets and main components was carried out by AutoDockTools 1.5.7. H9c2 cells were used to establish a oxygen-glucose deprivation model， and the protective effect of BXG on cells was investigated by detecting cell viability， cell survival rate and reactive oxygen species（ROS） level. The protein expression levels of signal transducer and activator of transcription 3（STAT3）， phosphorylation（p）-STAT3 and glutathione peroxidase 4（GPX4） were detected by Western blot to clarify the regulatory effect of BXG on ferroptosis. ResultsA total of 61 chemical components in BXG intestinal absorption fluid were identified， and network pharmacology obtained 27 potential targets of BXG for the treatment of HF， as well as 139 signaling pathways. BXG may act on core targets such as STAT3， tumor protein p53（TP53）， epidermal growth factor receptor（EGFR）， JUN and prostaglandin-endoperoxide synthase 2（PTGS2） through core components such as glabrolide and limonin， which in turn intervene in lipid and atherosclerosis， phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）， endocrine resistance and other signaling pathways to exert therapeutic effects on HF. Molecular docking showed that the docking results of multiple groups of targets and compounds were good. In vitro cell experiments showed that compared with the blank group， the cell viability and survival rate of the model group were significantly decreased， the level of ROS was significantly increased（P<0.01）， the expression levels of STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 proteins were significantly decreased（P<0.05， P<0.01）. Compared with the model group， the cell viability and survival rate of the BXG group were significantly increased， the ROS level was significantly decreased（P<0.01）， the STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 protein levels were significantly increased（P<0.05， P<0.01）. ConclusionBXG may inhibit the occurrence of ferroptosis by up-regulating the expression of STAT3 and GPX4， thus exerting a therapeutic effect on HF， and flavonoids may be the key components of this role.

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， network pharmacology， molecular docking and cell experiments， the active ingredients， possible targets and molecular mechanisms of Baoxin granules（BXG） regulating ferroptosis in the treatment of heart failure（HF） were explored. MethodsBXG intestinal absorption fluid was prepared by everted gut sac and the chemical composition contained therein were identified by UPLC-Q-TOF-MS. According to the obtained components， the potential targets of BXG were predicted， and the HF-related targets and related genes of ferroptosis were retrieved at the same time， and the intersecting targets were obtained by Venn diagram. In addition， the protein-protein interaction（PPI） network and the component-target network were constructed， and the core components and core targets were obtained by topological analysis. Then Gene Ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis were performed on the core targets， and molecular docking validation of the key targets and main components was carried out by AutoDockTools 1.5.7. H9c2 cells were used to establish a oxygen-glucose deprivation model， and the protective effect of BXG on cells was investigated by detecting cell viability， cell survival rate and reactive oxygen species（ROS） level. The protein expression levels of signal transducer and activator of transcription 3（STAT3）， phosphorylation（p）-STAT3 and glutathione peroxidase 4（GPX4） were detected by Western blot to clarify the regulatory effect of BXG on ferroptosis. ResultsA total of 61 chemical components in BXG intestinal absorption fluid were identified， and network pharmacology obtained 27 potential targets of BXG for the treatment of HF， as well as 139 signaling pathways. BXG may act on core targets such as STAT3， tumor protein p53（TP53）， epidermal growth factor receptor（EGFR）， JUN and prostaglandin-endoperoxide synthase 2（PTGS2） through core components such as glabrolide and limonin， which in turn intervene in lipid and atherosclerosis， phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）， endocrine resistance and other signaling pathways to exert therapeutic effects on HF. Molecular docking showed that the docking results of multiple groups of targets and compounds were good. In vitro cell experiments showed that compared with the blank group， the cell viability and survival rate of the model group were significantly decreased， the level of ROS was significantly increased（P<0.01）， the expression levels of STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 proteins were significantly decreased（P<0.05， P<0.01）. Compared with the model group， the cell viability and survival rate of the BXG group were significantly increased， the ROS level was significantly decreased（P<0.01）， the STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 protein levels were significantly increased（P<0.05， P<0.01）. ConclusionBXG may inhibit the occurrence of ferroptosis by up-regulating the expression of STAT3 and GPX4， thus exerting a therapeutic effect on HF， and flavonoids may be the key components of this role.

ObjectiveThe nucleolar protein PES1 (Pescadillo homolog 1) plays critical roles in ribosome biogenesis and cell cycle regulation, yet its involvement in cellular senescence remains poorly understood. This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role. MethodsInitially, we assessed PES1 expression patterns in two distinct senescence models: replicative senescent mouse embryonic fibroblasts (MEFs) and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells. Subsequently, PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types. Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays, respectively. The expression of senescence-associated proteins (p53, p21, and Rb) and SASP factors (IL-6, IL-1β, and IL-8) were analyzed by Western blot or qPCR. Furthermore, Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology. ResultsPES1 expression was significantly downregulated in senescent MEFs and HepG2 cells. PES1 knockdown resulted in decreased EdU-positive cells and increased SA‑β‑gal-positive cells, indicating proliferation inhibition and senescence induction. Mechanistically, PES1 suppression activated the p53-p21 pathway without affecting Rb expression, while upregulating IL-6, IL-1β, and IL-8 production. Notably, PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress, as evidenced by aberrant nucleolar morphology. ConclusionOur findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent (but Rb-independent) cellular senescence, highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.

This study aims to explore the mechanism through which Yishen Jiangtang Decoction(YSJTD) regulates endoplasmic reticulum stress(ERS)-mediated NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome to improve diabetic nephropathy(DN) in db/db mice. Thirty db/db mice were randomly divided into the model group, YSJTD group, ERS inhibitor 4-phenylbutyric acid(4-PBA) group, with 10 mice in each group. Additionally, 10 db/m mice were selected as the control group. The YSJTD group was orally administered YSJTD at a dose of 0.01 mL·g~(-1), the 4-PBA group was orally administered 4-PBA at a dose of 0.5 mg·g~(-1), and the control and model groups were given an equal volume of carboxylmethyl cellulose sodium. The treatments were administered once daily for 8 weeks. Food intake, water consumption, and body weight were recorded every 2 weeks. After the intervention, fasting blood glucose(FBG), glycosylated hemoglobin(HbA1c), urine microalbumin(U-mALB), 24-hour urine volume, serum creatinine(Scr), and blood urea nitrogen(BUN) were measured. Inflammatory markers interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected using the enzyme-linked immunosorbent assay(ELISA). Renal pathology was assessed through hematoxylin-eosin(HE), periodic acid-Schiff(PAS), and Masson staining, and transmission electron microscopy(TEM). Western blot was used to detect the expression levels of glucose-regulated protein 78(GRP78), C/EBP homologous protein(CHOP), NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), cysteinyl aspartate-specific proteinase(caspase-1), and gasdermin D(GSDMD) in kidney tissues. The results showed that compared to the control group, the model group exhibited poor general condition, increased weight and food and water intake, and significantly higher levels of FBG, HbA1c, U-mALB, kidney index, 24-hour urine volume, IL-1β, and IL-18. Compared to the model group, the YSJTD and 4-PBA groups showed improved general condition, increased body weight, decreased food intake, and lower levels of FBG, U-mALB, kidney index, 24-hour urine volume, and IL-1β. Specifically, the YSJTD group showed a significant reduction in IL-18 levels compared to the model group, while the 4-PBA group exhibited decreased water intake and HbA1c levels compared to the model group. Although there was a decreasing trend in water intake and HbA1c in the YSJTD group, the differences were not statistically significant. No significant differences were observed in BUN, Scr, and kidney weight among the groups. Renal pathology revealed that the model group exhibited more severe renal damage compared to the control group. Kidney sections from the model group showed diffuse mesangial proliferation in the glomeruli, tubular edema, tubular dilation, significant inflammatory cell infiltration in the interstitium, and increased glycogen staining and blue collagen deposition in the basement membrane. In contrast, the YSJTD and 4-PBA groups showed varying degrees of improvement in renal damage, glycogen staining, and collagen deposition, with the YSJTD group showing more significant improvements. TEM analysis indicated that the model group had extensive cytoplasmic edema, homogeneous thickening of the basement membrane, fewer foot processes, and widening of fused foot processes. In the YSJTD and 4-PBA groups, cytoplasmic swelling of renal tissues was reduced, the basement membrane remained intact and uniform, and foot process fusion improved.Western blot results indicated that compared to the control group, the model group showed upregulation of GRP78, CHOP, GSDMD, NLRP3, ASC, and caspase-1 expression. In contrast, both the YSJTD and 4-PBA groups showed downregulation of these markers compared to the model group. These findings suggest that YSJTD exerts a protective effect against DN by alleviating NLRP3 inflammasome activation through the inhibition of ERS, thereby improving the inflammatory response in db/db DN mice.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Diabetic Nephropathies/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Inflammasomes/drug effects* ; Male ; Kidney/pathology* ; Endoplasmic Reticulum Chaperone BiP ; Humans ; Interleukin-18/genetics* ; Mice, Inbred C57BL

OBJECTIVE: To compare the effectiveness of clavicular hook plate fixation in the treatment of acromioclavicular joint dislocation and distal clavicle fractures. METHODS: A clinical data of 90 patients, who underwent clavicular hook plate fixation between January 2014 and June 2023, was retrospectively analyzed. There were 40 patients with distal clavicle fractures (fracture group) and 50 with acromioclavicular joint dislocations (dislocation group). There was no significant difference in the baseline data of gender, age, cause of injury, side of injury, time from injury to operation, and constituent ratio of osteoporosis patients between the two groups ( P>0.05). The time to remove the internal fixators and the occurrence of complications were recorded. Before removing the internal fixator and at 3 months after removing, the visual analogue scale (VAS) score was used to evaluate the degree of pain, and the mobility of the shoulder joint in forward flexion, elevation, and abduction was measured. Before removing the internal fixators, the Constant-Murley score and the University of California, Los Angeles (UCLA) score were used to evaluate the function of the shoulder joint. X-ray films of the shoulder joint were taken during follow-up to observe the occurrence of subacromial osteolysis, acromioclavicular joint osteoarthritis, and distal clavicle bone atrophy. Subgroup comparison was conducted between patients with and without subacromial osteolysis in the two groups. RESULTS: All incisions healed by first intention in both groups. All patients were followed up 1-9 years, with a median of 5 years; the difference in follow-up time between the two groups was not significant ( P>0.05). During follow-up, subacromial osteolysis occurred in 74 cases, including 41 cases of typeⅠand 33 cases of type Ⅱ, distal clavicle bone atrophy in 15 cases, and acromioclavicular joint osteoarthritis in 8 cases. There were significant differences in the removal time of internal fixators, the incidence of bone atrophy, and the incidence of osteoarthritis between the two groups ( P<0.05). There was no significant difference in the incidence of subacromial osteolysis ( P>0.05). Before removing the internal fixators, there was no significant difference in VAS score, UCLA score, and Constant-Murley score between the two groups ( P>0.05), while there were significant differences in shoulder joint range of motion in all directions ( P<0.05). After removing the internal fixators, only the difference in elevation was significant ( P<0.05). Within the group comparison, the VAS score and mobility of shoulder joint in abduction and elevation after removing the internal fixators were significantly superior to those before removing ( P<0.05). In the fracture and dislocation groups, there was only a significant difference in plate length between the subgroup with and without subacromial osteolysis ( P<0.05), while there was no significant difference in the above other indicators ( P>0.05). CONCLUSION Clavicular hook plate is a good choice for treating acromioclavicular dislocation or distal clavicle fractures, but the incidence of subacromial osteolysis is higher, and the degree of bone resorption is more severe in fracture patients. After removal of the internal fixator, the shoulder functions significantly improve. It is recommended to remove the internal fixator as soon as possible within the allowable range of the condition.
Humans ; Clavicle/surgery* ; Acromioclavicular Joint/surgery* ; Bone Plates ; Fracture Fixation, Internal/instrumentation* ; Fractures, Bone/surgery* ; Male ; Retrospective Studies ; Female ; Adult ; Middle Aged ; Treatment Outcome ; Joint Dislocations/surgery* ; Aged ; Range of Motion, Articular ; Young Adult ; Postoperative Complications

Accurate timing of myelination is crucial for the proper functioning of the central nervous system. Here, we identified a de novo heterozygous mutation in TMEM63A (c.1894G>A; p. Ala632Thr) in a 7-year-old boy exhibiting hypomyelination. A Ca²⁺ influx assay suggested that this is a loss-of-function mutation. To explore how TMEM63A deficiency causes hypomyelination, we generated Tmem63a knockout mice. Genetic deletion of TMEM63A resulted in hypomyelination at postnatal day 14 (P14) arising from impaired differentiation of oligodendrocyte precursor cells (OPCs). Notably, the myelin dysplasia was transient, returning to normal levels by P28. Primary cultures of Tmem63a^-/- OPCs presented delayed differentiation. Lentivirus-based expression of TMEM63A but not TMEM63A_A632T rescued the differentiation of Tmem63a^-/- OPCs in vitro and myelination in Tmem63a^-/- mice. These data thus support the conclusion that the mutation in TMEM63A is the pathogenesis of the hypomyelination in the patient. Our study further demonstrated that TMEM63A-mediated Ca²⁺ influx plays critical roles in the early development of myelin and oligodendrocyte differentiation.
Animals ; Cell Differentiation/physiology* ; Oligodendroglia/metabolism* ; Mice, Knockout ; Mice ; Male ; Myelin Sheath/metabolism* ; Humans ; Child ; Cells, Cultured ; Oligodendrocyte Precursor Cells/metabolism*

Objective To investigate the effect of microglia activation regulated by C-X3-C motif chemokine ligand 1(CX3CL1)-C-X3-C motif chemokine receptor 1(CX3CR1)pathway on memory function in hemorrhagic shock/resuscitation rats.Methods The experiment was divided into two parts.In the first part,the rats were randomly divided into sham group,model-0.5 hour group,model-1.5 hour group,model-3 hour group,10 rats in each group.There were differences in the time of hemorrhagic shock among each group.In the second part,rats were randomly divided into control group and CX3CL1 group,10 rats in each group.The rats in CX3CL1 group were treated with CX3CL1 protein factor(intraventricular injection),and the rats in control group were treated with saline.All rats were trained in Morris water maze experiments before model construction,and tests of Morris water maze experiments were carried out after 4 days of model construction.After completion,the whole brains were taken for HE staining and immunohistochemical staining.Cerebrospinal fluid was taken for detection of inflammatory cytokines,and hippocampus tissues were taken for Real-time PCR detection and Western blotting detection.Results Compared with the sham group,the escape latency of rats in model group increased,the number of platform crossings and the resident time in the third quadrant decreased.The neuronal state was impaired in HE staining in model group.In addition,compared with the sham group,the expression of ionized calcium binding adaptor molecule-1(Iba1)in the brain of the rats in model group increased,the contents of tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 in the cerebrospinal fluid increased,and the M1-type microglia markers CD16,TNF-α,IL-1β and inducible nitric oxide synthase(iNOS)mRNA content increased.At the same time,compared with the sham group,the expressions of CX3CL1 and CX3CR1 in the brain of model group decreased,and the expressions of phosphorylated nuclear factor-κB(p-NF-κB)and nucleotide binding oligomerization domain(NOD)-like receptor protein 3(NLRP3)increased.However,compared with the control group,rats in CX3CL1 group had reduced escape latency,increased platform crossing times and quadrantⅢresident time,and recovered neuronal states.In addition,the expression of Iba1 in the brain of CX3CL1 group decreased,the contents of TNF-α and IL-6 in the cerebrospinal fluid decreased,the mRNA contents of M1-type microglia markers like CD16,TNF-α,IL-1β and iNOS decreased,and the mRNA contents of markers of M2-type microglia glial like CD206,transforming growth factor-β(TGF-β),arginase-1(Arg1),Chitinase 3-like protein 1(Ym 1)increased.Conclusion CX3CL1 can help inhibit the excessive activation of microglia,induce the polarization of microglia to M2 type,inhibit the polarization of M1 type,reduce the release of inflammatory cytokines,and alleviate the memory function damage induced by hemorrhagic shock/resuscitation.

Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.

Modern Chinese medicine studies have confirmed that the interaction between traditional Chinese medicine(TCM)and intestinal flora is the key to the treatment of diseases with tradi-tional Chinese medicine.This interplay includes such activities as:traditional Chinese medicine can be metabolized by intestinal flora into effective components with different biological activities from its precursors;TCM chemicals improve the composition of gut microbiota,consequently ameliorating its dysfunction as well as associated pathological conditions;and gut microbiota mediate the interactions between the multiple chemicals in TCM.There-fore,it becomes an important way to understand the modern sci-entific connotation of traditional Chinese medicine theory to study the pharmacological mechanism of the efficacy of traditional Chi-nese medicine by targeting Gut microbiota.