［Abstract］Objective: To investigate the intervention effect and potential mechanism of small extracellular vesicles (sEVs) derived from β-nicotinamide mononucleotide (NMN)-engineered human umbilical cord mesenchymal stem cells (hucMSCs) on the senescence of dermal fibroblasts (DFs). Methods: hucMSCs were isolated and cultured, and sEVs were extracted from cell culture supernatant. NMN-engineered hucMSC-sEVs (NMN-sEVs) were prepared by coincubation. A photoaging cell model of DFs was established by ultraviolet (UV) irradiation. Before UV irradiation, cells were pretreated with NMN, sEVs and NMN-sEVs, respectively. Senescence-associated β-galactosidase staining, reactive oxygen species detection and cellular immunofluorescence assay were used to evaluate cellular senescence status, oxidative stress level and type Ⅰ collagen synthesis. Transcriptome sequencing was performed on DFs in each group. Differentially expressed genes were screened combined with bioinformatics analysis, and verification experiments were carried out for key targets. Results: High-performance liquid chromatography showed that the encapsulation efficiency of NMN was (28.46±3.80)%, and the drug loading capacity was (95.13±12.69) μg/1010 sEV, indicating the successful construction of NMN-sEVs. Compared with the control group, the UV group exhibited an increased proportion of senescent cells, elevated oxidative stress level and down-regulated expression of collagen type Ⅰ α1 chain (COL1A1) (all P＜0.05). Compared with the UV group, interventions with NMN, sEVs and NMN-sEVs could effectively delay DF senescence, alleviate oxidative stress damage and promote COL1A1 synthesis, and the NMN-sEVs group showed the most remarkable effect. Transcriptome sequencing results showed that the expression of RNA-binding motif protein 39 (RBM39) was significantly higher in UV photoaging cells than that in the control group (P＜0.01); after NMN-sEVs intervention, its expression level RBM39 was significantly decreased compared with the UV group (P＜0.001). Conclusion: NMN-sEVs can effectively delay UV-induced senescence of DFs, and its mechanism may be attributed to the targeted inhibition of RBM39 expression.

Objective: To investigate the effects and mechanisms of fecal extracellular vesicles (FEVs) in dextran sulfate sodium (DSS)-induced murine colitis. Methods: A DSS-induced mouse colitis model was established to evaluate the effects of FEVs intervention on indicators including the disease activity index (DAI) and histopathological scores. qRT-PCR was performed to detect the mRNA expression of intestinal inflammatory factors, tight junction proteins and antioxidant enzymes in mice. Furthermore, 16S rRNA sequencing combined with untargeted metabolomics was applied to analyze the alterations in gut microbiota and metabolites. Results: FEVs significantly alleviated colitis in mice, as evidenced by reduced DAI, attenuated colon damage, diminished inflammation, and restored intestinal barrier function. Mechanistically, FEVs suppressed pro-inflammatory cytokines and oxidative stress while modulating gut microbiota composition and related metabolite levels. Conclusion: FEVs ameliorate DSS-indeced colitis in mice by regulating the microbiota-metabolism-immune axis.

Objective: To investigate the protein expression profiles of platelet-derived extracellular vesicles (pEVs) from experimental SjOgren′s syndrome (ESS) mice and explore potential molecular mechanisms of pEVs involved in SjOgren′s syndrome. Methods: An ESS mouse model was established. Platelets were isolated from mouse peripheral blood and activated by collagen to generate pEVs. LC-MS/MS-based proteomics was applied to screen differentially expressed proteins (DEPs). Bioinformatic analyses including GO enrichment, KEGG pathway enrichment, PPI network and protein domain enrichment were performed for DEPs. Results: The purity of isolated platelets reached 98%, and the isolated pEVs met the identification criteria for extracellular vesicles. A total of 417 DEPs were identified between ESS and wild-type groups, among which 235 proteins were upregulated and 182 were downregulated. The most significantly upregulated proteins included thrombospondin-2 (TSP2), orosomucoid-2 (ORM2), apolipoprotein F (Apof), cytochrome c and LIM senescent cell antigen-like domain-containing protein 1. Markedly downregulated proteins contained Serpina1c, Serpina3m, NME1, carboxylesterase 1B, COP9 signalosome subunit 7A (COPS7A) and ubiquitin-specific peptidase 14 (USP14). GO enrichment revealed obvious alterations in cellular component, molecular function and biological process of pEV-derived proteins from ESS mice. KEGG analysis indicated that upregulated DEPs were mainly enriched in cell adhesion and actin cytoskeleton regulation pathways, while downregulated DEPs were enriched in complement and coagulation cascades, proteasome and amino acid biosynthesis pathways. PPI network demonstrated that Abelson interactor 1, glutaredoxin, leucyl-tRNA synthetase, integrin β3 and LIM senescent cell antigen-like domain-containing protein 1 were hub proteins among upregulated DEPs. Gp1ba, von Willebrand factor, heat-shock protein 90 kD β member 1, proteasome subunit 4/14, retinol-binding protein 4 and transthyretin occupied core positions in downregulated DEPs network. PI showed that upregulated DEPs were enriched in calmodulin homology domain, TCP1/cpn60 chaperone domain and spectrin repeat domain. Downregulated DEPs were mainly enriched in serine protease inhibitor domain, importin-β N-terminal domain and Cullin sumoylation domain. Conclusion: The proteomic profiles of pEVs in ESS mice are characterized and key differential molecules and related signaling pathways are also identified, which provides experimental basis for understanding the role of PEVs in autoimmune injury of SjOgren′s syndrome.

目的： 探讨磷脂酶A2G16(phospholipase A2 group ⅩⅥ，PLA2G16)在胃癌中的表达及与患者预后的关系，以及其对胃癌细胞化疗敏感性的影响及潜在机制。方法： 通过TCGA数据库筛选出与胃癌相关的磷脂酶A2(phospholipase A2，PLA2)家族成员mRNA在胃癌组织和正常组织中的差异表达，结合数据库行Kaplan-Meier生存分析。收集96例胃癌组织及癌旁组织，采用qRT-PCR检测PLA2G16 mRNA表达水平，分析其与患者临床病理特征及总体生存期的关系，采用单因素和多因素Cox回归模型评估其预后价值。取对数生长期胃癌HGC-27细胞，转染特异性siRNA构建PLA2G16瞬时敲低模型，并通过qRT-PCR和蛋白免疫印迹验证敲低效率。在PLA2G16瞬时敲低模型基础上，给予胃癌HGC-27细胞不同浓度顺铂和5-氟尿嘧啶处理；采用CCK-8法检测细胞活力，并计算半数抑制浓度(IC₅₀)；分别采用流式细胞术、克隆形成实验、CCK-8实验、划痕愈合实验及Transwell实验检测细胞凋亡、克隆形成、细胞活力、增殖、迁移和侵袭能力。取对数生长期胃癌HGC-27细胞，构建PLA2G16和(或)自噬相关基因5（autophagy-related gene 5，ATG5）瞬时敲低模型，并予以顺铂处理，通过qRT-PCR与蛋白免疫印迹检测自噬等相关蛋白及mRNA表达水平，同时检测细胞活力、凋亡、克隆形成、迁移侵袭能力。结果： PLA2家族成员在胃癌组织和胃组织中呈现不同表达模式，其中PLA2G16、PLA2G7、PLA2G15 mRNA在胃癌组织中表达明显升高(P<0.05)，PLA2G1B mRNA表达明显降低(P<0.05)。qRT-PCR结果显示，与癌旁组织相比，PLA2G16 mRNA在胃癌组织中显著高表达(P<0.05)。Kaplan-Meier生存分析结果显示，PLA2G16 mRNA高表达患者总体生存期明显短于低表达患者(P<0.05)。PLA2G16 mRNA高表达与淋巴结转移阳性、TNM Ⅲ/Ⅳ期相关(P<0.05)。单因素Cox回归分析结果显示，淋巴结转移阳性、TNM Ⅲ/Ⅳ期及PLA2G16高表达与胃癌患者总体生存期缩短相关(P均<0.05)；多因素Cox回归分析显示，TNM Ⅲ/Ⅳ期和PLA2G16高表达是胃癌患者总体生存期缩短的独立危险因素(P<0.05)。敲低PLA2G16可显著增强顺铂和5-氟尿嘧啶对胃癌细胞HGC-27的毒性效应和促凋亡作用，并抑制其生长、克隆形成、迁移及侵袭能力，胃癌HGC-27细胞IC50分别为107.7 mg/L、1.868 mg/L；PLA2G16敲低后胃癌HGC-27细胞中ATG5 mRNA和蛋白表达水平明显降低(P<0.01)。PLA2G16和(或)ATG5瞬时敲低模型经顺铂处理后，胃癌HGC-27细胞ATG5等相关蛋白及mRNA表达明显降低，活力明显降低、凋亡率明显升高(P均<0.01)，集落形成数量、划痕愈合率及迁移和侵袭细胞数显著减少(P均<0.05)。结论： PLA2G16在胃癌组织及胃癌细胞中呈显著高表达且与患者不良预后相关，可能通过调控ATG5影响胃癌HGC-27细胞自噬，从而影响其对化疗的敏感性。

Objective: To investigate the core role of cellular senescence in radiation-induced cardiovascular injury and explore the regulatory effects of vitamin C. Methods: Twenty-eight C57BL/6 mice were used to establish a radiation-induced cardiac injury model and randomly divided into control and radiation groups. Cardiac ultrasound, hematoxylin-eosin (HE) staining and immunofluorescence were performed to verify the injury model. Cardiac microvascular endothelial cells (CMECs) were irradiated at 8 Gy to construct an in-vitro cellular injury model, which was validated by CCK-8 assay, Western blotting, tube formation assay and migration assay. RNA-sequencing and KEGG enrichment analysis were conducted on irradiated CMECs to explore potential mechanisms. Senescent phenotypes were validated at both cellular and animal levels using β-galactosidase staining, quantitative real-time PCR (qRT-PCR) and immunofluorescence co-localization. Four groups were set up as control group, radiation (8 Gy) group, vitamin C (200 μmol/L) group, and radiation (8 Gy) + vitamin C (200 μmol/L) group, to explore the regulatory effect of vitamin C on endothelial senescence and function. Results: Radiotherapy-treated mice exhibited decreased survival rate, left ventricular ejection fraction and left ventricular fractional shortening compared with control mice (all P<0.05). HE staining revealed disorganized arrangement of myocardial fibers, widened interstitial space, reduced microvessel density, thickened vascular walls and massive perivascular inflammatory cell infiltration in the radiotherapy group. Sirius-red staining demonstrated markedly increased collagen deposition in the myocardial interstitium, which was most prominent in the perivascular regions. Immunofluorescence showed significantly up-regulated fluorescence intensity of endothelial injury-related adhesion molecules ICAM-1 and VCAM-1 in the radiotherapy group (P<0.05 or P<0.01). Myocardial microvessel density was notably reduced after radiation exposure (P<0.05). The CCK-8 assay indicated that cell viability of CMECs gradually declined with prolonged radiation exposure (P<0.05 or P<0.01). Western blotting analysis revealed a time-dependent increase in the p-eNOS/eNOS ratio after irradiation (P<0.05 or P<0.01). Tube-formation assay showed shorter total tube length and fewer branching nodes in the radiotherapy group (all P<0.05). Transwell migration assay displayed a remarkable reduction in the migration rate of irradiated CMECs (all P<0.01). Volcano plot analysis identified significant up-regulation of Cdkn1a and Trp53 (all P<0.05). KEGG enrichment analysis indicated significant activation of multiple signaling pathways including the IL-17 and TNF signaling pathways. In cellular experiments, β-galactosidase staining demonstrated a substantial increase in senescent cells 7 d after 8 Gy irradiation (P<0.01), accompanied by enlarged cell volume, irregular morphology and flattened cellular shape. Western blotting showed that p21 protein expression in irradiated CMECs increased first and then decreased (P<0.05 or P<0.01), whereas the protein levels of p53 and γ-H2AX kept rising over time (P<0.05 or P<0.01). qRT-PCR revealed significant up-regulation of multiple senescence-associated secretory phenotype (SASP) mRNA at 72 h after 8 Gy irradiation (P<0.05 or P<0.01). In animal experiments, Western blotting analysis demonstrated elevated protein levels of p53, p21 and γ-H2AX in mouse cardiac tissues 6 months after 16 Gy thoracic irradiation (P<0.05 or P<0.01). β-galactosidase staining showed obvious accumulation of senescent cells within the myocardial interstitium of irradiated hearts. qRT-PCR confirmed significantly increased expression of Cdkn1a, Cdkn2a and SASP mRNA (P<0.05 or P<0.01). Immunofluorescence co-localization presented markedly higher expression of p21 and p16 in irradiated mouse hearts (all P<0.01), with obvious co-localization with the endothelial marker CD31. β-galactosidase staining showed that the number of senescent cells was significantly lower in the radiotherapy + vitamin C group than in the radiotherapy group (P<0.01). CCK-8 assay indicated that vitamin C intervention partially restored the proliferative capacity of radiation-injured CMECs (P<0.05). Conclusion: Cellular senescence serves as a critical mechanism underlying radiation-induced cardiac vascular endothelial injury. Vitamin C alleviates vascular endothelial senescence and improves endothelial dysfunction.

Objective: To investigate the intervention effect of esketamine (ESK) on cytomegalovirus-associated neuropathic pain (CMVANP) and explore the potential mechanisms involved in this process. Methods: The CMV-ANP mouse model was established by intraperitoneal injection of murine cytomegalovirus (MCMV) suspension. The model animals received intraperitoneal injection of ESK (10 mg/kg) for 10 consecutive days with corresponding controls. Mechanical allodynia was evaluated by Von-Frey filaments. Immunofluorescence was used to detect the number of TRPV1positive nociceptive neurons and glial activation in dorsal root ganglia. The protein levels of TRPV1, TRPA1 and phosphorylated Akt (p-Akt) in dorsal root ganglia were detected by Western blotting. Results: ESK significantly alleviated mechanical allodynia in MCMV-infected mice (P<0.05). After ESK intervention, the number of TRPV1-positive neurons in dorsal root ganglia decreased, the expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) declined, and glial activation was inhibited. ESK blocked MCMV-provoked TRPV1/TRPA1 pathway activation and reduced the protein abundance of TRPA1 and p-Akt in dorsal root ganglia. Conclusion: ESK inhibits the activity of TRPV1/TRPA1-p-Akt pathway in dorsal root ganglia and alleviates MCMV-induced peripheral sensitization, then relieves neuropathic pain.

Objective: To investigate the effect of rhubarb on gastrointestinal motility in patients with sepsis complicated with acute gastrointestinal injury (AGI), and to analyze its influence on nutritional status and levels of inflammatory factors. Methods: A total of 70 patients with sepsis complicated with AGI who were admitted to the Critical Care Medicine of the Affiliated Hospital of Jiangsu University, Zhenjiang Hospital of Traditional Chinese Medicine, and Jurong People′s Hospital from November 2021 to November 2024 were enrolled. The patients were divided into control group (n=35) and rhubarb group (n=35) using the random digital table method. The control group received routine comprehensive treatment, including treatment of the primary disease, antiinfective therapy, nutritional support, and prokinetics when necessary. On this basis, the rhubarb group received 10 g/d rhubarb via nasogastric feeding for 7 consecutive days. The antral motility index (AMI), AGI grade improvement, gastrointestinal function improvement rate, prealbumin and transferrin levels, C-reactive protein (CRP), procalcitonin (PCT), IL-6 levels, as well as ICU length of stay and total hospitalization duration were compared between the two groups before and after treatment. Results: Compared with before treatment, the AMI, prealbumin, and transferrin levels in the rhubarb group were significantly increased after treatment, while the CRP, PCT, and IL-6 levels were significantly decreased (all P<0.05). Compared with the control group, the rhubarb group demonstrated significantly improved AGI scores (P<0.05), higher prealbumin and transferrin levels, and lower PCT and IL-6 levels (all P<0.05). In the control group, CRP levels decreased significantly after treatment (P<0.05), while no statistically significant differences were observed in other parameters (all P＞0.05). There were no significant differences in ICU length of stay and total hospitalization duration between control group and rhubarb group (both P＞0.05). Conclusion: Rhubarb could significantly improve gastrointestinal function in patients with sepsis complicated by AGI, enhance nutritional status by increasing prealbumin and transferrin levels, and reduce systemic inflammation by lowering PCT and IL-6 levels. However, no significant benefit was observed regarding ICU or overall hospital length of stay.

Objective: To observe the changes of serum 25-hydroxyvitamin D3 ［25-(OH)D3］ at birth, corrected gestational age 40 weeks, corrected age 3 months and corrected age 6 months in premature infants, and to explore its correlation with physical growth. Methods: A total of 243 premature infants admitted to the Neonatal Intensive Care Unit of the Affiliated Hospital of Xuzhou Medical University from February 2021 to March 2024 were enrolled. According to gestational age, infants were divided into moderate-late preterm infant group (32 weeks≤gestational age＜37 weeks, n=113), extremely preterm infant group (28 weeks≤gestational age＜32 weeks, n=89), and ultra-preterm infant group (24 weeks≤gestational age＜28 weeks, n=41). Serum 25-(OH)D3 and physical growth indexes were detected at four time-points. Inter-group differences were compared, and Pearson correlation analysis was used to analyze the relationship between the growth rate of 25-(OH)D3 and physical indexes. Results: Significant inter-group, time and interaction effects were found in serum 25-(OH)D3, head circumference, body length and weight (P＜0.05). The levels of 25-(OH)D3, head circumference, body length and weight increased sequentially in three groups at each follow-up time-point (P＜0.05). Serum 25-(OH)D3 was highest in moderate-late preterm infants, followed by extremely preterm infants and ultra-preterm infants (P＜0.05). Serum 25-(OH)D3 was positively correlated with head circumference, body length and weight at each timepoint (P＜005). The growth rates of physical indexes and 25-(OH)D3 decreased with corrected postnatal age, and followed the order: moderatelate preterm infant group＞extremely preterm infant group＞ultra-preterm infant group (P＜0.05). The growth rate of 25-(OH)D3 was positively correlated with the growth rates of head circumference, body length and weight from corrected 40 weeks to 3 months and from 3 months to 6 months (P＜0.05); only body-length growth rate showed positive correlation from birth to corrected 40 weeks (P＜0.05). Conclusion: Serum 25-(OH)D3 level rises with gestational age and keeps positively correlated with physical growth in premature infants. Ultra-preterm infants suffer more serious vitamin D deficiency and worse catch-up growth. The increasing rate of 25-(OH)D3 is an important factor for catch-up growth. Enhanced vitamin D supplementation and dynamic monitoring up to corrected age of 6 months are recommended for ultra-preterm infants.