Osteoarthritis (OA) is a challenging degenerative joint disease to manage. Previous research has indicated that cell-free fat extract (CEFFE) may hold potential for OA treatment. This study investigated the role of Annexin A5 (AnxA5) within CEFFE in regulating macrophage polarization and protecting chondrocytes. In vitro experiments demonstrated that AnxA5 effectively inhibited M1 macrophage polarization by facilitating toll-like receptor (TLR) 4 internalization and lysosomal degradation through calcium-dependent endocytosis. This process decreased TLR4 expression, suppressed pro-inflammatory mediator release, and reduced the production of reactive oxygen species. Furthermore, AnxA5 displayed protective effects against chondrocyte necrosis and apoptosis. In vivo, studies revealed that intra-articular administration of AnxA5 ameliorated pain symptoms in a monosodium iodoacetate-induced osteoarthritis rat model. Histological analyses indicated a decrease in synovial inflammation and mitigation of cartilage damage following AnxA5 treatment. These results underscored the potential of AnxA5 as a therapeutic option for OA due to its capacity to regulate macrophage polarization and maintain chondrocyte viability. Further investigation into the specific mechanisms and clinical applications of AnxA5 may help improve the management of OA.

Cervical cancer is the fourth leading cause of cancer deaths in women globally. Combining gene therapy with chemo- and radiotherapy may improve cervical cancer treatment outcomes. This study evaluated the effects of Annexin A5(ANXA5) overexpression alongside 5-fluorouracil (5-FU) and irradiation on the viability of CaSki cervical squamous cell carcinoma (SCC) cells. pAdenoVator-CMV-ANXA5-IRES-GFP-plasmid and mock plasmid were transfected into CaSki cells using calcium-phosphate. Seventy-two hours post-transfection, GFP expression was quantified by fluorescence microscopy and flow cytometry to evaluate transfection efficiency. ANXA5 overexpression was confirmed via qPCR. Twenty-four hours post-transfection, cells received a single dose of 8 Gy and were treated with 1 and 2 µg/ml of 5-FU (IC50 = 2.783 µg/ml). Cell viability, apoptosis, cell cycle stage, and Bcl-2 and Bax gene expression were assessed via MTT, annexin V/7-AAD, PI staining, and qPCR assays, respectively. ANXA5 was overexpressed 31.5-fold compared to control (p < 0.0001). MTT assays showed ANXA5 overexpression dose-dependently reduced CaSki cell viability (p < 0.001). IC50 of 5-FU was reduced from 2.783 μg/mL to 1.794 μg/mL when combined with ANXA5 overexpression. Additive effects on cell death were observed for ANXA5 plus 5-FU or irradiation versus ANXA5 alone. Apoptosis assays indicated combinatorial treatment increased CaSki cell apoptosis over ANXA5 alone. Cell cycle analysis revealed ANXA5 arrested cell cycle at G1/S phases; the percentage of cells in the S phase further rose with combination treatment. Finally, combination therapy significantly decreased Bcl-2 expression and increased Bax versus control (p < 0.001). Altogether, ANXA5 overexpression alongside 5-FU and irradiation may improve cervical squamous cell carcinoma (SCC) treatment efficacy. Further, in vivo investigations are warranted to confirm these in vitro results.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is one of the causes of acute kidney injury. Annexin A5 (AnxA5), a calcium-dependent cell membrane-binding protein, shows protective effects in various organ IRI models. This study explored the therapeutic effect of exogenous AnxA5 monomer protein on renal IRI and its potential mechanism of action.
RESULTS: Different doses of AnxA5 were injected intravenously to treat bilateral renal IRI in SD rats. This model confirmed the protective effects of AnxA5 on kidney structure and function. In vitro, HK-2 cells were subjected to hypoxia for 12 h, followed by restoration of normal oxygen supply to simulate IRI. In vitro experiments demonstrated the mechanism of action of AnxA5 by measuring cellular activity and permeability. A comparison of the mutant AnxA5 protein M23 and the application of a calcium-free culture medium further validated the protective effect of AnxA5 by forming a network structure.
CONCLUSIONS: Exogenous AnxA5 monomers prevented renal IRI by binding to the damaged renal tubular epithelial cell membrane, forming a two-dimensional network structure to maintain cell membrane integrity, and ultimately prevent cell death.

UNASSIGNED: One of the key obstacles to the healing of diabetic wound is the persistence of active inflammation. We previously demonstrated the potential of cell-free fat extract (CEFFE) to promote the healing of diabetic wounds, and annexin A5 (A5) is a crucial anti-inflammatory protein within CEFFE. This study aimed to evaluate the therapeutic potential of A5 in diabetic wounds.
UNASSIGNED: A5 was loaded into GelMA hydrogels and applied to skin wounds of diabetic mice in vivo. The diabetic wounds with the treatment of GelMA-A5 were observed for 14 days and evaluated by histological analysis. Accessment of inflammation regulation were conducted through anti-CD68 staining, anti-CD86 and anti-CD206 staining, and qRT-PCR of wound tissue. In presence of A5, macrophages stimulated by lipopolysaccharide (LPS) in vitro, and detected through qRT-PCR, flow cytometry, and immunocytofluorescence staining. Besides, epithelial cells were co-cultured with A5 for epithelialization regulation by CCK-8 assay and cell migration assay.
UNASSIGNED: A5 could promote diabetic wound healing and regulate inflammations by promoting the transition of macrophages from M1 to M2 phenotype. In vitro experiments demonstrated that A5 exerted a significant effect on reducing pro-inflammatory factors and inhibiting the polarization of macrophages from M0 toward M1 phenotype. A5 significantly promoted the migration of epithelial cells.
UNASSIGNED: Annexin A5 has a significant impact on the regulation of macrophage inflammation and promotion of epithelialization.

Leukemia is a malignant disease of the hematopoietic system, in which clonal leukemia cells accumulate and inhibit normal hematopoiesis in the bone marrow and other hematopoietic tissues as a result of uncontrolled proliferation and impaired apoptosis, among other mechanisms. In this study, the anti-leukemic effect of a compound (SGP-17-S) extracted from Chloranthus multistachys, a plant with anti-inflammatory, antibacterial and anti-tumor effects, was evaluated. The effect of SGP-17-S on the viability of leukemic cell was demonstrated by MTT assay, cell cycle, and apoptosis were assessed by flow cytometry using PI staining and Annexin V/PI double staining. Combinations of network pharmacology and cellular thermal shift assay (CETSA) with western blot were used to validate agents that act on leukemia targets. The results showed that SGP-17-S inhibited the growth of leukemia cells in a time- and dose-dependent manner. SGP-17-S blocked HEL cells in the G2 phase, induced apoptosis, decreased Bcl-2 and caspase-8 protein expression, and increased Bax and caspase-3 expression. In addition, CETSA revealed that PARP1 is an important target gene for the inhibition of HEL cell growth, and SGP-17-S exerted its action on leukemia cells by targeting PARP1. Therefore, this study might provide new solutions and ideas for the treatment of leukemia.

OBJECTIVE: Cyclophosphamide (CP) is a widely used anti-cancer drug. It works by alkylation and is commonly used in cancer treatment. In this study, the goal was to create biodegradable drug delivery carriers with minimal side effects for breast cancer treatment by developing gold nanoparticles/reduced graphene oxide (Au-rGO) nanocomposites using a sustainable synthesis method and loading them with cyclophosphamide.
METHODS: Cyclophosphamide-loaded gold/reduced graphene oxide nanocomposites (Au-rGOCP) were synthesized and evaluated using FT-IR, XRD, release pattern, and FE-SEM techniques. Furthermore, the anticancer effect against breast cancer cells was evaluated through MTT and Annexin V assays. CAT, SOD, and GPx biomarkers were used to assess the antioxidant effect of the free and nano-formulated cyclophosphamide.
RESULTS: The characterization results showed the effective loading of cyclophosphamide in the nanocarriers. Additionally, Au-rGO had a higher drug loading capacity for cyclophosphamide during a 24-hour contact period (92.34%). The pH value affected the amount of cyclophosphamide released from the nanocarriers. Au-rGO/CP displayed significant in vitro anti-cancer activity against MCF-7 cancer cells relative to free CP and rGO/CP. According to Annexin V assay results, Au-rGO/CP induced a higher apoptosis rate in MCF-7 breast cancer cells than other forms.
CONCLUSIONS: In conclusion, our findings demonstrate that the gold-decorated reduced graphene oxide nanocomposite enhances treatment efficacy and significantly increases apoptosis and cell death induction. As a result, CP-loaded Au-rGO-based compounds could be a promising treatment for breast cancer.

Flow cytometry remains the most widely used method for detecting and quantifying apoptosis and related forms of cell death in mammalian cells. The multiparametric nature of flow cytometry allows multiple apoptotic characteristics to be labeled and analyzed in a single sample, making it a powerful tool for analyzing the complex progression of apoptotic death. This chapter provides methods for combining assays for single apoptotic characteristics like caspase activation, annexin V binding, and cell membrane permeability into multiparametric assays that provide deeper insights into the cell death process. This approach to analyzing multiple apoptotic characteristics simultaneously yields far more information than single-parameter assays. While more informative than single-parameter assays, these multicolor methods can still be analyzed on relatively simple flow cytometers, making them widely accessible.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a \"neck\"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. hAnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in hAnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.

Objective: To investigate the effect of plasminogen activator urokinase receptor (PLAUR) gene on neutrophil activation and apoptosis in neutrophil-like cell model. Methods: Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid (ATRA). Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells (siRNA group). The phosphate buffer saline (PBS) group (untransfected neutrophil-like cells) and normal blank control group (NC group) (neutrophil-like cells transfected with blank plasmid) were used as controls (n=3). After starvation culture and addition of interleukin-17 afterwards in these 3 groups, the expression of CD11b on the cell membrane was detected by flow cytometry, and the levels of myeloperoxide (MPO) and extracellular neutrophil traps (NETs) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA) to investigate the activation of neutrophil-like cells. The apoptosis was detected by flow cytometry with annexin V/propidium iodide (PI) double staining and the expressions of apoptosis-related proteins caspase-3, bax and bcl-2 were detected by Western blotting. Results: The expression of CD11b in siRNA group (32.37±8.17) was lower than that in PBS group (46.27±1.54) and NC group (53.07±8.14) (P<0.05) by flow cytometry. The levels of MPO and NETs (33.37±1.11, 57.69±3.03) in the supernatant of siRNA group were significantly lower than those in PBS group (41.64±2.20, 77.60±4.33) and NC group (40.84±5.11, 76.15±2.10) (P<0.05). Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group (20.42%±2.45%) was significantly higher than that in PBS group (11.91%±2.23%) and NC group (11.13%±2.56%) (P<0.05). The relative expression of caspase-3 protein and bax protein (0.84±0.05, 0.83±0.04) in siRNA group was significantly higher than that in PBS group (0.68±0.02, 0.63±0.08) and NC group (0.71±0.01, 0.66±0.10) (P<0.05), and the relative expression of anti-apoptosis protein bcl-2 decreased in siRNA group (0.38±0.02) than in PBS group (0.73±0.05) and NC group (0.69±0.06) (P<0.05). Conclusion: PLAUR promotes the activation of neutrophil-like cells and inhibits the apoptosis.
目的： 在类中性粒细胞模型中，探讨尿激酶型纤溶酶原激活物受体（PLAUR）基因对中性粒细胞活化及凋亡的影响。 方法： 采用体外培养的人急性髓系粒细胞白血病细胞株HL60作为研究对象，全反式维甲酸（ATRA）诱导HL60细胞分化为类中性粒细胞。构建干扰人PLAUR基因的慢病毒载体，转染入类中性粒细胞（siRNA组），并设置磷酸盐缓冲液（PBS）组（未转染）和空白对照（NC组，转染空白质粒）作为对照（n=3）。饥饿培养、加入白细胞介素-17干预后，流式细胞术检测细胞膜CD11b表达，以及酶联免疫吸附法检测细胞培养液上清中髓过氧化物（MPO）、中性粒细胞胞外诱捕网（NETs）水平，了解类中性粒细胞活化情况。Annexin V/PI染色流式细胞术检测细胞凋亡，并用Western蛋白印迹法检测凋亡相关蛋白caspase-3、bax、bcl-2表达。 结果： 流式细胞术检测siRNA组HL60细胞细胞膜CD11b表达（32.37±8.17）较另两组下降（PBS组46.27±1.54，NC组53.07±8.14）（P<0.05）。siRNA组细胞培养液上清中MPO和NETs水平（33.37±1.11、57.69±3.03）也均较PBS组（41.64±2.20、77.60±4.33）和NC组（40.84±5.11、76.15±2.10）下降，差异均有统计学意义（均P<0.05）。流式细胞术检测siRNA组凋亡率（20.42%±2.45%）较PBS组（11.91%±2.23%）和NC组（11.13%±2.56%）升高（P<0.05）。siRNA组caspase-3和bax蛋白表达（0.84±0.05、0.83±0.04）较PBS组（0.68±0.02、0.63±0.08）和NC组（0.71±0.01、0.66±0.10）高，差异有统计学意义（P<0.05）；抗凋亡蛋白bcl-2表达量减少（siRNA组：0.38±0.02，PBS组：0.73±0.05，NC组：0.69±0.06）（P<0.05）。 结论： PLAUR可促进类中性粒细胞活化，抑制其凋亡。.