Physical modulation of mesenchymal stem cell exosomes: A new perspective for regenerative medicine.

Mesenchymal stem cell-derived exosomes (MSC-Exo) offer promising therapeutic potential for various refractory diseases, presenting a novel therapeutic strategy. However, their clinical application encounters several obstacles, including low natural secretion, uncontrolled biological functions and inherent heterogeneity. On the one hand, physical stimuli can mimic the microenvironment dynamics where MSC-Exo reside. These factors influence not only their secretion but also, significantly, their biological efficacy. Moreover, physical factors can also serve as techniques for engineering exosomes. Therefore, the realm of physical factors assumes a crucial role in modifying MSC-Exo, ultimately facilitating their clinical translation. This review focuses on the research progress in applying physical factors to MSC-Exo, encompassing ultrasound, electrical stimulation, light irradiation, intrinsic physical properties, ionizing radiation, magnetic field, mechanical forces and temperature. We also discuss the current status and potential of physical stimuli-affected MSC-Exo in clinical applications. Furthermore, we address the limitations of recent studies in this field. Based on this, this review provides novel insights to advance the refinement of MSC-Exo as a therapeutic approach in regenerative medicine.

Apolipoprotein A-I Binding Protein Inhibits the Formation of Infantile Hemangioma through Cholesterol-Regulated Hypoxia-Inducible Factor 1α Activation.

Infantile hemangioma (IH) is the most frequent vascular tumor of infancy with unclear pathogenesis; disordered angiogenesis is considered to be involved in its formation. Apolipoprotein A-I binding protein (AIBP)-also known as NAXE (NAD [P]HX epimerase)-a regulator of cholesterol metabolism, plays a critical role in the pathological angiogenesis of mammals. In this study, we found that AIBP had much lower expression levels in both tissues from patients with IH and hemangioma endothelial cells (HemECs) than in adjacent normal tissues and human dermal vascular endothelial cells, respectively. Knockout of NAXE by CRISPR-Cas9 in HemECs enhanced tube formation and migration, and NAXE overexpression impaired tube formation and migration of HemECs. Interestingly, AIBP suppressed the proliferation of HemECs in hypoxia. We then found that reduced expression of AIBP correlated with increased hypoxia-inducible factor 1α levels in tissues from patients with IH and HemECs. Further mechanistic investigation demonstrated that AIBP disrupted hypoxia-inducible factor 1α signaling through cholesterol metabolism under hypoxia. Notably, AIBP significantly inhibited the development of IH in immunodeficient mice. Furthermore, using the validated mouse endothelial cell (ie, EOMA cells) and Naxe-/- mouse models, we demonstrated that both endogenous AIBP from tumors and AIBP in the tumor microenvironment limit the formation of hemangioma. These findings suggested that AIBP was a player in the pathogenesis of IH and could be a potential pharmacological target for treating IH.

Traditional Chinese Medicine formula, Sanwujiao granule, attenuates ischemic stroke by promoting angiogenesis through early administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke (IS) is one of the most lethal diseases with the insufficient pharmacology therapeutic approach. Sanwujiao granule (SW) is widely used for IS in China with little known about its underlying mechanism. AIM OF THE STUDY: To investigate the characteristics of therapeutic effects and potential mechanisms of SW against IS. MATERIALS AND METHODS: The fingerprint of SW was applied by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Three different drug treatment strategies, including prophylactic administration, early administration and delayed administration, were applied in rats' permanent middle cerebral occlusion (pMCAO) model. The Garcia neurological deficit test, adhesive removal test, rotarod test, TTC and TUNEL staining were performed to evaluate the pathological changes. The transcriptomic analysis was used to predict the potential mechanism of SW. The vascular deficiency model of Tg(kdrl:eGFP) zebrafish larvae and oxygen-glucose deprivation model on bEnd.3 cells were used to verify SW's pharmacological effect. qRT-PCR, immunofluorescent staining and Western Blot were applied to detect the expression of genes and proteins. The network pharmacology approach was applied to discover the potential bioactive compounds in SW that contribute to its pharmacological effect. RESULTS: SW early and delayed administration attenuated cerebral infarction, neurological deficit and cell apoptosis. The transcriptomic analysis revealed that SW activated angiogenesis-associated biological processes specifically by early administration. CD31 immunofluorescent staining further confirmed the microvessel intensity in peri-infarct regions was significantly elevated after SW early treatment. Additionally, on the vascular deficiency model of zebrafish larvae, SW showed the angiogenesis effect. Next, the cell migration and tube formation were also observed in the bEnd.3 cells with the oxygen-glucose deprivation induced cell injury. It's worth noting that both mRNA and protein levels of angiogenesis factor, insulin-like growth factor 1, were significantly elevated in the pMCAO rats' brains treated with SW. The network pharmacology approach was applied and chasmanine, karacoline, talatisamine, etc. were probably the main active compounds of SW in IS treatment as they affected the angiogenesis-associated targets. CONCLUSIONS: These results demonstrate that SW plays a critical role in anti-IS via promoting angiogenesis through early administration, indicating that SW is a candidate herbal complex for further investigation in treating IS in the clinical.

N4BP1 regulates keratinocytes development and plays protective role in burn- and adhesive-related skin injury via MMP9.

Extensive studies have demonstrated critical roles of Regnase-1 in skin inflammation; however the role of N4BP1, a member of Regnase-1 family, in skin is largely unexplored. Here, we found that N4BP1 was highly expressed in skin and its expression was further increased upon skin injury. Compared to wildtype mice, N4BP1 deficient mice showed severe skin injury upon tape-stripping and burns. Overexpression of N4BP1 in HaCaT cells caused more cuboidal with higher cell-to-cell packing, while reduced expression of N4BP1 made cells become more spindle shaped and loosely packed. Overexpression of N4BP1 promoted cell migration, while silence of N4BP1 reduced migration. N4BP1 deficient HaCaT cells were more sensitive to heats compared to control cells. RNA profiling in N4BP1 genetically modified cells demonstrated that N4BP1 broadly affects cellular behaviors such as epithelium development. RNA profiling, RT-PCR verification, WB analysis and RNA immunoprecipitation demonstrated that MMP9 was one of N4BP1 targets that significantly increased in N4BP1 deficient HaCaT cells and skin tissues. Collectively, our results demonstrate a protective role of N4BP1 in skin injury through broadly affecting cellular behaviors of keratinocytes. Furthermore, we identified MMP9 is a target of N4BP1 in keratinocytes. Our findings provide new insight to understand how N4BP1 protects skin under injury.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Super-enhancer receives signals from the extracellular matrix to induce PD-L1-mediated immune evasion via integrin/BRAF/TAK1/ERK/ETV4 signaling.

OBJECTIVE: PD-L1 and PD-L2 expression levels determine immune evasion and the therapeutic efficacy of immune checkpoint blockade. The factors that drive inducible PD-L1 expression have been extensively studied, but mechanisms that result in constitutive PD-L1 expression in cancer cells are largely unknown. METHODS: DNA elements were deleted in cells by CRISPR/Cas9-mediated knockout. Protein function was inhibited by chemical inhibitors. Protein levels were examined by Western blot, mRNA levels were examined by real-time RT-PCR, and surface protein expression was determined by cellular immunofluorescence and flow cytometry. Immune evasion was examined by in vitro T cell-mediated killing. RESULTS: We determined the core regions (chr9: 5, 496, 378-5, 499, 663) of a previously identified PD-L1L2-super-enhancer (SE). Through systematic analysis, we found that the E26 transformation-specific (ETS) variant transcription factor (ETV4) bound to this core DNA region but not to DNA surrounding PD-L1L2SE. Genetic knockout of ETV4 dramatically reduced the expressions of both PD-L1 and PD-L2. ETV4 transcription was dependent on ERK activation, and BRAF/TAK1-induced ERK activation was dependent on extracellular signaling from αvß3 integrin, which profoundly affected ETV4 transcription and PD-L1/L2 expression. Genetic silencing or pharmacological inhibition of components of the PD-L1L2-SE-associated pathway rendered cancer cells susceptible to T cell-mediated killing. CONCLUSIONS: We identified a pathway originating from the extracellular matrix that signaled via integrin/BRAF/TAK1/ERK/ETV4 to PD-L1L2-SE to induce PD-L1-mediated immune evasion. These results provided new insights into PD-L1L2-SE activation and pathways associated with immune checkpoint regulation in cancer.

Effect of porcine circovirus type 2 on the severity of lung and brain damage in piglets infected with porcine pseudorabies virus.

Porcine circovirus type 2 (PCV2) is widespread throughout Chinese farms, and the infection rate of porcine pseudorabies virus (PRV) is very high. The emergence of mixed infection involving PCV2 and PRV has been difficult to prevent and control and has caused considerable economic loss. The present study investigated lung and brain damage caused by PRV in piglets with PCV2 infection. Twenty piglets were divided randomly into two experiment groups (PRV group and PRV + PCV2 group; n = 10 per group). The pigs were observed for clinical signs at specified times. At necropsy, lung and brain tissue samples were collected for histopathological examination, and tissue virus load was determined using quantitative polymerase chain reaction. Severe pathogenicity due to PRV was evident in two-month-old piglets. PCV2 and PRV co-infection led to more severe neurological and respiratory symptoms and a higher mortality rate in the piglets. In addition, the pathological damage to the lung and brain was also aggravated. The co-infection was associated with a significant increase in the content of PRV in the brain and lung tissue. In conclusion, PCV2 and PRV co-infection could cause severe and irreversible damage to piglets.

Salidroside Attenuates Denervation-Induced Skeletal Muscle Atrophy Through Negative Regulation of Pro-inflammatory Cytokine.

Skeletal muscle atrophy is associated with pro-inflammatory cytokines. Salidroside is a biologically active ingredient of Rhodiola rosea, which exhibits anti-inflammatory property. However, there is little known about the effect of salidroside on denervation-induced muscle atrophy. Therefore, the present study aimed to determine whether salidroside could protect against denervation-induced muscle atrophy and to clarify potential molecular mechanisms. Denervation caused progressive accumulation of inflammatory factors in skeletal muscle, especially interleukin 6 (IL6) and its receptor, and recombinant murine IL6 (rmIL6) local infusion could induce target muscle atrophy, suggesting that denervation induced inflammation in target muscles and the inflammation may trigger muscle wasting. Salidroside alleviated denervation-induced muscle atrophy and inhibited the production of IL6. Furthermore, the inhibition of phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the decreased levels of suppressor of cytokine signaling (SOCS3), muscle RING finger protein-1 (MuRF1), atrophy F-box (atrogin-1), microtubule-associated protein light chain 3 beta (LC3B) and PTEN-induced putative kinase (PINK1) were observed in denervated muscles that were treated with salidroside. Finally, all of these responses to salidroside were replicated in neutralizing antibody against IL6. Taken together, these results suggest that salidroside alleviates denervation-induced inflammation response, thereby inhibits muscle proteolysis and muscle atrophy. Therefore, it was assumed that salidroside might be a potential therapeutic candidate to prevent muscle wasting.

Effect of the SSeCKS-TRAF6 interaction on gastrodin-mediated protection against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced astrocyte activation and neuronal death.

The ubiquitous environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to trigger neurotoxicity. In this study, we investigated the protective effects of gastrodin on TCDD-induced neurotoxicity and the underlying molecular mechanisms. The results show that gastrodin decreased cell viability, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release, and inducible nitrix oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) expression in TCDD-treated C6 cells. TCDD stimulated NF-κB signalling activation, demonstrated by increased p-NF-κB expression and translocation of nuclear Factor kappa B (NF-κB) to the nucleus. TCDD did not affect TRAF6 protein expression but enhanced the attenuated the Src-suppressed-C Kinase Substrate (SSeCKS)-tumor necrosis factor receptor-associated factor 6 (TRAF6) interaction, thereby triggering NF-κB signalling activation. Gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS-TRAF6 interaction in vitro. Gastrodin attenuated SSeCKS-TRAF6 interaction in vivo and protected mice from NF-κB signalling activation following TCDD exposure. Finally, gastrodin blocked the apoptosis of PC12 neuronal cells induced by medium conditioned with TCDD-treated astrocytes. In summary, gastrodin inhibited TCDD-induced NF-κB signalling activation by lessening the SSeCKS-TRAF6 interaction, resulting in attenuated astrocyte activation and subsequent neuronal apoptosis. These findings will contribute to an improved understanding of TCDD-induced neurotoxicity and strategies to antagonise it using gastrodin.

Endosomal/lysosomal location of organically modified silica nanoparticles following caveolae-mediated endocytosis.

Organically modified silica (ORMOSIL) nanoparticles (NPs) are widely used in biomedicine. However, their cell uptake process has not yet been characterised in detail. Here, we investigated the mechanism underlying endocytosis and subcellular localisation of ORMOSIL NPs. Exposure to ORMOSIL NPs induced a decrease in cell viability and increase in lactate dehydrogenase release in a dose-dependent manner in A549 cells. Once internalised, ORMOSIL NPs were translocated from early endosomes to the lysosomes, where they accumulated. Furthermore, deficiency of autophagosomal/lysosomal fusion failed to block lysosomal localisation of ORMOSIL NPs, suggesting that autophagy was not involved in the final lysosomal accumulation of ORMOSIL NPs. Meanwhile, an inhibitor of caveolae-mediated endocytosis, rather than inhibitors of phagocytosis or clathrin-mediated endocytosis, succeeded in blocking ORMOSIL NP cell uptake, indicating the involvement of caveolae-mediated endocytosis. Together, these results provide a new understanding of the toxicity, and suggest better biomedical applications, of ORMOSIL NPs.

Pyrroloquinoline quinone attenuates cachexia-induced muscle atrophy via suppression of reactive oxygen species.

BACKGROUND: Cachexia, a wasting syndrome, is most commonly observed in individuals with advanced cancer including lung cancer, esophageal cancer, liver cancer, etc. The characteristic sign of cachexia is muscle atrophy. To date, effective countermeasures have been still deficiency to alleviate muscle atrophy. Reactive oxygen species (ROS) are important regulators of muscle atrophy. Therefore, the effects of a naturally antioxidant, pyrroloquinoline quinone (PQQ), were explored on muscle atrophy induced by cachexia in the present study. METHODS: Tumor necrosis factor-α (TNF-α) induced C2C12 myotubes atrophy model was constructed. The atrophied C2C12 myotubes were dealt with the presence or absence of N-acetyl-L-cysteine (NAC, an antioxidant for ROS abolition) (5 mM) or PQQ (80 µM) for 24 hours. ROS content was determined by dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The diameter of myotubes was analyzed by myosin heavy chain (MHC) staining. The protein levels of MHC, muscle atrophy F-box (MAFbx) and muscle RING finger-1 (MuRF-1) in each group were observed by Western blotting. RESULTS: First, ROS generation was enhanced in C2C12 myotubes treated with TNF-α. NAC treatments significantly avoided the reduction in the diameter of C2C12 myotubes, and concomitantly increased MHC levels, and decreased ROS contents, MuRF1 and MAFbx levels. These data suggested that the increased ROS induced by TNF-α might play a central role in muscle wasting. PQQ (a naturally occurring antioxidant) administration inhibited C2C12 myotubes atrophy induced by TNF-α, as evidenced by the increase of the diameter of C2C12 myotubes, together with increased MHC levels and decreased ROS, MAFbx and MuRF-1 levels. CONCLUSIONS: PQQ resists atrophic effect dependent on, at least in part, decreased ROS in skeletal muscle treated with TNF-α.

In vitro Antichlamydial Activity of 1,2,3,5-Tetrasubstituted Pyrrole Derivatives.

BACKGROUND: Chlamydia is a group of bacterial pathogens distributed worldwide that can lead to serious reproductive and other health problems. The rise of antibiotic-resistant pathogens promotes the development of novel antichlamydial agents. The aim of this study is to assess in vitro antichlamydial activity of our previously synthesized 1,2,3,5- tetrasubstituted pyrroles. METHODS: The derivatives were screened for their antichlamydial activity against three Chlamydia strains by calculating IC50 values using concentration-response inhibition data between 1 and 32 µM. The action of the compounds on Chlamydia elementary body (EB) infectivity and the impact of the chemicals' administration time on their antichlamydial effect were evaluated to reveal the inhibitory mechanism. RESULTS: Some of the compounds moderately inhibited the Chlamydia strains. Compound 10 exhibited the strongest inhibitory activity, with IC50 values from 4.34 to 5.83 µM. These pyrrole derivatives inhibited Chlamydia infection by reducing EB infectivity during the early stage and disturbing Chlamydia growth by targeting the early-to-middle stage prior to 12 h of the chlamydial life cycle. CONCLUSION: Our findings highlight the potential of 1,2,3,5-tetrasubstituted pyrrole derivatives as promising lead molecules for the development of antichlamydial agents.

Mechanistic Role of Reactive Oxygen Species and Therapeutic Potential of Antioxidants in Denervation- or Fasting-Induced Skeletal Muscle Atrophy.

Skeletal muscle atrophy occurs under various conditions, such as disuse, denervation, fasting, aging, and various diseases. Although the underlying molecular mechanisms are still not fully understood, skeletal muscle atrophy is closely associated with reactive oxygen species (ROS) overproduction. In this study, we aimed to investigate the involvement of ROS in skeletal muscle atrophy from the perspective of gene regulation, and further examine therapeutic effects of antioxidants on skeletal muscle atrophy. Microarray data showed that the gene expression of many positive regulators for ROS production were up-regulated and the gene expression of many negative regulators for ROS production were down-regulated in mouse soleus muscle atrophied by denervation (sciatic nerve injury). The ROS level was significantly increased in denervated mouse soleus muscle or fasted C2C12 myotubes that had suffered from fasting (nutrient deprivation). These two muscle samples were then treated with N-acetyl-L-cysteine (NAC, a clinically used antioxidant) or pyrroloquinoline quinone (PQQ, a naturally occurring antioxidant), respectively. As compared to non-treatment, both NAC and PQQ treatment (1) reversed the increase in the ROS level in two muscle samples; (2) attenuated the reduction in the cross-sectional area (CSA) of denervated mouse muscle or in the diameter of fasted C2C12 myotube; (3) increased the myosin heavy chain (MHC) level and decreased the muscle atrophy F-box (MAFbx) and muscle-specific RING finger-1 (MuRF-1) levels in two muscle samples. Collectively, these results suggested that an increased ROS level was, at least partly, responsible for denervation- or fasting-induced skeletal muscle atrophy, and antioxidants might resist the atrophic effect via ROS-related mechanisms.