Resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into esophageal fibroblasts via AKT signaling pathway.

Objectives: Resveratrol has been implicated in the differentiation and development of human umbilical cord mesenchymal stem cells. The differentiation of into esophageal fibroblasts is a promising strategy for esophageal tissue engineering. However, the pharmacological effect and underlying mechanism of resveratrol on human umbilical cord mesenchymal stem cells differentiation are unknown. Here, we investigated the effects and mechanism of resveratrol on the differentiation of human umbilical cord mesenchymal stem cells. Methods: Using a transwell-membrane coculture system to culture human umbilical cord mesenchymal stem cells and esophageal fibroblasts, we examined how resveratrol act on the differentiation of human umbilical cord mesenchymal stem cells. Immunocytochemistry, Sirius red staining, quantitative real-time PCR, and Western blotting were performed to examine collagen synthesis and possible signaling pathways in human umbilical cord mesenchymal stem cells. Results: We found that resveratrol promoted collagen synthesis and AKT phosphorylation. However, co-treatment of cells with resveratrol and the PI3K inhibitor LY294002 inhibited collagen synthesis and AKT phosphorylation. We demonstrated that resveratrol down-regulated the expression of IL-6, TGF-ß, caspase-9, and Bax by activating the AKT pathway in human umbilical cord mesenchymal stem cell. Furthermore, resveratrol inhibited phosphorylated NF-ĸB in human umbilical cord mesenchymal stem cells. Conclusion: Our data suggest that resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into fibroblasts. The underlying mechanism is associated with the downregulation of IL-6 and TGF-ß via the AKT pathway and by inhibiting the NF-ĸB pathway. Resveratrol may be useful for esophageal tissue engineering.

Intratumoral Lactate Depletion Based on Injectable Nanoparticles-Hydrogel Composite System Synergizes with Immunotherapy against Postablative Hepatocellular Carcinoma Recurrence.

Thermal ablation is a crucial therapeutic modality for hepatocellular carcinoma (HCC), but its efficacy is often hindered by the high recurrence rate attributed to insufficient ablation. Furthermore, the residual tumors following insufficient ablation exhibit a more pronounced immunosuppressive state, which accelerates the disease progression and leads to immune checkpoint blockade (ICB) resistance. Herein, evidence is presented that heightened intratumoral lactate accumulation, stemming from the augmented glycolytic activity of postablative residual HCC cells, may serve as a crucial driving force in exacerbating the immunosuppressive state of the tumor microenvironment (TME). To address this, an injectable nanoparticles-hydrogel composite system (LOX-MnO2 @Gel) is designed that gradually releases lactate oxidase (LOX)-loaded hollow mesoporous MnO2 nanoparticles at the tumor site to continuously deplete intratumoral lactate via a cascade catalytic reaction. Using subcutaneous and orthotopic HCC tumor-bearing mouse models, it is confirmed that LOX-MnO2 @Gel-mediated local lactate depletion can transform the immunosuppressive postablative TME into an immunocompetent one and synergizes with ICB therapy to significantly inhibit residual HCC growth and lung metastasis, thereby prolonging the survival of mice postablation. The work proposes an appealing strategy for synergistically combining antitumor metabolic therapy with immunotherapy to combat postablative HCC recurrence.

HBV suppresses macrophage immune responses by impairing the TCA cycle through the induction of CS/PDHC hyperacetylation.

BACKGROUND: It is now understood that HBV can induce innate and adaptive immune response disorders by affecting immunosuppressive macrophages, resulting in chronic HBV infection. However, the underlying mechanism is not fully understood. Dysregulated protein acetylation can reportedly influence the differentiation and functions of innate immune cells by coordinating metabolic signaling. This study aims to assess whether HBV suppresses macrophage-mediated innate immune responses by affecting protein acetylation and to elucidate the underlying mechanisms of HBV immune escape. METHODS: We investigated the effect of HBV on the acetylation levels of human THP-1 macrophages and identified potential targets of acetylation that play a role in glucose metabolism. Metabolic and immune phenotypes of macrophages were analyzed using metabolomic and flow cytometry techniques. Western blot, immunoprecipitation, and immunofluorescence were performed to measure the interactions between deacetylase and acetylated targets. Chronic HBV persistent infected mice were established to evaluate the role of activating the tricarboxylic acid (TCA) cycle in macrophages for HBV clearance. RESULTS: Citrate synthase/pyruvate dehydrogenase complex hyperacetylation in macrophages after HBV stimulation inhibited their enzymatic activities and was associated with impaired TCA cycle and M2-like polarization. HBV downregulated Sirtuin 3 (SIRT3) expression in macrophages by means of the toll-like receptor 2 (TLR2)-NF-κB- peroxisome proliferatoractivated receptor γ coactivator 1α (PGC-1α) axis, resulting in citrate synthase/pyruvate dehydrogenase complex hyperacetylation. In vivo administration of the TCA cycle agonist dichloroacetate inhibited macrophage M2-like polarization and effectively reduced the number of serum HBV DNA copies. CONCLUSIONS: HBV-induced citrate synthase/pyruvate dehydrogenase complex hyperacetylation negatively modulates the innate immune response by impairing the TCA cycle of macrophages. This mechanism represents a potential therapeutic target for controlling HBV infection.

Qing`e Pill Inhibits Osteoblast Ferroptosis via ATM Serine/Threonine Kinase (ATM) and the PI3K/AKT Pathway in Primary Osteoporosis.

Osteoporosis (OP) is an aging-related disease that is the main etiology of fragility fracture. Qing'e Pill (QEP) is a mixture of traditional Chinese medicine (TCM) consisting of Eucommia ulmoides Oliv., Psoralea corylifolia L., Juglans regia L., and Allium sativum L. QEP has an anti-osteoporosis function, but the underlying mechanism remains unclear. In this study, online databases were employed to determine the chemical compounds of QEP and potential target genes in osteoporosis. Potential pathways associated with genes were defined by Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. A compound-target-disease network was constructed. Hub genes screened through Cytoscape were intersected with the FerrDB database. The potential key genes were validated in HFOB 1.19 cells, and rat models were ovariectomized through Western blot, RT-qPCR, ELISA, HE staining, immunohistochemistry, and immunofluorescence analyses. The intersection targets of QEP and osteoporosis contained 121 proteins, whereas the target-pathway network included 156 pathways. We filtered five genes that stood out in the network analysis for experimental verification. The experiments validated that QEP exerted therapeutic effects on osteoporosis by inhibiting ferroptosis and promoting cell survival via the PI3K/AKT pathway and ATM. In conclusion, combining the application of network analysis and experimental verification may provide an efficient method to validate the molecular mechanism of QEP on osteoporosis.

Integrative analysis of multiomics data identified acetylation as key variable of excessive energy metabolism in hyperthyroidism-induced osteoporosis rats.

BACKGROUND: Results from the previous experiment have demonstrated bone loss and excess metabolism in Hyperthyroidism-induced rats. Thus, an underlying relationship between metabolism and bone loss was speculated. In addition, previous studies have shown the influence of acetylation on metabolism in tissues and diseases. The hypothesis from this case study suggests that excessive metabolism is induced by acetylation of vital metabolism enzymes. RESULTS: In the case study, a HYP-induced osteoporosis rat model was used and the glucose metabolite was tested through the acetylation of proteins by the mass spectrometer. The results showed that pivotal enzymes of Glycolysis-Tricarboxylic acid cycle-Oxidative phosphorylation were acetylated along with upregulated metabolites. With all acetyly-lysine sites of related enzymes listed, the results in this study showed that bone loss in HYP rats was accompanied by the upregulation of CREB-binding protein (Crebbp, CBP). Furthermore, it is also indicated that CBP has a close relationship with the enhancement of LDHA which promotes glucose metabolism. CONCLUSIONS: Acetylation is highly correlated with excessive energy metabolism in HYP-induced osteoporotic rats, where a representation relationship between CBP and LDHA is demonstrated. SIGNIFICANCE: Hyperthyroidism may lead to osteoporosis. Our study found an interesting phenomenon of hyperthyroidism induced-osteoporosis is that osteoporosis is accompanied by excessive glucose metabolism. In this process, some molecular mechanisms are still unclear. This study indicates a high degree of acetylation of metabolic enzymes, which may be closely related to excessive glucose metabolism. The relationship between CBP and LDHA was also investigated in this study, which showed that CBP and LDHA had some extent interaction. Glucose metabolism and acetylation maybe all associated with hyperthyroidism induced-osteoporosis. This data provides new insights into the molecular mechanisms of hyperthyroidism induced-osteoporosis.

Sublethal heat stress-induced O-GlcNAcylation coordinates the Warburg effect to promote hepatocellular carcinoma recurrence and metastasis after thermal ablation.

The malignant transformation of residual hepatocellular carcinoma (HCC) cells after thermal ablation is considered as the main factor promoting postoperative HCC progression, which greatly limits the improvement of long-term survival, and at present there is no effective targeted therapeutic strategies. The Warburg effect is a metabolic feature correlated highly with malignant transformation (e.g. epithelial-to-mesenchymal transition [EMT]). Here, we showed that sublethal heat stress triggered a stronger Warburg effect of HCC cells, which contributed to the thermotolerance and invasion of HCC cells. Sublethal heat stress-induced O-GlcNAcylation was involved in this process. Such enhanced Warburg effect in HCC cells may be eliminated through O-GlcNAcylation inhibition, resulting in impaired thermotolerance and EMT, and thereby preventing tumor recurrence and metastasis of HCC-bearing mice after insufficient thermal ablation. Finally, we present evidence that sublethal heat stress-induced O-GlcNAcylation regulates the Warburg effect in HCC cells by promoting hypoxia-inducible factor 1α (HIF-1α) stability. In conclusion, the present study suggests that O-GlcNAcylation coordinates the Warburg effect to promote HCC progression after thermal ablation, which may serve as a novel potential target for controlling postoperative HCC recurrence and metastasis.

Ginsenoside Rb1 does not halt osteoporotic bone loss in ovariectomized rats.

Osteoporosis (OP) is a systemic skeletal disorder, manifesting with a reduction in bone mass and deterioration of the microarchitecture. Mesenchymal stem cells (MSCs) have an innate ability to differentiate into several cell types, including osteoblasts (OB). Ginsenoside Rb1 (GRb1) is an ethanol extract from ginseng and contains a highly concentrated form of ginsenoside. GRb1 shows extensive beneficial health effects such as anti-oxidative and anti-inflammatory functions, modulating the immune system and inhibiting osteoclastogenesis. We hypothesized that GRb1 can promote MSC differentiation into OBs and inhibit bone loss. In the present study, we aimed to address two questions: (1) Will GRb1 have a positive effect on osteogenic differentiation of MSCs? and (2) Will GRb1 halt bone loss in ovariectomized (OVX) rats? We investigated the effects of GRb1 on viability and osteogenic differentiation of rat mesenchymal stem cells (rMSCs). Our results showed that GRb1 at concentrations of 10-8 M and 10-6 M can increase alkaline phosphatase activity, mineralization and the expression of osteogenic related proteins, such as osteopontin and osteoprotegerin, while incubating rMSCs with osteogenic induction medium and GRb1. Adding GRb1 into the medium can prevent rMSCs from Oxidative damage at the concentration of 25µM H2O2. Furthermore, 40 4-month-old rats were assigned to 5 groups(8 rats per group): the basal group, the sham group, the OVX group, the high dose of GRb1 group (6 mg/kg/day) and the low dose of GRb1 group (3 mg/kg/day). Rats recrived treatment 3days after surgery and last for 14 weeks. Examinations included serum analysis, mechanical testing, Masson-Goldner trichrome staining and bone histomorphometry analysis. The results showed that OVX can lead to dyslipidemia and excessive oxidative stress, whereas GRb1 cannot significantly halt dyslipidemia and excessive oxidative stress in OVX rats. In addition, the bone density of the lumbar vertebra and femur were decreased significantly in the OVX rats, and GRb1 could not inhibit bone loss. Bone histomorphometry analysis showed that the number and width of bone trabecula of the tibia were reduced in OVX rats, and GRb1 could not prevent their occurrence. A bone biomechanics assay showed that GRb1 cannot improve the ability of bone structure to resist fracture of the femur in OVX rats. The current study demonstrated that GRb1 has an obvious effect on osteogenic differentiation in rMSCs but no obvious effect on bone loss in OVX rats. These findings indicate GRb1 has a positive effect on rMSCs but does not have an effect on bone loss in OVX rats at the concentration we used.

Energy Metabolism in the Bone is Associated with Histomorphometric Changes in Rats with Hyperthyroidism.

BACKGROUND/AIMS: In this study we assessed histomorphometric changes induced by thyroxine (T4) in 3-month-old hyperthyroid male rats and examined whether the potential mechanism of these changes is related to bone changes. METHODS: Rats were classified as either hyperthyroid following administration of 250 µg/kg/day freshly prepared T4 by gavage for 2 months or euthyroid following administration of vehicle alone (n = 8 per group). We measured bone mineral density (BMD), bone biomechanical properties, and bone histomorphometric changes. Levels of serum indicators were also measured, and three right femurs from the two groups were selected for proteomic investigation. RESULTS: Compared with the control rats, hyperthyroid rats showed a reduction in the fifth lumbar vertebral BMD as well as in the entire femoral BMD (p = 0.033 and 0.026, respectively). Histomorphometric analysis of the proximal tibial metaphysis showed that the percentage of the trabecular area, trabecular number, and percentage of the cortical bone area in the hyperthyroid rats significantly decreased compared with those of the control rats. Conversely, bone formation rate (per unit of bone surface and bone volume), percentage of the osteoclast perimeter, trabecular separation, and endosteal mineral apposition rate in the hyperthyroid rats significantly increased compared with the control rats (all p < 0.05). Except for stiffness (p = 0.24), all bone biomechanical properties of the femur showed a significant decreasing trend in the hyperthyroid rats versus the control rats (all p < 0.05). Serum levels of osteocalcin, alkaline phosphatase, terminal telopeptides of type ß collagen, and tartrate-resistant acid phosphatase were higher in the hyperthyroid rats than in the control rats (all p < 0.05). Using isobaric tags for relative and absolute quantification (iTRAQ), the expression levels of 1,310 proteins were found to be significantly different between the hyperthyroid and control rats (711 proteins were upregulated and 599 were downregulated in hyperthyroid rats). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that most of the enzymes in the glycolysis-tricarboxylic acid (TCA) cycle-oxidative phosphorylation signalling pathway were upregulated in hyperthyroid rats, and seven differentially expressed proteins were selected to verify the iTRAQ results using western blotting. CONCLUSION: Energy metabolism via the glycolysis-TCA cycle-oxidative phosphorylation pathway is positively associated with T4-induced bone histomorphometric changes in rats.