Research progress of fullerenes and their derivatives in the field of PDT.

In contemporary studies, the predominant utilization of C60 derivatives pertains to their role as photosensitizers or agents that scavenge free radicals. The intriguing coexistence of these divergent functionalities has prompted extensive investigation into water-soluble fullerenes. The photodynamic properties of these compounds find practical applications in DNA cleavage, antitumor interventions, and antibacterial endeavors. Consequently, photodynamic therapy is progressively emerging as a pivotal therapeutic modality within the biomedical domain, owing to its notable levels of safety and efficacy. The essential components of photodynamic therapy encompass light of the suitable wavelength, oxygen, and a photosensitizer, wherein the reactive oxygen species generated by the photosensitizer play a pivotal role in the therapeutic mechanism. The remarkable ability of fullerenes to generate singlet oxygen has garnered significant attention from scholars worldwide. Nevertheless, the limited permeability of fullerenes across cell membranes owing to their low water solubility necessitates their modification to enhance their efficacy and utilization. This paper reviews the applications of fullerene derivatives as photosensitizers in antitumor and antibacterial fields for the recent years.

The role of DRP1 mediated mitophagy in HT22 cells apoptosis induced by silica nanoparticles.

Silica nanoparticles (SiNPs) are widely used in the biomedical field and can enter the central nervous system through the blood-brain barrier, causing damage to hippocampal neurons. However, the specific mechanism remains unclear. In this experiment, HT22 cells were selected as the experimental model in vitro, and the survival rate of cells under the action of SiNPs was detected by MTT method, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and adenosine triphosphate (ATP) were tested by the kit, the ultrastructure of the cells was observed by transmission electron microscope, membrane potential (MMP), calcium ion (Ca2+) and apoptosis rate were measured by flow cytometry, and the expressions of mitochondrial functional protein, mitochondrial dynein, mitochondrial autophagy protein as well as apoptosis related protein were detected by Western blot. The results showed that cell survival rate, SOD, CAT, GSH-Px, ATP and MMP gradually decreased with the increase of SiNPs concentration, while intracellular ROS, Ca2+, LDH and apoptosis rate increased with the increase of SiNPs concentration. In total cellular proteins,the expressions of mitochondrial functional proteins VDAC and UCP2 gradually increased, the expression of mitochondrial dynamic related protein DRP1 increased while the expressions of OPA1 and Mfn2 decreased. The expressions of mitophagy related proteins PINK1, Parkin and LC3Ⅱ/LC3Ⅰ increased and P62 gradually decreased, as well as the expressions of apoptosis related proteins Apaf-1, Cleaved-Caspase-3, Caspase-3, Caspase-9, Bax and Cyt-C. In mitochondrial proteins, the expressions of mitochondrial dynamic related proteins DRP1 and p-DRP1 were increased, while the expressions of OPA1 and Mfn2 were decreased. Expressions of mitochondrial autophagy associated proteins PINK1, Parkin, LC3II/LC3I increased, P62 decreased gradually, as well as the expressions of apoptosis related proteins Cleaved-Caspase-3, Caspase-3, and Caspase-9 increased, and Cyt-C expressions decreased. To further demonstrate the role of ROS and DRP1 in HT22 cell apoptosis induced by SiNPs, we selected the ROS inhibitor N-Acetylcysteine (NAC) and Dynamin-related protein 1 (DRP1) inhibitor Mdivi-1. The experimental results indicated that the above effects were remarkably improved after the use of inhibitors, further confirming that SiNPs induce the production of ROS in cells, activate DRP1, cause excessive mitochondrial division, induce mitophagy, destroy mitochondrial function and eventually lead to apoptosis.

Stearic acid promotes lipid synthesis through CD36/Fyn/FAK/mTORC1 axis in bovine mammary epithelial cells.

Stearic acid (C18:0, SA) is a saturated long-chain fatty acid (LCFA) that has a prominent function in lactating dairy cows. It is obtained primarily from the diet and is stored in the form of triacylglycerol (TAG) molecules. The transmembrane glycoprotein cluster of differentiation 36 (CD36) is also known as fatty acid translocase, but whether SA promotes lipid synthesis through CD36 and FAK/mTORC1 signaling is unknown. In this study, we examined the function and mechanism of CD36-mediated SA-induced lipid synthesis in bovine mammary epithelial cells (BMECs). SA-enriched supplements enhanced lipid synthesis and the FAK/mTORC1 pathway in BMECs. SA-induced lipid synthesis, FAK/mTORC1 signaling, and the expression of lipogenic genes were impaired by anti-CD36 and the CD36-specific inhibitor SSO, whereas overexpression of CD36 effected the opposite results. Inhibition of FAK/mTORC1 by TAE226/Rapamycin attenuated SA-induced TAG synthesis, inactivated FAK/mTORC1 signaling, and downregulated the lipogenic genes PPARG, CD36, ACSL1, SCD, GPAT4, LIPIN1, and DGAT1 at the mRNA and protein levels in BMECs. By coimmunoprecipitation and yeast two-hybrid screen, CD36 interacted directly with Fyn but not Lyn, and Fyn bound directly to FAK; FAK also interacted directly with TSC2. CD36 linked FAK through Fyn, and FAK coupled mTORC1 through TSC2 to form the CD36/Fyn/FAK/mTORC1 signaling axis. Thus, stearic acid promotes lipogenesis through CD36 and Fyn/FAK/mTORC1 signaling in BMECs. Our findings provide novel insights into the underlying molecular mechanisms by which LCFA supplements promote lipid synthesis in BMECs.

Chlorogenic acid enhances PPARγ-mediated lipogenesis through preventing Lipin 1 nuclear translocation in Staphylococcus aureus-exposed bovine mammary epithelial cells.

Chlorogenic acid (CGA) as one of the most ubiquitously dietary polyphenolic compounds, has been reported to have various antimicrobial effects and exhibit strong anti-inflammatory ability. Staphylococcus aureus is a gram-positive bacterium that can induce mastitis. However, the mechanism through which S. aureus infection affects lipid synthesis and whether CGA have protective effect on S. aureus reduced lipid synthesis is not fully understood. In this study, the internalization of S. aureus reduced intracellular lipid droplet formation, decreased the levels of intracellular triacylglycerol, total cholesterol and 7 types of fatty acid and downregulated the expression of lipogenic genes FAS, ACC, and DGAT1 in bovine mammary epithelial cells (BMECs). In addition, we found that S. aureus intracellular infection attenuated mTORC1 activation resulting in Lipin 1 nuclear localization. Remarkablely, S. aureus infection-mediated repression of lipid synthesis related to the mTORC1 signaling and Lipin 1 nuclear localization can be alleviated by CGA. Thus, our findings provide a novel mechanism by which lipid synthesis is regulated under S. aureus infection and the protective effects of CGA on lipid synthesis in BMECs.

VDAC1 Protein Regulation of Oxidative Damage and Mitochondrial Dysfunction-Mediated Cytotoxicity by Silica Nanoparticles in SH-SY5Y Cells.

Silica nanoparticles (SiNPs) have been widely used in industry, electronics, and pharmaceutical industries. In addition, it is also widely used in medicine, tumor treatment and diagnosis, as well as other biomedical and biotechnology fields. The opportunities for people to contact SiNPs through iatrogenic, occupational, and environmental exposures are gradually increasing. The damage and biological effects of SiNPs on the nervous system have attracted widespread attention in the field of toxicology. Central nerve cells are rich in mitochondria. It is suggested that the effects of SiNPs on mitochondrial damage of nerve cells may involve the maintenance of neuronal membrane potential, the synthesis and operation of neurotransmitters, and the transmission of nerve pulses, and so on. We established an experimental model of SH-SY5Y cells to detect the cell survival rate, apoptosis, changes of reactive oxygen species and mitochondrial membrane potential, and the expression of mitochondrial function-related enzymes and proteins, so as to reveal the possible mechanism of SiNPs on neuronal mitochondrial damage. It was found that SiNPs could cause oxidative damage to cells and mitochondria, destroy some normal functions of mitochondria, and induce apoptosis in SH-SY5Y cells. The voltage-dependent anion channel 1(VDAC1) protein inhibitor DIDS could effectively reduce intracellular oxidative stress, such as the reduction of ROS content, and could also usefully restore some functional proteins of mitochondria to normal levels. The inhibition of VDAC1 protein may play an important role in the oxidative damage and dysfunction of neuronal mitochondria induced by SiNPs.

Monofunctional dimetallic Ru(η6-arene) complexes inhibit NOTCH1 signaling pathway and synergistically enhance anticancer effect in combination with cisplatin or vitamin C.

ONS donor ligands L1-L4 were utilized in the preparation of monofunctional dimetallic Ru(η6-arene) complexes (C1-C4). These ONS donor ligand based novel tricoordinated Ru(II) complexes bearing η6-arene co-ligand were prepared for the first time. The current methodology resulted in excellent isolated yields and these complexes were characterized in detail by different spectroscopic and spectrometric techniques. The structures of C1-C2 and C4 were characterized in solid state by single crystal X-ray analysis. The in vitro anticancer analyses showed these novel complexes suppressed the growth of breast (MCF-7), liver (HepG2) and lung (A549) cancer cells. C2 suppressed the growth of these cells in dose-dependent manner revealed form the MTT and crystal violet cell viability assays. Moreover, C2 was observed the most potent complex that was used further in detailed mechanistic analyses in cancer cells. C2 showed good cytotoxic activity at 10 µM dose level as compared to cisplatin or oxaliplatin in these cancer cells. We observed morphological changes in cancer cells upon treatment with C2. Moreover, C2 suppressed the invasion and migration ability of cancer cells. C2 induced cellular senescence to retard cell growth and suppressed the formation of cancer stem cells. Importantly, C2 showed synergistic anticancer effect in combination with cisplatin and Vitamin C to further inhibit cell growth which suggested the potential role of C2 in cancer therapy. Mechanistically, C2 inhibited NOTCH1 dependent signaling pathway to suppress cancer cell invasion, migration and cancer stem cells formation. Thus, these data suggested potential role of C2 in cancer therapy by targeting NOTCH1-dependent signaling to suppress tumorigenesis. The results obtained in this study for these novel monofunctional dimetallic Ru(η6-arene) complexes showed their high anticancer potency and this study will pave to further cytotoxicity exploration on this class of complexes.

CXCL8 and the peritoneal metastasis of ovarian and gastric cancer.

CXCL8 is the most representative chemokine produced autocrine or paracrine by tumor cells, endothelial cells and lymphocytes. It can play a key role in normal tissues and tumors by activating PI3K-Akt, PLC, JAK-STAT, and other signaling pathways after combining with CXCR1/2. The incidence of peritoneal metastasis in ovarian and gastric cancer is extremely high. The structure of the peritoneum and various peritoneal-related cells supports the peritoneal metastasis of cancers, which readily produces a poor prognosis, low 5-year survival rate, and the death of patients. Studies show that CXCL8 is excessively secreted in a variety of cancers. Thus, this paper will further elaborate on the mechanism of CXCL8 and the peritoneal metastasis of ovarian and gastric cancer to provide a theoretical basis for the proposal of new methods for the prevention, diagnosis, and treatment of cancer peritoneal metastasis.

FAK/IL-8 axis promotes the proliferation and migration of gastric cancer cells.

Gastric cancer (GC) is one of the most common malignancies in China and is associated with high mortality. The occurrence and development of gastric cancer are related to genetic and environmental factors. Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor protein tyrosine kinase that is activated by the extracellular matrix and growth factors. FAK is highly expressed in cancer and promotes its development by regulating cancer cell proliferation, migration, and angiogenesis. The expression of IL-8 is increased in many types of malignant tumor cells and is linked to their proliferation, migration, invasion, angiogenesis, and EMT. In this study, we found FAK to be essential for the proliferation, migration, and peritoneal metastasis of gastric cancer cells. To examine the molecular regulatory mechanisms of FAK in the peritoneal dissemination of gastric cancer, we performed RNA-seq analysis of MKN-45-FAK-/- and MKN45 cells and demonstrated that IL-8 was downregulated in FAK-deficient cells. Conversely, we confirmed that IL-8 activates FAK activity. We established that IL-8 promotes the proliferation, colony formation, and migration of gastric cancer cells that are partially mediated by FAK. Thus, we propose that an IL-8-FAK-IL-8 positive feedback loop effects the proliferation and migration of gastric cancer cells.

The role and mechanism of unfolded protein response signaling pathway in methylmercury-induced apoptosis of mouse spermatocytes germ cell-2 cells.

The study aimed to explore the role and mechanism of unfolded protein response (UPR) in methylmercury (MeHg)-induced Mouse Spermatocytes (GC-2spd[ts]) apoptosis. Methods such as MTT, flow cytometry, and Western Blot were used to evaluate the cell viability, membrane potential (MMP), reactive oxygen species (ROS), calcium ion (Ca2+ ), rate of cell apoptosis, and the expression of apoptosis-related and UPR-related protein. The results showed that with the increase of MeHg concentration, cell viability and MMP decreased, ROS, Ca2+ , rate of cell apoptosis, and the expression of apoptosis-related protein and UPR-related protein increased. To further explore the effect of ROS-induced oxidative damage on it, the ROS inhibitor N-acetyl-L-cysteine (NAC) was used. The effects of MeHg on germ cell (GC-2) cells were partially inhibited after NAC pretreatment. Our present study proved that MeHg might induce cell apoptosis by activating the UPR signaling pathway in GC-2 cells and affect normal reproductive function.

Research progress on polysaccharide components of Cistanche deserticola as potential pharmaceutical agents.

Cistanche deserticola is a traditional and precious Chinese herbal medicine, known as "desert ginseng", with anti-inflammatory, anti-oxidant, improving immunity, nourishing the kidneys and other pharmacological effects. Its chemical components mainly include phenylethanol glycosides, iridoids, polysaccharides and volatile components, among which polysaccharides have received extensive attention due to their biological activities such as regulating immune activity, anti-aging, anti-spleen deficiency and antitumor. In recent years, a large number of research have been carried out on the extraction and isolation, chemical structure analysis and biological activity of Cistanche deserticola polysaccharides. The methods of polysaccharide extraction mainly include traditional extraction method, ultrasonic assisted method, microwave assisted method and enzyme assisted method, etc. The extracted polysaccharides were analyzed by chemical methods including methylation, acid hydrolysis and Smith degradation and spectroscopy methods such as NMR and IR. A variety of polysaccharides with new structures were obtained, and some polysaccharides with known structures were also investigated for their biological activities and their structure-activity relationships. However, the relationship between polysaccharides structure and their biological activities is still unclear due to the large number of polysaccharide components, their complex structures and the lack of systematic research and analysis on them. It is expected that the subsequent study of polysaccharide structure and active conformational relationship will be highly valuable for the application of Cistanche deserticola in pharmaceutical sciences and health food.

Bacterial outer membrane vesicles as a candidate tumor vaccine platform.

Cancer represents a serious concern for human life and health. Due to drug resistance and the easy metastasis of tumors, there is urgent need to develop new cancer treatment methods beyond the traditional radiotherapy, chemotherapy, and surgery. Bacterial outer membrane vesicles (OMVs) are a type of double-membrane vesicle secreted by Gram-negative bacteria in the process of growth and life, and play extremely important roles in the survival and invasion of those bacteria. In particular, OMVs contain a large number of immunogenic components associated with their parent bacterium, which can be used as vaccines, adjuvants, and vectors to treat diseases, especially in presenting tumor antigens or targeted therapy with small-molecule drugs. Some OMV-based vaccines are already on the market and have demonstrated good therapeutic effect on the corresponding diseases. OMV-based vaccines for cancer are also being studied, and some are already in clinical trials. This paper reviews bacterial outer membrane vesicles, their interaction with host cells, and their applications in tumor vaccines.

Protective effects of chlorogenic acid on inflammatory responses induced by Staphylococcus aureus and milk protein synthesis in bovine mammary epithelial cells.

Staphylococcus aureus (S. aureus) is a major mastitis-causing pathogen in dairy cows. Dairy cows with mastitis suffer from a decrease in milk yield and protein content. Chlorogenic acid (CGA) is a natural product with anti-inflammatory effects. In this study, we examined the function and mechanism of CGA with regard to its anti-inflammatory effects and evaluated its protective function in milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were cultured with and without infection by S. aureus and CGA, and extracellular inflammatory cytokines and amino acids in the medium and milk proteins were determined by ELISA. The function of IL-10RA in anti-inflammatory processes and of SF-1 in milk protein synthesis was assessed by gene silencing. The activity of mTORC1, NF-κB, and STAT5 was examined by western blot. S. aureus caused intracellular infection and upregulated TNF-α, IL-1ß, IL-6, and IL-8, whereas uptake of amino acids and milk protein synthesis were suppressed. CGA mitigated the S. aureus-induced inflammatory response and milk protein synthesis in vitro and in vivo. CGA alleviated S. aureus-induced inhibition of mTORC1 and STAT5 and upregulated IL-10 and IL-10RA. In addition, SF-1 was predicted to be a transcription factor of the milk protein-encoding genes α-LA, ß-LG, and CSN2. S. aureus downregulated SF-1 and CGA reversed the decline in milk protein synthesis due to SF-1 knockdown. Thus, CGA mitigates the inflammatory response that is induced by S. aureus and protects the uptake of amino acids and milk protein synthesis in BMECs.

Methylmercury induced apoptosis of human neuroblastoma cells through the reactive oxygen species mediated caspase and poly ADP-ribose polymerase/apoptosis-inducing factor dependent pathways.

Methylmercury (MeHg) is an environmental neurotoxic substance, which can easily cross the blood-brain barrier, causing irreversible damage to the human central nervous system. Reactive oxygen species (ROS) are involved in various ways of intracellular physiological or pathological processes including neuronal apoptosis. This study attempted to explore the role of ROS-mediated poly ADP-ribose polymerase (PARP)/apoptosis-inducing factor (AIF) apoptosis signaling pathway in the process of MeHg-induced cell death of human neuroblastoma cells (SH-SY5Y). Here, we found that SH-SY5Y cells underwent apoptosis in response to MeHg, which was accompanied by the increased levels of ROS and calcium ion, and the activation of caspase cascades and PARP. Inhibiting the production of ROS can reduce the apoptosis rate to a certain extent. PARP/AIF apoptotic pathway is independent of caspase dependent signaling pathway and regulates it. In conclusion, these results suggest that ROS mediated activation of caspase pathway and PARP/AIF signaling pathway are involved in MeHg induced apoptosis, and these two pathways interact with each other.

Inhibition of SREBP-mediated lipid biosynthesis and activation of multiple anticancer mechanisms by platinum complexes: Ascribe possibilities of new antitumor strategies.

Cancer is one of the most aggressive diseases with poor prognosis and survival rates. Lipids biogenesis play key role in cancer progression, metastasis and tumor development. Suppression of SREBP-mediated lipid biogenesis pathway has been linked with cancer inhibition. Platinum complexes bearing good anticancer effect and multiple genes activation properties are considered important and increase the chances for development of new platinum-based drugs. In this study, we synthesized pyridine co-ligand functionalized cationic complexes and characterized them using multiple spectroscopic and spectrophotometric methods. Two of these complexes were studied in solid state by single crystal X-ray analysis. The stability of these complexes were measured in solution state using 1H NMR methods. These complexes were further investigated for their anticancer activity against human breast, lung and liver cancer cells. MTT assay showed potential cytotoxic activity in dose-dependent manner and decrease survival rates of cancer cells was observed upon treatment with these complexes. Biological assays results revealed higher cytotoxicity as compared to cisplatin and oxaliplatin. Further we studied C2, C6 and C8 in detailed mechanistic anticancer analyses. Clonogenic assay showed decrease survival of MCF-7, HepG2 and A549 cancer cells treated with C2, C6 and C8 as compared to control cells treated with DMSO. TUNEL assay showed more cell death, these complexes suppressed invasion and migration ability of cancer cells and decreased tumor spheroids formation, thus suggesting a potential role in inhibition of cancer metastasis and cancer stem cells formation. Mechanistically, these complexes inhibited sterol regulatory element-binding protein 1 (SREBP-1) expression in cancer cells in dose-dependent manner and thereby reduced lipid biogenesis to suppress cancer progression. Furthermore, expression level was decreased for the key genes LDLR, FASN and HMGCR, those required for sterol biosynthesis. Taken together, these complexes suppressed cancer cell growth, migration, invasion and spheroids formation by inhibiting SREBP-1 mediated lipid biogenesis pathway.

Platinum complexes inhibit HER-2 enriched and triple-negative breast cancer cells metabolism to suppress growth, stemness and migration by targeting PKM/LDHA and CCND1/BCL2/ATG3 signaling pathways.

Triple-negative-breast cancer (TNBC) and HER-2 enriched positive aggressive types of breast cancer and are highly metastatic in nature. Anticancer agents those target TNBC and HER-2 enriched positive breast cancers are considered important in the field of breast cancer research. In search of the effective anticancer agents, we synthesized Pt(II) complexes to target these cancers. Platinum complexes (C1-C8) were prepared in single step by the reaction of commercially available K2PtCl4 with the readily prepared ligands (L1-L8). All these compounds were characterized successfully by different spectroscopic and spectrophotometric analyses. Structures of C1, C3 and C8 were characterized by single crystal X-ray analysis that confirmed the exact chelation mode of the SNO-triply coordinated ligand. All these complexes inhibited the in vitro growth of MCF-7 (luminal-like), MDA-MB-231 (TNBC) and SKBR3 (HER-2 enriched) breast cancer cells. C1, C3 and C7 induced cell death and suppressed the clonogenic potential of these cancer cells. Importantly, C1, C3 and C7 showed potentials to suppress cancer stem cells/mammosphere formation and cell migration ability of MDA-MB-231 and SKBR3 breast cancer cells. These complexes also induced cellular senescence in MDA-MB-231 and SKBR3 cells, thus suggesting a cell retardation mechanism. Similarly, these complexes induced DNA damage by activating p-H2AX expression and promoted autophagy via ATG3/LC3B axis activation in MDA-MB-231 and SKBR3 cells. Furthermore, these complexes decreased the expression of oncogenic proteins such as BCL2 and cylin-D1 those are involved in cancer cell survival and cell cycle progression. To further gain insight, we found that C1 and C7 targeted glycolytic pathways by regulating PKM and LDHA expression, which are involved in glycolysis. Moreover, C1 and C7 suppressed the formation of ATP production that is required for cancer cell growth. Taken together, the easy synthesis and biological assays results point towards the importance of these complexes in MDA-MB-231 (TNBC) and SKBR3 (HER-2 enriched) breast cancer cells by targeting multiple signaling pathways those are considered important during breast cancer progression. This study produces bases for further deeper in vitro or in vivo study that could lead to the effective breast cancer agents which we are working on.

Inhibition of the mTORC1/NF-κB Axis Alters Amino Acid Metabolism in Human Hepatocytes.

In addition to serving as the building blocks for protein synthesis, amino acids can be used as an energy source, through catabolism. The transamination, oxidative deamination, and decarboxylation processes that occur during amino acid catabolism are catalyzed by specific enzymes, including aspartate aminotransferase (AST), glutamate dehydrogenase (GDH), glutamic acid decarboxylase (GAD), and ornithine decarboxylase (ODC); however, the overall molecular mechanisms through which amino acid catabolism occurs remain largely unknown. To examine the role of mechanistic target of rapamycin complex 1 (mTORC1) on amino acid catabolism, mTORC1 was inactivated by rapamycin or shRNA targeting Raptor, versus activated by overexpressing Rheb or amino acids in human hepatocytes. The expression of amino acid catabolic genes and related transcription factor was investigated by RT/real-time PCR and western blot analysis. A few types of amino acid metabolite were examined by ELISA and HPLC analysis. The data showed that inactivated mTORC1 resulted in inhibition of NF-κB and the expression of AST, GDH, GAD, and ODC, whereas activated mTORC1 enhanced NF-κB activation and the expression levels of the catabolism-associated genes. Further, inhibition of NF-κB reduced the expression levels of AST, GDH, GAD, and ODC. mTORC1 upregulated NF-κB activation and the expression of AST and ODC in response to glutamate and ornithine treatments, whereas rapamycin inhibited the utilization of glutamate and ornithine in hepatocytes. Taken together, these results indicated that the mTORC1/NF-κB axis modulates the rate of amino acid catabolism by regulating the expression of key catabolic enzymes in hepatocytes.

Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

BACKGROUND: Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established. METHODS: Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM. RESULTS: S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1ß and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages. CONCLUSIONS: S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages. Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.

The mTORC1/4EBP1/PPARγ Axis Mediates Insulin-Induced Lipogenesis by Regulating Lipogenic Gene Expression in Bovine Mammary Epithelial Cells.

4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with lipid metabolism. However, it is unknown which effector protein connects mTORC1 and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as part of the underlying mechanism by which insulin-induced lipid synthesis and secretion are regulated by mTORC1 in primary bovine mammary epithelial cells (pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1 phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin 1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1 by shRaptor or shRheb attenuated the synthesis and secretion of TAG and PA. In contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1 phosphorylation and PPARγ expression and upregulated the mRNA and protein levels of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ. Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ. These results demonstrate that mTORC1 regulates lipid synthesis and secretion by inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid synthesis and secretion are regulated in pBMECs.

Marek's disease virus type 1 encoded analog of miR-155 promotes proliferation of chicken embryo fibroblast and DF-1 cells by targeting hnRNPAB.

Marek's disease virus type 1 (MDV-1) is a representative oncogenic Alpha herpesvirus that causes an immunosuppressive and neoplastic lymphoproliferative avian disease, namely Marek's disease (MD). The rapid-onset T-cell lymphoma in chickens induced by MDV-1 has been historically regarded as an ideal natural model for herpesvirus-related cancer research. As a viral analog of cellular miR-155, the MDV-1-encoded miR-M4-5p has been shown to be crucial for the virally-induced MD tumorigenesis. Our previous studies demonstrated that miR-M4-5p induces an over-expression of oncogene c-Myc by targeting LTBP1 and suppressing the TGF-ß signaling pathway during MDV-1 infection. We have now further identified the chicken heterogeneous nuclear ribonucleoprotein AB (hnRNPAB) as a new cellular biological target for miR-M4-5p. Suppression of hnRNPAB expression mediated by miR-M4-5p promotes the proliferation, but not the apoptosis, of both primary chicken embryo fibroblasts (CEFs) and transformed chicken fibroblast DF-1 cell line. HnRNPAB is a member of the hnRNP family of proteins that play important roles in normal biological processes as well as cancer development. Our data suggests that the recognition and down-regulation of hnRNPAB by miR-M4-5p may be one of the important strategies for MDV-1 to trigger the development of MD lymphomas.

mTORC1 mediates peptidoglycan induced inflammatory cytokines expression and NF-κB activation in macrophages.

Peptidoglycan (PGN) is the major structural component of the bacterial cell wall, especially gram positive bacteria, which induces inflammatory responses. Mammalian target of rapamycin (mTOR) regulates the production of inflammatory cytokines induced by antigens, while the function of mTORC1 in peptidoglycan induced inflammatory response is unknown. This study aims to examine the role and the regulatory mechanism of mTOR signaling pathway in peptidoglycan induced cytokine expression in mouse macrophages. We observed that peptidoglycan upregulated the secretion of proinflammatory cytokines IL-6, TNF-α and anti-inflammatory cytokine IL-10 in a dose- and time-dependent manner. mTORC1 positively regulates IL-6 and TNF-α, but negatively regulates IL-10 secretion. mTORC1 regulates NF-κB p65 activation by degrading IκB-α in response to peptidoglycan. mTOR, NF-κB and STAT3 signaling pathways are involved in peptidoglycan induced inflammatory cytokines expression via a TLR1/TLR2-dependent mechanism in macrophages. Thus, mTORC1 pathway regulates the innate immune response to bacterial peptidoglycan.