Apoptosis In RAW264 Research Articles

Cigarette smoke extracts induce apoptosis in Raw264.7 cells via endoplasmic reticulum stress and the intracellular Ca2+/P38/STAT1 pathway

Cigarette smoke (CS) exposure is a risk factor for chronic obstructive pulmonary disease (COPD). CS exposure impairs the ability of killing pathogens in macrophages, which might be due to the abnormal apoptosis induced by CS. This study explored the effects and mechanisms of cigarette smoke extract (CSE) on the apoptosis of macrophages in vitro. Raw264.7 cells were treated with CSE at different concentrations, and viability and apoptosis of cells was accessed. The protein expression was detected by western blot. The intracellular Ca2+ level was evaluated by Fluo-4 AM probe assay.CSE induced the apoptosis and increased the expression of cleaved caspase 3, which were attenuated by a caspase inhibitor. CSE increased the expression of CHOP, BiP and P-eif2α, and the inhibitor of endoplasmic reticulum stress (ERS) decreased the apoptosis induced by CSE. Phosphorylation levels of P38, JNK and ERK1/2 were increased following incubation with CSE. Only P38 inhibitor significantly reduced apoptosis induced by CSE, while ERK1/2 inhibitor promoted apoptosis. Phosphorylation of STAT1 at Ser727 was activated by CSE and attenuated by the P38 inhibitor. Finally, CSE increased the level of intracellular Ca2+, and calcium chelator partly attenuated the apoptosis and phosphorylation of P38 and STAT1 induced by CSE.CSE induced a caspase 3-dependent apoptosis in Raw264.7 cells via ERS and intracellular Ca2+/P38/STAT1 pathway.

Thymol and Piperine-Loaded Poly(D,L-lactic-co-glycolic acid) Nanoparticles Modulate Inflammatory Mediators and Apoptosis in Murine Macrophages

AbstractThis study aimed at preparing and characterizing thymol, eugenol, and piperine-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles and evaluating the effect on inflammatory mediators secretion and apoptosis in Raw 264.7 macrophage cells. Nanoparticles were produced by the solvent evaporation technique. Dynamic light scattering and scanning electron microscopy were used to study the physicochemical characteristics. Raw 264.7 macrophage cells were used as a model for in vitro assays. The 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium assay was used to determine the cytotoxicity of the formulated nanoparticles. An annexin V apoptosis detection kit was used to assess apoptosis. Nitric oxide production was determined using the Griess reagent, and the inflammatory mediators level was evaluated with Th1/Th2 cytokine and fluorometric cyclooxygenase kits. The loaded nanoparticles showed a particle size around 190 nm with a low polydispersity between 0.069 and 0.104 and a zeta potential between–1.2 and–9.5 mV. Reduced cytotoxicity of nanoparticles compared to free molecules against Raw 264.7 macrophage cells was observed and seemed to occur through a mechanism associated with apoptosis. A decrease in cyclooxygenase enzyme activity with an increasing concentration was observed. Both free molecules and nanoparticles showed their capacity to modulate the inflammatory process mostly by inhibiting the investigated inflammatory cytokines. The data presented in this study indicate that thymol and piperine-loaded poly(D,L-lactic-co-glycolic acid nanoparticles could serve as a novel anti-inflammatory colloidal drug delivery system with reduced toxicity. However, further study should be considered to optimize the formulation’s loading capacity and thereby probably enhance their bioactivity in treating inflammatory diseases.

Mesenchymal stem cells-derived extracellular vesicles carrying microRNA-17 inhibits macrophage apoptosis in lipopolysaccharide-induced sepsis

ObjectiveSepsis, as a disease affecting the microcirculation and tissue perfusion, results in tissue hypoxia and multiple organ dysfunctions. Bone mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) have been demonstrated to transfer trivial molecules (proteins/peptides, mRNA, microRNA and lipids) to alleviate sepsis. We sought to define the function of microRNA (miR)-17 carried in BMSC-EVs in sepsis. MethodsThe purity of the extracted BMSCs was identified and confirmed by detection of the surface markers by flow cytometry, followed by osteoblastic, adipogenic, and chondrocyte differentiation experiments. Subsequently, EVs were collected from the medium of BMSCs. The uptake of PKH-67-labeled BMSC-EVs or EVs carrying cy3-miR-17 by RAW264.7 cells was observed under laser confocal microscopy. Furthermore, a series of gain- and loss-of-function approaches were conducted to test the effects of LPS, miR-17 and BRD4 on the inflammatory factors (IL-1β, IL-6 and TNF-α), number of M1 macrophages and M2 macrophages, inflammatory-related signal pathway factors (EZH2, c-MYC and TRAIL), macrophage proliferation, and apoptosis in sepsis. The survival rates were measured in vivo. ResultsBMSC-EVs was internalized by the RAW264.7 cells. BDR4 was verified as a target of miR-17, while the expression pattern of miR-17 was upregulated in BMSC-EVs. MiR-17 carried by BMSC-EVs inhibited LPS-induced inflammation and apoptosis of RAW264.7 cells, but improved the viability of RAW264.7 cells. Next, in vitro experiments supported that miR-17 inhibited LPS-induced inflammation in RAW264.7 cells through BRD4/EZH2/TRAIL axis. BRD4 overexpression reversed the effects of miR-17. Moreover, the therapeutic function of BMSC-EVs carried miR-17 was verified by in vivo experiments. ConclusionsMiR-17 derived from BMSCs-EVs regulates BRD4-mediated EZH2/TRAIL axis to essentially inhibit LPS-induced macrophages inflammation.

RETRACTED: mtROS-mediated Akt/AMPK/mTOR pathway was involved in Copper-induced autophagy and it attenuates Copper-induced apoptosis in RAW264.7 mouse monocytes

Copper (Cu) is a trace element necessary in animals as well as human beings. However, excessive Cu is toxic to immunocytes, but the precise mechanism is largely unclear so far. This work was conducted aiming to examine the Cu-mediated autophagy mechanism together with its role in Cu toxicology in RAW264.7 cells. Here, we demonstrated that CuSO4 reduced the cell viability depending on its dose. CuSO4 could obviously increase autophagy in RAW264.7 cells. According to the obtained results, CuSO4 induced autophagy through Akt/AMPK/mTOR pathway which characterized by down regulation of p-Akt (Ser473)/Akt, p-mTOR/mTOR, p-ULK1(Ser757)/ULK1 and subsequent up-regulation of p-AMPKα/AMPKα and p-ULK1(Ser555)/ULK1. Furthermore, CuSO4 significantly induced the production of mitochondrial reactive oxygen species (mtROS). In addition, CuSO4-mediated apoptosis and autophagy might be suppressed through suppressing mtROS generation by exposure to Mito-TEMPO. Intriguingly, autophagy promotion with rapamycin could decrease the apoptosis and the inhibition of autophagy with knock down Atg5 could enhance the apoptosis induced by CuSO4. Moreover, our results suggested that mtROS is the original cause in CuSO4-induced apoptosis and autophagy. Additionally, CuSO4 induced autophagy through mtROS-dependent Akt/AMPK/mTOR signalling pathwayin RAW264.7 cells. Moreover, autophagy activation might potentially generate a protection mechanism for improving CuSO4-induced RAW264.7 cell apoptosis.

<p><strong>Inhibition of cystathionine-γ-lyase-mediated hydrogen sulfide production in LPS-stimulated RAW 264.7 macrophages by polyherbal extract</strong></p>

The polyherbal formulation chandraprabha vati (CV) comprising 29 herbs is traditionally used as an anti-inflammatory agent for arthritis and urinary ailments in Indian Siddha medicine. Earlier, we reported that lipopolysaccharide (LPS) induced apoptosis in RAW 264.7 macrophages is mediated by hydrogen sulfide (H 2 S), a potential target for many inflammatory diseases. Here, we hypothesized that pretreatment with CV decreases H 2 S level and thereby alleviate the inflammatory conditions induced by LPS in RAW 264.7 macrophages, and this protective effect occurs through alterations in cystathionine-γ-lyase (CSE), a H 2 S synthesizing enzyme. Accordingly, we evaluated the anti-inflammatory effect of CV in LPS-stimulated RAW 264.7 macrophages in vitro . The CV pretreatment followed by 12 h of LPS-stimulation showed significantly decreased TNF-α, H 2 S production possibly through CSE gene expression and NF-кB activation compared to the non pretreated macrophages. Our results further confirm that the polyherbal extract chandraprabha vati may be a useful therapy for inflammatory disorders by reducing H 2 S levels.

Inhibition of immunotoxicity of Pb2+-induced RAW264.7 macrophages by selenium species in selenium-enriched rice

Previous studies have revealed that Selenium-enriched rice protein hydrolysates (SPHs) could alleviate Pb2+-induced apoptosis in RAW264.7 macrophages. The purpose of the current study was to detect the effect of different selenium (Se) species on immunotoxicity of the Pb2+-induced RAW264.7 macrophages and explore the potential immunomodulatory mechanism. Herein, SPHs, an isolated SPHs fraction (SPHs-2), selenomethionine (SeMet), selenite (SeIV) were used to investigate their inhibitory effect and the impacts on the expression of cytokines and related protein kinases in immunomodulatory pathways. The results showed that, compared with Pb2+-only group, Se-containing components significantly enhanced the cell viability and effectively decrease nitric oxide (NO) content in Pb2+-induced RAW264.7 cells. Furthermore, compared with other Se species, SPHs-2 markedly decreased the secretion levels of pro-inflammatory cytokines TNF-α, NF-κB, IL-1β, MyD88, IL-6 and IL-8. Western blot results demonstrated that SPHs-2 effectively downregulated the expressions of IκB, IKKα, p38, and Erk1/2, and also successfully blocked the phosphorylation of these protein kinases. Our findings suggested that SPHs-2 effectively attenuate inflammatory response and inhibit the immunotoxicity of Pb2+ on RAW264.7 macrophages via regulating NF-κB/MAPK signaling pathways.

Effect of Liposome-Encapsulated Zoledronic Acid on Microenvironment of Hepatocellular Carcinoma May Depend on the Ratio Between M1 and M2 Polarized Macrophages.

We studied the effect of zoledronic acid encapsulated into liposomes (L-ZOL) on tumorassociated macrophages in the stroma of hepatocellular carcinoma xenograft. Liposomes were prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-snglycero-3-phospho-sn-1-glycerol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] using thin film method and loaded with zoledronic acid. It was shown that L-ZOL promoted apoptosis of RAW264.7 cells, eliminate much more protumoral M2 macrophages than antitumoral M1 macrophages in the tumor xenograft, and did not significantly reduce the size of xenograft in 6 days. Thus, the effect of treatment depends on the ratio between antitumoral M1 and protumoral M2 polarized macrophages in the tumor.

Mycobacterium smegmatis Bacteria Expressing Mycobacterium tuberculosis-Specific Rv1954A Induce Macrophage Activation and Modulate the Immune Response

Mycobacterium tuberculosis (M. tb), the intracellular pathogen causing tuberculosis, has developed mechanisms that endow infectivity and allow it to modulate host immune response for its survival. Genomic and proteomic analyses of non-pathogenic and pathogenic mycobacteria showed presence of genes and proteins that are specific to M. tb. In silico studies predicted that M.tb Rv1954A is a hypothetical secretory protein that exhibits intrinsically disordered regions and possess B cell/T cell epitopes. Treatment of macrophages with Rv1954A led to TLR4-mediated activation with concomitant increase in secretion of pro-inflammatory cytokines, IL-12 and TNF-α. In vitro studies showed that rRv1954A protein or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) activates macrophages by enhancing the expression of CD80 and CD86. An upregulation in the expression of CD40 and MHC I/II was noted in the presence of Rv1954A, pointing to its role in enhancing the association of APCs with T cells and in the modulation of antigen presentation, respectively. Ms_Rv1954A showed increased infectivity, induction of ROS and RNS, and apoptosis in RAW264.7 macrophage cells. Rv1954A imparted protection against oxidative and nitrosative stress, thereby enhancing the survival of Ms_Rv1954A inside macrophages. Mice immunized with Ms_Rv1954A showed that splenomegaly and primed splenocytes restimulated with Rv1954A elicited a Th1 response. Infection of Ms_Rv1954A in mice through intratracheal instillation leads to enhanced infiltration of lymphocytes in the lungs without formation of granuloma. While Rv1954A is immunogenic, it did not cause adverse pathology. Purified Rv1954A or Rv1954A knock-in M. smegmatis (Ms_Rv1954A) elicited a nearly two-fold higher titer of IgG response in mice, and PTB patients possess a higher IgG titer against Rv1954A, also pointing to its utility as a diagnostic marker for TB. The observed modulation of innate and adaptive immunity renders Rv1954A a vital protein in the pathophysiology of this pathogen.

Knockdown of fatty acid binding protein 4 exacerbates Bacillus Calmette-Guerin infection-induced RAW264.7 cell apoptosis via the endoplasmic reticulum stress pathway.

Mycobacterial infection can induce alveolar macrophage apoptosis, which plays a vital role in the pathogenesis of tuberculosis. Accumulating evidence has demonstrated that fatty acid oxidation is involved in apoptosis during various pathological processes, including bacterial infection. However, whether fatty acid oxidation regulates mycobacterial infection-induced macrophage apoptosis remains unclear. Hence, the present study aimed to investigate the role of fatty acid binding protein 4 (FABP4) which is a carrier protein for fatty acids, in regulating apoptosis in RAW264.7 cells infected with Bacillus Calmette-Guerin (BCG). In our study, the impact of BCG infection on apoptosis and fatty acid oxidation in RAW264.7 cells was examined. Notably, we found that FABP4 was overexpressed during this process. Furthermore, small interfering RNAs targeting FABP4 were used to investigate the role of FABP4 in regulating apoptosis and fatty acid oxidation in BCG-infected RAW264.7 cells. The results indicated that mycobacterial infection promoted apoptosis and enhanced fatty acid oxidation in RAW264.7 cells. Moreover, FABP4 knockdown exacerbated BCG-induced apoptosis and upregulated the expression of p-PERK, p-eIF2α and chop, which are endoplasmic reticulum (ER) stress markers. In addition, FABP4 knockdown promoted fatty acid oxidation and ROS production, which result in the activation of ER stress. Our data suggested that FABP4 knockdown exacerbated BCG-induced apoptosis in RAW264.7 cells via the ER stress pathway.

Autophagy plays a protective role during Pseudomonas aeruginosa-induced apoptosis via ROS-MAPK pathway.

Pseudomonas aeruginosa infection can induce alveolar macrophage apoptosis and autophagy, which play a vital role in eliminating pathogens. These two processes are usually not independent. Recently, autophagy has been found to interact with apoptosis during pathogen infections. Nevertheless, the role of autophagy in P. aeruginosa-infected cell apoptosis is unclear. In this study, we explored the impact of P. aeruginosa infection on autophagy and apoptosis in RAW264.7 cells. The autophagy activator rapamycin was used to stimulate autophagy and explore the role of autophagy on apoptosis in P. aeruginosa-infected RAW264.7 cells. The results indicated that P. aeruginosa infection induced autophagy and apoptosis in RAW264.7 cells, and that rapamycin could suppress P. aeruginosa-induced apoptosis by regulating the expression of apoptosis-related proteins. In addition, rapamycin scavenged the cellular reactive oxygen species (ROS) and diminished p-JNK, p-ERK1/2 and p-p38 expression of MAPK pathways in RAW264.7 cells infected with P. aeruginosa. In conclusion, the promotion of autophagy decreased P. aeruginosa-induced ROS accumulation and further attenuated the apoptosis of RAW264.7 cells through MAPK pathway. These results provide novel insights into host–pathogen interactions and highlight a potential role of autophagy in eliminating P. aeruginosa.

Upregulation of miR-150-5p alleviates LPS-induced inflammatory response and apoptosis of RAW264.7 macrophages by targeting Notch1.

Circulating miR-150-5p has been identified as a prognostic marker in patients with critical illness and sepsis. Herein, we aimed to further explore the role and underlying mechanism of miR-150-5p in sepsis. Quantitative real-time-PCR assay was performed to detect the expression of miR-150-5p upon stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The levels of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β were measured by ELISA assay. Cell apoptosis was determined using flow cytometry. Western blot was used to assess notch receptor 1 (Notch1) expression in LPS-induced RAW264.7 cells. Dual-luciferase reporter assay was employed to validate the target of miR-150-5p. Our data showed that miR-150-5p was downregulated and Notch1 was upregulated in LPS-stimulated RAW264.7 cells. miR-150-5p overexpression or Notch1 silencing alleviated LPS-induced inflammatory response and apoptosis in RAW264.7 cells. Moreover, Notch1 was a direct target of miR-150-5p. Notch1 abated miR-150-5p-mediated anti-inflammation and anti-apoptosis in LPS-induced RAW264.7 cells. miR-150-5p alleviated LPS-induced inflammatory response and apoptosis at least partly by targeting Notch1 in RAW264.7 cells, highlighting miR-150-5p as a target in the development of anti-inflammation and anti-apoptosis drugs for sepsis treatment.

The protective effect of soybean protein-derived peptides on apoptosis via the activation of PI3K-AKT and inhibition on apoptosis pathway.

Soybean protein‐derived peptides (SBP) are a rich source of various bioactive peptides with multiple health benefits. However, the prospective effects of SBP on human cells are still unclear. Therefore, this article investigated the effects of small molecular weight SBP on MG132‐induced apoptosis in RAW264.7 cells. SBP inhibited MG132‐induced apoptosis of RAW264.7 cells in a dose‐dependent manner by flow cytometry. To further study its molecular mechanisms, Western blot analysis demonstrated that SBP could activate the PI3K‐AKT pathway by increasing the phosphorylation of PI3K and AKT and inhibiting apoptosis pathway by downregulating the expressions of pro‐apoptotic proteins of Bim, Bax, Fas, and Fasl and promoting the expressions of anti‐apoptotic proteins of Bcl‐xL and Bcl‐2. These results indicated the protective effect of SBP on MG132‐induced apoptosis in RAW264.7 cells.

3-Acetyldeoxynivalenol induces lysosomal membrane permeabilization-mediated apoptosis and inhibits autophagic flux in macrophages

3-Acetyldeoxynivalenol (3-Ac-DON), the acetylated derivative of deoxynivalenol (DON), has been reported to be coexisted with DON in various cereal grains. Ingestion of grain-based food products contaminated by 3-Ac-DON might exert deleterious effects on the health of both humans and animals. However, the biological toxicity of 3-Ac-DON on macrophages and the underlying mechanisms remain largely unknown. In the present study, we showed that RAW 264.7 macrophages treated with 0.75 or 1.50 μg/mL of 3-Ac-DON resulted in DNA damage and the related cell cycle arrest at G1 phase and cell death, activation of the ribotoxic stress and the endoplasmic reticulum (ER) stress responses. The 3-Ac-DON-induced cell death was accompanied by a protective autophagy, because gene silencing of Atg5 using the small interfering RNA enhanced cell death. Results of further experiments revealed a role for lysosomal membrane permeabilization in the 3-Ac-DON triggered inhibition of autophagic flux. Additional work also showed that increased lysosomal biogenesis and leakage of cathepsin B (CTSB) from lysosomes to cytosol was critical for the 3-Ac-DON-induced cell death. Importantly, 3-Ac-DON-induced DNA damage and cell death were rescued by CA-074-me, a CTSB inhibitor. Collectively, these results indicated a critical role of lysosomal membrane permeabilization in the 3-Ac-DON-induced apoptosis of RAW 264.7 macrophages.

Foxc2 Alleviates Ox-LDL-Induced Lipid Accumulation, Inflammation, and Apoptosis of Macrophage via Regulating the Expression of Angptl2.

The present study aimed to investigate the role of Forkhead box protein C2 (Foxc2) in oxidized low-density lipoprotein (ox-LDL)-induced macrophages and identify the potential mechanisms. RAW264.7 cells, the murine macrophage cell line, were stimulated by ox-LDL, and cell proliferation was examined. The levels of inflammation- and oxidative stress-related markers were detected using kits after induction with ox-LDL. Subsequently, the expression of Foxc2 was measured using Western blotting. After transfection with Foxc2 pcDNA3.1, intracellular lipid droplets were examined using oil red O staining. The levels of total cholesterol (TC), free cholesterol (FC), inflammatory cytokines, and oxidative stress markers were determined. Moreover, apoptosis of RAW264.7 cells was detected using flow cytometry, and apoptosis-related proteins were measured using Western blotting. Angiopoietin-like protein 2 (Angptl2) was predicted as a target gene of Foxc2. Therefore, the expression of Angptl2 was examined after Foxc2 overexpression in ox-LDL-induced RAW264.7 cells. Then, the changes of intracellular lipid droplets, TC, FC, inflammatory cytokines, oxidative stress factors, and cell apoptosis were detected after Angptl2 overexpression or co-transfection with Foxc2 and Angptl2 pcDNA3.1. The results revealed that ox-LDL induction inhibited proliferation of RAW264.7 cells and promoted the release of inflammatory factors. Importantly, the expression of Foxc2 was obviously decreased after stimulation by ox-LDL. Foxc2 overexpression suppressed lipid accumulation, TC, FC levels, inflammation, oxidative stress, and apoptosis induced by ox-LDL, whereas these inhibitory effects were relieved after co-transfection with Angptl2 pcDNA3.1. These findings demonstrated that Foxc2 can alleviate ox-LDL-induced lipid accumulation, inflammation, and apoptosis of macrophage via regulating the expression of Angptl2.

Dexmedetomidine Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Regulation of microRNA-223-3p and HDAC4

Objective: Acute lung injury (ALI) mainly causes acute respiratory distress syndrome, pneumonia, and acute respiratory failure. Due to the high lethality of ALI, this study was for the investigation of the mechanism by which dexmedetomidine (DEX) alleviated lipopolysaccharide (LPS)-induced ALI by regulating histone deacetylase 4 (HDAC4) and microRNA-223-3p (miR-223-3p). Methods: After LPS induction of ALI, the mice were treated with DEX (low, medium and high doses). Then the mice treated with LPS-induced ALI and high dose of DEX were transfected with miR-223-3p mimics or sh-HDAC4 to figure their roles in inflammation, and apoptosis in lung tissue of mice with ALI. Mouse monocyte RAW264.7 was treated with LPS-induced ALI and different doses of DEX and transfected with miR-223-3p mimics or sh-HDAC4 to explore their roles in proliferation, cycle distribution, inflammation, and apoptosis of RAW264.7 cells. RT-qPCR and western blot analysis were functioned to detect the expression of miR-223-3p, HDAC4, and TLR4/MyD88/NF-κB signaling pathway-related factors in lung tissue and RAW264.7 cells of mice with ALI. Results: Lowly expressed miR-223-3p and over-expressed HDAC4 were detected in lung tissue of mice with ALI and RAW264.7 cells. MiR-223-3p mimics or sh-HDAC4 decreased TLR4/MyD88/NF-κB signaling pathway-related factors in lung tissue of mice and RAW264.7 cells. MiR-223-3p mimics or sh-HDAC4 inhibited inflammation and cell apoptosis in lung tissue of mice with ALI, as well as facilitated proliferation, cycle entry and inhibited inflammation and apoptosis of RAW264.7 cells. Conclusion: This study suggests that DEX can alleviate LPS-induced ALI by mediating miR-223-3p and HDAC4 expression. This finding has key implications for developing the mechanism of ALI. Funding Statement: This work was supported by Science and Technology Department of Jilin Province (No. 20190701068GH). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: This study was approved and supervised by the animal ethics committee of China-Japan Union Hospital of Jilin University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

LncRNA NEAT1 facilitates the progression of sepsis through up-regulating TSP-1 via sponging miR-370-3p.

Sepsis is a systemic inflammatory disease. LncRNA NEAT1 has been reported to be up-regulated in sepsis patients. Nevertheless, the modulatory network of NEAT1 in sepsis remains to be revealed. The abundance of long noncoding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1), miR-370-3p, and thrombospondin-1 (TSP-1) were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) in sepsis patients and lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the concentration of cytokines in RAW 264.7 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry assay, and Western blot assay were conducted to detect the proliferation and apoptosis of RAW 264.7 cells. Dual-Luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and RNA-pull down assay were conducted to confirm the combination between miR-370-3p and NEAT1 or TSP-1 in RAW 264.7 cells. The enrichment of NEAT1 was enhanced in sepsis patients and LPS-stimulated RAW 264.7 cells. NEAT1 contributed to LPS-induced inflammation and apoptosis of RAW 264.7 cells. MiR-370-3p bound to NEAT1, and it was negatively regulated by NEAT1 in RAW 264.7 cells. LPS promoted the inflammation and apoptosis while restrained the proliferation of RAW 264.7 cells via NEAT1/miR-370-3p axis. TSP-1 was a target of miR-370-3p in RAW 264.7 cells, and miR-370-3p suppressed the inflammation and apoptosis while it facilitated the proliferation of LPS-induced RAW 264.7 cells via TSP-1. LncRNA NEAT1 promoted the inflammation and apoptosis while restrained the proliferation of LPS-stimulated RAW 264.7 cells through the miR-370-3p/TSP-1 axis.

Dexamethasone induces aberrant macrophage immune function and apoptosis.

Glucocorticoids (GCs) are known potent clinical drugs, however, their mode of action is still complex and debatable. Macrophages are the most important target of GCs and play a key role in tumor immunity in vivo, but their relationship is also controversial. In the present study, the lentivirus system was used to overexpress and knock down the level of transcription factor Krüppel-like factor 9 (KLF9). The results revealed that dexamethasone (Dex) induced ROS generation and mitochondria-dependent apoptosis in RAW 264.7 cells via the KLF9. In addition, overexpression of KLF9 significantly increased apoptosis of RAW 264.7 cells. Notably, ELISA assay revealed that increased expression of KLF9 inhibited LPS-induced COX-2 expression and reduced COX-2-derived prostaglandin E2 and pro-inflammatory cytokine secretion. Furthermore, a co-culture system was used to reveal that overexpression of KLF9 in RAW 264.7 cells promoted HepG2 cell survival. In summary, it is reported that KLF9 promoted apoptosis of proinflammatory macrophages, and suppressed the antitumor effects, which can be selectively targeted by GCs as a novel mechanism to suppress antineoplastic activity.

The role of the miR-21-5p-mediated inflammatory pathway in ulcerative colitis.

Ulcerative colitis (UC), a major type of inflammatory bowel disease, is also a chronic non-specific intestinal inflammation condition of unknown etiology. The pathogenesis of UC is closely associated with immune abnormalities, inflammatory damage and genetics. The present study aimed to explore the effects of microRNA (miR)-21-5p on the interleukin-6 (IL-6) receptor (IL6R)/signal transducer and activator of transcription (STAT3) signal pathway in UC, in order to identify a highly effective treatment for UC. A total of 45 patients with UC and 45 healthy controls were recruited for the present study. The expression levels of miR-21-5p and STAT3 in the sera of patients with UC and healthy controls were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A UC rat model was established using dextran sulfate sodium. Following lipopolysaccharide (LPS) treatment, RAW264.7 cells were transfected with a miR-21-5p inhibitor. The levels of morphological damage and apoptosis of the colonic mucosal epithelial tissue were investigated using hematoxylin and eosin staining and a TUNEL staining assay, and then the colon macroscopic damage index and disease activity index were measured in rats. Western blot analysis was used to detect the protein expression levels of IL6R, STAT3, intracellular adhesion molecule 1 (ICAM-1), NF-κB, cleaved caspase-3, cleaved caspase-9 and Fas ligand (FasL). RT-qPCR detected the mRNA expression levels of miR-21-5p, IL6R, STAT3, ICAM-1, NF-κB, caspase-3, caspase-9 and FasL. An ELISA was performed to measure the levels of inflammatory cytokines. The viability and apoptosis levels of RAW264.7 cells were examined using MTT and flow cytometry assays. Additionally, STAT3 was investigated as a direct target of miR-21-5p in RAW264.7 cells using a dual-luciferase reporter assay. The results of the present study demonstrated that inflammation and apoptotic markers were revealed to be significantly downregulated following transfection with miR-21-5p inhibitors in RAW264.7 cells induced by LPS, and that cell viability was increased. Furthermore, STAT3 was confirmed to be a target of miR-21-5p in RAW264.7 cells. Collectively, these data demonstrated that miR-21-5p inhibition mediated the IL-6/STAT3 pathway in UC rats to decrease the levels of inflammation and apoptosis in RAW264.7 cells, and suggested that miR-21-5p may be an important therapy target in human UC.

JAK2/STAT1-mediated HMGB1 translocation increases inflammation and cell death in a ventilator-induced lung injury model

Janus kinase 2/signal transducer and activators of transcription 1 (JAK2/STAT1) signaling is a common pathway that contributes to numerous inflammatory disorders, including different forms of acute lung injury (ALI). However, the role of JAK2/STAT1 in ventilator-induced lung injury (VILI) and its underlying mechanism remain unclear. In this study, using lipopolysaccharide (LPS) inhalation plus mechanical ventilation as VILI mouse model, we found that the administration of JAK2 inhibitor AZD1480 markedly attenuated lung destruction, diminished protein leakage, and inhibited cytokine release. In addition, when mouse macrophage-like RAW 264.7 cells were exposed to LPS and cyclic stretch (CS), AZD1480 prevented cell autophagy, reduced apoptosis, and suppressed lactate dehydrogenase release by downregulating JAK2/STAT1 phosphorylation levels and inducing HMGB1 translocation from the nucleus to the cytoplasm. Furthermore, HMGB1 and STAT1 knockdown attenuated LPS+CS-induced autophagy and apoptosis in RAW 264.7 cells. In conclusion, these findings reveal the connection between the JAK2/STAT1 pathway and HMGB1 translocation in mediating lung inflammation and cell death in VILI, suggesting that these molecules may serve as novel therapeutic targets for VILI.

Endoplasmic Reticulum Stress Cooperates in Silica Nanoparticles-Induced Macrophage Apoptosis via Activation of CHOP-Mediated Apoptotic Signaling Pathway.

While silica nanoparticles (SiNPs) have wide applications, they inevitably increase atmospheric particulate matter and human exposure to this nanomaterial. Numerous studies have focused on how to disclose SiNP toxicity and on understanding its toxic mechanisms. However, there are few studies in the literature reporting the interaction between endoplasmic reticulum (ER) stress and SiNP exposure, and the corresponding detailed mechanisms have not been clearly determined. In this study, CCK-8 and flow cytometry assays demonstrated that SiNPs gradually decreased cell viability and increased cell apoptosis in RAW 264.7 macrophage cells in dose- and time-dependent manners. Western blot analysis showed that SiNPs significantly activated ER stress by upregulating GRP78, CHOP, and ERO1α expression. Meanwhile, western blot analysis also showed that SiNPs activated the mitochondrial-mediated apoptotic signaling pathway by upregulating BAD and Caspase-3, and downregulating the BCL-2/BAX ratio. Moreover, 4-phenylbutyrate (4-PBA), an ER stress inhibitor, significantly decreased GRP78, CHOP, and ERO1α expression, and inhibited cell apoptosis in RAW 264.7 macrophage cells. Furthermore, overexpression of CHOP significantly enhanced cell apoptosis, while knockdown of CHOP significantly protected RAW 264.7 macrophage cells from apoptosis induced by SiNPs. We found that the CHOP-ERO1α-caspase-dependent apoptotic signaling pathway was activated by upregulating the downstream target protein ERO1α and caspase-dependent mitochondrial-mediated apoptotic signaling pathway by upregulating Caspase-3 and downregulating the ratio of BCL-2/BAX. In summary, ER stress participated in cell apoptosis induced by SiNPs and CHOP regulated SiNP-induced cell apoptosis, at least partly, via activation of the CHOP-ERO1α-caspase apoptotic signaling pathway in RAW 264.7 macrophage cells.