Cholesterol-autoxidation metabolites in host defense against infectious diseases.

Cholesterol plays essential roles in biological processes, including cell membrane stability and myelin formation. Cholesterol can be metabolized to oxysterols by enzymatic or nonenzymatic ways. Nonenzymatic cholesterol metabolites, also called cholesterol-autoxidation metabolites, are formed dependent on the oxidation of reactive oxygen species (ROS) such as OH• or reactive nitrogen species, such as ONOO- . Cholesterol-autoxidation metabolites are abundantly produced in diseases such as inflammatory bowel disease and atherosclerosis, which are associated with oxidative stress. Recent studies have shown that cholesterol-autoxidation metabolites can further regulate the immune system. Here, we review the literature and summarize how cholesterol-autoxidation metabolites, such as 25-hydroxycholesterol (25-OHC), 7α/ß-OHC, and 7-ketocholesterol, deal with the occurrence and development of infectious diseases through pattern recognition receptors, inflammasomes, ROS production, nuclear receptors, G-protein-coupled receptor 183, and lipid availability. In addition, we include the research regarding the roles of these metabolites in COVID-19 infection and discuss our viewpoints on the future research directions.

Nickel oxide nanoparticles induce apoptosis and ferroptosis in airway epithelial cells via ATF3.

Exposure to nickel oxide nanoparticles (NiONPs), which have been widely produced and applied in industry, leads to adverse pulmonary and systemic effects. The aim of this study is to investigate the involvement of apoptosis and ferroptosis in NiONPs-induced acute lung injury (ALI). Intratracheal instillation of NiONPs into mice elevated the levels of pro-inflammatory cytokines, neutrophils, and proteins in the bronchoalveolar lavage fluid, and triggered apoptosis and ferroptosis in the lung tissues. Consistently, NiONPs-induced apoptosis and ferroptosis were observed in in vitro experiments using human lung epithelial cells. Activating transcription factor 3 (ATF3), a stress-inducible transcription factor, was upregulated by NiONPs exposure in both murine lung tissues and human lung epithelial cells. Moreover, human lung epithelial cells with ATF3 deficiency exhibited a lower level of apoptosis and ferroptosis when exposed to NiONPs. Collectively, our findings demonstrated that ATF3 was responsive to NiONPs exposure, and promoted NiONPs-induced apoptosis and ferroptosis in lung epithelial cells, indicating that ATF3 is a potential biomarker and therapeutic target for NiONPs-associated ALI.

LncRNA MALAT1 Participates in Protection of High-Molecular-Weight Hyaluronan against Smoke-Induced Acute Lung Injury by Upregulation of SOCS-1.

Smoke-induced acute lung injury (ALI) is a grievous disease with high mortality. Despite advances in medical intervention, no drug has yet been approved by the Food and Drug Administration (FDA) for ALI. In this study, we reported that pretreatment with high-molecular-weight hyaluronan (1600 kDa, HA1600) alleviated pulmonary inflammation and injury in mice exposed to smoke and also upregulated long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), as well as suppressor of cytokine signaling-1 (SOCS-1), in the lung tissues. Next, we overexpressed MALAT1 in the lungs by intratracheal administration of adenovirus cloned with MALAT1 cDNA and found that the survival of mice after smoke exposure was improved. Moreover, pulmonary overexpression of MALAT1 ameliorated smoke-induced ALI in mice and elevated the level of SOCS-1 in the lungs. In conclusion, the results pointed out that HA1600 exerted a protective effect against smoke-induced ALI through increasing the MALAT1 level and the subsequent SOCS-1 expression. Our study provides a potential therapeutic approach to smoke-induced ALI and a novel insight into the mechanism of action of HA1600.

ATF3 Promotes Arsenic-Induced Apoptosis and Oppositely Regulates DR5 and Bcl-xL Expression in Human Bronchial Epithelial Cells.

Arsenic is one of the most common environmental pollutants eliciting serious public health issues; however, it is also a well-recognized chemotherapeutic agent for acute promyelocytic leukemia. The association between arsenic exposure and lung diseases has been established, but underlying molecular mechanisms are poorly defined. Here we investigated the toxicology of arsenic in airway epithelium. Arsenic rapidly induced the activating transcription factor ATF3 expression through the JNK and p38 pathways. The ATF3-deficient BEAS-2B cells were relatively resistant to apoptosis upon arsenic exposure, indicating a facilitatory role of ATF3 in arsenic-induced apoptosis. We further showed that ATF3 oppositely regulated the transcription of death receptor (DR5) and Bcl2-like 1 (Bcl-xL) by directly binding to the promoter DR5 and Bcl-xL. Altogether, our findings establish ATF3 as a pro-apoptotic protein in arsenic-induced airway epithelial apoptosis through transcriptionally regulating DR5 and Bcl-xL, highlighting the potential of ATF3 as an early and sensitive biomarker for arsenic-caused lung injury.

High Molecular Weight Hyaluronan Suppresses Macrophage M1 Polarization and Enhances IL-10 Production in PM2.5-Induced Lung Inflammation.

PM2.5 is particulate matter with a diameter of 2.5 µm or less. Airway macrophages are the key players regulating PM2.5-induced inflammation. High molecular weight hyaluronan (HMW-HA) has previously been shown to exert protective effects on PM2.5-induced acute lung injury and inflammation. However, little is known about the detailed mechanism. In this study, we aimed to determine whether HMW-HA alleviates PM2.5-induced pulmonary inflammation by modulating macrophage polarization. The levels of M1 biomarkers TNF-α, IL-1ß, IL-6, CXCL1, CXCL2, NOS2 and CD86, as well as M2 biomarkers IL-10, MRC1, and Arg-1 produced by macrophages were measured by ELISA, qPCR, and flow cytometry. In addition, the amount of M1 macrophages in lung tissues was examined by immunofluorescence of CD68 and NOS2. We observed a decline in PM2.5-induced M1 polarization both in macrophages and lung tissues when HMW-HA was administered simultaneously. Meanwhile, western blot analysis revealed that PM2.5-induced JNK and p38 phosphorylation was suppressed by HMW-HA. Furthermore, in vitro and in vivo studies showed that co-stimulation with HMW-HA and PM2.5 promoted the expression and release of IL-10, but exhibited limited effects on the transcription of MRC1 and ARG1. In conclusion, our results demonstrated that HMW-HA ameliorates PM2.5-induced lung inflammation by repressing M1 polarization through JNK and p38 pathways and promoting the production of pro-resolving cytokine IL-10.

SOCS-1 ameliorates smoke inhalation-induced acute lung injury through inhibition of ASK-1 activity and DISC formation.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of apoptosis and pro-inflammatory cytokine signaling, two major contributors to the pathogenesis of ALI. We have found that SOCS-1 protects lung epithelial cells from smoke-induced apoptosis through two mechanisms. One is that SOCS-1 enhances degradation of ASK-1 and diminishes cleavage of pro-caspase-3 to repress smoke-triggered apoptosis in lung epithelial cells. The other is that SOCS-1 represses smoke-triggered DISC formation through altering TRADD-caspase-8 interaction rather than TNFR-1-TRADD interaction or TNFR-1-TRAF-2 interaction. In conclusion, SOCS-1 relieves smoke inhalation-induced lung injury by repressing ASK-1 and DISC-mediated epithelium apoptosis.

Isoliquiritigenin attenuates high glucose-induced proliferation, inflammation, and extracellular matrix deposition in glomerular mesangial cells by suppressing JAK2/STAT3 pathway.

To investigate the effect of isoliquiritigenin (ISL) on high glucose (HG)-induced glomerular mesangial cells (GMCs) proliferation, extracellular matrix (ECM) deposition and inflammation, and the underlying mechanisms. Mouse GMCs (SV40-MES-13) were cultured in HG medium, with or without ISL. The proliferation of GMCs was determined by MTT assay. The production of proinflammatory cytokines was detected by qRT-PCR and ELISA. The expression of connective tissue growth factor (CTGF), TGF-ß1, collagen IV, and fibronectin was measured by qRT-PCR and western blot. The phosphorylation of JAK2 and STAT3 was examined by western blot. Next, JAK2 inhibitor AG490 was applied to HG-exposed GMCs. The levels of JAK2/STAT3 phosphorylation and pro-fibrotic markers were analyzed by western blot, and the secretion of TNF-α and IL-1ß was evaluated by ELISA. GMCs were treated with HG, HG plus ISL or HG plus ISL, and recombinant IL-6 (rIL-6) which is a JAK2 activator. The levels of JAK2/STAT3 activation, ECM formation, and proinflammatory cytokines secretion were determined by western blot and ELISA, respectively. In mouse GMCs, ISL successfully repressed HG-induced hyperproliferation; production of TNF-α and IL-1ß; expression of CTGF, TGF-ß1, collagen IV, and fibronectin; and activation of JAK2/STAT3. Similar to ISL, AG490 was able to reverse the inflammation and ECM generation caused by HG. Moreover, rIL-6 impeded the amelioration of ISL on HG-induced adverse effects. Our study demonstrated that ISL displayed preventive effects on HG-exposed GMCs through inhibiting JAK2/STAT3 pathway and provided an insight into the application of ISL for diabetic nephropathy (DN) treatment.

Regulatory effect of orexin system on various diseases through mTOR signaling pathway.

Orexin (OX)A and OXB are a pair of neuropeptides secreted by orexin-producing neurons in the lateral hypothalamus. The orexin system can regulate many physiological processes through these two receptor pathways, such as feeding behavior, sleep/wake state, energy homeostasis, reward, and the coordination of emotion. Mammalian target of rapamycin (mTOR) can coordinate upstream signals with downstream effectors, thereby regulating fundamental cellular processes and also plays an essential role in the signaling network downstream of the orexin system. In turn, the orexin system can activate mTOR. Here, we review the association of the orexin system with the mTOR signaling pathway mainly by discussing that drugs in various diseases exert their effects on the orexin system, indirectly affecting the mTOR signaling pathway.

The Protective Mechanism of TFAM on Mitochondrial DNA and its Role in Neurodegenerative Diseases.

Mitochondrial transcription factor A (TFAM) is a mitochondrial protein encoded by nuclear genes and transported from the cytoplasm to the mitochondria. TFAM is essential for the maintenance, expression, and delivery of mitochondrial DNA (mtDNA) and can regulate the replication and transcription of mtDNA. TFAM is associated with the formation of mtDNA nucleomimetic structures, mtDNA repair, and mtDNA stability. However, the mechanism by which TFAM protects mtDNA is still being studied. This review provides a summary of the protective mechanism of TFAM on mtDNA including the discrete regulatory effects of TFAM acetylation and phosphorylation on mtDNA, the regulation of Ca2+ levels by TFAM to activate transcription in mitochondria, and the increased binding of TFAM to mtDNA damage hot spots. This review also discusses the association between TFAM and some neurodegenerative diseases.

KPNB1-mediated nuclear import in cancer.

Dysregulation of nucleocytoplasmic shuttling impairs cellular homeostasis and promotes cancer development. KPNB1 is a member of karyopherin ß family, mediating the transportation of proteins from the cytoplasm to the nucleus. In a variety of cancers, the expression of KPNB1 is upregulated to facilitate tumor growth and progression. Both downregulation of KPNB1 level and inhibition of KPNB1 activity prevent the entry of cancer-related transcription factors into the nucleus, subsequently suppressing the proliferation and metastasis of cancer cells. Currently, five KPNB1 inhibitors have been reported and exhibited good efficacy against cancer. This paper provides an overview of the role and mechanism of KPNB1 in different cancers and KPNB1-targeted anticancer compounds which hold promise for the future.

Design and biological features of platinum (II) complexes with 3-hydroxy-3-(Trifluoromethyl)cyclobutane-1,1-Dicarboxylate as a leaving ligand.

A series of platinum compounds 2a-5a and 2b-5b with fluoro-functional groups are designed and synthesized. Among them, complex 2b is the most effective agent with 3-hydroxy-3-(trifluoromethyl)cyclobutane-1,1-dicarboxylate as a leaving ligand, which showed better cytotoxic activity than compounds containing only CF3 or OH group at 3-position of cyclobutane-1,1-dicarboxylate. The water solubility of 2a is better than that of carboplatin (32 mg/mL vs. 16 mg/mL), and its antitumor activity on A549 is 4.6-fold higher than that of carboplatin. The IC50 value of 2b on A549 cells is 4.73 ± 0.64 µM, which is comparable to that of oxaliplatin and higher than that of carboplatin. Meanwhile, 2a and 2b are less toxic than oxaliplatin and cisplatin toward BEAS-2B cells. Moreover, 2a and 2b induce cell apoptosis in vitro by the Bax-Bcl-2-caspase-3 pathway and ferroptosis through inhibiting GPx-4 and elevating COX2. Results from in vivo experiment show that the inhibition rate of A549 xenograft tumor is cisplatin > 2b > oxaliplatin > 2a > carboplatin.

ATF3 inhibits arsenic-induced malignant transformation of human bronchial epithelial cells by attenuating inflammation.

Arsenic is a naturally occurring metalloid strongly associated with the incidence of lung cancer. Understanding the mechanisms of arsenic-induced carcinogenesis favors the development of effective interventions to reduce the incidence and mortality of lung cancer. In this study, we investigated the role of activating transcription factor 3 (ATF3) in arsenic-induced transformation of human bronchial epithelial cells. ATF3 was upregulated during chronic exposure to 0.25 µM arsenic, and loss of ATF3 promoted arsenic-induced transformation. Moreover, arsenic-transformed ATF3 knockout (ATF3 KO-AsT) cells exhibited more aggressive characteristics, including acceleration in proliferation, resistance to chemotherapy and increase in migratory capacity. RNA-seq revealed that pathways involved in inflammation, cell cycle, EMT and oncogenesis were affected due to ATF3 deficiency during chronic arsenic exposure. Further experiments confirmed the overproduction of IL-6, IL-8 and TNFα as well as enhanced phosphorylation of AKT and STAT3 in ATF3 KO-AsT cells. Our results demonstrate that ATF3 upregulated by chronic low-dose arsenic exposure represses cell transformation and acquisition of malignant characteristics through inhibiting the production of proinflammatory cytokines and activation of downstream proteins AKT and STAT3, providing a new strategy for the prevention of carcinogen-induced lung cancer.

Resveratrol-Responsive CArG Elements from the Egr-1 Promoter for the Induction of GADD45α to Arrest the G2/M Transition.

Suicide gene therapy is based on the introduction of a foreign gene into tumor cells to sensitize cells to treatment, to convert a nontoxic compound into a lethal drug, or to produce a cytotoxic effect. We have constructed a suicide gene therapy vector that contains resveratrol-responsive CArG elements from the Egr-1 promoter and the GADD45α open reading frame. CArG elements are utilized as a "molecular switch" to drive the expression of GADD45α. When transfected into lung cancer cells, the vector is able to express GADD45α upon resveratrol treatment, and subsequently leads to cell cycle arrest at the G2/M transition. In this chapter, we describe a detailed protocol for vector construction, transfection, cell viability assay, and cell cycle analysis.

Astragalus polysaccharide strengthens the inflammatory and immune responses of Brucella suis S2-infected mice and macrophages.

Brucella infection is one of the most serious zoonoses worldwide, affecting humans and domestic and wild animals. Astragalus polysaccharide (APS) is extracted from astragalus, which exhibits bioactive properties, including immunomodulation and anti-tumour and antiviral activity. The present study revealed that APS treatment promoted macrophage activation, the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-12 and interferon-γ, and Brucella clearance in murine macrophages and spleens. APS treatment was also demonstrated to protect the integrity of macrophages during infection with live attenuated Brucella suis strain 2 (B. suis S2). The results from in vitro experiments were consistent with the findings from the in vivo study, showing the elevated secretion of TNF-α and nitric oxide in APS-treated murine peritoneal macrophages following B. suis S2 infection. The current study demonstrated the potential of APS in the control and treatment of Brucella infection, and the enhancement of host inflammatory and immune responses.

Improving the anticancer activity of platinum(iv) prodrugs using a dual-targeting strategy with a dichloroacetate axial ligand.

Four novel platinum(iv) complexes, characteristic of DCA/TFA and with chloride ions as axial ligands, were designed and synthesized. This type of platinum(iv) complexes 1a-2b exhibited significant cytotoxic activity, and the cytotoxicity of 1b was the greatest among these four complexes, which was 20.61 fold and 7.65 fold higher than that of cisplatin against HepG-2 and NCI-H460 cancer cells, respectively. The result from the apoptosis assay of 1b was consistent with the result from the cytotoxicity assay. In addition, complexes 1a and 1b induced cell cycle arrest at the S phase on HepG-2 cells. Taken together, our data showed that Pt(iv) complex 1b released the corresponding Pt(ii) complex and DCA, and induced apoptosis as well as disruption of the mitochondrial membrane potential, establishing Pt(iv) complex 1b as a potential dual-targeting anticancer agent.

GADD45α-targeted suicide gene therapy driven by synthetic CArG promoter E9NS sensitizes NSCLC cells to cisplatin, resveratrol, and radiation regardless of p53 status.

Background: GADD45α is a tumor suppressor protein often upregulated by environmental stresses and DNA-damage agents to cause growth arrest, apoptosis, tumor growth inhibition, and anti-angiogenesis. A novel suicide gene therapy vector pE9NS.G45α was engineered by cloning GADD45α opening reading frame downstream to the synthetic CArG promoter E9NS, which contains nine repeats of CArG element with modified core A/T sequence and functions as a molecular switch to drive the expression of GADD45α. The current study aims to determine the efficacy of this suicide gene therapy vector in combination with cisplatin, resveratrol, and radiation in NSCLC cell lines with various p53 statuses. Methods: Three NSCLC cell lines, H1299 (deleted p53), A549 (wild-type p53), and H23 (mutated p53), were examined in the present investigation to represent NSCLC with different p53 functions. MTT assay was conducted to select suitable doses of cisplatin, resveratrol, and radiation for gene therapy, and dual luciferase assay was performed to validate the activation of promoter E9NS. The efficacy of gene therapy combinations was evaluated by the amount of GADD45α expression, cell survival, and apoptosis. Results: All the combinations successfully activated promoter E9NS to elevate intracellular GADD45α protein levels and subsequently enhanced cell viability reduction and apoptosis induction regardless of p53 status. Conclusion: Our study demonstrates that GADD45α-targeted suicide gene therapy controlled by synthetic promoter E9NS sensitizes NSCLC cells to cisplatin, resveratrol, and radiation and is effective against NSCLC at least in vitro.

High molecular weight hyaluronan attenuates fine particulate matter-induced acute lung injury through inhibition of ROS-ASK1-p38/JNK-mediated epithelial apoptosis.

Inhalation of fine particulate matter (PM2.5) is asscoiated with lung injury. High molecular weight hyaluronan (HMW-HA) is an essential constituent of extracellular matrix (ECM), exhibiting anti-oxidative and anti-inflammatory properties when administered by injection, inhalation, nebulization or gene delivery of HA synthases. The aim of the present study is to determine whether HMW-HA alleviates PM2.5-induced acute lung injury (ALI) and investigate the underlying mechanisms. We observed that HMW-HA suppressed pathological injury, inflammation, oxidative stress, edema and epithelial damage caused by PM2.5 in the lungs of the rats. The protective mechanism of HMW-HA was further explored in vitro. The results elucidated that reactive oxygen species (ROS) was involved in PM2.5-induced cell apoptosis, and HMW-HA mitigated the oxidative potential of PM2.5, subsequently inhibiting phosphorylation of ASK1 at Thr845, downstream phosphorylation of p38 and JNK, and eventual apoptosis. Our study indicates that HMW-HA is a promising strategy in the prevention of PM2.5-induced pulmonary damage.

SOCS-1 Suppresses Inflammation Through Inhibition of NALP3 Inflammasome Formation in Smoke Inhalation-Induced Acute Lung Injury.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality rates. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of proinflammatory cytokine signaling. We have found that adenoviral gene transfer of SOCS-1 ameliorates smoke inhalation-induced lung injury in C57BL/6 mice. We also found that the release of adenosine triphosphate (ATP) was increased post smoke exposure, while oxidized ATP, an inhibitor of purinergic P2X7 receptor, suppressed smoke-induced NALP3 inflammasome assembly, caspase-1 activation, and K+ efflux. Similar to oxidized ATP, high protein level of SOCS-1 dampened the formation of NALP3 inflammasome and the activation of caspase-1 and IL-1ß induced by smoke exposure in mouse alveolar macrophages. In conclusion, SOCS-1 relieves smoke inhalation-induced pulmonary inflammation and injury by inhibiting NALP3 inflammasome formation.

CArG-driven GADD45α activated by resveratrol inhibits lung cancer cells.

We report anticarcinogenic effects of suicide gene therapy that relies on the use of resveratrol-responsive CArG elements from the Egr-1 promoter to induce GADD45α. In A549 lung cancer cells, endogenous GADD45α was not induced upon resveratrol treatment. Therefore, induction of exogenous GADD45α resulted in growth inhibition. Resveratrol transiently induced Egr-1 through ERK/JNK-ElK-1. Hence, we cloned natural or synthetic Egr-1 promoter upstream of GADD45α cDNA to create a suicide gene therapy vector. Since natural promoter may have antagonized effects, we tested synthetic promoter that contains either five, six or nine repeats of CArG elements essential in the Egr-1 promoter to drive the expression of GADD45α upon resveratrol treatment. Further analysis confirmed that both synthetic promoter and natural Egr-1 promoter were able to "turn on" the expression of GADD45α when combined with resveratrol, and subsequently led to suppression of cell proliferation and apoptosis.

Sequential activation of Elk-1/Egr-1/GADD45α by arsenic.

Long-term exposure to arsenic, an environmental contaminant, leads to increased risks of cancers. In the present study, we investigated the sequential regulation of Elk-1 and Egr-1 on As3+-induced GADD45α, an effector of G2/M checkpoint. We found that As3+ transcriptionally induced both Elk-1 and Egr-1, and NF-κB binding site was necessary for As3+-induced Egr-1 promoter activity. However, specific inhibition of JNK, ERK, and Elk-1 inhibited Egr-1 induction. Furthermore, silencing of Egr-1 downregulated As3+-induced expression of GADD45α and ChIP assay confirmed the direct binding of Egr-1 to GADD45α promoter. Taken together, our data indicated that the increase of GADD45α in response to As3+ was mediated sequentially by Elk-1 and Egr-1.