Total flavones of Abelmoschus manihot improve diabetic nephropathy by inhibiting the iRhom2/TACE signalling pathway activity in rats

Context: Total flavones extracted from Abelmoschus manihot L. (Malvaceae) medic (TFA) have been proven clinically effective at improving renal inflammation and glomerular injury in chronic kidney disease (CKD).

Objective: This study evaluated the function of TFA as an inhibitor of iRhom2/TACE (tumour necrosis factor-α converting enzyme) signalling and investigated its anti-DN (diabetic nephropathy) effects in a DN rat model.

Materials and methods: In vitro, cells were treated with 200?μg/mL advanced glycation end products (AGEs), and then co-cultured with 20?μg/mL TFA for 24?h. Real time PCR, western blotting and co-immunoprecipitation assays were performed. In vivo, DN was induced in 8 week old male Sprague-Dawley rats via unilateral nephrectomy and intraperitoneal injection of streptozotocin, then TFA were administered to rats by gavage for 12 weeks at three different doses (300, 135 and 75?mg/kg/d). 4-Phenylbutanoic acid (2.5?mg/kg/d) was used as a positive control.

Results: IC₅₀ of TFA is 35.6?μM in HK2 and 39.6?μM in HRMC. TFA treatment (20?μM) inhibited the activation of iRhom2/TACE signalling in cultured cells induced by AGEs. LD₅₀>26?g/kg and ED₅₀=67?mg/kg of TFA in rat by gavage, TFA dose-dependently downregulated the expression of proinflammatory cytokines and exerted anti-inflammatory effects significantly though inhibiting the activation of iRhom2/TACE signalling.

Discussion and conclusions: Our results show that TFA could dose-dependently ameliorate renal inflammation by inhibiting the activation of iRhom2/TACE signalling and attenuating ER stress. These results suggest that TFA has potential therapeutic value for the treatment of DN in humans.     

Association of systemic inflammation with marked changes in particulate air pollution in Beijing in 2008

Many studies have linked ambient fine particulate matter (aerodynamic diameters less than 2.5 μm, PM_2.5) air pollution to increased morbidity and mortality of cardiovascular diseases in the general population, but the biologic mechanisms of these associations are yet to be elucidated. In this study, we aimed to evaluate the relationship between daily variations in exposure to PM_2.5 and inflammatory responses in mice during and for 2 months after the Beijing Olympic Games. Male C57BL/6 mice were exposed to Beijing PM_2.5 or filtered air (FA) in 2008 during the 2 months of Beijing Olympic and Paralympic Games, and for 2 months after the end of the Games. During the Games, circulating monocyte chemoattractant protein 1 and interleukin 6 were increased significantly in the PM_2.5 exposure group, when compared with the FA control group, although there were no significant inter-group differences in tumor necrosis factor-α or interferon-γ, or in macrophages, neutrophils or lymphocytes in the spleen or thymus between these 2 groups. However, macrophages were significantly increased in the lung and visceral fat with increasing PM_2.5. After the Olympic Games, there were no significant PM_2.5-associated differences for macrophages, neutrophils or lymphocytes in the thymus, but macrophages were significantly elevated in the lung, spleen, subcutaneous and visceral fat with increasing PM_2.5, and the numbers of macrophages were even higher after than those during the Games. Moreover, the number of neutrophils was markedly higher in the spleen for the PM_2.5-exposed- than the FA-group. These data suggest that short-term increases in exposure to ambient PM_2.5 leads to increased systemic inflammatory responses, primarily macrophages and neutrophils in the lung, spleen, and visceral adipose tissue. Short-term air quality improvements were significantly associated with reduced overall inflammatory responses.     

Impact of probable interaction of low temperature and ambient fine particulate matter on the function of rats alveolar macrophages

The present study aimed to explore the probable interaction of low temperature and ambient fine particulate matter (PM_2.5) on rat alveolar macrophages (AMs). AMs were separated from rat BALF and exposed to PM_2.5 (0, 25, 50, 100 μg/ml) under different temperature (18, 24, 30, 37 °C) for 8 h. Results indicated that viability and phagocytosis function of AMs decreased with the decline of temperature and the rise of PM_2.5 dose, and the strongest toxicity was shown in the highest PM_2.5 (100 μg/ml) exposure group at 18 °C. Both PM_2.5 and lower temperature increased the releasing of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 1α (MIP-1α) and interleukin-6 (IL-6), while significant interaction was only found in MIP-1α production. No obvious change was found in granulocyte-macrophage colony-stimulating factor (GM-CSF) detection. These results indicated that both the two factors are harmful to rat AMs and lower temperature could increase the toxicity of PM_2.5 on the AMs.     

Ginsenoside Rg1 reduces toxicity of PM2.5 on human umbilical vein endothelial cells by upregulating intracellular antioxidative state

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5 μm (PM_2.5) and protective effect of Ginsenoside Rg1 (Rg1) against PM_2.5 on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM_2.5 on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm–nucleus location were assayed. The present results showed that PM_2.5 (50–800 μg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40 μg/ml) diminished PM_2.5-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM_2.5 increase oxidative stress and decrease cell viability; Rg1 antagonize PM_2.5-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM_2.5 and Rg1 on HUVECs.     

Resveratrol relieves particulate matter (mean diameter < 2.5 μm)‐induced oxidative injury of lung cells through attenuation of autophagy deregulation

Oxidative stress and inflammation are critically implicated in ambient fine particulate matter (mean diameter < 2.5 μm; PM_2.5)‐induced lung injury. Autophagy, playing a crucial role in various physiopathological conditions, modulates cellular homeostasis and stress adaptation. Resveratrol is a phytoalexin that exerts potent antioxidant effects on cardiopulmonary diseases. To date, the mechanisms by which resveratrol protects against PM_2.5 remain to be elucidated. In the present study, we investigated the effect of resveratrol on PM_2.5‐induced oxidative injury. The potential role of nuclear factor erythroid‐2‐related factor 2 and autophagy in this progress was explored. Human bronchial epithelial cells were treated with PM_2.5 and the cytotoxicity and oxidative stress markers were determined. The results showed that PM_2.5 decreased cell viability and elevated the level of lactate dehydrogenase. The levels of malondialdehyde and reactive oxygen species were increased by PM_2.5 exposure. PM_2.5 also induced a significant increase of the inflammatory cytokines including interleukin (IL)‐6, IL‐8, IL‐1β and tumor necrosis factor α. Meanwhile, PM_2.5 triggered autophagy formation and alteration of the nuclear factor erythroid‐2‐related factor 2 pathway. Furthermore, human bronchial epithelial cells were co‐treated with PM_2.5 and resveratrol in the presence or absence of 3‐methylamphetamine, an inhibitor of autophagic formation. It was revealed that resveratrol intervention abolished PM_2.5‐induced oxidative injury partially through the suppression of autophagy deregulation. Findings from this study could provide new insights into the molecular mechanisms of pulmonary intervention during PM_2.5 exposure.     

Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway

Sulforaphane (SFN) is a natural isothiocyanate that is present in cruciferous vegetables such as broccoli and cabbage. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents, which is related in part to its potent anti-inflammation properties. In the present study, we compared the anti-inflammatory effect of SFN on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 (−/−) mice. Pretreatment of SFN in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNF-α, IL-1β, COX-2 and iNOS. HO-1 expression was significantly augmented in LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by SFN. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (−/−) primary peritoneal macrophages. We concluded that SFN exerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages.     

NLRP3 inflammasome activation is associated with PM2.5‐induced cardiac functional and pathological injury in mice

Growing evidences indicate that inflammation induced by PM_2.5 exposure has been considered as a major driving force for the development of cardiovascular diseases. However, the mechanisms underlying PM_2.5‐induced cardiac injury remain unclear. This study aims to investigate the role of NLRP3 inflammasome in PM_2.5‐induced cardiac functional and pathological injury in mice. In this study, BALB/c mice were intratracheally instilled with PM_2.5 suspension (4.0 mg/kg BW) for 5 days to set up a cardiac injury model, which was evaluated by electrocardiogram monitoring, HE and Masson staining. Then, the effects of PM_2.5 on the expression of α‐SMA, NLRP3, IL‐1β, and IL‐18 proteins and the activation of caspase‐1 and IL‐1β were investigated. The results showed that PM_2.5 exposure induced characteristic abnormal ECG changes such as the abnormality of heart rhythm, tachycardia, and T‐wave reduction. Inflammatory cell infiltration and fibrosis were observed in the heart tissues of PM_2.5‐exposed mice. Meanwhile, PM_2.5 exposure increased the expression of α‐SMA. And, NLRP3 activation‐associated proteins of NLRP3, IL‐1β, IL‐18, Cleaved caspase‐1 p10, and Cleaved IL‐1β were upregulated in heart tissue of PM_2.5‐induced mice. In summary, PM_2.5 exposure could induce cardiac functional and pathological injury, which may be associated with the activation of NLRP3 inflammasome.     

The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non‐allergic families in Wuhan on mouse peritoneal macrophages

Recent studies have shown that fine particulate matter (PM_2.5) is associated with multiple adverse health outcomes and PM_2.5‐induced oxidative stress is now commonly known as a proposed mechanism of PM_2.5‐mediated toxicity. However, the association between allergic symptoms in children and exposure to PM_2.5 has not been fully elucidated, particularly the role of PM_2.5 on the indoor environment involved in allergy or non‐allergy is unknown. The aim of the present study was to explore whether indoor PM_2.5 from the homes of children with allergic symptoms had more increased risks of allergy than that of healthy ones and then compare the toxicity and inflammatory response of them. In this study, indoor PM_2.5 was collected from the homes of schoolchildren with allergic symptoms and those of healthy ones respectively, and components of PM_2.5 were analyzed. PM_2.5‐mediated oxidative damage and inflammatory response were further evaluated in mouse peritoneal macrophages based on its effects on the levels of reactive oxygen species accumulation, lipid peroxidation, DNA damage or cytokine production. It seems that oxidative stress may contribute to PM_2.5‐induced toxicity, and PM_2.5 from the allergic indoor environment produced more serious toxic effects and an inflammatory response on mouse peritoneal macrophages than that from a non‐allergic indoor environment. Copyright © 2015 John Wiley & Sons, Ltd.     

Treg responses are associated with PM2.5-induced exacerbation of viral myocarditis

Abstract

The adverse cardiovascular events induced by ambient fine particles (PM_2.5) are paid more attention in the world. The current study was conducted to explore the mechanisms of T regulatory cells (Treg) responses in PM_2.5-induced exacerbation of viral myocarditis. The male BALB/c mice were administered an intratracheal (i.t.) instillation of 10?mg/kg b.w. PM_2.5 suspension. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100?μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, serum, splenetic, and cardiac tissues were examined. The results showed that pre-exposure to PM_2.5 aggravated the cardiac inflammation in the CVB3-infected mice along with an increase of Treg cells in the spleen. The mRNA expressions of interleukin-6 (IL-6), TNF-α, transforming growth factor-β (TGF-β), and Foxp3 were up-regulated in the PM_2.5-pretreated mice than that in the CVB3-treated mice. Similar results were found in the sera. In addition, compared with the CVB3-treated mice, the cardiac protein expression of TGF-β increased in the PM_2.5-pretreated mice. These results demonstrated that preexposure to PM_2.5 exacerbated virus-induced myocarditis possibly through the depression of the immune response and increase of inflammation in myocardium through the Treg responses.     

5种炎性刺激剂对小鼠腹腔巨噬细胞生成肿瘤坏死因子的影响

肿瘤坏死因子α（ＴＮＦα）主要由单核巨噬细胞分泌,近年研究表明,它在炎症中起重要作用,如诱发皮肤炎症反应,引起过敏性变态反应等。慢性炎症性疾病类风湿性关节炎与ＴＮＦα关系密切［１,２］。ＴＮＦα对血管内皮细胞、白细胞、单核巨噬细胞、破骨细胞、滑膜细胞等具广泛的调节效应［３］。ＴＮＦα能直接诱导中性多核白细胞释放嗜天青颗粒、氧自由基和溶酶体酶,既有杀伤病原体的有利作用,又有引起炎症部位组织细胞损伤的有害作用。本文研究了５种外源性刺激剂脂多糖（ＬＰＳ）、佛波脂（ＰＭＡ）、钙离子载体Ａ３２１８７、甲…     

The role of pro-/anti-inflammation imbalance in Aβ42 accumulation of rat brain co-exposed to fine particle matter and sulfur dioxide

Taiyuan is a center of coal-based electricity production and many chemicals industries, where mixtures of sulfur dioxide (SO₂) and particulate matter may be more prominent. The focus of the present study was to determine if there is a link between adverse effects in the brain and the combined-exposure to SO₂ and fine particulate matter (PM_2.5). Rats were exposed alternately to PM_2.5 with different dosages (1.5, 6.0 and 24.0?mg/kg body weight) and SO₂ at the level of 5.6?mg/m³. The results showed that the combined exposure to PM_2.5 and SO₂ enhanced the mRNA expression and protein level of TNF-α and IL-6 in rat cortex and hippocampus relative to the control, SO₂ and PM_2.5 alone. Instead, TGF-β1 mRNA and protein level were down-regulated in the brain. Additionally, PM_2.5 at medium and/or high dose caused marked increase in Aβ42 level and PM_2.5?+?SO₂ induced further increase of Aβ42 level in the cortex and hippocampus. It suggests that SO₂ and PM_2.5 can synergistically exert inflammation responses and induce Aβ42 accumulation in the brain. Also, it is notable that the Aβ42 accumulation of rat cortex and hippocampus were closely associated with pro-/anti-inflammatory cytokines ratio. These results clearly demonstrated that the combined exposure to PM_2.5 and SO₂ can induce the imbalance of pro-/anti-inflammatory cytokine, resulting in Aβ42 accumulation of rat brain cortex and hippocampus.     

Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells

Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM_2.5) and cardiovascular disorders, but how PM_2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen‐activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM_2.5. H9c2 cells were incubated with PM_2.5 at 100–800 µg ml^–1 to evaluate the effects of PM_2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis‐related proteins as well as activation of MAPKs. PM_2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration‐dependent manner. PM_2.5 decreased the Bcl‐2/Bax ratio and increased cleaved caspase‐3 levels. A Western blots study showed up‐regulation of phosphorylated MAPKs including extracellular signal‐regulated protein kinases (ERKs), c‐Jun NH₂‐terminal kinases (JNKs) and p38 MAPK in the PM_2.5‐treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM_2.5 on apoptosis and the expression of related proteins. In conclusion, PM_2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM_2.5‐induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd.     

Hydrogen gas reduces HMGB1 release in lung tissues of septic mice in an Nrf2/HO-1-dependent pathway

BackgroundLung injury is a vital contributor of mortality in septic patients. Our previous studies have found that molecular hydrogen (H₂), which has anti-oxidant, anti-inflammatory, and anti-apoptosis effects, had a therapeutic effect on a septic animal model through increasing expression of nuclear factor-erythroid 2-related factor 2 (Nrf2). The aim of this research was to investigate the effects of 2% H₂ gas inhalation on sepsis-induced lung injury and its underlying mechanisms.MethodsMale wild-type (WT) and Nrf2-knockout (Nrf2-KO) ICR mice underwent sham or cecal ligation and puncture (CLP) operation. Two percent of H₂ gas was inhaled for 60 min beginning at both 1 h and 6 h after sham or CLP surgery. To assess the severity of septic lung injury, the 7-day survival rate, wet/dry (W/D) weight ratio of lung tissue, lung histopathologic score, pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), high-mobility group box 1 (HMGB1)), anti-inflammatory cytokine (interleukin 10 (IL-10)), antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and heme oxygenase 1 (HO-1)), and an oxidative product (malondialdehyde (MDA)) were detected after sham or CLP operation. The histopathologic changes were observed in lung tissues by hematoxylin and eosin (HE) staining, and pro-inflammatory cytokines (TNF-α and IL-6), anti-inflammatory cytokine (IL-10), antioxidant enzymes (SOD and CAT), and MDA were detected in lung tissues by an enzyme-linked immunosorbent assay (ELISA).ResultsThe results indicated that 2% H₂ gas treatment increased the survival rates, decreased the W/D weight ratio and the lung injury score, alleviated the injuries caused by oxidative stress and inflammation, and induced HO-1 level but reduced HMGB1 level in WT but not Krf2-KO mice. These data reveal that H₂ gas could suppress lung injury in septic mice through regulation of HO-1 and HMGB1 expression and that Nrf2 plays a main role in the protective effects of H₂ gas on lung damage caused by sepsis.     

TNF-alpha preserves lysosomal stability in macrophages: A potential defense against oxidative lung injury

Iron-catalyzed oxidative damage on the respiratory epithelium is prevented by alveolar macrophages depositing iron inside their lysosomes. Bound in an un-reactive state to various metalloproteins, e.g. ferritin, most lysosomal iron is kept separated from reactive oxygen species (ROS) by intracellular anti-oxidative enzyme systems. Some ROS may, however, escape this protective shield of antioxidants, react with small amounts of free redox-active iron within lysosomes, thereby causing peroxidative damage on lysosomes and possibly also ensuing cell death. Since macrophages, containing large amounts of lysosomal iron, are very resistant to TNF-α, we hypothesized that this cell type has developed specific defense mechanisms against TNF-α-induced ROS generation. Murine macrophages were exposed (or not) to non-toxic concentrations of TNF-α and/or iron and were then challenged with H₂O₂. Iron-exposed oxidatively stressed cells exhibited extensive lysosomal disruption resulting in pronounced cell death. In contrast, TNF-α stabilized lysosomes and protected cells, particularly those iron-exposed, by reducing cellular iron and increasing H-ferritin. Intracellular generation of H₂O₂ under oxidative stress was kept unchanged by TNF-α and/or iron. However, TNF-α increased basal levels of glutathione by up-regulating the synthesis of γ-glutamylcystein synthetase, thereby strengthening the anti-oxidative capacity. TNF-α inhibitors would block this novel anti-oxidative defense system, possibly explaining their adverse effects on the lung.     

TLR-2 is involved in airway epithelial cell response to air pollution particles

Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of several oxidant stress response genes. Components of ambient air PM responsible for stimulating epithelial cells have not been conclusively identified, although metal contaminants, benzo[a]pyrene and biological matter have been implicated. Stimulation of IL-8 release from NHBE with coarse (PM_2.5-10), fine (PM_2.5), and UF particle fractions has shown that the coarse particle fraction has the greatest effect on the epithelial cells as well as alveolar macrophages (AM). Since this fraction concentrates fugitive dusts and particle-associated microbial matter, it was hypothesized that NHBE may recognize PM through microbial pattern recognition receptors TLR2 and TLR4, as has been previously shown with AM. NHBE were shown to release IL-8 when exposed to a Gram-positive environmental isolate of Staphylococcus lentus, and lower levels when exposed to Gram-negative Pseudomonas spp. Comparison of TLR2 and TLR4 mRNA expression in NHBE and AM showed that NHBE express similar levels of TLR2 mRNA as the AM, but expressed very low levels of TLR4. When NHBE were stimulated with PM_2.5-10, PM_2.5, and UF PM, in the presence or absence of inhibitors of TLR2 and TLR4 activation, a blocking antibody to TLR2 inhibited production of IL-8, while TLR4 antagonist E5531 or the LPS inhibitor Polymixin B had no effect. Furthermore, effects on expression of TLR2 and TLR4 mRNA, as well as the stress protein HSP70 was assessed in NHBE exposed to PM. TLR4 expression was increased in these cells while TLR2 mRNA levels were unchanged. Hsp70 was increased by PM_2.5-10 > PM_2.5 > UF PM suggesting the possibility of indirect activation of TLR pathway by this endogenous TLR2/4 agonist.     

Inhibitory effect of particulate matter on toll-like receptor 9 stimulated dendritic cells by downregulating mitogen-activated protein kinase and NF-κB pathway

ABSTRACT

Ambient particulate matter (PM) is associated with adverse health consequences. However, the influence of PM on the innate immune system is poorly understood. The aim of the present study was to examine the effect of diesel particulate matter 2.5 μm (PM_2.5, SRM1650b) on dendritic cells. PM_2.5 significantly reduced cytokine levels of interleukin (IL)-12 p40, IL-6 and TNF-α levels in CpG-DNA (TLR9 ligand)-stimulated dendritic cells. To determine the mechanisms underlying this observed inhibition induced by PM_2.5, western blot analysis was conducted. PM_2.5 was found to downregulate ERK1/2, JNK1/2, p38 MAPKs, and NF-κB pathways. PM_2.5 exposure decreased TLR9-dependent NF-κB and activator protein (AP-1) reporter luciferase activities. Our findings demonstrate that PM_2.5 reduced the production of cytokines which may be associated with inhibition of MAPK and NF-κB signaling pathway. Further, data suggest the immunosuppressive effect of PM_2.5 on the innate immune cells may lead to serious damage to the host immune system.