Nrf2 attenuates inflammatory response in COPD/emphysema: Crosstalk with Wnt3a/β‐catenin and AMPK pathways

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2^?/? mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.     

Inhibition of MD2‐dependent inflammation attenuates the progression of non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD) can progress to the more serious non‐alcoholic steatohepatitis (NASH), characterized by inflammatory injury and fibrosis. The pathogenic basis of NAFLD progressing to NASH is currently unknown, but growing evidence suggests MD2 (myeloid differentiation factor 2), an accessory protein of TLR4, is an important signalling component contributing to this disease. We evaluated the effectiveness of the specific MD2 inhibitor, L6H21, in reducing inflammatory liver injury in a relevant high‐fat diet (HFD) mouse model of NASH and in the palmitic acid (PA)‐stimulated human liver cell line (HepG2). For study, genetic knockout (MD2^?/?) mice were fed a HFD or control diet for 24 weeks, or wild‐type mice placed on a similar diet regimen and treated with L6H21 for the last 8 or 16 weeks. Results indicated that MD2 inhibition with L6H21 was as effective as MD2 knockout in preventing the HFD‐induced hepatic lipid accumulation, pro‐fibrotic changes and expression of pro‐inflammatory molecules. Direct challenge of HepG2 with PA (200 μM) increased MD2‐TLR4 complex formation and expression of pro‐inflammatory and pro‐fibrotic genes and L6H21 pre‐treatment prevented these PA‐induced responses. Interestingly, MD2 knockout or L6H21 increased expression of the anti‐inflammatory molecule, PPARγ, in liver tissue and the liver cell line. Our results provide further evidence for the critical role of MD2 in the development of NASH and conclude that MD2 could be a potential therapeutic target for NAFLD/NASH treatment. Moreover, the small molecule MD2 inhibitor, L6H21, was an effective and selective investigative agent for future mechanistic studies of MD2.     

Zingerone attenuates aortic banding‐induced cardiac remodelling via activating the eNOS/Nrf2 pathway

Cardiac remodelling refers to a series of changes in the size, shape, wall thickness and tissue structure of the ventricle because of myocardial injury or increased pressure load. Studies have shown that cardiac remodelling plays a significant role in the development of heart failure. Zingerone, a monomer component extracted from ginger, has been proven to possess various properties including antioxidant, anti‐inflammatory, anticancer and antidiabetic properties. As oxidative stress and inflammation contribute to acute and chronic myocardial injury, we explored the role of zingerone in cardiac remodelling. Mice were subjected to aortic banding (AB) or sham surgery and then received intragastric administration of zingerone or saline for 25 days. In vitro, neonatal rat cardiomyocytes (NRCMs) were treated with zingerone (50 and 250 μmol/L) when challenged with phenylephrine (PE). We observed that zingerone effectively suppressed cardiac hypertrophy, fibrosis, oxidative stress and inflammation. Mechanistically, Zingerone enhanced the nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2)/antioxidant response element (ARE) activation via increasing the phosphorylation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Additionally, we used Nrf2‐knockout (KO) and eNOS‐KO mice and found that Nrf2 or eNOS deficiency counteracts these cardioprotective effects of zingerone in vivo. Together, we concluded that zingerone may be a potent treatment for cardiac remodelling that suppresses oxidative stress via the eNOS/Nrf2 pathway.     

Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR‐δ/AMPK pathway

Clinical trials have shown that angiotensin II receptor blockers reduce the new onset of diabetes in hypertensives; however, the underlying mechanisms remain unknown. We investigated the effects of telmisartan on peroxisome proliferator activated receptor γ (PPAR‐δ) and the adenosine monophosphate (AMP)‐activated protein kinase (AMPK) pathway in cultured myotubes, as well as on the running endurance of wild‐type and PPAR‐δ‐deficient mice. Administration of telmisartan up‐regulated levels of PPAR‐δ and phospho‐AMPKα in cultured myotubes. However, PPAR‐δ gene deficiency completely abolished the telmisartan effect on phospho‐AMPKαin vitro. Chronic administration of telmisartan remarkably prevented weight gain, enhanced running endurance and post‐exercise oxygen consumption, and increased slow‐twitch skeletal muscle fibres in wild‐type mice, but these effects were absent in PPAR‐δ‐deficient mice. The mechanism is involved in PPAR‐δ‐mediated stimulation of the AMPK pathway. Compared to the control mice, phospho‐AMPKα level in skeletal muscle was up‐regulated in mice treated with telmisartan. In contrast, phospho‐AMPKα expression in skeletal muscle was unchanged in PPAR‐δ‐deficient mice treated with telmisartan. These findings highlight the ability of telmisartan to improve skeletal muscle function, and they implicate PPAR‐δ as a potential therapeutic target for the prevention of type 2 diabetes.     

Pirfenidone attenuates nonalcoholic fatty liver disease through activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway

Nonalcoholic fatty liver disease (NAFLD) originates from the hepatopathy of fatty liver. Pirfenidone is a novel broad-spectrum anti-fibrosis agent used for treating various kinds of tissue fibrosis. The present study will evaluate the effects of Pirfenidone on liver injury in high-fat diet (HFD)-fed mice to evaluate the value of Pirfenidone in treating NAFLD. The pathology of NAFLD was simulated by feeding mice with an HFD in the present study, followed by treating the HFD mice with 150 and 300 mg/kg/day Pirfenidone once a day. The pathological state of HFD mice was identified by the elevated liver weight, promoted serum triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels, declined serum high-density lipoprotein cholesterol (HDL-C) levels, increased alanine aminotransferase and aspartate aminotransferase activity, and histopathological changes to the liver tissues, all of which were dramatically ameliorated by 150 and 300 mg/kg Pirfenidone administration. Furthermore, the excessive production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, as well as upregulated phosphorylated nuclear factor kappa-B (p- NF-κB p65), were observed in HFD-fed mice, but significantly reversed by Pirfenidone. Finally, activated oxidative stress, identified by promoted malondialdehyde (MDA) levels and declined catalase (CAT) activity, was observed in HFD-fed mice, accompanied by the downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and sterol-regulatory element-binding proteins-1c (SREBP-1c). After the treatment with Pirfenidone, oxidative stress was greatly mitigated. Our results imply that Pirfenidone ameliorated the progression of NAFLD by mediating inflammation and oxidative stress.     

Intracellular role of exchangeable apolipoproteins in energy homeostasis,obesity and non‐alcoholic fatty liver disease

Exchangeable apolipoproteins play an important role in systemic lipid metabolism, especially for lipoproteins with which they are associated. Recently, emerging evidence has suggested that exchangeable apolipoproteins, such as apolipoprotein A4 (apoA4), apolipoprotein A5 (apoA5), apolipoprotein C3 (apoC3) and apolipoprotein E (apoE), also exert important effects on intracellular lipid homeostasis. There is a close link between lipid metabolism in adipose tissue and liver because the latter behaves as the metabolic sensor of dysfunctional adipose tissue and is a main target of lipotoxicity. Given that the energy balance between these two major lipogenic organs is intimately involved in the pathogenesis of obesity and non‐alcoholic fatty liver disease (NAFLD), we here review recent findings concerning the intracellular function of exchangeable apolipoproteins in triglyceride metabolism in adipocytes and hepatocytes. These apolipoproteins may act as mediators of crosstalk between adipose tissue and liver, thus influencing development of obesity and hepatosteatosis. This review provides new insights into the physiological role of exchangeable apolipoproteins and identifies latent targets for therapeutic intervention of obesity and its related disorders.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Protective effect of heme oxygenase induction in ethinylestradiol‐induced cholestasis

Estrogen‐induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase‐1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol‐induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down‐regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up‐regulated Mrp3 (348%, P ≤ 0.05). Heme pre‐treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up‐regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol‐treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme‐induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up‐regulation (Mrp2/Mrp4) or down‐regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2‐dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol‐induced cholestasis.     

A network pharmacology‐based approach to explore the effects of Chaihu Shugan powder on a non‐alcoholic fatty liver rat model through nuclear receptors

The pathogenesis of non‐alcoholic fatty liver disease (NAFLD) is still not fully understood, and currently, no effective pharmacotherapy is available. Nuclear receptors (NRs) are important biological participants in NAFLD that exhibit great therapeutic potential. Chaihu Shugan powder (CSP) is a traditional Chinese medicine (TCM) formula that has a wide therapeutic spectrum including NAFLD, but the effective components and functional mechanisms of CSP are unclear. We adopted a network pharmacology approach using multiple databases for Gene Ontology (GO) enrichment analysis and the molecular complex detection (MCODE) method for a protein‐protein interaction (PPI) analysis, and we used molecular docking method to screen the NR targets and determine the corresponding CSP components. The screening results were validated through a NAFLD rat model that was used to explain the possible relationship between CSP and NAFLD. Finally, we screened PPARγ, FXR, PPARα, RARα and PPARδ as target genes and quercetin, kaempferol, naringenin, isorhamnetin and nobiletin as target compounds. The five components were detected through high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS), the results of which aligned with the docking experiments of PPARγ, PPARα and PPARδ. After CSP intervention, the NAFLD rat model showed ameliorated effects in terms of bodyweight, hepatic histopathology, and serum and liver lipids, and the mRNA levels of PPARγ, FXR, PPARα and RARα were significantly changed. The results from this study indicate that CSP exhibits healing effects in an NAFLD model and that the network pharmacology approach to screening NR targets and determining the corresponding CSP components is a practical strategy for explaining the mechanism by which CSP ameliorates NAFLD.     

Curcumol inhibits ferritinophagy to restrain hepatocyte senescence through YAP/NCOA4 in non‐alcoholic fatty liver disease

ObjectivesIn recent years, cellular senescence has attracted a lot of interest in researchers due to its involvement in non‐alcoholic fatty liver disease (NAFLD). However, the mechanism of cellular senescence is not clear. The purpose of this study was to investigate the effect of curcumol on hepatocyte senescence in NAFLD and the molecular mechanisms implicated.Materials and methodsLVG Golden Syrian hamsters, C57BL/6J mice and human hepatocyte cell line LO2 were used. Cellular senescence was assessed by analyses of senescence marker SA‐β‐gal, p16 and p21, H3K9me3, γ‐H2AX and telomerase activity.ResultsThe results showed that curcumol could inhibit hepatocyte senescence in both in vivo and in vitro NAFLD models, and the mechanism might be related to its regulation of ferritinophagy and subsequent alleviation of iron overload. Moreover, overexpression of nuclear receptor coactivator 4 (NCOA4) weakened the effect of curcumol on ferritinophagy‐mediated iron overload and cellular senescence. Furthermore, we demonstrated that curcumol reduced the expression of NCOA4 by Yes‐associated protein (YAP). In addition, depression of YAP could impair the effect of curcumol on iron overload and cellular senescence.ConclusionOur results clarified the mechanism of curcumol inhibition of hepatocyte senescence through YAP/NCOA4 regulation of ferritinophagy in NAFLD. These findings provided a promising option of curcumol to regulate cellular senescence by target YAP/NCOA4 for the treatment of NAFLD.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.