Nephropreventing effect of hypoxia‐inducible factor 1α in a rat model of ischaemic/reperfusion acute kidney injury

Acute kidney injury (AKI) occurs in 5% of hospitalized patients and in 50% of sepsis patients with acute renal dysfunction. However, there have been no safe and effective therapeutic strategies. The hypoxia condition is closely related to renal injury and function under AKI. As hypoxia‐inducible factor 1α (HIF‐1α) is critical for the cellular response to hypoxia, we investigated the protective effect of HIF‐1α in a rat AKI model. We found that HIF‐1α injection improved the survival of rat with AKI, and the level of creatinine and blood urea nitrogen (BUN) was also increased. Our data showed that HIF‐1α treatment significantly alleviated ischaemic/reperfusion injury to kidney tubules and nephrocytes. We also found the downstream factors, such as EPOR, VEGF, and PHD3, were also upregulated by HIF‐1α. Finally, it was observed that HIF‐1α treatment also increased the percentage of adult resident progenitor cells (ARPC) in vitro and in vivo. In conclusion, HIF‐1α plays a protective role in the ischaemic AKI model through stimulating the proliferation of ARPC, and our study provided a potential therapeutic strategy for AKI.     

Silencing of hypoxia inducible factor‐1α by RNA interference inhibits growth of SK‐NEP‐1 Wilms tumour cells in vitro,and suppresses tumourigenesis and angiogenesis in vivo

Wilms tumour is the most common tumour of the pediatric kidney. Elevation of hypoxia‐inducible factor 1α (HIF‐1α) has been detected in 93% to 100% of human Wilms tumour specimens, suggesting a potential value of HIF‐1α as a therapeutic target for Wilms tumour. In the present study, a stable HIF‐1α‐silenced Wilms tumour cell strain was established by introducing HIF‐1α short‐hairpin RNA (shRNA) into SK‐NEP‐1 cells. Silencing of HIF‐1α significantly reduced single‐cell growth capacity, suppressed proliferation and arrested cell cycle of SK‐NEP‐1 cells. In addition, reduction of HIF‐1α expression induced apoptosis in SK‐NEP‐1 cells, which was accompanied by increased levels of cleaved caspase‐3, cleaved poly (ADP‐ribose) polymerase (PARP) and Bax as well as downregulation of Bcl‐2 in the cells. Furthermore, when inoculated subcutaneously in nude mice, HIF‐1α‐silenced SK‐NEP‐1 cells displayed retarded tumour growth and impaired tumour angiogenesis. In summary, the findings of this study suggest that HIF‐1α plays a critical role in the development of Wilms tumour, and it may serve as a candidate target of gene therapy for Wilms tumour.     

Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia‐inducible factors HIF1α and HIF2α

Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia‐inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC‐associated drug resistance. Both HIFαs contribute to CSC survival but play different roles –HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self‐renewal – and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse.     

Andrographolide inhibits hypoxia‐induced hypoxia‐inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP‐5 pathways in EA.hy926 cells

Andrographolide is a potent anti‐inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET‐1) is an endothelium‐derived vasoconstrictor with pro‐inflammatory properties secreted in response to hypoxia. Mitogen‐activated protein kinase phosphatase 5 (MKP‐5) is a dual‐specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia‐induced HIF‐1α expression and ET‐1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP‐5 plays in andrographolide's inhibition of hypoxia‐induced expression of HIF‐1α and ET‐1. Hypoxic conditions were created using the hypoxia‐mimetic agent CoCl₂. Andrographolide enhanced HO‐1 and MKP‐5 expression and cellular cGMP content in addition to inhibiting hypoxia‐induced ROS generation. Concomitantly, the HO‐1 byproduct CO and the cGMP analogue 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) increased MKP‐5 expression, and pretreatment with CO and 8‐Br‐cGMP inhibited hypoxia‐induced HIF‐1α and ET‐1 expression. Transfection of HO‐1 siRNA or pretreatment with the HO‐1 inhibitor ZnPP‐9 or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide‐induced MKP‐5 expression. Moreover, silencing MKP‐5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia‐induced p38 MAPK activation and HIF‐1α expression. The inhibition of hypoxia‐induced HIF‐1α and ET‐1 expression by andrographolide is likely associated with HO‐1/CO/cGMP/MKP‐5 pathways, which is involved in inhibiting hypoxia‐induced p38 MAPK activation.     

RNAi Silencing of IL‐1β and TNF‐α in the Treatment of Post‐traumatic Arthritis in Rabbits

Post‐traumatic arthritis is a secondary complication to severe joint trauma. With the disease progression, it may eventually lead to osteoarthritis in patients whose age is considerably younger than patients with traditional bone arthritis. The main objective of this study was to explore the feasibility of using lentiviral‐mediated RNA interference silencing of IL‐1β and TNF‐α to treat post‐traumatic arthritis in rabbits. About 48 New Zealand rabbits underwent bilateral knee joint surgery to stimulate traumatic arthritis. They were then randomly divided into four groups of 12 rabbits each. The histopathology of the cartilage was observed, and the changes were assessed by Mankin scoring. ELISA was used to detect the expression of IL‐1β and TNF‐α in the synovial fluid. (i) Compared with the control group, the transfection and co‐transfected groups displayed reduced cartilage damage and speed of degeneration. The co‐transfected group showed the greatest alleviation of symptoms. The Mankin score was statistically different (p < 0.01). (ii) Compared with the control group, the expression of IL‐1β or TNF‐α was reduced in the respective transfection groups (p < 0.01 in both groups) and IL‐1β and TNF‐α were reduced in the co‐transfected group (p < 0.01). The co‐transfected group showed the lowest expression of the three experimental groups of both IL‐1β and TNF‐α (p < 0.01). Lentivirus‐mediated RNA interference can knock down the expression of IL‐1β and TNF‐α in joint fluids and, in a synergistic effect when two siRNAs are co‐transfected, ease cartilage degeneration.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Discrimination of skin sensitizers from non‐sensitizers by interleukin‐1α and interleukin‐6 production on cultured human keratinocytes

In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non‐sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non‐sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin‐1α (IL‐1α) and IL‐6 were measured. The sensitivity of IL‐1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL‐6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro‐inflammatory cytokines IL‐1α and IL‐6 by human HaCaT cells may potentially classify skin sensitizers from non‐sensitizers. Copyright © 2015 John Wiley & Sons, Ltd.     

Up‐regulation of Gadd45α after exposure to metal nanoparticles: The role of hypoxia inducible factor 1α

The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as cobalt and titanium dioxide (Nano‐Co and Nano‐TiO₂) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage, and apoptosis. The growth arrest and DNA damage‐inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non‐toxic doses of Nano‐Co and Nano‐TiO₂ were selected to treat cells. Our results showed that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression, but Nano‐TiO₂ did not. To investigate the potential pathways involved in Nano‐Co‐induced Gadd45α up‐regulation, we measured the expression of hypoxia inducible factor 1α (HIF‐1α) in PW cells exposed to Nano‐Co and Nano‐TiO₂. Our results showed that exposure to Nano‐Co caused HIF‐1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock‐out cells [HIF‐1α (?/?)] and its wild‐type cells [HIF‐1α (+/+)] were used. Our results demonstrated that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression in wild‐type HIF‐1α (+/+) cells, but only a slight increase in HIF‐1α (?/?) cells. Pre‐treatment of PW cells with heat shock protein 90 inhibitor, 17‐(Allylamino)?17‐demethoxygeldanamycin (17‐AAG), prior to exposure to Nano‐Co significantly abolished Nano‐Co‐induced Gadd45α expression. These results suggest that HIF‐1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano‐Co. These findings may have important implications for understanding the potential health effects of metal nanoparticle exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 490–499, 2015.     

Surgery‐induced changes in rat IL‐1β and acetylcholine metabolism: role of physostigmine

Pharmacological enhancement of cholinergic activity following administration of physostigmine is known to induce protective effects generally. However, it is unclear whether the effect of physostigmine on inflammation and acetylcholine (ACh) metabolism is related to different types of surgical intervention or anaesthesia alone. To investigate this, rats were subjected to partial liver resection (PLR) or sham surgery, with a control group receiving anaesthesia alone. Half of each treatment group received a single intra‐operative dose of physostigmine (0.04 mg/kg); the others received placebo. Acetylcholinesterase (AChE) activity and plasma and brain concentrations of interleukin (IL)‐1β and ACh were determined. Both PLR and sham operation induced a time‐dependent increase in plasma concentrations of IL‐1β compared with rats receiving anaesthesia alone (3.9‐ and 4.8‐fold increases, respectively). In the brain, IL‐1β concentrations had increased approximately twofold after surgery compared with the control group. Blood AChE was transiently decreased after surgery. Brain AChE activity increased 1.3‐fold (P = 0.014) only after PLR; consequently, cerebral ACh concentrations were significantly reduced. Physostigmine administration significantly reduced IL‐1β and AChE levels. Cerebral ACh concentrations were markedly increased from 544 ± 122 ng/mg protein following placebo administration to 654 ± 93 ng/mg protein after physostigmine administrations (P < 0.001). We conclude that a single dose of physostigmine intra‐operatively has a sustained anti‐inflammatory effect (up to 120 min after injection) that is especially pronounced under the conditions of PLR surgery. In addition to its protective peripheral action, physostigmine exerts a neuroprotective action by increasing levels of the neurotransmitter ACh.     

Suppression of tumour necrosis factor‐α by Schizonepeta tenuifolia water extract via inhibition of IκBα degradation and Jun N‐terminal kinase/stress‐activated protein kinase activation

Objectives The anti‐inflammatory effects of an aqueous extract of Schizonepeta tenuifolia on lipopolysaccharide (LPS)‐induced tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) in vivo and in vitro have been investigated. Methods C57BL/6 mice were orally administered phosphate‐buffered saline (control) or S. tenuifolia water extract (50, 200, 500 or 1000 mg/kg) for 10 days before intraperitoneal administration of LPS (1.3 mg/kg). Blood samples were obtained 1 h after LPS challenge, followed by determination of TNF‐α and IL‐6 levels. Peritoneal macrophages from thioglycollate‐injected mice were obtained and stimulated with LPS and S. tenuifolia water extract for viability assay, cytokine analysis, real‐time RT PCR and Western blotting. Key findings Oral administration of S. tenuifolia water extract to mice significantly reduced LPS‐induced serum levels of TNF‐α, but not IL‐6. When peritoneal macrophages were treated in vitro with S. tenuifolia water extract, the inhibition of LPS‐induced TNF‐α was more pronounced than that of IL‐6 at the level of secreted protein and mRNA. S. tenuifolia water extract reduced the degradation of IκBα and the nuclear relocation of p65 NF‐κB, but the phosphorylation of IκBα was not affected. Inhibition of c‐Jun N‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) by S. tenuifolia water extract led secondarily to the inhibition of phospho‐c‐Jun and phospho‐ATF‐2. Conclusions These results indicated that the downregulation of TNF‐α by S. tenuifolia water extract may have involved the inhibition of both IκBα degradation and activation of c‐Jun and ATF‐2 involving suppression of JNK/SAPK.     

Acute effect of β‐sitosterol on calcium uptake mediates anti‐inflammatory effect in murine activated neutrophils

Objectives To evaluate the effect of β‐sitosterol on ⁴⁵Ca²⁺ uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, and interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) levels, in carrageenan‐induced inflammation in the mouse air pouch model. Methods Dried Esenbeckia leiocarpa bark was macerated and extracted resulting in a crude hydroalcoholic extract (CHE) that was partitioned to obtain an alkaloid fraction. The alkaloid was then partitioned in polar and nonpolar subfractions. β‐Sitosterol was isolated from the nonpolar subfraction and identified by comparison with the literature. The effect of β‐sitosterol on ⁴⁵Ca²⁺ uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, IL‐1β and TNF‐α levels in carrageenan‐induced inflammation in mice were evaluated. Key findings β‐Sitosterol promoted a time‐ and dose‐dependent increase of the calcium uptake in activated neutrophils that was promptly reversed by nifedipine, BAPTA‐AM, LY294002, and colchicine. β‐Sitosterol inhibited myeloperoxidase and adenosine deaminase activity, and IL‐1β and TNF‐α levels. Conclusions β‐Sitosterol inhibited either myeloperoxidase and adenosine deaminase activity or IL‐1β and TNF‐α levels. This effect seemed to be mediated by the calcium uptake in activated neutrophils in a time‐ and concentration‐dependent manner through L‐type voltage dependent calcium channels, intracellular calcium, phosphoinositide kinase‐3, and microtubule modulation.     

Discovery of naphthyl‐N‐acylhydrazone p38α MAPK inhibitors with in vivo anti‐inflammatory and anti‐TNF‐α activity

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen‐activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti‐inflammatory profile of new naphthyl‐N‐acylhydrazone derivatives using animal models. Although all tested compounds ( 3a–d ) have been characterized as p38α MAPK inhibitors and have showed in vivo anti‐inflammatory action, LASSBio‐1824 ( 3b ) presented the best performance as p38α MAPK inhibitor, with IC₅₀ = 4.45 μm , and also demonstrated to be the most promising anti‐inflammatory prototype, with good in vivo anti‐TNF‐α profile after oral administration.     

Exposures to the environmental contaminants pentachlorophenol and dichlorodiphenyltrichloroethane increase production of the proinflammatory cytokine,interleukin‐1β, in human immune cells

Pentachlorophenol (PCP) and dichlorodiphenyltrichloroethane (DDT) are organochlorine environmental contaminants found in human blood at very significant levels (as high as 5 μm for PCP and 260 nm for DDT). Cancers of the blood (lymphoma and myeloma) and kidney as well as others have been associated with exposure to these contaminants. Interleukin (IL)‐1β is a proinflammatory cytokine and is involved in stimulating cell proliferation. High levels of IL‐1β are associated with inflammatory diseases and tumor progression. Previous studies showed that PCP and DDT at certain concentrations were able to stimulate secretion of IL‐1β. This study shows that the increased secretion of IL‐1β seen with both contaminants is due to compound‐induced increases in the production of this cytokine. Increased production began within 6 hours of exposure to PCP and continued to increase up to 24 hours. DDT‐induced stimulation of IL‐1β appeared to be maximal after 6 hours of exposure and then diminished by 24 hours. The increases seen in IL‐1β production stimulated by PCP appear to be at least partially due to compound‐induced increases in IL‐1β mRNA. Although DDT caused increased production of IL‐1β, it did not appear to cause consistent increases in its mRNA. PCP‐ and DDT‐induced increases in IL‐1β production were dependent primarily on the p38 mitogen‐activated protein kinase pathway. These results indicate that both PCP and DDT are able to increase IL‐1β production in a p38 mitogen‐activated protein kinase‐dependent manner, which may have the potential to influence chronic inflammation.     

Synthesis of 1,4‐Anthracene‐9,10‐dione Derivatives and Their Regulation of Nitric Oxide,IL‐1β and TNF‐α in Activated RAW264.7 Cells

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono‐ and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL‐1β, TNF‐α and nitric oxide production by LPS/IFN‐γ‐stimulated RAW264.7 cells. The disubstituted 1,4‐anthracene‐9,10‐dione 10 showed significant inhibition of nitric oxide, TNF‐α and IL‐1β production at the concentration of 5 μg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3 , 4 , 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL‐1β production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF‐α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.     

Comparative assessment of HIF‐1α and Akt responses in human lung and skin cells exposed to benzo[α]pyrene: Effect of conditioned medium from pre‐exposed primary fibroblasts

Exposure to atmospheric pollutants has been accused for many adverse health effects. Benzo[α]pyrene (Β[α]Ρ) in particular, the most extensively studied member of pollutants, is implicated in both cancer initiation and promotion. In the present study, we compared the effects of noncytotoxic doses of Β[α]Ρ, between human skin and lung epithelial cells A431 and A549, respectively, focusing on Akt kinase and HIF‐1α, as it is well known that these proteins are upregulated in various human cancers promoting survival, angiogenesis and metastasis of tumor cells. Also, taking into consideration that fibroblasts are involved in cancer progression, we tested the possible modulation of epithelial cell response by paracrine factors secreted by Β[α]Ρ‐treated fibroblasts. Low doses of Β[α]Ρ were found to enhance epithelial cell proliferation and upregulate both Akt kinase and HIF‐1α, with A549 cells exhibiting a more sustained profile of upregulation. It is to notice that, the response of HIF‐1α was remarkably early, acting as a sensitive marker in response to airborne pollutants. Also, HIF‐1α was induced by Β[α]Ρ in both lung and skin fibroblasts indicating that this effect may be conserved throughout different cell types and tissues. Interestingly however, the response of both proteins was differentially modified upon treatment with conditioned medium from Β[α]Ρ‐exposed fibroblasts. This is particularly evident in A459 cells and confirms the critical role of intercellular and paracrine factors in the modulation of the final response to an extracellular signal. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1103–1112, 2016.     

Deleterious effects of non‐synonymous single nucleotide variants of human IL‐1β gene

The IL‐1β gene is currently topic of interest for its important role in the pathogenesis of intervertebral disk degeneration. The new sequencing technology makes it crucial to study the effects of variants in IL‐1β. Thus, 714 IL‐1β variants with evidence supporting were collected from the EMBL database. Among them, 62 were non‐synonymous single nucleotide variants (nsSNVs). Furthermore, six common nsSNVs were predicted to have damaging effects by SIFT, PolyPhen, PROVEAN and SNPs&GO. Based on the constructed three‐dimensional structure of pro‐IL‐1β, rs375479974 with a mutation of Phe to Ser was proposed to reduce the stability of the pro‐IL‐1β protein. The rs375479974 variant was found to cause least common stabilizing amino acid residues, decrease hydrophilic and increase hydrophobic surface areas in the greatest degree, and have the lowest free energy alterations in I‐Mutant 2.0 sequence analysis. When analyzing the interaction between the experimental 3D structure of mature IL‐1β and its neutralizing McAb canakinumab complex, the rs775174784 substitution of Leu with Phe was found to attenuate this interaction by reducing binding energy, while rs375479974 not. Molecular dynamics simulation results in intervertebral disk environment supported rs775174784's effects. These results suggest that both rs375479974 and rs775174784 may have potential clinical and drug target implications.