Xiaoxiao Meng

HIF-1α regulates EMT via the Snail and β-catenin pathways in paraquat poisoning-induced early pulmonary fibrosis.

Paraquat (PQ) poisoning‐induced pulmonary fibrosis is one of the primary causes of death in patients with PQ poisoning. Hypoxia‐inducible factor‐1α (HIF‐1α) and epithelial‐mesenchymal transition (EMT) are involved in the progression of pulmonary fibrosis. Snail and β‐catenin are two other factors involved in promoting EMT. However, the relationship among HIF‐1α, Snail and β‐catenin in PQ poisoning‐induced pulmonary fibrosis is not clear. Our research aimed to determine whether the regulation of HIF‐1α in EMT occurs via the Snail and β‐catenin pathways in PQ poisoning‐induced pulmonary fibrosis. Sixty‐six Sprague–Dawley rats were randomly and evenly divided into a control group and a PQ group. The PQ group was treated with an intragastric infusion of a 20% PQ solution (50 mg/kg) for 2, 6, 12, 24, 48 and 72 hrs. A549 and RLE‐6TN cell lines were transfected with HIF‐1α siRNA for 48 hrs before being exposed to PQ. Western blotting, real‐time quantitative PCR, immunofluorescence, immunohistochemistry and other assays were used in our research. In vivo, the protein levels of HIF‐1α and α‐SMA were increased at 2 hrs and the level of ZO‐1 (Zonula Occluden‐1) was reduced at 12 hrs. In vitro, the transient transfection of HIF‐1α siRNA resulted in a decrease in the degree of EMT. The expression levels of Snail and β‐catenin were significantly reduced when HIF‐α was silenced. These data demonstrate that EMT may be involved in PQ poisoning‐induced pulmonary fibrosis and regulated by HIF‐1α via the Snail and β‐catenin pathways. Hypoxia‐inducible factor‐1α may be a therapeutic target for the treatment of PQ poisoning‐induced pulmonary fibrosis.

Expression and significance of HIF-1α in pulmonary fibrosis induced by paraquat.

It is commonly accepted that epithelial–mesenchymal transition contributes to fibrotic remodeling, but the molecular pathways involved in paraquat (PQ)-induced epithelial–mesenchymal transition remain uncharacterized. The objective of this study was to evaluate the potential involvement of HIF-1α in TGF-β1/β-Catenin and Snail pathway after PQ poisoning. In our study, 86 Spragne-Dawley rats were randomly divided into control group and PQ group, which received intragastric infusion of 20% PQ solution 50 mg/kg. Rats in the PQ group were subsequently divided into eight subgroups (10 for each subgroup) and samples were collected at different predetermined time points (2, 6, 12, 24, 48, 72, 96 h and 7 d). Fibrosis markers, including β-catenin, snail and α-SMA, were measured by western blot. The activity of HIF-1α was determined by western blot and immunofluorescence. We found that in PQ-induced pulmonary fibrosis, the level of PaO2 was significantly reduced in the 6-h subgroup, when compared to the control group (P < 0.01). Interestingly, between 6 and 72 h, there was no significant difference in PaO2. On the other hand, the level of PaCO2 started to increase from 72-h subgroup (P < 0.01). Fibrosis markers including β-catenin, snail and α-SMA, measured by western blot, were significantly increased at 2 h, while the level of p-GSK-3β was increased at 6 h. And the level of GSK-3β showed significant reduction beginning at 24 h. The activity of HIF-1α measured by western blot assays was significantly increased starting from 2 h with sustained expression. The result of Pearson coefficient analysis showed that HIF-1α was positively correlated with Snail (r = 0.935, P < 0.01) and β-catenin (r = 0.761, P < 0.05). Meanwhile, immunofluorescent analysis of HIF-1α revealed partial staining appearing from 2 h. Our data illustrated a positive correlation between Snail, β-catenin signaling and HIF-1α, suggesting a potential synergistic role of HIF-1α in PQ-induced pulmonary fibrosis, which may be independent of GSK-3β. It might also represent a potential therapeutic window for treatment of paraquat poisoning.

CARD9 gene silencing with siRNA protects rats against severe acute pancreatitis: CARD9-dependent NF-κB and P38MAPKs pathway.

We previously reported the up‐regulation of caspase recruitment domain 9 (CARD9) expressions in severe acute pancreatitis (SAP) patients, but little is known about its regulation. In this study, small interfering RNA (siRNA) was used to reduce the levels of CARD9 expression in sodium taurocholate‐stimulated SAP rats. CARD9 was overexpressed in SAP rats, which correlated with the severity of pancreatitis. When compared to the untreated group, the cohort that received the siRNA treatment demonstrated a significant reduction in pancreatic injury, neutrophil infiltration, myeloperoxidase activity and pro‐inflammatory cytokines. Furthermore, siRNAs showed that the reduction of CARD9 in SAP rats down‐regulated the expression of NF‐κBp65 and P38MAPK which are involved in the transcription and release of a wide variety of inflammatory cytokines. These findings provide evidence that CARD9 is up‐regulated in SAP rats and acts as a potential therapeutic target for the treatment thereof. Blocking the activation of NF‐κB and P38MAPK via siRNA‐mediated gene knock‐down of CARD9 appears to reduce the inflammatory response in pancreatic tissue.

NLRP3 participates in the regulation of EMT in bleomycin-induced pulmonary fibrosis

&NA; Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and irreversible lung disease. Studies have shown that epithelial‐mesenchymal transition (EMT) plays an important role in the development of IPF. The NLRP3 inflammasome is reported to be activated and play an important role in many respiratory diseases. However, whether the NLRP3 inflammasome is activated in alveolar epithelial cells as well as the regulatory role of NLRP3 in EMT have not been reported. In this study, we transfected NLRP3 siRNA into A549 and RLE‐6TN cells and treated them with bleomycin (BLM) for 24 h. Then, we detected the expression of NLRP3 inflammasome‐related proteins, EMT‐related proteins and transforming growth factor‐&bgr;1 (TGF‐&bgr;1) via western blotting, immunofluorescence and real‐time quantitative PCR. The mRNA and protein level of NLRP3, ASC and caspase‐1 increased after treatment with BLM. The IL‐1&bgr; levels were significantly decreased after inhibition of NLRP3 and caspase‐1. E‐cadherin expression increased and &agr;‐SMA was reduced in the BLM group when inhibited by NLRP3. The level of TGF‐&bgr;1 was reduced after NLRP3 silencing. These results indicated that the NLRP3 inflammasome was activated in alveolar epithelial cells and that NLRP3 may regulate EMT through TGF‐&bgr;1. These results may extend our understanding of the mechanism of pulmonary fibrosis and provide a new therapeutic target for pulmonary fibrosis. Graphical abstract Figure. No caption available. HighlightsEMT participated in the process of BLM‐induced pulmonary fibrosis.The NLRP3 inflammasome was activated in the type II alveolar epithelial cells.The NLRP3 may modulate EMT through TGF‐&bgr;1 signaling pathway.

A positive feedback loop promotes HIF-1α stability through miR-210-mediated suppression of RUNX3 in paraquat-induced EMT

Irreversible pulmonary fibrosis induced by paraquat (PQ) poisoning is the major cause of death in patients with PQ poisoning. The epithelial–mesenchymal transition (EMT) is postulated to be one of the main mechanisms of pulmonary fibrosis. Here, we investigated the role of miR‐210 in PQ‐induced EMT and its relationship with hypoxia‐inducible factor‐1α (HIF‐1α). Western blotting, immunofluorescence, immunoprecipitation and other methods were used in this study. We found that miR‐210 expression was significantly increased after PQ poisoning, and it may be regulated by HIF‐1α. Overexpression of miR‐210 further increased the HIF‐1α protein level and promoted EMT. Moreover, miR‐210 knock‐down reduced the HIF‐1α protein level and decreased the degree of EMT. Runt‐related transcription factor‐3 (RUNX3), a direct target of miR‐210, was inhibited by miR‐210 in response to PQ poisoning. RUNX3 increased the hydroxylation ability of prolyl hydroxylase domain‐containing protein 2 (PHD2), a key enzyme that promotes HIF‐1α degradation. PHD2 immunoprecipitated with RUNX3 and its level changed similarly to that of RUNX3. The expression of the HIF‐1α protein was significantly reduced when RUNX3 was overexpressed. HIF‐1α protein levels were markedly increased when RUNX3 was silenced. Based on these results, a positive feedback loop may exist between miR‐210 and HIF‐1α. The mechanism may function through miR‐210‐mediated repression of RUNX3, which further decreases the hydroxylation activity of PHD2, enhances the stability of HIF‐1α, and promotes PQ‐induced EMT, aggravating the progression of pulmonary fibrosis. This study further elucidates the mechanism of PQ‐induced pulmonary fibrosis and may provide a new perspective for the future development of therapies.

Porous Se@SiO 2 nanospheres treated paraquat-induced acute lung injury by resisting oxidative stress

Acute paraquat (PQ) poisoning is one of the most common forms of pesticide poisoning. Oxidative stress and inflammation are thought to be important mechanisms in PQ-induced acute lung injury (ALI). Selenium (Se) can scavenge intracellular free radicals directly or indirectly. In this study, we investigated whether porous Se@SiO2 nanospheres could alleviate oxidative stress and inflammation in PQ-induced ALI. Male Sprague Dawley rats and RLE-6TN cells were used in this study. Rats were categorized into 3 groups: control (n=6), PQ (n=18), and PQ + Se@SiO2 (n=18). The PQ and PQ + Se@SiO2 groups were randomly and evenly divided into 3 sub-groups according to different time points (24, 48 and 72 h) after PQ treatment. Porous Se@SiO2 nanospheres 1 mg/kg (in the PQ + Se@SiO2 group) were administered via intraperitoneal injection every 24 h. Expression levels of reduced glutathione, malondialdehyde, superoxide dismutase, reactive oxygen species (ROS), nuclear factor-κB (NF-κB), phosphorylated NF-κB (p-NF-κB), tumor necrosis factor-α and interleukin-1β were detected, and a histological analysis of rat lung tissues was performed. The results showed that the levels of ROS, malondialdehyde, NF-κB, p-NF-κB, tumor necrosis factor-α and interleukin-1β were markedly increased after PQ treatment. Glutathione and superoxide dismutase levels were reduced. However, treatment with porous Se@SiO2 nanospheres markedly alleviated PQ-induced oxidative stress and inflammation. Additionally, the results from histological examinations and wet-to-dry weight ratios of rat lung tissues showed that lung damage was reduced after porous Se@SiO2 nanosphere treatment. These data indicate that porous Se@SiO2 nanospheres may reduce NF-κB, p-NF-κB and inflammatory cytokine levels by inhibiting ROS in PQ-induced ALI. This study demonstrates that porous Se@SiO2 nanospheres may be a therapeutic method for use in the future for PQ poisoning.

Nuclear translocation of HIF-1α induced by influenza A (H1N1) infection is critical to the production of proinflammatory cytokines

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection. Emerging Microbes & Infections (2017) 6, e39; doi:10.1038/emi.2017.21; published online 24 May 2017

Effect of ulinastatin on paraquat-induced-oxidative stress in human type II alveolar epithelial cells

BACKGROUND: Ulinastatin (UTI) is a urinary trypsin inhibitor extracted and purified from urine of males. This study aimed to explore the effects of UTI on paraquat-induced-oxidative stress in human type II alveolar epithelial cells. METHODS: The human type II alveolar epithelial cells, A549 cells, were cultured in vitro. The A549 cells were treated with different concentrations of paraquat (200, 400, 600, 800, 1 000, 1 200 µmol/L) and ulinastatin(0, 2 000, 4 000, 6 000, 8 000 U/mL) for 24 hours, the cell viability was measured by cell counting kit-8 and the median lethal concentration was selected. In order to establish an in vitro model of paraquat intoxication and to determine the safe dose of ulinastatin, we calculated LD50 using cell counting kit-8 to determine the survival rate of the cells. A549 cells were divided into normal control group, paraquat group and paraquat+ulinastatin group. The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by biochemistry colorimetry, while the level of reactive oxygen spies (ROS) was detected by DCFH-DA assay. RESULTS: The survival rate of A549 cells treated with different concentrations of paraquat decreased in a concentration-dependent manner. Whereas there was no decrease in the survival rate of cells treated with 0–4 000 U/mL ulinastatin. The levels of MDA, MPO, and ROS were significantly higher in the paraquat group than in the normal control group after 24-hour-exposure. And the survival rate of the paraquat+ulinastatin group was higher than that of the paraquat group, but lower than that of the normal control group. The levels of MDA, MPO, and ROS were lower than those of the paraquat group. CONCLUSION: Ulinastatin can alleviate the paraquat-induced A549 cell damage by reducing oxidative stress.

The KCa3.1 blocker TRAM-34 inhibits proliferation of fibroblasts in paraquat-induced pulmonary fibrosis.

KCa3.1, a Ca2+-activated K+ channel, plays an important role in modulating calcium signaling and maintaining membrane potential during cell activation. It has been reported to promote fibroblast function in many fibrotic diseases. However, the role of KCa3.1 in the pathophysiology of pulmonary fibrosis after paraquat (PQ) poisoning has not been studied. A rat model of PQ poisoning was used. After treatment with TRAM-34, which is a highly selective KCa3.1 blocker, the expression of KCa3.1, TGF-β1 and α-SMA were evaluated via Western blot, histology and other assays. Bromodeoxyuridine (BrdU) marking and MTT assay were used to measure primary rat pulmonary fibroblast proliferation. The results showed that KCa3.1 expression was elevated after PQ poisoning. Blockade of KCa3.1 alleviated PQ-induced pulmonary inflammation and fibrosis. Blockade of KCa3.1 also attenuated the level of collagen I and α-SMA and the proliferation of fibroblasts. However, TGF-β1 expression remained unaffected by blockade of KCa3.1 in rat lung tissues after PQ poisoning. The present study suggests that KCa3.1 expression increased and might promote pulmonary fibroblast proliferation in PQ-induced pulmonary fibrosis. In addition, we confirmed that TRAM-34 attenuates proliferation and collagen secretion of fibroblasts. Our findings indicated that TRAM-34 might inhibit PQ-induced proliferation of pulmonary fibroblasts and prevent progression of lung fibrosis.

Atorvastatin attenuates paraquat poisoning-induced epithelial-mesenchymal transition via downregulating hypoxia-inducible factor-1 alpha

Aim: This study investigated the effects of atorvastatin (ATS) on the paraquat (PQ)‐induced epithelial‐mesenchymal transition (EMT) and the potential mechanism through hypoxia‐inducible factor‐1 alpha (HIF‐1&agr;). Main methods: Sprague–Dawley (SD) rats were randomly divided into a control group (n = 5), PQ group (n = 20), PQ + ATS L group (n = 20, ATS 20 mg/kg daily) and PQ + ATS H group (n = 20, ATS 40 mg/kg daily). All treated rats were given a 20% PQ solution (50 mg/kg) once by gavage and then sacrificed 12, 24, 72 and 168 h after PQ exposure. The A549 and RLE‐6TN cell lines were treated with ATS, PQ or both for 24 h. Mesenchymal (&agr;‐SMA and vimentin) and epithelial (E‐cadherin and ZO‐1) cell marker expression was tested both in vivo and in vitro. The effects of ATS on HIF‐1&agr; and &bgr;‐catenin expression were also evaluated. Key findings: ATS alleviated PQ poisoning‐induced lung injury and pulmonary fibrosis in vivo. This effect was dose‐dependent. ATS treatment attenuated the EMT by increasing the levels of the epithelial markers E‐cadherin and ZO‐1 and by decreasing the expression of the mesenchymal markers &agr;‐SMA and vimentin in both lung tissues and in vitro cell culture. In addition, ATS treatment may decrease the HIF‐1&agr; and &bgr;‐catenin levels both in vivo and in vitro. Significance: In conclusion, ATS can attenuate PQ‐induced pulmonary fibrosis. The mechanism may involve the downregulation of the HIF‐1&agr;/&bgr;‐catenin pathway and the inhibition of the PQ‐induced EMT by ATS. ATS may be considered as a therapeutic agent for PQ poisoning‐induced pulmonary fibrosis.