CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury

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Background: Heat causes airway damage during inhalation injury because of bronchial epithelial cell damage. Accumulating evidence shows that mitochondrial uniporter (MCU) is involved in cell damage. We investigated the MCU activity after heat treatment and assessed whether Astragaloside-IV (AS-IV) suppresses heat-induced apoptosis in bronchial epithelial cells by inhibiting the activation of the mitochondrial Ca²⁺ uniporter (MCU), mitochondrial depolarisation and reactive oxygen species (ROS) production. Methods: The bronchial epithelial cell line 16HBE14o- was heat treated, and cell apoptosis was induced in vitro and in vivo. AS-IV was inorganically administered to Wistar rats twice a day after thermal inhalation injury, and 16HBE140- cells were treated with AS-IV after incubation at 47°C for 5 min. Protein expression was determined using Western blotting and commercial kits, apoptosis with TUNEL staining, mitochondrial channel activity by patch clamp, reactive oxygen species by MitoSOX^TM fluorescence, ATP levels and enzyme activities by commercial kits as well as mitochondrial respiration and calcium by fluorescence. Results: AS-IV markedly inhibited heat-induced apoptosis, as indicated by the increased expression of the pro-apoptotic genes Bak, Bik and Bmf and increased expression of the apoptosis markers Bax, cleaved parp, cleaved caspase3 and cytochrome C. We found that MCU activation promoted mitochondrial Ca²⁺ overload, ATP depletion, mitochondrial ROS production and cytochrome c release and rapidly induced apoptosis. However, AS-IV treatment reduced excessive MCU activation and led to resistance against mitochondrial Ca²⁺ overload and excessive cytochrome C release; these effects were blocked by the MCU activator spermine. AS-IV treatment elevated ATP production and decreased ROS activity. Conclusions: MCU plays crucial roles in heat-induced mitochondrial apoptosis in 16HBE140- cells, suggesting a potential target for AS-IV treatment.

Section: Resultssupporting

confidence: 81%

“…Damage from heat exposure can result in various pathological changes in multiple tissues and organs, and it primarily affects bronchial epithelial cells and lung cells [1]. The main factors underlying this injury include reduced enzyme activity and changes in cell signalling.…”

Section: Introductionmentioning

confidence: 99%

Astragaloside-IV Protects Against Heat-induced Apoptosis by Inhibiting Excessive Activation of Mitochondrial Ca2+ Uniporter

Dong

Zhang

Chen

et al. 2017

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“…The thermal inhalation injury model was carried out as previously described [15]. Curcumin (10 mg/kg body weight) was administered intragastrically two days before the thermal inhalation and administered twice daily after the surgery.…”

Section: Methodsmentioning

confidence: 99%

“…Aliquots of the lung lysates (30 µg protein/well) and cell lysates (20 µg protein/well) were separated on 4–12% Bis-Tris gels. Western blotting was carried out as previously described [15]. Antibodies against Bcl2, Bax, a-ATP, cleaved PARP, cleaved caspase-3, gp91 phox , p47 phox , p67 phox , p22 phox , p40 phox , and Rac were all obtained from Abcam (Cambridge, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Curcumin Inhibits Heat-Induced Apoptosis by Suppressing NADPH Oxidase 2 and Activating the Akt/mTOR Signaling Pathway in Bronchial Epithelial Cells

Yuan

Tang

et al. 2017

Background: Heat causes bronchial epithelial cell apoptosis, which is a known factor contributing to airway damage during inhalation injury. Accumulating evidence has shown the effect of curcumin on inhibiting apoptosis. In this study, we investigated whether curcumin suppresses heat-induced apoptosis in bronchial epithelial cells and the underlying mechanism. Methods: Bronchial epithelial cell line 16HBE140 cells were incubated at either 42 °C, 47 °C, 52 °C, or 57 °C for 5 min in a cell incubator and then returned back to normal culture conditions (37 °C). An in vivo thermal inhalation injury rat model was established with a heat gun blowing hot air into the airway of rats. 16HBE140 cells and lung tissue were obtained for further study with or without curcumin treatment. Cell viability was determined by measuring the absorbance of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). 2',7'-dichlorofluorescein diacetate fluorescence was used as a measure of reactive oxygen species (ROS) production. Levels of Bcl2, Bax, α-ATP, cleaved Poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, gp91^phox, p47^phox, p67^phox, p22^phox, p40^phox, and Rac were determined by Western blotting. TUNEL staining was used to determine apoptosis. Results: Heat treatment triggered the apoptosis of 16HBE140 cells as shown by the increase in apoptosis molecular markers, including Bcl-2, Bax, cleaved PARP, and cleaved caspase-3. Administration of curcumin significantly inhibited apoptosis of 16HBE140 cells and suppressed the membrane translocation of NADPH oxidase 2 cytosolic components, as well as ROS production. Downregulation of Akt and mTOR phosphorylation induced by heat was also reversed by curcumin. Furthermore, we demonstrated that NADPH oxidase 2 is upstream of Akt/mTOR in heat-induced apoptosis. The protective role of curcumin on bronchial epithelia apoptosis was also confirmed in vivo by a rat inhalation injury model. Conclusion: This study demonstrates that one of the critical mechanisms underlying curcumin inhibiting heat-induced apoptosis is through suppressing NADPH Oxidase 2 and activating the Akt/mTOR signaling pathway in bronchial epithelial cells.

“…In addition, they are a main source of various cytokines and chemokines, which can initiate a series of inflammatory responses [3]. Heat stress is a common factor that induces inflammation [4]; however, its underlying mechanism remains obscure. Therefore, clarifying the molecular mechanisms involved in heat-induced alterations in bronchial epithelial cells may offer clues regarding thermal inhalation lung injury pathogenesis and treatment.…”

Section: Introductionmentioning

confidence: 99%

Astragaloside-IV Alleviates Heat-Induced Inflammation by Inhibiting Endoplasmic Reticulum Stress and Autophagy

Dong

Zhou

Zhang

et al. 2017

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Background: Thermal injury is the main cause of pulmonary disease in stroke after burn and can be life threatening. Heat-induced inflammation is an important factor that triggers a series of induces pathological changes. However, this mechanism underlying heat-induced inflammation in thermal inhalation injury remains unclear. Studies have revealed that astragaloside-IV (AS-IV), a natural compound extracted from Astragalus membranaceus, has protective effects in inflammatory diseases. Here, we investigated whether the protective effects of AS-IV occur because of the suppression of heat-induced endoplasmic reticulum (ER) stress and excessive autophagy Methods: AS-IV was administered to Wistar rats after thermal inhalation injury and 16HBE140-cells were treated with AS-IV. TNF-α, IL-6, and IL-8 levels were determined by ELISA and real-time PCR. ER stress and autophagy were determined by western blot. Autophagic flux was measured by recording the fluorescence emission of the fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. Results: AS-IV had protective effects against heat-induced reactive oxygen species production and attenuated ER stress. AS IV alleviated heat-induced excessive autophagy in vitro and in vivo. Excessive autophagy was attenuated by the PERK inhibitor GSK2656157 and eIF2α siRNA, suggesting that heat stress-induced autophagy can activate the PERK-eIF2α pathway. Beclin 1 and Atg5 siRNAs inhibited the upregulation of the inflammatory cytokines TNF-α, IL-6, and IL-8 after heat exposure. Conclusions: Thus, AS-IV may attenuate inflammatory responses by disrupting the crosstalk between autophagy and the PERK-eIF2α pathway and may be an ideal agent for treating inflammatory pulmonary diseases.