Abstract
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.
Introduction
Thermal inhalation injury is quite common in fire victims and is the leading cause of mortality in heat-related injuries. Thermal inhalation injury induces an abnormal inflammatory response in the small airways and alveoli, contributing to pneumonia, SARS, and sepsis and resulting in multiple organ dysfunction and even death [1, 2]. The bronchial epithelium acts as the main barrier for protecting the lungs from endogenous and exogenous damage. 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.
Astragalosides IV (AS-IV), a major active compound extracted from the Astragalus root, possesses comprehensive pharmacological activities such as anti-inflammatory [5], anti-oxidative [6], and anti-cancer functions [7]. AS-IV also increases T and B lymphocyte proliferation and antibody production and inhibits IL-1 and TNF-α production from peritoneal macrophages. Furthermore, TNF-α- and LPS-induced inflammatory reactions may be inhibited by AS-IV treatment via the NF-kB pathway in endothelial cells [8]. Evidence from pharmacological research and clinical practice suggests that AS-IV possesses effective anti-inflammatory properties and the ability to treat infectious diseases [9, 10].
Reactive oxygen species (ROS) induces cell apoptosis, cell death, and inflammation. The endoplasmic reticulum (ER) is an important organelle in cells that is highly responsive to the cell changes and plays an important role in the folding of newly synthesized proteins [11]. Under hypoxic conditions, the increased ROS production leads to oxidative damage, possibly affecting ER homeostasis and causing ER stress [12]. ER stress elicits the unfolded protein response (UPR) to facilitate repair toward reestablishing normal ER function through the mediation of three ER-resident transducers: activating the pancreatic ER stress kinase (PERK), transcription factor 6 (ATF6), and IRE1 [13, 14]. However, the role of ER stress in heat exposure has not been completely defined. ER stress is involved in regulating various cell processes, including apoptosis and autophagy. A recent study suggested that GRP78 and PERK/eukaryotic initiation factor 2 alpha (eIF2α) pathway are closely involved in activating autophagy.
Under normal conditions, basal autophagy is a key cellular mechanism that provides a protective role for maintaining cell homeostasis. In mammalian cells, autophagy can eliminate damaged proteins and organelles by lysosome degradation and recycle the resulting amino acids, free sugars, and fatty acids for the energy needs of the cell [15]. Under pathological conditions, autophagy is induced in response to stress conditions including cell starvation, hypoxia, oxidant injury, and heat [13]. In these cases, elevated autophagy degrades damaged material to produce metabolic substrates and to meet the bioenergy needs of the cell [16]. However, excessive and impaired autophagy can result in cell damage. Several studies have focused on the functional link between autophagy and inflammation-associated disease; findings suggest a critical role of autophagy in inflammation regulation in diseases caused by acute lung injury (ALI) [17]. Autophagy has been demonstrated to induce inflammation, such as the activation of NF-kB, the polarization of immune cells, and the regulation of cytokine production [18, 19]. Emerging investigations have demonstrated that autophagy can be activated by heat in lung cells, acting as a deleterious process during lung injury [20]. Moreover, some reports have indicated that ER stress is responsible for the subsequent induction of autophagy, with a loss of the functional link between autophagy and the UPR. Recent studies have shown that AS-IV protects cells from injury via the inhibition of ER stress and the production of ROS. In the present study, we hypothesized that the crosstalk between ER stress and various autophagy pathways is one of the primary mechanisms underlying the pathogenesis and progression of inflammation in heat-induced lung inflammation. In particular, we investigated whether AS-IV could attenuate heat-induced inflammatory responses by affecting the activation of the crosstalk between ER stress and autophagy cellular pathways.
Materials and Methods
Cell culture and treatment
The 16HBE140 cells were cultured at 37°C under a 5% CO2 humidified-atmosphere in Dulbeccos' modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated at 47°C for 5 min to as the in vitro heat exposure model. Cultured cells were treated with AS-IV (2.5, 5, or 10 µmol/L) (Sigma-Aldrich, Shanghai, China) 24 h after heat treatment.
Animals and thermal inhalation injury
Adult male Wistar rats were used to induce thermal inhalation injury. All procedures were approved the Animal Ethical Committee of Shanghai University and Traditional Chinese Medicine, and were conducted according to the approved guidelines. The animals were anesthetized with 2.5% isoflurane in 95% oxygen and 5% CO2 with a face mask. Animals were placed on controlled heating pads, and the core temperature was maintained at 37°C. Tracheotomy was performed and a 1 cm catheter was gentle inserted into the trachea. We used a heat gun (TAK-3316E, Shenzhen Takgiko Techonology Shenzhen, China) to produce heated air at 47°C. Air was then blown into the trachea, which was maintained at a flow rate of 190 L/min for a total of 10 min, with 1 min intervals between each minute. After that, we sutured the wounds and intraperitoneally injected Acetylpromazine (0.75 mg/kg body weight) to relieve pain. The tracheotomy was also performed in a sham group. The AS-IV treatment group involved the intragastric administration of AS-IV two times everyday (12.5, 25, or 50 mg/kg/day). The model group and the sham group were given a gavage of distilled water of the same volume as the drug.
Determination of ROS
To evaluate ROS generation, the dye 2',7'-dichlorofluorescein diacetate (DCFDA) (Thermo fisher scientific, Waltham, MA, USA) was used to measure changes in ROS levels. Briefly, cells were cultured in glass bottom dishes and incubated with 5 µmol/L of H2DCFDA in the dark for 30 min. Cells were then washed three times with pre-warmed phosphate buffered saline (PBS) and resuspended in PBS. The relative levels of fluorescence were determined by confocal microscopy at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Pictures were taken at 10 frames/s using a 4× objective at 510 nm.
Western blot analysis
Cells were lysed in RIPA buffer containing 0.5% sodium deoxycholate, 1% NonidetP-40,150 mM NaCl, 50 mM Tris pH 7.5, 0.1% sodium dodecylsulfate, 1 mM PMSF, and a mixture of protease inhibitors. The cell lysates were then centrifuged at 12,000 × g for 15 minat4ºC. The protein concentrations of the supernatants were determined using the BCA protein assay (Thermo Fisher Scientific, Shanghai, China). Cell lysates were loaded onto SDS–polyacrylamide gels and transferred to PVDF membranes. Nonspecific binding sites were blocked with 10% fat-free milk for 1 h at room temperature. Membranes were then incubated with primary antibody. Antibodies against GRP78, CHOP, PERK, phosphate PERK, eIF2α, phosphate eIF2α, LC3, P62, IRE1, XBPls ,ATF6, and Beclin 1 were all obtained from Abeam. Phosphate PERK, eIF2α, and phosphate eIF2α were purchased from Cell Signaling Technology.
mRFP-GFP-LC3 Assay
The 16HBE140 cells was plated on the cover slip of the 6-well plate and added with the mRFP-GFP-LC3 virus (MOI15) (Hanbio. Co. LTD, Shanghai, China). New media was changed after 12 h. After 48 h of infection, confocal images were obtained at 561 and 488 nm excitation using a 63× oil objective (iXon Ultra 897 CCD). The puncta of each cell was counted and 10 pictures were taken for each sample. The GFP signal is sensitive to the acidic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, colocalization of both GFP and mRFP fluorescence indicates a compartment that has not fused with a lysosome. In contrast, an mRFP signal without GFP indicates a compartment fused with a lysosome. Thus, autophagy flux was then measured by confocal counting of GFP+/mRFP+(yellow) and GFP-/mRFP+(red) puncta.
siRNA transfection assay
eIF2α siRNA, CHOP siRNA, ATF4 siRNA, Beclin 1 siRNA, and ATG5 siRNA were purchased from Santa Cruz Biotechnology. They were used for transient transfection of 16HBE140 cells cells with Lipofectin 2000 (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) to suppress the expression of targeted gene. After 48 h of the initial transfection and treatment, cell samples were collected and analyzed using Western blotting and Real-time PCRto confirm the expressions of proteins.
Real-time PCR
cDNA samples were mixed with primers and the SGExcel UltraSYBR Mixture (Thermo Scientific, Fremont, USA). PCR was conducted using the following parameters: 50°C for 2 min, 95°C for 10 min,and40 cycles at 95°C for 15 s and 60°C for 1 min. Quantitative real-time PCR was performed for IL-lβ, IL-6, IL-10 ‚TGF-β and TNF-α, and normalized to the copies of GAPDH mRNA from the same sample. All PCR assays were performed in triplicate and results are represented as the mean values. The specificity of primers was confirmed by melt curve analysis. The sequences of primers used were as follows:
1L-6 forward primer sequence: ACAACCACGGCCTTCCCTACTT;
1L-6 reverse primer sequence: CACGATTTCCCAGAGAACATGTG;
1L-8 forward primer sequence: ATGACTTCCAAGCTGGCCGTGGCT;
1L-8 reverse primer sequence: TCTCAGCCCTCTTCAAAAACTTCTC;
TNF-α forward primer sequence: CAGAGGGAAGAGTTCCCCAG;
TNF-α reverse primer sequence: CCTTGGTCTGGTAGGAGACG;
1L-10 forward primer sequence: CTTAATGCAGGACTTTAAGGGTTA;
1L-10 reverse primer sequence: ATTCATGGCCTTGTAGACACC;
TGF-β forward primer sequence: AGAGACGTGGGGACTTCTTG;
TGF-β reverse primer sequence: GAATAGGGGCGTCTGAGGAA.
ELISA of lL-10‚ TGF-β‚IL-6, IL-8, and TNF-α Protein Production
1L-6, IL-8, IL-10 ‚TGF-β and TNF-α secretion amounts in the culture medium and supernatant homogenated from the lung tissue were analyzed by an Immunoassay Kit (BioSource International, Inc., Camarillo, CA) and used according to manufacturer's instructions. The MPO activities in the lung tissue were assayed by MPO and MDA kits (Jiancheng Bioengineering Institute of Nanjing (Nanjing, China).), following the manufacturers instructions.
Statistical analysis
Statistical analysis was performed by using one-way AN OVA and t-tests. Data are presented as means ± SD of three independent experiments for each group and were analyzed with GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). All tests were two tailed. A P value of <0.05 was considered significant.
Results
AS-IV treatment exerted a protective effect by inhibiting the production of ROS and mitigating ER stress
The 16HBE140 cells were incubated at 47°C for 5 min as the heat exposure damage model. To examine whether damage from heat exposure could lead to ER stress and the overproduction of ROS, we assessed the expression of the ER stress molecular chaperones GRP78 and the ER stress-associated apoptosis protein CHOR which are produced in part by ER stress. The intracellular ROS were labeled with H2DCFDA fluorescence. As shown in Fig. 1A, 16HBE140 cells were incubated at 47°C and the expression of GRP78 and CHOP reached the highest levels at 12 h; levels returned to normal on day two. The trend of ROS production also peaked at 12 h and decreased on day two in comparison with the control group (Fig. 1B). Furthermore, given that heat exposure lead to the increased expression of GRP78 and CHOP, hence we further investigated all branches of ER stress induced by heat exposure including PERK⁄eIF2α ‚IREl/XBPls and ATF6 axis. As shown in Fig. 1C, the expression of phosphorylated PERK and phosphorylated eIF2α reached the highest levels at 12 h; levels returned to normal on day two. However, the expression of IRE1, XBPls and ATF6 had no significant changes in heat exposure damage in comparison with control group(Fig. 1D). To confirm the effect of AS-IV in heat exposure damage, the cells were treated with AS-IV (2.5, 5, and 10 µmol/L). Compared with the heat group, the expression of GRP78 and CHOP was significantly reduced with both the middle and high dose AS-IV treatment, with no changes observed with low dose AS-IV treatment. Decreased production of ROS provided further evidence of the protection of AST in heat-induced oxidative damage(Fig. 1E and F). To confirm that the effect of AS-IV on ER stress involved the PERK/eIF2α pathway, we determined the expression of PERK and eIF2cdnterestingly AS-IV treatment remarkably downregulated the expression of phosphorylated PERK and phosphorylated eIF2α after heat exposure damage(Fig. 1G), indicating that PERK/eIF2α are closely involved in the protective mechanism of AS-IV in ER stress.
AS-IV treatment exerted a protective effect by inhibiting the production of ROS and mitigating endoplasmic reticulum (ER) stress. (A) and (E) ROS production was detected by H2DCFDA. ns‚no significant difference,***P<0.001 vs. Ctrl. n=3. ***P<0.001 vs. heat control. n=3. (B) and (F) Representative Western blot and quantification data of the ER stress marker GRP78 and CHOP, ns‚no significant difference ,P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group (n ≥ 3). P*<0.05, **P<0.01, ***P<0.001 vs. heat control. (C) and (G) Representative Western blot and quantification data of PERK, phosphorylated PERK, eIF2α and phosphorylated eIF2α. P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group. P*<0.05, **P<0.01, and ***P< 0.0001 vs. heat group (n ≥3).(D) Representative Western blot and quantification data of IREl‚ XBPls and ATF6. P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group. P*<0.05, **P<0.01, and ***P< 0.0001 vs. heat group (n ≥3).
AS-IV treatment exerted a protective effect by inhibiting the production of ROS and mitigating endoplasmic reticulum (ER) stress. (A) and (E) ROS production was detected by H2DCFDA. ns‚no significant difference,***P<0.001 vs. Ctrl. n=3. ***P<0.001 vs. heat control. n=3. (B) and (F) Representative Western blot and quantification data of the ER stress marker GRP78 and CHOP, ns‚no significant difference ,P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group (n ≥ 3). P*<0.05, **P<0.01, ***P<0.001 vs. heat control. (C) and (G) Representative Western blot and quantification data of PERK, phosphorylated PERK, eIF2α and phosphorylated eIF2α. P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group. P*<0.05, **P<0.01, and ***P< 0.0001 vs. heat group (n ≥3).(D) Representative Western blot and quantification data of IREl‚ XBPls and ATF6. P*<0.05, **P<0.01, and ***P< 0.0001 vs. control group. P*<0.05, **P<0.01, and ***P< 0.0001 vs. heat group (n ≥3).
AS-IV downregulated heat-induced excessive activation ofautophagy in 16HBE140 cells
Autophagy has been shown to play a crucial role in the progression of heat exposure damage and is closely associated with ER stress; excessive autophagy activation leads to autophagic death [17]. To determine the effects of AST on autophagy in heat exposure damage, we examined the expression of autophagy-associated proteins, including LC3, p62, and Beclin 1. Surprisingly, as shown in Fig. 2A, the expression of LC3 and Beclin 1 were significantly increased at 12 h and peaked on day one, while the expression of p62 was remarkably decreased on day one. However, AS-IV treatment exhibited significant downregulation of the expression of LC3 and Beclin 1, and upregulation of p62 after heat exposure (Fig. 2C). To further demonstrate these results, 16HBE140 cells were infected with Ad-LC3-GFP-RFP, and heat exposure exhibited elevated LC3 puncta and autophagy markers from 12 h to 2 days (Fig. 2B). This effect was remarkably inhibited by AS-IV treatment (5 and 10 µmol/L) (Fig. 2D), suggesting that AS-IV may exert protective effects against heat exposure damage through the inhibition of excessive autophagy.
AS-IV downregulated heat-induced excessive activation of autophagy in 16HBE140 cells. (A) and (C) Representative Western blots and quantification data of the autophagy markers, Beclin 1, P62, and LC3. P* < 0.05, ***P < 0.0001 vs. control group (n ≥ 3). P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group. (B) and (D) Representative confocal fluorescent images of autophagy flux; 16HBE140-cells were infected with Ad-LC3-GFP-RFP‚and autolysosome (red) and autophagosome (yellow) puncta were quantified in each group of cells (original magnification 400 x).
AS-IV downregulated heat-induced excessive activation of autophagy in 16HBE140 cells. (A) and (C) Representative Western blots and quantification data of the autophagy markers, Beclin 1, P62, and LC3. P* < 0.05, ***P < 0.0001 vs. control group (n ≥ 3). P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group. (B) and (D) Representative confocal fluorescent images of autophagy flux; 16HBE140-cells were infected with Ad-LC3-GFP-RFP‚and autolysosome (red) and autophagosome (yellow) puncta were quantified in each group of cells (original magnification 400 x).
AS-IV treatment alleviated the heat-induced release of inflammatory factors in l6HBE140 cells
It is accepted that, under stressed conditions, cells release danger signals to notify the immune system of urgent situations. The activation of innate immune receptors leads to inflammatory cytokine production. To determine whether heat exposure could induce inflammation, the expression of TNF-α, IL-6, IL-10 ‚TGF-β and IL-8 were detected by ELISA and real-time PCR. As shown in Fig. 3 A and B, compared with the heat group, the expression of TNF-α, IL-6, IL-10 ‚TGF-β and IL-8 were significantly increased after heat exposure on day one, and returned to normal levels on day five. To determine whether AS-IV can alleviate heat-induced inflammation, we examined the expression of inflammatory factors. Interestingly, AS-IV treatment also showed clear effective protection against the increased expression of TNF-α, IL-6, IL-10 ‚TGF-β and IL-8, as determined by ELISA and real-time PCR (Fig. 3C and D), suggesting that AS-IV has a protective effect against heat exposure-induced inflammatory reactions.
AS-IV treatment alleviated the heat-induced release of inflammatory factors in 16HBE140 cells. (A) and (C) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and IL-8 on 16HBE140 cells. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments, ns‚no significant difference, **P < 0.01, and ***P < 0.0001 vs. control group. (B) and (D) TNF-α, IL-6.IL-10, TGF-β and IL-8 production in culture supernatants of 16HBE140 cells were measured by ELISA. Data are means ± SD of three independent experiments, ns‚no significant difference, ***P < 0.0001 vs. control group. P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group.
AS-IV treatment alleviated the heat-induced release of inflammatory factors in 16HBE140 cells. (A) and (C) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and IL-8 on 16HBE140 cells. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments, ns‚no significant difference, **P < 0.01, and ***P < 0.0001 vs. control group. (B) and (D) TNF-α, IL-6.IL-10, TGF-β and IL-8 production in culture supernatants of 16HBE140 cells were measured by ELISA. Data are means ± SD of three independent experiments, ns‚no significant difference, ***P < 0.0001 vs. control group. P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group.
Crosstalk between ER stress, autophagy, and inflammatory reactions after heat exposure
To determine the role of crosstalk between ER stress and autophagy after heat exposure, the ROS scavenger NAC, the ER stress inhibitor GSK2656157, ER stress activator tunicamycin and eIF2α siRNA were administrated to 16HBE140 cells. As shown in Fig. 4A and B, the expression of phosphorylated PERK and eIF2α were significantly decreased in the NAC group as compared with the heat group, while the expression of GRP78 and CHOP were also downregulated after NAC treatment, suggesting that ROS are involved in regulating ER stress in heat exposure damage. Next, we found that the expression of LC3 and Beclin 1 were further decreased with GSK2656157 treatment and eIF2α siRNA, while increased with tunicamycin treatment compared with the heat group, respectively (Fig. 4C and Fig. 4D). These data indicated that the inhibition of ER stress by GSK2656157 and eIF2α siRNA abolishes excessive autophagy induced by heat exposure. To determine if PERK is inducing autophagy through ATF4 and CHOP, we used CHOP siRNA and ATF4 siRNA to treat the cells, respectively. Interestingly, both of the CHOP siRNA and ATF4 siRNA also inhibited autophagy induced by heat exposure, as determined by the expression of LC3 and Beclin l (Fig. 4E). In order to detect the role of ER stress and autophagy on inflammation after heat exposure, the ER stress inhibitor GSK2656157 and eIF2α siRNA, and the autophagy inhibitor Beclin 1 siRNA and ATG5 siRNA, were treated to 16HBE140 cells, respectively. Interestingly, as shown in Fig. 4F and 4G, compared with the heat group, the protein levels and transcriptional levels of IL-8, IL-6, IL-10, TGF-β and TNF-α were significantly inhibited in the GSK2656157, eIF2α siRNA, CHOP siRNA, ATF4 siRNA, Beclin 1 siRNA, and ATG5 siRNA groups, while remarkably activated in the tunicamycin group, respectively. Taken together, our results indicated that the inhibition of ER stress not only abolishes the activation of autophagy but also attenuates the release of inflammatory factors after heat exposure.
Crosstalk between ER stress, autophagy, and inflammatory reactions after heat exposure. (A) Representative Western blot and quantification data of PERK, phosphorylated PERK, eIF2α, and phosphorylated eIF2α. ***P< 0.0001 vs. heat control (n ≥ 3). (B) Representative Western blot and quantification data of the ER stress markers GRP78 and CHOP. ***P < 0.0001 vs. heat control (n ≥ 3). (C)-(E) Representative Western blots and quantification data of the autophagy markers, Beclin 1, P62, and LC3. P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat control (n ≥ 3). (F) TNF-α, IL-6, IL-10, TGF-β and IL-8 production in culture supernatants of 16HBE140 cells were measured by ELISA. P* < 0.05, **P < 0.01, and ***P< 0.0001 vs. heat group (n ≥ 3). (G) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and 1L-8 on 16HBE140 cells. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments. P*< 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group.
Crosstalk between ER stress, autophagy, and inflammatory reactions after heat exposure. (A) Representative Western blot and quantification data of PERK, phosphorylated PERK, eIF2α, and phosphorylated eIF2α. ***P< 0.0001 vs. heat control (n ≥ 3). (B) Representative Western blot and quantification data of the ER stress markers GRP78 and CHOP. ***P < 0.0001 vs. heat control (n ≥ 3). (C)-(E) Representative Western blots and quantification data of the autophagy markers, Beclin 1, P62, and LC3. P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. heat control (n ≥ 3). (F) TNF-α, IL-6, IL-10, TGF-β and IL-8 production in culture supernatants of 16HBE140 cells were measured by ELISA. P* < 0.05, **P < 0.01, and ***P< 0.0001 vs. heat group (n ≥ 3). (G) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and 1L-8 on 16HBE140 cells. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments. P*< 0.05, **P < 0.01, and ***P < 0.0001 vs. heat group.
AS-IV treatment ameliorates heat-induced inflammation by inhibiting ER stress and reducing autophagy activation in vivo
To further confirm the effect of AS-IV on ER stress, autophagy, and inflammation after heat exposure in vivo, intragastric administration of AS-IV two times every day (12.5, 25, or 50 mg/kg/day) in heat-injured rats. First, we detected lung injury in each group by Hematoxylin and eosin (HE)staining and myeloperoxidase (MPO) activity (a marker of neutrophil infiltration and an indicator of ALI). As shown in Fig. 5A and B, the inflammatory exudation was obviously increased after heat injury, and MPO activity was significantly enhanced at 12 h, peaked on day one, and returned to normal levels on day five. In addition, the expression levels of TNF-α, IL-8, IL-10, TGF-β and IL-6 after heat injury were consistent with the MPO activity assay results. The activity of MPO and the expressions of TNFα, IL-8, IL-10, TGF-β and IL-6 were significantly decreased after treatment with AS-IV (25 mg/kg and 50 mg/kg) when compared with the heat injury group (Fig. 5C and D). Moreover, we investigated the effect of tauroursodeoxycholic acid (TUDCA), a ER stress inhibitor on heat induced inflammation. Unsurprisingly, TUDCA treatment obviously inhibited the activity of MPO and the expressions of TNFα, IL-8, IL-10, TGF-β and IL-6 after heat exposure(Fig. 5B and E).
AS-IV treatment ameliorates heat-induced inflammation through the inhibition of ER stress and by reducing autophagy activation in vivo. (A) Representative images of immunohistochemistry show the airway morphology of rats with heat-inhalation (heat) or without heat-inhalation (sham), and those treated with AS-IV (25 mg/kg and 50 mg/kg) or DMSO (blank control group). (B) Assay of myeloperoxidase (MPO) activity in rat lung tissues, ns‚no significant difference ‚ ***P < 0.0001 vs. control group (n ≥ 3). ***P < 0.0001, vs. heat group. TNF-α, IL-6, IL-10, TGF-β and IL-8 production in rat lung tissues were measured by ELISA. ns‚ no significant difference ,P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. control group (n ≥ 3). **P < 0.01, and ***P < 0.0001 vs. heat control. (D) and (E) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and IL-8 on rat lung tissues. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments, ns‚ no significant difference ,P*<0.05, **P<0.01, and ***P < 0.0001 vs. control group. **P < 0.01, and ***P < 0.0001 vs. heat group.(F) and (H) Representative Western blots and quantification data of GRP78, CHOP, Beclin 1, P62, and LC3. ns‚ no significant difference, P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. control group .**P < 0.01, and ***P < 0.0001 vs.heat group (n ≥ 3).(G) Representative Western blot and quantification data of IREl‚XBPls and ATF6. ns‚no significant difference vs. control group. (n ≥3).
AS-IV treatment ameliorates heat-induced inflammation through the inhibition of ER stress and by reducing autophagy activation in vivo. (A) Representative images of immunohistochemistry show the airway morphology of rats with heat-inhalation (heat) or without heat-inhalation (sham), and those treated with AS-IV (25 mg/kg and 50 mg/kg) or DMSO (blank control group). (B) Assay of myeloperoxidase (MPO) activity in rat lung tissues, ns‚no significant difference ‚ ***P < 0.0001 vs. control group (n ≥ 3). ***P < 0.0001, vs. heat group. TNF-α, IL-6, IL-10, TGF-β and IL-8 production in rat lung tissues were measured by ELISA. ns‚ no significant difference ,P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. control group (n ≥ 3). **P < 0.01, and ***P < 0.0001 vs. heat control. (D) and (E) Real-time PCR of genes coding for TNF-α, IL-6, IL-10, TGF-β and IL-8 on rat lung tissues. Relative mRNA levels were normalized to the levels of 18s. Results are the average of three independent experiments, ns‚ no significant difference ,P*<0.05, **P<0.01, and ***P < 0.0001 vs. control group. **P < 0.01, and ***P < 0.0001 vs. heat group.(F) and (H) Representative Western blots and quantification data of GRP78, CHOP, Beclin 1, P62, and LC3. ns‚ no significant difference, P* < 0.05, **P < 0.01, and ***P < 0.0001 vs. control group .**P < 0.01, and ***P < 0.0001 vs.heat group (n ≥ 3).(G) Representative Western blot and quantification data of IREl‚XBPls and ATF6. ns‚no significant difference vs. control group. (n ≥3).
Consistent with the results in vitro, the expressions of GRP78 and CHOP reached the highest levels at 12 h, and returned to normal levels on day two after heat injury (Fig. 5F). Compared with the heat group, the expressions of GRP78 and CHOP were significantly decreased in the AS-IV (25 mg/kg and 50 mg/kg) group (Fig. 5E and F). We also detected the expression levels of LC3, p62, and Beclin 1 by Western blot. As shown in Fig. 5F and Fig. 5H ‚ the expression of LC3 and Beclin 1 were further increased in the heat injury group. In contrast, decreased expression of P62 was observed in the heat injury group, which was partly inhibited in the AS-IV (25 mg/kg and 50 mg/kg) group. In addition, we found that the expression of IRE1, XBPls and ATF6 had no significant changes in heat exposure damage compared with sham group in vivo (Fig. 5G).These data confirmed that the protective effect of AS-IV on heat-induced inflammation is through the inhibition of ER stress and autophagy activation.
Discussion
In the clinic, heat treatment is beneficial for cancer therapy because heat can kill cancer cells. However, heat stress can also be harmful to normal tissues and cells. In this regard, heat stress is cytotoxic to cells and can induce many cellular changes, including a loss of cellular homeostasis. While there have been some reports on the effects of long-time hyperthermia in cells [21, 22], additional research is needed in the area of cell damage and cell signaling pathway after heat treatment in short time, which may be useful for the prevention or treatment of.
In present study, we observed increased levels of ROS-dependent oxidants and the accumulation of ER stress, autophagy, and inflammation after heat treatment. To the best of our knowledge, the present study is the first report demonstrating that the crosstalk between ER stress and autophagy is involved in heat-induced inflammation. Our findings illustrate the potential protective effect of AS-IV on heat exposure-induced inflammatory response. The mechanism of action of AS-IV may be through the regulation of crosstalk between ER stress and autophagy. Oxidative stress is a leading cause in the development and genesis of multisystem disorders. The antioxidant capacity of AS-IV has been shown through its beneficial effects on the antioxidant system. The results of the present study indicate that, after AS-IV treatment (2.5, 5, and 10 µmol/L), the levels of ROS significantly decreased, with a more evident decrease following 10 µmol/L AS-IV treatment. AS-IV has been widely studied for its anti-inflammatory function in vivo. In the present study, three doses of AS-IV (12.5, 25, and 50 mg/kg body weight) were used. The data demonstrated that AS-IV (25 and 50 mg/kg body weight] had potential protective effects against heat-induced inflammation. However, the 50 mg/kg dose exhibited a marked response against the antioxidant and anti-inflammatory parameters. Furthermore, since a lower dose may induce less potential adverse events, more studies are required to investigate low doses of AS-IV.
Astragalus membranaceus is one of the most widely used Chinese medicinal herbs for the treatment of many diseases, and AS-IV administration has been used for various inflammatory conditions [5, 8, 23-25]. In addition, the therapeutic potential of AS-IV in several pulmonary disease models has recently attracted attention. Consistent with our previous study in thermal inhalation injury, which showed that AS-IV could dampen lung inflammatory responses and oxidative stress, the present study further demonstrated the anti-inflammatory effect of AS-IV and the possible underlying mechanisms in heat-treated 16HB140 cells.
Even the temperature of the air around the fire source in the conflagration can reach to as high as 800°C [26], the temperature of the air in the bronchi airway can be about 48.3°C because over 90% of the heat inhalation is released after passing the upper airway. High temperature (above 43°C) can also cause pathological changes ‚ affecting cell biological function [27]. Accordingly, we implemented our research using 47°C in short time which was consist with the pathological change in inhalation injury after burn. A sensitive increase in temperature (40-46°C) can cause protein unfolding, entanglement, and aggregation, and further induce ER stress [27]. ER stress was induced with increased eIF2α phosphorylation and XBP1 splicing, resulting in the increased expression of GADD34, CHOP and BiP genes [14, 28]. We obtained similar ER stress responses in heat-treated 16HB140 cells. It was accepted that PERK/eIF2α, ATF6, and IRElα/XBP1 can activate ER chaperone genes to increase the folding capacity of the ER by leading the expression of the X-Box binding protein 1 (XBP1). However, in our models, we didn'tfind the change of the ATF6 and IRE1α/XBP1 after the heat treatment. According to different kinds of cell types, treatment, animal models, it is possible that different branches of ER stress could be activated. Excessive ER stress may result in apoptosis by activating CHOP and other apoptotic signals. It was reported that hyperthermia will induce ER stress via the phosphorylation of PERK, eIF2α, and IRE lain cancer cells [29]. Our study has also found that ER stress was significantly induced by heat exposure, and AST attenuated heat-induced ER stress in 16HB140 cells, as shown by reduced expression of GRP78, CHOP, PERK, and eIF2α.
Autophagy is a lysosome-dependent dynamic intracellular process that involves the delivery and degradation of misfolded or long-lived proteins and damaged organelles. It is activated by extracellular or intracellular stress such as starvation, heat, infection, and ER stress. There is some evidence that ER stress-induced autophagy can also keep cells from oxidants, ATP depletion, and IR injury by eliminating damaged organelles and protein aggregates formed during the injury [30, 31]. However, in some conditions, excessive autophagy caused by stress may exceed the cell-bearing capacity and lead to inflammation, apoptosis, and cell death. Although autophagy has recently been demonstrated to play an important role on the regulation of inflammation, the role of autophagy in the process of lung inflammation has been controversial [32-34]. A decrease in the autophagosome has been shown to induce inflammatory responses and the rescue of autophagy may be used as a novel anti-inflammatory target [35]. In avian influenza A-infected lung epithelial cells and PM-induced airway inflammation, autophagy activation is required for the production of proinflammatory cytokines [15]. Whether autophagy is protective or detrimental in the process of lung inflammation seems to be different according to the specific circumstances and cell types. Consistent with the latter studies, our data implies that autophagy is deleterious in heat-induced inflammation, and the inhibition of autophagy resulted in the decreased release of inflammatory cytokines. Thus, targeting autophagy signals may be effective for heat-induced airway inflammation and the treatment of thermal lung diseases. A recent study indicates that the activity of autophagy is in response to ER stress and inflammation in adipocytes. In addition, ER stress inhibitors can inhibit the activation of autophagy and this might aggravate brain ischemia. In this study, we found that the PERK inhibitor and eIF2α siRNA significantly reduced the activation of autophagy in heat-treated 16HB140 cells. Collectively, the data highlight the effect of AS-IV on heat-induced autophagy and indicates that it is primarily dependent on the PERK-eIF2α pathway. Furthermore, we observed that induction of ROS by heat treatment rapidly activated ER stress, while the inhibition of ER stress can inhibit the activation of autophagy, which can attenuate heat-induced inflammation. AST treatment attenuated ER stress and inhibited excessive the activation of autophagy, providing protection against heat-induced inflammation both in vivo and in vitro.
This study also has some limitations. The most important is that the experiments in vitro used an immortal cell line. However, it is not ethically possible to do an in vivo study on humans, and there is a controversial issue regarding whether autophagy detrimental or protective. Our present data provide novel evidence of a positive association between heat stroke upregulated autophagy to severe heat stress injury. Additionally, heat-induced cell injury can be attenuated with autophagy inhibitors. Our present results are, in part, supported by the findings of another study on heat-induced autophagy in several cell lines without nutrient depletion. Heat treatment increased the light chain 3 of the microtubule associated protein 1 (LC3-I) and LC3-II autophagy markers at the protein levels. These findings provide evidence that heat is a potent inducer of autophagy in mammalian cells. Furthermore, there should be more signaling pathway in crosstalk between ER stress, autophagy, and inflammatory reactions after heat exposure which we should focus in the furture. And the immune system was reported to be closely related with the inflammation and involved in the anti-inflammatory effect of AS-IV [36], which was needed the further research in the future.
In conclusion, the current study respectively determined the roles of ER stress and autophagy inheat exposure damage. Heat exposure-induced ROS and ER stress accelerates the excessive activity of autophagy and further leads to inflammation both in vitro and in vivo (Fig. 6). Thus, AS-IV may attenuate inflammatory responses through the intervention of the crosstalk between autophagy and the ER strees, which may provide a new therapeutic target for heat induced inflammatory pulmonary disease.
AS-IV alleviates heat-induced inflammation by inhibiting endoplasmic reticulum stress and autophagy.
AS-IV alleviates heat-induced inflammation by inhibiting endoplasmic reticulum stress and autophagy.
Acknowledgement
This work was supported by a grant from the Key Program of Logistics Scientific Research of the PLA (BWS11J039) and by the National Natural Science Foundation of China (No. 81571896).
Disclosure Statement
The authors of this article declare no conflict of interests.