Calcitonin gene‑related peptide alleviates hyperoxia‑induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis
- Authors:
- Published online on: May 1, 2024 https://doi.org/10.3892/mmr.2024.13234
- Article Number: 110
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Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Hyperoxia therapy is often used to treat premature infants and children with acute hypoxic respiratory failure, and although it may improve their survival rate, it can also easily cause acute and chronic lung injury (1,2). Hyperoxia-induced lung injury (HILI) is a common neonatal emergency and a major cause of severe permanent lung injury and death (3,4). However, the exact pathogenesis of HILI remains unclear, thus hindering the development of effective treatments. HILI primarily affects alveolar epithelial cells, especially alveolar type II epithelial cells (AECII), a subgroup that functions as stem cells for growth and damage repair (5). Therefore, it is of significant interest and clinical value to study the regeneration and repair of AECII and to explore the underlying pathological mechanisms of early intervention strategies for HILI.
Calcitonin gene-related peptide (CGRP) is a neuropeptide transmitter that is synthesized primarily by neurons, but AECII can also synthesize CGRP (6–9). CGRP exerts its biological functions by binding to its specific CGRP receptor (CGRPR), which is a G protein-coupled receptor (GPCR) (10). CGRP is not only a potent vasodilator (11), but it is also involved in the occurrence and development of pain (12,13), inflammation (14,15), cardiovascular disease (16,17) and wound healing (18). It has been reported that CGRP can alleviate lung injury by inhibiting ischemia-reperfusion and lipopolysaccharide-induced inflammation (19–21), but its role in the development and progression of HILI remains unclear. Previous studies by the authors revealed that CGRP plays an important role in repairing damage caused by HILI by inhibiting apoptosis and promoting cell proliferation in AECII (22–25), but the underlying molecular mechanisms are largely unclear. Therefore, further investigations on the role and mechanisms of CGRP in the repair of damage caused by HILI may assist in the development of effective strategies for the prevention and treatment of HILI.
Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cationic channel protein that can be activated by physical and chemical stimuli, such as traction stimulation (for example, lung ventilation) or capsaicin, to facilitate the entry of Ca2+-dominant cations, thus participating in a variety of physiological or pathological processes (26,27). Although several studies have revealed that activation of TRPV1 channels promotes Ca2+-dependent CGRP production and release from sensory endings (28,29), whether CGRP plays a role by inversely regulating TRPV1 channels is unknown, to the best of the authors' knowledge. Furthermore, although TRPV1 channels expressed in lung epithelial cells play a protective role in ischemia-reperfusion injury (19,30), its role in AECII has not been reported, to the best of the authors knowledge, let alone its potential involvement in the pathogenesis of HILI.
Therefore, the aim of the present study was to explore the protective effects of CGRP against HILI and its underlying molecular mechanisms, focusing on its downstream signaling pathway. It was revealed that CGRP exerted a protective role in HILI via a novel and unique CGRPR/TRPV1/Ca2+ signaling axis, highlighting a potential target for the prevention and treatment of HILI.
Materials and methods
Cell culture and induction of hyperoxia
Human alveolar A549 cells were purchased from Haixing Biological Technology Co., Ltd. (cat. no. TCH-C116). Cells were cultured in DMEM-HIGH Glucose medium supplemented with 10% fetal bovine serum (AusGeneX Pty Ltd.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Beyotime Institute of Biotechnology). The cells were plated in 6- or 96-well plates for subsequent experiments. When the cell confluence reached 50–60%, the cells were treated with 10 nM CGRP (cat. no. HY-P1548A; MedChemExpress) or 1 µM capsaicin (cat. no. HY-10448; MedChemExpress), and then stimulated with hyperoxia. Cells were pretreated with CGRP8-37 (cat. no. HY-P1014), SB-705498 (cat. no. HY-10633), BAPTA-AM (cat. no. HY-100545), U-73122 (cat. no. HY-13419), U-73343 (cat. no. HY-108630), or Go6976 (cat. no. HY-10183; all from MedChemExpress) for 2 h before CGRP treatment. Hyperoxia exposure was defined as cells cultured in a closed oxygen chamber (Billups-Rothenberg, Inc.) supplied with 95 O2 and 5% CO2 for 24 h (31). The cells of the control group were cultured under normal air conditions. All cells were cultured at a constant temperature of 37°C and supplied with 5% CO2.
Lentivirus infection
Lentivirus infection was performed as previously described (32). Lentivirus was purchased from OBiO Technology Corp., Ltd. The TRPV1 shRNA and shNC primer sequences are shown in Table I. A549 cells were infected with lentivirus according to the manufacturer's protocol, and stable cells were screened with puromycin 72 h later.
Immunofluorescence assay
A549 cells were plated on 24-well coverslips at a density of 5×104/ml. When the confluence reached 60–70%, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed with PBS three times. Cells were blocked with 1% bovine serum albumin (Beyotime Institute of Biotechnology) at room temperature for 30 min, then incubated with anti-TRPV1 antibody (1:200; cat. no. ACC-030; Alomone Labs) for 2 h at 4°C or anti-CGRPR antibody (1:200; cat. no. A8533; ABclonal) overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor 488-labeled anti-rabbit secondary antibody (1:500; cat. no. A0423; Beyotime Institute of Biotechnology) at room temperature for 1 h. Cell nuclei were counterstained with 5 µg/ml DAPI at room temperature for 5 min, and images were captured using a confocal microscope (Nikon Corporation).
Cell counting kit-8 (CCK-8) proliferation assay
Cell proliferation assay was performed as previously described (31,32). Cell proliferation was measured using a CCK-8 assay (cat. no. HY-K0301; MedChemExpress). A549 cells were plated in 96-well plates at a density of 4×104/ml. After 24 h, the media was replaced, the treatments were applied, and cells were cultured in either normal air or hyperoxic conditions for 24 h. Next, 100 µl medium containing 10% CCK-8 solution was added to each well and incubated for 1–2 h at 37°C. The optical density was measured at 450 nm using a spectrometer. Cell viability was calculated using the absorbance method to express cell proliferation (33).
Apoptosis assay
Apoptosis assay was performed as previously described (31). Apoptosis detection kits were purchased from BD Biosciences (cat. no. 556547) or Shanghai Yeasen Biotechnology Co., Ltd. (cat. no. 40302ES). Cells were plated in 6-well plates for 24 h at 37°C and then treated with the various drugs for another 24 h. The cells were digested using EDTA-free trypsin for 2 min, transferred to flow tubes, centrifuge at 300 × g for 5 min at room temperature, and washed twice with PBS. Subsequently, cells were resuspended in 100 µl Annexin V Binding Buffer, and 5 µl FITC Annexin V and 5 µl PI were added. Then, the cells were gently rocked and incubated for 15 min in the dark at room temperature, after which, 400 µl Binding Buffer was added to the cell suspension. Apoptosis was measured using a flow cytometer (Beckman Coulter Life, Inc.) and analyzed using FlowJo software (version 10.8.1; FlowJo LLC). Apoptosis cells (%)=Q2 (late apoptotic cells) + Q3 (early apoptotic cells).
Calcium measurement
A549 cells were plated on coverslips and incubated with 5 µM Fura-2 AM (cat. no. F1221; Invitrogen; Thermo Fisher Scientific, Inc.) in physiological salt solution (PSS) at 37°C for 60 min, then washed with PSS with or without antagonist for 20 min. Cells on coverslips were then mounted in a standard perfusion chamber on a Nikon microscope table. The fluorescence ratio of Fura-2 (F340/380) was tracked over time at an excitation wavelength of 340 or 380 nm and captured using an intensified CCD camera (Hamamatsu Photonics K.K.) and a MetaFluor Imaging system (Version 7.10.4.407; Molecular Devices, LLC). The ratio of F340/380 represented the intracellular Ca2+ levels and was quantified using Δ(F340/380), that is, the difference between the baseline and maximum values after stimulation. The PSS for Ca2+ measurement consisted of the following: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES and 10 mM glucose at pH 7.3. The osmolality of the solution was ~300 mOsmol/kg H2O.
Electrophysiological recordings
Whole-cell membrane currents of A549 cells were recorded with an EPC 10 USB Double Patch Clamp Amplifier (HEKA). Data were digitized at 10 kHz and filtered at 5 kHz. The cell voltage was fixed at 0 mV to inactivate voltage-gated calcium and sodium channels, and a 100 ms linear ramp protocol (−100 to 100 mV) was applied every 2 sec. The amplitude of the current was recorded. The extracellular buffer contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES at pH 7.3. The pipette solution contained 140 mM CsCl, 5 mM EGTA, 3 mM Mg-ATP, and 10 mM HEPES at pH 7.3. The osmolality of all the solutions was ~300 mOsmol/kg H2O.
Reverse transcription-quantitative PCR (RT-qPCR)
RT-qPCR was performed as previously described (34). Total RNA was extracted using an RNA isolation kit (cat. no. R0027; Beyotime Institute of Biotechnology). Then, cDNA synthesis was performed using PrimeScript™ RT MasterMix (cat. no. RR036A; Takara Bio, Inc.) according to the manufacturer's instructions. Finally, using 1,000 ng cDNA as the template, SYBR Green qPCR MasterMix (cat. no. HY-K0523; MedChemExpress) was added in a 10 µl reaction system for qPCR. The amplification procedure was as follows: Heating at 95°C for 5 min to activate the hot-start DNA polymerase, followed by 40 cycles of 10 sec at 95°C and 30 sec at 60°C. The data were quantified using the 2−ΔΔCq relative quantification method (35), and β-actin was used as the internal control. The primers were designed and purchased from Sangon Biotech Co., Ltd. The primer sequences are shown in Table II.
Western blotting
RIPA Lysis Buffer (cat. no. HY-K1001; MedChemExpress) containing 1% protease inhibitor cocktail (cat. no. HY-K0010; MedChemExpress), 1% phosphatase inhibitor cocktail (cat. no. HY-K0021; MedChemExpress) and 1% PMSF Solution (cat. no. ST507; Beyotime Institute of Biotechnology) was used to extract proteins from the pretreated cells. The protein concentration was determined using a bicinchoninic acid assay (cat. no. P0010; Beyotime Institute of Biotechnology), and then the total protein was mixed with SDS-PAGE loading buffer and boiled for 10 min. Equal amounts of 30 µg protein were loaded on a 10% SDS-PAGE gel (cat. no. PG112; EpiZyme, Inc.) for electrophoretic separation, and transferred to PVDF membranes (cat. no. ISEQ00010; MilliporeSigma). Blots were blocked for 10 min at room temperature using rapid blot blocking buffer (cat. no. P30500; New Cell & Molecular Biotech Co., Ltd.), and then incubated at 4°C overnight with the following specific primary antibodies: anti-TRPV1 (1:500), anti-CGRPR (1:500), anti-PCNA (1:1,000; cat. no. 13110; Cell Signaling Technology, Inc.), anti-Cyclin D1 (1:1,000; cat. no. 55506; Cell Signaling Technology, Inc.), anti-Bcl-2 (1:1,000; cat. no. 60178-1-Ig, ProteinTech Group, Inc.), anti-Bax (1:5,000; cat. no. 60267-1-Ig; ProteinTech Group, Inc.), anti-β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.), or anti-GAPDH (1:1,000; cat. no. 2118; Cell Signaling Technology, Inc.).
After the primary antibody was recovered, TBST (0.05% Tween-20) was used to wash the membrane three times, 8 min each time. Then the membranes were incubated with goat anti-rabbit (cat. no. ZB-2301) or goat anti-mouse (cat. no. ZB-2305; ZSGB Biotechnology, Inc.) secondary antibody for 1 h at room temperature. The dilution ratio of the secondary antibodies was 1:5,000. Signals were visualized using enhanced chemiluminescence reagent (MilliporeSigma) and densitometric analysis was performed using ImageJ software (version Fiji; National Institutes of Health).
ELISA
ELISA was performed as previously described (34). The levels of CGRP in the supernatant of A549 cells treated with air or hyperoxia were detected using a human CGRP ELISA kit (cat. no. CB-E08210h; Cusabio Technology, LLC) according to the manufacturer's protocol.
Statistical analysis
SPSS version 25.0 (IBM Corp.) was used to analyze the data. All data are presented as the mean ± SD of at least three repeats. The unpaired Student's t-test was used for comparison between two groups. A one-way ANOVA was used for comparison among multiple groups; if the variances were homogeneous, a Fisher's least significant difference test was used for analysis; otherwise, Dunnett's T3 analysis was used. P<0.05 was considered to indicate a statistically significant difference.
Results
Hyperoxia downregulates CGRPR and TRPV1 channels expression in A549 cells
Since CGRP exerts its biological effects by binding to its specific CGRPR (10), to confirm whether CGRP exerted its protective effects through TRPV1, immunofluorescence was first performed to detect the expression and localization of CGRPR and TRPV1 in A549 cells. CGRPR and TRPV1 were expressed and were primarily located in the cytoplasm and cell membrane (Fig. 1A). Whether hyperoxia affects CGRP release is unknown, thus, A549 cells were cultured either in normal air or hyperoxic conditions, after which, the release of CGRP was detected. ELISA results revealed that there was no difference in CGRP release between the air and hyperoxia groups (Fig. 1B). At the same time, the effect of hyperoxia on the expression of CGRPR and TRPV1 channels in A549 cells was examined. After incubation under hyperoxic conditions, the mRNA and protein expression levels of CGRPR were significantly decreased compared with cells cultured with normal air (Fig. 1C and D). Similarly, the mRNA and protein expression levels of TRPV1 were also considerably reduced (Fig. 1E and F). Therefore, these results showed that hyperoxia downregulates the expression of CGRPR and TRPV1 channels in A549 cells without altering CGRP release.
CGRP/CGRPR and capsaicin/TRPV1 promote cell proliferation but inhibit apoptosis under hyperoxic conditions
Next, the effects of hyperoxia on the proliferation and apoptosis of A549 cells were investigated. First, compared with the normal air group, the proliferation of A549 cells was significantly decreased in the hyperoxia group (Fig. 2A), consistent with a previous report on HILI (36). It is well established that capsaicin, a selective TRPV1 agonist (37,38), induces CGRP release from nerve endings (39). Additionally, TRPV1 channels exert a protective role in ischemia-reperfusion injury (19,30); however, the role of TRPV1 channels in HILI is unknown. Therefore, whether CGRP/CGRPR and capsaicin/TRPV1 exerted a protective effect in hyperoxia-induced alveolar cell injury was investigated. Indeed, CGRP and capsaicin enhanced cell proliferation of cells in both the normal air and hyperoxia groups in a dose-dependent manner (Fig. 2B and C). Based on these findings, 10 nM CGRP and 1 µM capsaicin were used in subsequent experiments. Moreover, hyperoxia significantly induced apoptosis in A549 cells, which was attenuated by CGRP and capsaicin (Fig. 2D). Taken together, CGRP/CGRPR and capsaicin/TRPV1 may play protective roles in hyperoxia-induced alveolar cell injury by promoting cell proliferation and inhibiting apoptosis.
CGRP promotes proliferation but inhibits apoptosis via a CGRPR/TRPV1 axis under hyperoxic conditions
Since CGRP/CGRPR and capsaicin/TRPV1 exerted protective effects in parallel, whether CGRP acted via a CGRPR/TRPV1 axis was next assessed. First, it was revealed that CGRP8-37 (100 nM), a selective CGRPR inhibitor, inhibited CGRP-induced A549 cell proliferation with no toxic effects (Fig. 3A). Second, the selective TRPV1 channel blocker SB-705498 (10 µM) also attenuated CGRP-induced cell proliferation (Fig. 3B). Third, CGRP8-37 and SB-705498 attenuated the inhibitory effect of CGRP on apoptosis, and they themselves had no significant effect on apoptosis (Fig. 3C and D). Therefore, CGRP may promote cell proliferation but inhibit apoptosis of A549 cells via a CGRPR/TRPV1 axis.
The CGRPR/TRPV1 axis affects transcription and protein expression levels of proliferation and apoptotic factors
Since Cyclin D1 and PCNA are critical factors for cell proliferation (40,41), while Bcl-2 and Bax are key targets of apoptosis (42,43), whether they were involved in the protective role of the CGRPR/TRPV1 axis in HILI was assessed. At the transcriptional level, compared with the normal air group, the mRNA expression levels of Cyclin D1 and PCNA in the hyperoxia group were significantly downregulated, and this was rescued by CGRP. However, CGRP8-37 or SB-705498 significantly attenuated the CGRP-induced increase in Cyclin D1 and PCNA levels in the hyperoxic cells (Fig. 4A). Similarly, it was found that the mRNA expression levels of Bcl-2 were downregulated but Bax was upregulated in the hyperoxia group. However, CGRP increased the Bcl-2 expression, and this was reversed by either CGRP8-37 or SB-705498 (Fig. 4B, left panel). By contrast, CGRP decreased the Bax expression, and this was reversed by either CGRP8-37 or SB-705498 (Fig. 4B, right panel). Meanwhile, western blotting was performed to detect the expression of proliferation-related and apoptosis-related proteins after pretreatment with CGRP, CGRP8-37 and SB-705498. The changes in these factors at the protein level reflected what was observed at the transcription level (Fig. 4C and D). Taken together, these data suggested that CGRP promotes proliferation but inhibits apoptosis of A549 cells via a CGRPR/TRPV1 axis.
Role of TRPV1 channels in CGRP-mediated cell proliferation and apoptosis in hyperoxia
First, lentiviral infection was used to knock down TRPV1 channels expression in A549 cells, and knockdown was confirmed at both the mRNA and protein levels (Fig. 5A and B). The shTRPV1-2 sequence exhibited the optimal result out of the three shRNAs used, and thus it was used for all subsequent experiments. ShTRPV1 significantly reduced CGRP-induced proliferation of A549 cells in hyperoxia (Fig. 5C). Similarly, CGRP had no inhibitory effect on apoptosis after shTRPV1 in hyperoxia (Fig. 5D). Finally, western blotting was used to analyze the effect of shTRPV1 on the protein expression levels of Cyclin D1, PCNA, Bcl-2 and Bax in hyperoxia. As demonstrated in Fig. 5E, shTRPV1 significantly attenuated the CGRP-induced upregulation of Cyclin D1, PCNA and Bcl-2 protein expression, and reversed the CGRP-induced downregulation of Bax expression. The results following the knockdown of TRPV1 in A549 cells were consistent with those of the selective TRPV1 blocker treatment (Fig. 4D). Therefore, these data confirmed the role of TRPV1 channels in CGRP-mediated proliferation and apoptosis of A549 cells in hyperoxia.
CGRP induces Ca2+ entry via TRPV1 channels
Since TRPV1 channels are plasma membrane Ca2+-permeable channels (32,44), whether CGRP protected against hyperoxia-induced alveolar cell injury via the TRPV1/Ca2+ signaling pathway was examined. Patch-clamp and calcium imaging were performed using A549 cells. As demonstrated in the left panel of Fig. 6A, the membrane currents increased significantly with the addition of CGRP, while SB-705498 inhibited the CGRP-induced membrane currents. The middle panel of Fig. 6A shows a voltage-current diagram of the same cell, highlighting CGRP-induced membrane non-selective cation currents after SB-705498 treatment. Summary data on the right panel of Fig. 6A demonstrates that SB-705498 significantly inhibited CGRP-induced membrane currents. Subsequently, Ca2+ imaging experiments were performed in A549 cells. As revealed in Fig. 6B and D, CGRP induced a significant increase in intracellular Ca2+ signaling, which was attenuated by SB-705498. To further examine the importance of CGRP-mediated Ca2+ signaling in A549 cells, BAPTA-AM, a cell-permeable calcium chelator was used. It significantly attenuated the CGRP-induced intracellular Ca2+ signaling (Fig. 6C and E). In addition, it was found that BAPTA-AM (1 µM) attenuated the CGRP-induced proliferation of A549 cells in hyperoxia, while BAPTA-AM itself did not affect cell proliferation (Fig. 6F). By contrast, BAPTA-AM reversed the CGRP-induced decrease in apoptosis under hyperoxic conditions (Fig. 6G and H). These data suggested that CGRP protects against hyperoxia-induced alveolar cell injury via the TRPV1/Ca2+ signaling pathway.
The phospholipase C (PLC)/protein kinase C (PKC) pathway is involved in CGRP-mediated TRPV1/Ca2+ signaling
Activation of GPCR can stimulate TRPV1 channels to mediate inflammation via the PLC/PKC pathway (45–47). CGRPR acts as a GPCR, thus whether PLC/PKC was involved in the CGRPR/TRPV1/Ca2+ axis in A549 cells was examined. First, the role of PLC in CGRPR activation was examined. It was revealed that the selective PLC inhibitor U-73122 (1 µM) reduced CGRP-induced Ca2+ signaling, whereas the inactive analog U-73343 did not reduce CGRP-induced Ca2+ signaling (Fig. 7A). In addition, whether PKC was involved in the activation of CGRPR/TRPV1 channels was assessed. CGRP-induced Ca2+ signaling was attenuated by the selective PKC inhibitor Go6976 (200 nM) (Fig. 7B). These results suggested that PLC/PKC is involved in activating CGRPR/TRPV1 channels.
CGRP/CGRPR regulates TRPV1/Ca2+ via the PLC/PKC pathway in hyperoxia
Further applying the PLC inhibitor U-73122 (1 µM) and PKC inhibitor Go6976 (200 nM), it was revealed that both inhibitors attenuated CGRP-induced cell proliferation in hyperoxia (Fig. 8A and B). In addition, both PLC and PKC inhibitors attenuated the CGRP-induced increase in Cyclin D1, PCNA and Bcl-2 protein expression, but reversed the CGRP-induced decrease in Bax expression (Fig. 8C-F). Therefore, these data suggested that CGRPR activates TRPV1 channels via the PLC/PKC pathway.
Discussion
In the present study, the protective role of CGRP in hyperoxia-induced human alveolar cell injury was verified, and the underlying molecular mechanisms were elucidated. The primary findings were as follows: i) Although hyperoxia treatment did not alter CGRP release from human alveolar cells, it significantly downregulated the mRNA and protein expression levels of CGRPR and TRPV1 channels; ii) CGRP promoted proliferation but inhibited apoptosis of human alveolar cells via the CGRPR/TRPV1/Ca2+ signaling axis in hyperoxia; iii) CGRP/CGRPR activated TRPV1 channels to induce Ca2+ entry via the PLC/PKC pathway; iv) CGRP protected against hyperoxia-induced human alveolar cell injury via regulation of proliferation and apoptotic factors Cyclin D1, PCNA, Bcl-2 and Bax.
It is well established that activation of TRPV1 channels at sensory nerve endings can induce the release of Ca2+-dependent CGRP to exert biological effects by acting on CGRPR, indicating that the TRPV1/Ca2+ signaling pathway plays a key role in regulating CGRP release from sensory nerve endings (28,29). The present study investigated whether CGRP acted inversely on TRPV1 channels to exert a protective effect against HILI from the perspective of CGRP. Previous studies have revealed that TRPV1 channels are functionally expressed in human bronchial epithelial cells, with increased expression in patients with asthma (48,49). However, there was no significant change in the expression of TRPV1 in pulmonary artery smooth muscle cells of rats under hypoxic conditions (50). Since the expression of TRPV1 under hyperoxia has not yet been reported to the best of the authors' knowledge, in the present study it was demonstrated for the first time that the expression of TRPV1 in human alveolar cells was downregulated under hyperoxic treatment. CGRPR expression is significantly downregulated in LPS-induced acute lung injury in rats (20). The expression of CGRPR under hyperoxic conditions was unknown however, in the present study, it was also demonstrated that hyperoxia downregulated CGRPR expression in A549 cells.
Although CGRP secretion from human alveolar cells was not altered by hyperoxia treatment compared with the control culture conditions, the exogenous addition of CGRP induced cell proliferation and inhibited cell apoptosis under hyperoxia, indicating that exogenous CGRP, but not endogenous CGRP, played a protective role in hyperoxia-induced human alveolar cell injury. Therefore, the protective role of exogenous CGRP and molecular mechanisms involved were further assessed. First, it was revealed that the promotion of cell proliferation by CGRP was attenuated by selective inhibitors of CGRPR and TRPV1. In addition, CGRP inhibited apoptosis in hyperoxia, but selective inhibitors for both CGRPR and TRPV1 reversed this inhibitory effect of CGRP on apoptosis. These results suggested that CGRPR/TRPV1 was involved in CGRP-mediated alveolar cell proliferation and apoptosis in hyperoxia. After applying selective inhibitors of CGRPR and TRPV1 channels or using shTRPV1 to knock down TRPV1 expression, the proliferation and apoptotic factors Cyclin D1, PCNA, Bcl-2 and Bax were assessed. Inhibition of CGRPR and TRPV1 channels reduced the protective effects of CGRP against hyperoxia-induced alveolar cell injury. Taken together, it was hypothesized that a CGRPR/TRPV1 axis plays a critical role in protecting alveolar cells under hyperoxic conditions.
It was also revealed that the TRPV1 agonist capsaicin promoted alveolar cell proliferation, but inhibited apoptosis under hyperoxia-induced injury, further highlighting the protective role of TRPV1. Since TRPV1 channels are plasma membrane Ca2+-permeable channels (32,44), patch clamp and Ca2+ imaging were used to confirm that CGRP induced an increase in membrane non-selective currents and intracellular Ca2+ signaling, while SB-705498 inhibited these changes. The intracellular calcium chelator BAPTA-AM was also used to elucidate the role of TRPV1/Ca2+ signaling in CGRP/CGRPR protection against hyperoxia-induced alveolar cell injury.
CGRPR is a GPCR, its activation can regulate Gs/Gi to promote or inhibit adenylate cyclase to generate cAMP and can regulate the Gq/11-Ca2+ pathway (51). Since the CGRPR/cAMP pathway has been extensively studied (52–55), a focus was placed on the largely undefined CGRPR/Ca2+ pathway. Since activation of the Gq/11 protein promotes PLC activity (51), in the present study it was revealed that a PLC inhibitor reduced CGRP-induced intracellular Ca2+ signaling while attenuating the effects of CGRP-mediated protection against cell proliferation and apoptosis under hyperoxic conditions, suggesting that CGRP/CGRPR regulates Gq/11-PLC pathway. Due to PLC activating the PKC pathway, it was further confirmed that CGRP/CGRPR regulates PKC in alveolar cells. It was revealed that a PKC selective inhibitor attenuated the CGRP-induced increase in intracellular Ca2+ signaling in alveolar cells, while reversing the changes of CGRP-mediated proliferation and apoptotic factors Cyclin D1, PCNA, Bcl-2 and Bax. These data suggested that the PLC/PKC pathway plays a role in CGRP-mediated protection against hyperoxia-induced alveolar cell injury. However, the exact mechanisms of how the CGRPR/TRPV1/Ca2+ axis regulates proliferation-related and apoptosis-related factors need to be further elucidated.
Finally, the limitations of the present study will be discussed. Since the present study was performed at the in vitro cellular level, it lacks animal experiments or human in vivo experiments; thus, further in vivo experiments are required to verify the role of the identified CGRPR/TRPV1/Ca2+ axis. In addition, the human alveolar A549 cells were used to establish the cell model; not using primary AECII is also a potential limitation of the present study. The reasons for not using primary AECII are that primary human AECII are difficult to isolate and culture; and primary AECII isolated from rats have high background levels of apoptosis (56). Moreover, primary cultured AECII are prone to mutation and are unsuitable for transfection. However, the A549 cells, an AECII line, have similar biological properties to AECII, are frequently used in studies on HILI, making them a valuable research subject for hyperoxia (57–61).
In conclusion, CGRP protected against hyperoxia-induced alveolar cell injury via a novel CGRPR/TRPV1/Ca2+ axis (Fig. 9). CGRP/CGRPR activated TRPV1 channels via the PLC/PKC pathway, inducing extracellular Ca2+ entry to promote cell proliferation but inhibit apoptosis following hyperoxic injury, ultimately protecting against HILI. Therefore, although activation of TRPV1 channels promotes Ca2+-dependent CGRP release from sensory endings (28,29), in the present study it was revealed that exogenous CGRP could also inversely regulate the function of TRPV1 channels in alveolar cells. Importantly, the CGRPR/TRPV1/Ca2+ axis protected against hyperoxia-induced alveolar cell injury, highlighting a potential target for the management of HILI.
Since TRPV1 channels are located both on sensory endings to promote CGRP release and on alveolar cells to protect against hyperoxia-induced alveolar cell injury, the findings of the present study strongly suggested that capsaicin is a potential candidate to effectively prevent/treat HILI given its alveolar cell protective and anti-inflammatory effects (62–64), although this requires further study. Meanwhile, TRPV1 can be used as a drug development target in future studies to explore its role in the prevention and treatment of HILI.
Acknowledgements
Not applicable.
Funding
The present study was supported (grant no. 82273115) by research grants from the National Natural Science Foundation of China.
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
FX conceived the study and designed some experiments. HD designed all experiments, wrote and finalized the manuscript. JL performed most experiments and data analysis, and drafted the manuscript. HW, LW, FF and JW performed some experiments and revising the manuscript. All authors read and approved the final the manuscript. JL and FX confirm the authenticity of all the raw data.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
|
CGRP |
calcitonin gene-related peptide |
|
CGRPR |
CGRP receptor |
|
GPCR |
G protein-coupled receptor |
|
TRPV1 |
transient receptor potential vanilloid 1 |
|
AECII |
alveolar type II epithelial cells |
|
HILI |
hyperoxia-induced lung injury |
|
PLC |
phospholipase C |
|
PKC |
protein kinase C |
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