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 CGRP_8-37 (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 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.

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 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 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).

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.

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.

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.

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 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.

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.

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.

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.

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 CGRP_8-37 (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, CGRP_8-37 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.

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, CGRP_8-37 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 CGRP_8-37 or SB-705498 (Fig. 4B, left panel). By contrast, CGRP decreased the Bax expression, and this was reversed by either CGRP_8-37 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, CGRP_8-37 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.

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.

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.

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.

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.