PM2.5 induces autophagy-mediated cell apoptosis via PI3K/AKT/mTOR signaling pathway in mice bronchial epithelium cells.

SUBJECT:han-mouse-c57bl6

Male C57BL/6 mouse housed in a PM2.5 or normal survival environment for 21 days

20 eight-week-old male C57BL/6 mice, weight 20-23 g, from Tianjin Medical University, housed at 23 +/- 1 C and 50 +/- 5% humidity on a 12 h light/dark cycle with food and water ad libitum. Randomly divided into two groups (n=10 each): PM2.5 environment and normal survival (Control) environment, for 21 days; euthanized by cervical decapitation on day 22.

Pulmonary function measured on day 22 under 40 mg/kg sodium pentobarbital anaesthesia with mechanical ventilation

soma:han-mbec-001

Mouse bronchial epithelium cells (MBECs)

LF:han-mouse-pm25-21d

FlexiVent pulmonary function in mice after 21 days of PM2.5 exposure

Pulmonary function measured on day 22 in mice housed 21 days in a PM2.5 or a normal survival environment (n=10 per group). PM2.5 significantly decreased lung function - reported as total lung capacity, residual volume, vital capacity and airway resistance in the Results and the abstract, and as lung capacity, residual volume, tidal volume and airway resistance in the Methods. Body weight was also significantly decreased and H&E staining showed PM2.5-induced lung injury. All values are reported only in Fig. 1A-F, so no numeric measurement, spread or p-value is recorded. Note that 'decreased airway resistance' is the paper's own wording; it is grouped with the other parameters under a single statement of decreased lung function and is not separately quantified or discussed, so no direction is asserted here beyond the quoted sentence.

Exposure conditions

Outputs

Evidence

OX:han-mbec-ros-24h

ROS production in PM2.5-exposed MBECs (DCFH-DA)

Intracellular reactive oxygen species imaged with a DCFH-DA probe in MBECs placed for 24 h in air containing PM2.5 or in 95% air / 5% CO2. ROS production was upregulated by PM2.5 relative to control, which the authors read as the source of oxidative injury in lung cells. Fluorescence intensity was calculated with analysis LS 5.0 but reported only in Fig. 2C, so no numeric value is recorded.

Exposure conditions

Outputs

Evidence

GE:han-serum-il1b-il6

Serum IL-1beta and IL-6 by ELISA in PM2.5-exposed mice

IL-1beta (Bio-Rad MLB00C) and IL-6 (Bio-Rad M6000B) measured by ELISA in plasma collected on day 22 from both mouse groups. PM2.5 increased IL-1beta and IL-6 expression levels in serum relative to control. Concentrations are reported only in Fig. 2A, so no numeric concentration, spread or p-value is recorded.

Exposure conditions

Outputs

Evidence

GE:han-mbec-il1b-il6

IL-1beta and IL-6 expression in PM2.5-exposed MBECs

IL-1beta and IL-6 expression in MBECs placed for 24 h in air containing PM2.5 or in 95% air / 5% CO2. Both were markedly increased by PM2.5 relative to control. The paper does not state which method was used for the MBEC cytokine measurement - the ELISA kits in the Methods are described for plasma only - so gene_expression_method is omitted. Values are reported only in Fig. 2B; none recorded.

Exposure conditions

Outputs

Evidence

GE:han-mbec-pi3k-akt-mtor

PI3K, AKT and mTOR expression and PI3K/AKT phosphorylation in MBECs

Western blot for PI3K, phospho-PI3K, AKT and phospho-AKT (beta-actin loading control) in MBECs after 24 h in air containing PM2.5, PM2.5 plus the PI3K inhibitor PI3KIR, PI3KIR alone, or 95% air / 5% CO2. PM2.5 increased PI3K, AKT and mTOR expression, and a statistical difference in PI3K and AKT phosphorylation was identified between the PM2.5 and control groups. PI3KIR significantly decreased PI3K, AKT and mTOR expression. The paper reports mTOR among the three targets in the text but lists no mTOR antibody in the Methods, so the antibody list on PROTOCOL:han-wb-001 records only the four that are named. Densitometry was performed with Quantity-One 1.2 but reported only in Fig. 4A and Fig. 5A, so no numeric value, spread or p-value is recorded.

Exposure conditions

Outputs

Evidence

GE:han-mbec-lc3b-autophagy

LC3B immunofluorescence for autophagy in MBECs

Autophagy assessed by anti-LC3B immunofluorescence in MBECs after 24 h in air containing PM2.5, PM2.5 plus PI3KIR, PI3KIR alone, or 95% air / 5% CO2. All four Methods arms are recorded; the paper describes only three of them, so the inhibitor-only arm carries no direction. PM2.5 induced autophagy of MBECs, and the PI3K inhibitor blocked PM2.5-induced autophagy. Results are reported only in Fig. 4B and Fig. 5B-C; the paper reports no LC3B puncta count, no fluorescence intensity and no p-value, so the output records carry no measurement value.

Exposure conditions

Outputs

Evidence

GE:han-lung-tunel-apoptosis

TUNEL apoptosis in lung tissue of PM2.5-exposed mice

TUNEL staining of 4 um lung sections from both mouse groups. PM2.5 increased lung cell apoptosis in lung tissue relative to control. The apoptosis rate was measured with Developer XD 1.2 across six fields of view but reported only in Fig. 3A, so no apoptosis rate, spread or p-value is recorded. This is not a gene expression measurement; it is recorded under gene_expression_assays because the schema has no apoptosis assay class, and the output records therefore carry no mrna_level, protein_level or percentage_positive_cells value.

Exposure conditions

Outputs

Evidence

GE:han-mbec-tunel-apoptosis

TUNEL apoptosis in MBECs with and without PI3K inhibition

TUNEL staining with DAPI counterstain in MBECs after 24 h in air containing PM2.5, PM2.5 plus PI3KIR, PI3KIR alone, or 95% air / 5% CO2. All four Methods arms are recorded; the paper describes only three of them, so the inhibitor-only arm carries no direction. PM2.5 induced apoptosis of MBECs relative to control, and the PI3K inhibitor blocked PM2.5-induced apoptosis. The apoptosis rate was measured with Developer XD 1.2 across six fields of view but reported only in Fig. 3B and Fig. 5B-C, so no apoptosis rate, spread or p-value is recorded. As with the lung tissue arm, this is not a gene expression measurement and is recorded here only because the schema has no apoptosis assay class.

Exposure conditions

Outputs

Evidence

KER:han-pi3k-to-autophagy

PI3K/AKT/mTOR signaling activation leads to increased autophagy

PM2.5 raised PI3K, AKT and mTOR expression and PI3K/AKT phosphorylation in MBECs and induced autophagy in the same cells; the PI3K inhibitor lowered pathway expression and blocked the autophagy, so the pathway is upstream of the autophagy response.

KER:han-autophagy-to-apoptosis

Increased autophagy leads to increased epithelial cell apoptosis

The paper's central claim, stated in its title and conclusion: PM2.5-induced autophagy mediates the apoptosis of MBECs. Both responses were blocked together by the PI3K inhibitor. The paper does not manipulate autophagy independently of the pathway, so the ordering of autophagy before apoptosis rests on the authors' interpretation rather than on a separate experiment.

KER:han-apoptosis-to-lung-function

Increased epithelial cell apoptosis leads to decreased lung function

The same mice that lost lung function showed increased apoptosis in lung tissue, and the paper frames MBEC apoptosis as the mechanism of PM2.5-induced lung disease. No experiment separates the two, so this is recorded as an indirect relationship with moderate support.

KER:han-oxidative-stress-to-apoptosis

Increased oxidative stress is associated with increased epithelial cell apoptosis

ROS production rose in the same PM2.5-exposed MBECs that underwent apoptosis, and the authors suggest the ROS rise induced oxidative injury. No antioxidant or ROS-scavenging experiment was performed, and the PI3K inhibitor arms were not assayed for ROS, so this relationship is recorded as weakly supported association rather than a tested causal step.

PROTOCOL:han-flexivent-001

FlexiVent pulmonary function testing in anaesthetized mice

On day 22 all experimental mice were anesthetized with 40 mg/kg sodium pentobarbital, intubated with a custom-made laryngoscope blade and mechanically ventilated with a rodent ventilator. Pulmonary functions - lung capacity, residual volume, tidal volume and airway resistance - were analysed using the FlexiVent system (SCIREQ) according to the manufacturer's protocol. The Results section lists the same panel as total lung capacity, residual volume, vital capacity and airway resistance.

Equipment: FlexiVent system (SCIREQ); Rodent ventilator; Custom-made laryngoscope blade

PROTOCOL:han-elisa-il1b-001

ELISA for IL-1beta in mouse plasma

On day 22 blood was collected from each group and plasma obtained by centrifugation at 10,000 x g for 10 min at 4 C. IL-1beta was measured with an ELISA kit (cat. no. MLB00C; Bio-Rad Laboratories, Inc.).

Equipment: Microplate reader; IL-1beta ELISA kit (Bio-Rad cat. no. MLB00C); Refrigerated centrifuge

PROTOCOL:han-elisa-il6-001

ELISA for IL-6 in mouse plasma

On day 22 blood was collected from each group and plasma obtained by centrifugation at 10,000 x g for 10 min at 4 C. IL-6 was measured with an ELISA kit (cat. no. M6000B; Bio-Rad Laboratories, Inc.).

Equipment: Microplate reader; IL-6 ELISA kit (Bio-Rad cat. no. M6000B); Refrigerated centrifuge

PROTOCOL:han-he-001

Hematoxylin and eosin staining of mouse lung sections

Lung tissues were fixed with 4% formaldehyde overnight at room temperature, embedded in paraffin and cut into 4 um sections. Sections underwent hematoxylin and eosin staining for 30 min at room temperature, were washed with PBS three times and observed under a light microscope at x100 magnification (Olympus Corporation).

Equipment: Microtome; Light microscope (Olympus Corporation)

PROTOCOL:han-dcfhda-001

DCFH-DA fluorescence detection of ROS production in MBECs

A 2',7'-dicholorofluorescein-diacetate (DCFH-DA) probe was used to evaluate ROS production in PM2.5-treated MBECs in six-well plates. DCFH-DA (10 uM) was added and cells cultured for 30 min at 37 C in the dark, then washed three times with ice-cold PBS. ROS production was examined at x50 magnification on a fluorescence microscope (Olympus Corporation) and fluorescence intensity calculated with analysis LS 5.0 soft image solution (Olympus Imaging America Inc.).

Equipment: Fluorescence microscope (Olympus Corporation); DCFH-DA probe; analysis LS 5.0 soft image solution

PROTOCOL:han-wb-001

Western blotting for PI3K, phospho-PI3K, AKT and phospho-AKT

MBECs (5x10^6) were lysed in RIPA buffer (M-PER reagent for cells, T-PER for tissues; Thermo Fisher Scientific). Protein concentration was measured with a BCA protein assay kit. Protein (20 ug) was electrophoresed on a 15% SDS-PAGE gel and transferred to PVDF (EMD Millipore). Membranes were blocked with 5% BSA at 4 C overnight, probed with primary rabbit anti-rat antibodies, washed three times in PBST, incubated with HRP-conjugated goat anti-rabbit IgG mAb (1:2,000; cat. no. PV-6001; OriGene Technologies) for 1 h at 37 C, and developed with a chemiluminescence assay system (EMD Millipore). Densitometric quantification used Quantity-One 1.2 software (Bio-Rad Laboratories).

Equipment: SDS-PAGE apparatus; Chemiluminescence imaging system (EMD Millipore)

PROTOCOL:han-tunel-tissue-001

TUNEL apoptosis staining of mouse lung tissue sections

TUNEL analysis used an In Situ Cell Death Detection kit (DeadEnd Colorimetric Tunel System; Promega Corporation). Lung tissue sections (4 um) were deparaffinized in xylene, rehydrated in graded ethanol and rehydrated for 3 min, then incubated with TUNEL for 2 h at 37 C per the manufacturer's protocol. Images were captured in six fields of view with a ZEISS LSM 510 confocal microscope at 488 nm, x100 magnification, and the apoptosis rate measured with Developer XD 1.2 software (Definiens AG). Note the tissue incubation is 2 h, versus 1 h for cultured cells, so the two TUNEL runs are recorded as separate protocols.

Equipment: ZEISS LSM 510 confocal microscope; In Situ Cell Death Detection kit (DeadEnd Colorimetric Tunel System, Promega); Developer XD 1.2 software (Definiens AG)

PROTOCOL:han-tunel-cells-001

TUNEL apoptosis staining of cultured MBECs

MBECs were treated with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBST at room temperature, and incubated with TUNEL (DeadEnd Colorimetric Tunel System, Promega) for 1 h at 37 C per the manufacturer's protocol. Cells were washed three times with PBS and incubated with 5% DAPI (Sigma-Aldrich) under Antifade mounting medium (cat. no. P0126; Beyotime Institute of Biotechnology) for 30 min at 37 C. Images were captured in six fields of view with a ZEISS LSM 510 confocal microscope at 488 nm, x100 magnification, and the apoptosis rate measured with Developer XD 1.2 software (Definiens AG).

Equipment: ZEISS LSM 510 confocal microscope; In Situ Cell Death Detection kit (DeadEnd Colorimetric Tunel System, Promega); Developer XD 1.2 software (Definiens AG)

PROTOCOL:han-lc3b-if-001

LC3B immunofluorescence for autophagy in MBECs

MBECs in culture dishes were fixed with 4% paraformaldehyde for 15 min at room temperature and blocked with 0.5% BSA for 30 min at 37 C. Cells were washed three times with PBST at room temperature and incubated with anti-LC3B (1:1,000; cat. no. ab48394; Abcam) for 12 h at 4 C, then with Alexa Fluor 488-labeled goat anti-rabbit secondary antibodies (1:2,000; Beyotime Institute of Biotechnology) for 12 h at 4 C. Images were captured on a confocal microscope (ZEISS GmbH; LSM510 META; x100 magnification) and analysed with AxioVision Rel. 4.6 software (Zeiss GmbH).

Equipment: Confocal microscope (ZEISS LSM510 META); Anti-LC3B antibody (Abcam ab48394); AxioVision Rel. 4.6 software