AECII-specific HMGCS2 overexpression in mouse pulmonary fibrosis

Phase 1: Animal Model Preparation & Gene Delivery (Days 0-14)

• Animals: C57BL/6 mice, 8-10 weeks old, male, ~22-25g (Envigo or Jackson Laboratory)
• Housing: SPF facility, 12h light/dark cycle, ad libitum access to standard chow
• Bleomycin-induced pulmonary fibrosis model:
• Mice receive 1.5 U/kg bleomycin (Biafra T001, in 50 μL sterile PBS) or equal volume vehicle (PBS) via oropharyngeal aspiration under isoflurane anesthesia
• Non-blinded operator performs all Installations; endpoint measurements performed by blinded operator
• AAV6-based AECII-specific HMGCS2 overexpression:
• Use AAV6 particles with Sftpc promoter驱动 (Addgene custom serotype, titer ≥1×10¹² gc/mL)
• Construct: AAV6-Sftpc-HMGCS2-3×FLAG (overexpression) or AAV6-Sftpc-scrambled-gRNA-3×FLAG (scr

Phase 1: Animal Model Preparation & Gene Delivery (Days 0-14)

• Animals: C57BL/6 mice, 8-10 weeks old, male, ~22-25g (Envigo or Jackson Laboratory)
• Housing: SPF facility, 12h light/dark cycle, ad libitum access to standard chow
• Bleomycin-induced pulmonary fibrosis model:
• Mice receive 1.5 U/kg bleomycin (Biafra T001, in 50 μL sterile PBS) or equal volume vehicle (PBS) via oropharyngeal aspiration under isoflurane anesthesia
• Non-blinded operator performs all Installations; endpoint measurements performed by blinded operator
• AAV6-based AECII-specific HMGCS2 overexpression:
• Use AAV6 particles with Sftpc promoter驱动 (Addgene custom serotype, titer ≥1×10¹² gc/mL)
• Construct: AAV6-Sftpc-HMGCS2-3×FLAG (overexpression) or AAV6-Sftpc-scrambled-gRNA-3×FLAG (scrambled control)
• Administer 30 μL via oropharyngeal aspiration at Day 3 post-bleomycin
• Vehicle group receives 30 μL AAV6-Sftpc-eGFP at equivalent titer

Phase 2: In Vivo Verification of Transduction (Days 7, 14)

• Tissue collection: Left lung flash-frozen in liquid nitrogen; right lung inflation-fixed with 4% PFA at 25 cmH₂O pressure
• Verification by qRT-PCR:
• RNA extracted with RNeasy Mini Kit (Qiagen 74104)
• Reverse transcription: SuperScript IV (Thermo 18090050)
• qRT-PCR primers: HMGCS2 F: 5′-GCCTGGAGCACAATCGACTT-3′, R: 5′-GCTGCTTCCACATGCGGATA-3′; GAPDH as endogenous control
• Expected overexpression: ≥3-fold increase in AAV-HMGCS2 vs AAV-scrambled groups
• Immunofluorescence validation:
• 5 μm paraffin sections; antigen retrieval with citrate buffer pH 6.0
• Primary: anti-FLAG M2 (1:500, Sigma F1804), anti-SFTPC (1:200, Abcam ab229639)
• Secondary: anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 647
• Confocal imaging (Zeiss LSM 980); co-localization confirmed with Pearson's r ≥0.75

Phase 3: Fibrosis Induction & Monitoring (Days 14-42)

• Body weight & clinical scoring (twice weekly):
• Weight loss >20% baseline, hunching, reduced mobility, piloerection =moribund criteria
• Bronchoalveolar lavage (BAL) at Day 28:
• 3×700 μL PBS lavages via tracheal cannula
• Total cell count (hemocytometer); differential on Diff-Quik (Siemens)-stained cytospins (300 cells counted)
• Expected: bleomycin groups show ≥50% neutrophils, ≥15% lymphocytes
• Lung function assessment at Day 42 (terminal):
• FlexiVent FX module (SCIREQ): dynamic compliance (Cdyn), elastance (H), resistance (R)
• Single-frequency oscillation (PVAN software); measurements averaged from ≥5 consecutive breaths

Phase 4: Terminal Tissue Collection & Histopathology (Day 42)

• Perfusion: Under isoflurane anesthesia, systemic perfusion with 10 mL cold PBS via left ventricle
• Right lung: Inflation-fixed with 4% PFA at 25 cmH₂O; paraffin-embedded
• Left lung: Flash-frozen in liquid nitrogen; stored at -80°C for biochemistry
• Histological scoring (Ashcroft modified):
• 5 μm H&E sections; 20 random fields at 200× magnification
• Score 0-8 scale: 0=normal, 8=fibrous obliteration of alveolar architecture
• Blinded scoring by two independent pathologists
• Masson's trichrome staining: Collagen content quantified by colorimetry (ImageJ) as % fibrosis area
• Immunohistochemistry:
• α-SMA (1:200, Abcam ab5694) — myofibroblast burden
• Phospho-SMAD3 (1:100, Cell Signaling 9520) — TGF-β pathway activation
• TUNEL assay (ApopTag, Chemicon) — AECII apoptosis

Phase 5: Molecular & Biochemical Endpoints (Day 42)

• HMGCS2 activity assay:
• Mitochondria isolated from left lung (Mitochondria Isolation Kit, Abcam ab110168)
• HMGCS2 enzymatic activity measured by HMG-CoA production (colorimetric assay, Sigma MAK097)
• Normalized to citrate synthase activity (CS activity kit, Abcam ab239712)
• Western blot:
• 20 μg lung protein per lane; SDS-PAGE 10% gel
• Antibodies: anti-HMGCS2 (1:500, Abcam ab137074), anti-FLAG-HRP (1:1000), anti-β-actin (1:5000, Sigma A2228)
• ECL detection (Bio-Rad Clarity); densitometry (ImageJ)
• qRT-PCR for fibrosis markers:
• Col1a1, Col3a1, Fn1, Acta2, Il6, Tgfbi
• ΔΔCt method; normalized to Gapdh and sham group

Phase 6: Statistical Analysis & Sample Size Justification

• Power calculation:
• Primary outcome (Ashcroft score): expected Δ=2.0, SD=1.5, α=0.05, β=0.20
• Per group n=12 → total N=48 (accounting for 15% mortality)
• ANOVA with Tukey's post-hoc (GraphPad Prism 10)
• Non-parametric alternatives: Kruskal-Wallis with Dunn's correction where appropriate