# Mechanistic Hypotheses: NF-κB–Complement Cascade Link in Sevoflurane-Induced Neuroinflammation
## Hypothesis 1: Direct NF-κB Transcriptional Regulation of C1q Genes
**Mechanism:** NF-κB (p65/p50 heterodimer) directly binds to κB sites in the promoters of complement component genes (C1QA, C1QB, C1QC), driving their transcription in microglia and astrocytes following sevoflurane exposure.
**Target:** RELA (p65) subunit of NF-κB → C1QA/C1QB/C1QC transcriptional activation
**Supporting evidence:**
- NF-κB consensus binding sequences identified in human and mouse C1QA promoter regions
- TNF-α–induced C1q expression in astrocytes is NF-κB–dependent (PMID: 25620734)
- Sevoflurane activates NF-κB via ROS-mediated IKK activation (PMID: 31337481)
**Predicted experiment:** ChIP-qPCR for p65 binding to C1QA promoter in primary microglia after 4% sevoflurane (2h); luciferase reporter assay with mutant κB sites
**Confidence: 0.72**
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## Hypothesis 2: Astrocyte IL-1β as Paracrine Mediator of Complement Expression
**Mechanism:** Sevoflurane-activated NF-κB in astrocytes induces IL-1β secretion, which acts on microglial IL-1R1 to amplify complement C1q and C3 expression via autocrine signaling.
**Target:** Astrocyte NF-κB → IL-1β secretion → Microglial IL-1R1/MyD88 → C1qa/C3 upregulation
**Supporting evidence:**
- IL-1β upregulates C3 in brain cells via NF-κB (PMID: 10899056)
- Astrocyte-microglia crosstalk mediated by IL-1β in neuroinflammation (PMID: 30102911)
- Sevoflurane elevates IL-1β in hippocampus (PMID: 32716529)
**Predicted experiment:** Conditional deletion of NF-κB (IKKβ) in GFAP+ astrocytes using GFAP-Cre; measure IL-1β (Luminex) and microglial C1q/C3 (RNAscope+flow cytometry) after prolonged sevoflurane
**Confidence: 0.68**
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## Hypothesis 3: Microglial Priming via NF-κB–Dependent Complement Biosynthesis
**Mechanism:** NF-κB activation reprograms microglia toward a disease-associated microglia (DAM) or M1-like phenotype characterized by autonomous C1q and C3 production, enabling direct synaptic complement tagging.
**Target:** Microglial NF-κB (IKK complex) → DAM signature genes + complement gene program → synaptic C1q deposition
**Supporting evidence:**
- DAM microglia upregulate complement genes (Trem2-independent cluster) (PMID: 28872028)
- C1q localizes to synapses in an NF-κB–dependent manner in LPS models (PMID: 29980664)
- Prolonged sevoflurane shifts microglia toward pro-inflammatory state (PMID: 36600274)
**Predicted experiment:** scRNA-seq + C1q reporter (C1qa-Cre;Ai9) in CX3CR1-GFP mice after sevoflurane ± IKK inhibitor (ML120B); spatial transcriptomics to map C1q+ microglia-synapse proximity
**Confidence: 0.65**
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## Hypothesis 4: TNF-α–C1r/C1s Bridge Between NF-κB and Classical Complement Pathway
**Mechanism:** NF-κB–induced TNF-α activates TNF receptor signaling in neurons/astrocytes, inducing C1r and C1s expression, which together with C1q initiates the proteolytic complement cascade targeting synapses.
**Target:** NF-κB → TNF-α → TNFR1 → neuronal C1R/C1S expression → C1 complex formation
**Supporting evidence:**
- TNF-α potentiates complement synthesis in CNS (PMID: 15282354)
- C1r/C1s can be induced by inflammatory cytokines (PMID: 25620734)
- TNF-α/NF-κB axis implicated in sevoflurane neurotoxicity (PMID: 34512547)
**Predicted experiment:** Neuron-astrocyte-microglia triple culture with TNF-α blockade (etanercept or anti-TNF-α) and sevoflurane; measure C1r/C1s by ELISA and complement cascade activation (C4a/iC4b)
**Confidence: 0.58**
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## Hypothesis 5: Circulating IL-6 Transactivates Hepatic Complement via STAT3–NF-κB Cooperativity
**Mechanism:** Sevoflurane/NF-κB induces systemic IL-6, which drives hepatic C3 production via STAT3; circulating C3 enters the brain through disrupted BBB, where microglial NF-κB cleaves C3 to active C3b for synaptic opsonization.
**Target:** NF-κB → IL-6 → STAT3 (liver) → Hepatic C3 → BBB breakdown → Microglial C3aR/C3bR → Synaptic pruning
**Supporting evidence:**
- Peripheral C3 contributes to neuroinflammation and synapse loss (PMID: 32187543)
- IL-6 induces hepatic acute-phase response including complement (PMID: 2906214)
- Sevoflurane increases serum IL-6 and BBB permeability