Nicotine, THC, and Dolutegravir Modulate E-Cigarette-Induced Changes in Addiction- and Inflammation-Associated Genes in Rat Brains and Astrocytes

Abstract

E-cigarette use has been marketed as a safer alternative to traditional cigarettes, as a means of smoking cessation, and are used at a higher rate than the general population in people with HIV (PWH). Early growth receptor 2 (EGR2) and Activity-Regulated Cytoskeleton-Associated Protein (ARC) have a role in addiction, synaptic plasticity, inflammation, and neurodegeneration. This study showed that 10 days of exposure to e-cigarette vapor altered gene expression in the brains of 6-month-old, male, Sprague Dawley rats. Specifically, the e-cigarette solvent vapor propylene glycol (PG) downregulated EGR2 and ARC mRNA expression in frontal cortex, an effect which was reversed by nicotine (NIC) and THC, suggesting that PG could have a protective role against NIC and cannabis dependence. However, in vitro, PG upregulated EGR2 and ARC mRNA expression at 18 h in cultured C6 rat astrocytes suggesting that PG may have neuroinflammatory effects. PG-induced upregulation of EGR2 and ARC mRNA was reversed by NIC but not THC. The HIV antiretroviral DTG reversed the effect NIC had on decreasing PG-induced upregulation of EGR2, which is concerning because EGR2 has been implicated in HIV latency reversal, T-cell apoptosis, and neuroinflammation, a process that underlies the development of HIV-associated neurocognitive disorders.

Keywords: THC; antiretroviral drugs; neuroinflammation; nicotine.

Figure 1

E-cigarette vapor, nicotine, and THC alter gene expression in rat frontal cortex. Heatmaps demonstrating significant differential gene expression (p < 0.05) for mRNA extracted and sequenced from whole brain lysate of rats exposed to e-cigarette vapor for 10 days. (a) PG (100% propylene glycol) vs. AIR (air), (b) PG vs. NIC (PG + Nicotine 30 mg/mL), (c) NIC vs. THC (PG + THC 200 mg/mL).

Figure 2

Propylene glycol (PG) decreases EGR2 and ARC mRNA and protein expression in frontal cortex of rats exposed to e-cigarette vapor for 10 days and these changes are reversed by THC and nicotine. (a) Fold change in EGR2 and (b) ARC mRNA normalized to ACTB. (c) Immunoblot of frontal cortex with an antibody specific for EGR2, ARC, and Actin. Quantification of immunoblot for (d) EGR2 and (e) ARC band intensity normalized to actin. AIR = Air, PG = 100% propylene glycol, THC = PG + THC 200 mg/mL. NIC = PG + nicotine 30 mg/mL. Mean ± SEM. Statistical significance was determined by one-way ANOVA, post hoc Tukey’s. ** p < 0.01 vs. Air; ^ p < 0.05, ^^^ p < 0.001 vs. PG.

Figure 3

PG (propylene glycol) alters EGR2 and ARC mRNA expression at 6 h and increases EGR2 and ARC mRNA expression in a dose-dependent manner at 18 h in cultured C6 rat astrocytes. Fold change in (a) EGR2 and (b) ARC mRNA levels normalized to ACTB following 18 h treatment with 5 mM, 50 mM, or 150 mM PG. Fold in of (c) EGR2 and (d) ARC mRNA levels normalized to ACTB following 6 h or 18 h treatment with 150 mM PG. Mean ± SEM. Statistical significance was determined by one-way ANOVA, post hoc Tukey’s. (a,b) * p < 0.05, ** p < 0.01 vs. vehicle; ^^ p <0.01 vs. 150 mM PG (c,d) **** p < 0.0001 vs. 0 h; ^^ p < 0.005 vs. 6 h, ^^^^ p < 0.00001 vs. 6 h.

Figure 4

PG (propylene glycol) increases EGR2 and ARC mRNA expression in C6 rat astrocytes at 18 h and is reversed by Nicotine (NIC), an effect that is lost with the addition of dolutegravir (DTG). Fold change in (a) EGR2 and (b) ARC mRNA levels normalized to ATCB following 18 h treatment with Vehicle, PG (150 mM), PG + THC (10µM), PG + NIC (10µM), DTG (200 nM), DTG, PG + DTG, PG + THC + DTG, or PG + NIC + DTG. Mean ± SEM. Statistical significance was determined by one-way ANOVA, post hoc Tukey’s. ** p < 0.01, *** p <0.001, **** p < 0.0001 vs. vehicle, ^ p <0.05, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. DTG.