ABSTRACT
Objective:To investigate the inhibitory effect and mechanism of heme oxygenase-1 (HO-1) on the inflammatory response of macrophages.Methods:Mouse macrophage strain RAW264.7 was cultured in vitro, and the cells in the logarithmic growth phase were used for the experiment. The RAW264.7 cells were divided into four groups. In blank control group, the cells were continuously incubated and received no treatment (cultured at 37 ℃, 95% air, 5% CO 2). In lipopolysaccharide (LPS) model group, 1 mg/L LPS was added to the medium to prepare LPS challenge model. In HO-1 inducer group, the cells were incubated with 30 μmol/L HO-1 inducer hemin for 1 hour, and then 1 mg/L LPS was added for incubation. In HO-1 inhibition group, the cells were incubated with 5 μmol/L HO-1 specific antagonist Zinc protoporphyrin Ⅸ (ZnPPⅨ) for 0.5 hour, and then 1 mg/L LPS was added for incubation. After 48 hours of incubation with LPS, the supernatant of each group was taken, and the protein expressions of HO-1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3) and mitochondrial autophagy marker microtubule-associated protein 1 light chain 3B (LC-3B) were detected by Western blotting. The expression of reactive oxygen species (ROS) was detected by immunofluorescence staining. Results:Compared with the blank control group, the cells in the LPS model group had a certain stress response, and autophagy occurred in mitochondria, but the expression of some inflammatory factors was restricted, which was related to the impairment of cell function. The protein expressions of HO-1, IL-1β, LC-3B, ROS were significantly increased, the protein expressions of TNF-α, TXNIP, and NLRP3 were decreased significantly, indicating that the cells were seriously injured after LPS challenge, and the model was successfully established. Compared with the LPS model group, HO-1 protein expression in the HO-1 inducer group was significantly increased (HO-1/GAPDH: 0.31±0.03 vs. 0.22±0.03, P < 0.05), the protein expressions of TNF-α, IL-1β, TXNIP, NLRP3, LC-3B and ROS were significantly inhibited [TNF-α protein (TNF-α/GAPDH): 0.08±0.01 vs. 0.45±0.05, IL-1β protein (IL-1β/GAPDH): 0.50±0.01 vs. 0.82±0.03, TXNIP protein (TXNIP/GAPDH): 0.21±0.02 vs. 0.28±0.02, NLRP3 protein (NLRP3/GAPDH): 0.11±0.01 vs. 0.17±0.02, LC-3B protein (LC-3B/GAPDH): 0.67±0.04 vs. 0.92±0.12, ROS (fluorescence intensity): 80.9±12.5 vs. 94.1±19.5, all P < 0.05], indicating that HO-1 could inhibit inflammatory response and oxidative stress, and reduce mitochondrial autophagy. Antagonizing HO-1 could increase inflammatory response, oxidative stress and mitochondrial autophagy, the inhibitory degree of TNF-α and IL-1β expression was significantly reduced as compared with the HO-1 inducer group [TNF-α protein (TNF-α/GAPDH): 0.26±0.02 vs. 0.08±0.01, IL-1β protein (IL-1β/GAPDH): 0.76±0.01 vs. 0.50±0.01, both P < 0.05], the protein expressions of TXNIP, NLRP3, LC-3B and ROS were significantly increased as compared with the LPS model group [TXNIP protein (TXNIP/GAPDH): 0.43±0.02 vs. 0.28±0.02, NLRP3 protein (NLRP3/GAPDH): 0.24±0.02 vs. 0.17±0.02, LC-3B protein (LC-3B/GAPDH): 1.12±0.07 vs. 0.92±0.12, ROS (fluorescence intensity): 112.0±17.0 vs. 94.1±19.5, all P < 0.05]. Conclusion:HO-1 can reduce the inflammatory response by inhibiting the activation of TXNIP/NLRP3 inflammasome and reducing the release of inflammatory mediators.
ABSTRACT
Dengue virus (DENV) infection causes life-threatening diseases such as dengue hemorrhagic fever and dengue shock syndrome. Currently, there is no effective therapeutic agent or vaccine against DENV infection; hence, there is an urgent need to discover anti-DENV agents. The potential therapeutic efficacy of lucidone was first evaluated in vivo using a DENV-infected Institute of Cancer Research (ICR) suckling mouse model by monitoring body weight, clinical score, survival rate, and viral titer. We found that lucidone effectively protected mice from DENV infection by sustaining survival rate and reducing viral titers in DENV-infected ICR suckling mice. Then, the anti-DENV activity of lucidone was confirmed by western blotting and quantitative-reverse-transcription-polymerase chain reaction analysis, with an EC50 value of 25 ± 3 µM. Lucidone significantly induced heme oxygenase-1 (HO-1) production against DENV replication by inhibiting DENV NS2B/3 protease activity to induce the DENV-suppressed antiviral interferon response. The inhibitory effect of lucidone on DENV replication was attenuated by silencing of HO-1 gene expression or blocking HO-1 activity. In addition, lucidone-stimulated nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in transactivation of HO-1 expression for its anti-DENV activity. Taken together, the mechanistic investigations revealed that lucidone exhibits significant anti-DENV activity in in vivo and in vitro by inducing Nrf2-mediated HO-1 expression, leading to blockage of viral protease activity to induce the anti-viral interferon (IFN) response. These results suggest that lucidone is a promising candidate for drug development.
