Maqui Berry and Ginseng Extracts Reduce Cigarette Smoke-Induced Cell Injury in a 3D Bone Co-Culture Model

Abstract

Cigarette smoking-induced oxidative stress has harmful effects on bone metabolism. Maqui berry extract (MBE) and ginseng extract (GE) are two naturally occurring antioxidants that have been shown to reduce oxidative stress. By using an osteoblast and osteoclast three-dimensional co-culture system, we investigated the effects of MBE and GE on bone cells exposed to cigarette smoke extract (CSE). The cell viability and function of the co-culture system were measured on day 14. Markers of bone cell differentiation and oxidative stress were evaluated at gene and protein levels on day 7. The results showed that exposure to CSE induced osteoporotic-like alterations in the co-culture system, while 1.5 µg/mL MBE and 50 µg/mL GE improved CSE-impaired osteoblast function and decreased CSE-induced osteoclast function. The molecular mechanism of MBE and GE in preventing CSE-induced bone cell damage is linked with the inhibition of the NF-κB signaling pathway and the activation of the Nrf2 signaling pathway. Therefore, MBE and GE can reduce CSE-induced detrimental effects on bone cells and, thus, prevent smoking-induced alterations in bone cell homeostasis. These two antioxidants are thus suitable supplements to support bone regeneration in smokers.

Keywords: cigarette smoke; co-culture system; ginseng; maqui berry; osteoblast; osteoclast.

Figure 1

The effects of MBE and GE on SCP-1/THP-1 co-culture system exposed to CSE. The co-culture system was co-incubated with 5% CSE and (or) antioxidants (1.5 μg/mL MBE, 50 μg/mL GE, and 3.5 mM NAC) for 14 days. (a) Mitochondrial activity of the co-culture system was measured on day 14. (b) Total DNA content of the co-culture system was measured on day 14. For osteoclast function, CAII activity (c) and TRAP activity (d) were compared after 14 days of co-culture. Statistical differences were determined using two-way ANOVA test followed by Turkey’s multiple comparisons. Data are present as means ± SEM, and the significances are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. CSE group. (N = 3–4, n = 3).

Figure 2

The expression levels of osteoblastic and osteoclastic genes in the co-culture system under exposure to CSE and antioxidants. SCP-1/THP-1 co-culture system was exposed to 5% CSE with or without antioxidants for 7 days. The gene expression levels were evaluated by RT-PCR. 18s rRNA served as a housekeeper gene. (a) Representative RT-PCR image showed the gene expression of Collagen 1, Runx2, NFATc1, TRAP 5b, and MMP9 in 3D co-culture system with different treatments. Expression levels of Collagen 1 (b), Runx2 (c), NFATc1 (d), TRAP 5b (e), and MMP9 (f) mRNA were measured on day 7. Statistical differences were determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are presented as means ± SEM, and the significance is shown as * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. CSE group. N = 3, n = 2.

Figure 3

The influence of MBE and GE on CSE-impaired bone-forming function. After co-culture with osteogenic medium (2% FCS, 200 μM L-ascorbic acid 2-phosphate, 5 mM β-glycerolphosphate, 25 mM HEPES, 1.5 mM CaCl₂, and 5 μM cholecalciferol) for 14 days, dot blot was performed to detect the secreted AP, OCN, and PINP in the supernatant. (a) A representative dot blot image showed the secreted protein levels of AP, OCN, and PINP in the 3D co-culture system with different treatments. (b) Secreted level of AP protein was measured in the co-culture system by day 14. (c) Secreted levels of OCN protein were measured in the co-culture system by day 14. (d) Secreted levels of PINP protein were measured in the co-culture system by day 14. Statistical differences were determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are presented as means ± SEM, and the significance is shown as * p < 0.05 and ** p < 0.01, vs. CSE group. N = 3, n = 3.

Figure 4

The sRANKL/OPG ratio and NF-κB expression in the co-culture system. Dot blot was used to detect the secreted sRANKL and OPG levels in the co-culture system by day 14 (N = 3, n = 3). Western blot (N = 3, n = 2) and immunofluorescence staining (N = 3, n = 3) were performed to detect the NF-κB protein level in both thee co-culture system and nucleus on day 7. (a) A representative dot blot image showing the secreted protein levels of sRANKL and OPG in the supernatant. (b) The ratio of sRANKL and OPG in the co-culture system. (c) A representative Western blot image showing the total protein levels of NF-κB and p-ERK1/2 in the co-culture system. (d,e) Quantitative analysis of total NF-κB and p-ERK1/2 protein levels in the co-culture system. (f) A representative immunofluorescence image showing the protein expressions of NF-κB (scale bar  =  20 μm). (g) The NF-κB protein level in the nucleus of the osteoclast. Statistical differences were determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are presented as means ± SEM, and the significance is shown as * p < 0.05, ** p < 0.01 and **** p < 0.0001 vs. CSE group.

Figure 5

Influence of MBE and GE on antioxidant signaling pathway. Western blot was performed to detect the protein expression levels of p-Nrf2 and SOD1 in the co-culture system by day 7. (a) Representative Western blot image showing the protein levels of p-Nrf2 and SOD1 among different groups. The levels of p-Nrf2 (b) and SOD1 proteins (c) were measured in the co-culture system on day 7. Statistical differences were determined using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Data are presented as means ± SEM, and the significance is shown as * p < 0.05 vs. CSE group. N = 3, n = 2.

Figure 6

Diagram of the effects of MBE and GE on preventing CSE-induced bone cell damage. Both CSE and antioxidants could activate the Nrf2 signaling pathway. The dissociation of Keap1/Nrf 2 complexes prevents the degradation of transcription factor Nrf2 and, consequently, results in the activation of Nrf2. Combined with antioxidant response element (ARE) promoter, phosphorylated Nrf2 (p-Nrf2) triggers the antioxidative transcription of SOD1. Then, the antioxidative defense system enhances osteogenesis in the SCP-1/THP-1 co-culture system. However, the slight activation of the Nrf2 signaling pathway by CSE could not inhibit the oxidative damage in osteoblasts. This led to decreased OPG secretion by osteoblasts and, thus, an increase in the RANKL: OPG ratio. Through the interaction between RANKL and RANK (RANKL receptor), increased RANKL activated the NF-κB pathway. In addition, CSE also upregulated the NF-κB pathway in the co-culture system. CSE promotes the expression of phosphorylation ERK 1/2 via the production of reactive oxygen species (ROS). ROS-induced inhibitor of kappa B kinase beta (IKKβ) results in the degradation of IκB-α and the activation of NF-κB. Transcriptional factor NF-κB promoted the expression of osteoclast-related genes and osteoclastogenesis. The black and green arrows represent the activity of CSE and antioxidants (MBE or GE), respectively.