Cadmium decreases human gingival fibroblast viability and induces pro-inflammatory response associated with Akt and MAPK pathway activation

Abstract

Smoking and particulate matter 2.5 (PM2.5) expose millions to cadmium (Cd), a toxic heavy metal linked to pro-inflammatory responses, oxidative stress, and disease pathogenesis. In the oral cavity, chronic Cd exposure contributes to the progression of periodontal diseases and oral cancers. However, the direct effect of Cd on oral tissues and the underlying mechanisms remains unclear. This study explored the impact of environmentally relevant concentrations of Cd on human gingival fibroblasts (HGFs) by evaluating cell viability, pro-inflammatory cytokine secretion (IL-6 and IL-8), COX-2 expression, and the activation of key signaling pathways: Akt, ERK1/2, and JNK. Cd exposure significantly reduced HGF viability, elevated IL-6 and IL-8 secretion, and upregulated COX-2 expression. These effects were attenuated by inhibitors targeting Akt, ERK1/2, and JNK pathways. By integrating cytokine profiling, COX-2 expression, and inhibitor-based pathway analysis, our study provides mechanistic insights into how low-level Cd exposure triggers early inflammatory responses in gingival fibroblasts. Our findings reveal that Cd exerts pro-inflammatory and cytotoxic effects on HGFs, which may play a role as one of the factors in the pathogenesis of smoking-related oral diseases. Targeting Akt, ERK1/2, and JNK signaling pathways could offer therapeutic strategies to attenuate Cd-induced oral pro-inflammatory responses and tissue damage.

Keywords: cadmium; cyclooxygenase; fibroblasts; interleukin-6; interleukin-8.

FIGURE 1

Dose-response curve of CdCl₂-induced cytotoxicity in HGF and cell viability of HGFs following exposure to Cd and inhibitors. Cell viability was assessed after exposure to increasing concentrations of CdCl₂ (0, 0.1, 1, 3, 6, 10, and 100 μM) for 24 h using MTT assays. The IC₅₀ value, representing the concentration of CdCl₂ required to reduce cell viability by 50%, was determined from the dose-response curve (A). The viability of HGFs was also assessed after treatment with 1 µM CdCl₂ alone or in combination with specific pathway inhibitors: 10 µM LY294002 (Akt), 25 µM U0126 (ERK1/2), 25 µM SB203580 (p38), and 25 µM SP600125 (JNK) (B). HGFs cultured in control (DMEM) without treatment were the control group representing 100% cell viability. Data are shown as mean ± SEM (n = 3).

FIGURE 2

CdCl₂-induced Akt, ERK 1/2, and JNK expression activation in cultured HGFs. HGF cells were incubated with 1 μM CdCl₂ or co-treatment of 1 μM CdCl₂ and each inhibitor: 10 µM LY294002 (Akt), 25 µM U0126 (ERK1/2), 25 µM SB203580 (p38), and 25 µM SP600125 (JNK). HGF cells were also treated with the inhibitor alone to determine its effect. Control refers to cells cultured in DMEM without CdCl₂ treatment. Cells were incubated for 5 min for phosphorylated Akt measurement and 30 min for phosphorylated ERK1/2, p38, and JNK measurement. Levels of phospho-Akt, total Akt (A), phospho-ERK 1/2, ERK (B), phospho-p38 MAPK, p38 MAPK (C), and phospho-JNK, JNK (D) were investigated by Western blotting. Beta actin, GAPDH, and tubulin were used as housekeeping genes based on molecular weights of proteins of interest. Data are shown as mean ± SEM (n = 4). Statistical significance was determined using One-way ANOVA followed by Tukey’s multiple comparisons test represented by *P ≤ 0.05, **P ≤ 0.01.

FIGURE 3

IL-6 and IL-8 secretion response to CdCl₂ exposure and the effect of inhibitors. HGF cells were co-treated with 1 µM CdCl₂ and specific inhibitors: 10 µM LY294002 (Akt), 25 µM U0126 (ERK1/2), and 25 µM SP600125 (JNK) prior to the measurement of IL-6 (A) and IL-8 levels (B) by ELISA. Control refers to cells cultured in DMEM without CdCl₂ treatment. Data are shown as mean ± SEM. For all groups n = 3. Statistical significance was determined using One-way ANOVA followed by Dunnett’s multiple comparison test represented by *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

FIGURE 4

Effect of CdCl₂ on COX-2 expression in HGFs. Western blot analysis of COX-2 expression in HGFs after 1-h incubation with 1 μM CdCl₂, with and without specific pathway inhibitors: 10 µM LY294002 (Akt), 25 µM U0126 (ERK1/2), and 25 µM SP600125 (JNK) were determined. Control refers to cells cultured in DMEM without CdCl₂ treatment. The upper bands represent COX-2, and the lower bands correspond to GAPDH which was used as housekeeping genes. Data are shown as mean ± SEM (n = 4). Statistical significance was determined using One-way ANOVA followed by Dunnett’s multiple comparison test represented by *P ≤ 0.05, **P ≤ 0.01.