Endoplasmic reticulum (ER) stress, caused by the accumulation of misfolded or unfolded proteins, activates the unfolded protein response to maintain cellular homeostasis and is implicated in bacterial infections. This study investigated ER stress activation in THP-1-derived macrophages infected with oral bacteria Porphyromonas gingivalis , Prevotella intermedia , Aggregatibacter actinomycetemcomitans , and Streptococcus oralis at an multiplicity of infection of 50 for 4 hours. mRNA and protein expressions related to ER stress were analyzed by real-time polymerase chain reaction and Western blot, while pro-inflammatory cytokines were measured using enzymelinked immunosorbent assay. P. gingivalis induced the highest mRNA expression of XBP1 and PERK, whereas A. actinomycetemcomitans showed elevated GRP78, ATF6, IRE1α, ATF4, and CHOP. P. intermedia strongly expressed PERK, while S. oralis showed higher GRP78, PERK, ATF4, and CHOP expression. Protein analysis revealed S. oralis had the highest phosphorylation levels of eIF2α and IRE1α, while CHOP was most highly expressed in P. intermedia . Pro-inflammatory cytokine expression showed P. intermedia and P. gingivalis elicited the most TNF-α, while P. gingivalis induced the highest IL-1β levels. These findings suggest oral bacteria induce varying levels of ER stress, influencing the progression of oral infectious diseases. Targeting ER stress could offer therapeutic potential for managing inflammatory conditions like periodontitis.

Introduction
Materials and Methods
    1. Bacterial culture
    2. Cell culture
    3. MTT assay
    4. Real-time polymerase chain reaction
    5. Western blotting
    6. Cytokine analysis
    7. Statistics
Results
    1. MTT assay confirms viability at MOI 50 for bacterialinfections
    2. Differential mRNA expression of ER stress markersinduced by oral bacterial infections
    3. Western blot analysis of ER stress-related proteinexpression following bacterial infections
    4. ELISA analysis of pro-inflammatory cytokineproduction following bacterial infections
Discussion
Funding
Conflicts of Interest
References

• Si Yeong Kim(Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea)
• Jung Hwa Park(Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea)
• Hee Sam Na(Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea)
• Jin Chung(Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea) Corresponding author