Inhibition of Virus Replication by Zinc Acetate In Vitro

Exploring the Potential of Japanese Rice-Koji Miso in Combating HAV Replication

• Emerging research underscores the potential of Japanese rice-koji miso extracts in inhibiting hepatitis A virus (HAV) replication, likely through the unfolded protein response, a cellular stress-activated mechanism.

• Zinc compounds, such as zinc sulfate and zinc chloride, have also demonstrated effectiveness in suppressing HAV activity.

• Meanwhile, patients with acute liver failure (ALF) Type A and diabetes face unique metabolic challenges that may influence treatment outcomes.

• This study examines the impact of miso extracts on hepatocyte mRNA expression and explores the role of zinc acetate in modulating HAV replication under varying glucose conditions.

Experimental Approach:

• Human hepatoma cell line Huh7 was treated with three types of Japanese rice-koji miso-Kurasaigetsuusujiomiso, Igoumiso, and a control miso—all prepared using rice, soy, salt, and specialized Yurara yeast, at a concentration of 5% for 24 hours.

• RNA sequencing (RNA-Seq) analysis was conducted using next-generation sequencing technology on the Illumina HiSeq1500 platform.

• To investigate the impact of glucose levels and zinc treatment, Huh7 cells or African green monkey kidney GL37 cells were cultured in either low glucose medium (1000 mg/dL) or high glucose medium (4500 mg/dL) and subsequently infected with hepatitis A virus (HAV) strain HA11-1299 (genotype IIIA) at a multiplicity of infection (MOI) of 0.01.

• At 24 hours post-infection, the cells were treated with zinc acetate at concentrations of 10 µM and 20 µM. Total cellular RNA was extracted at 48 hours post-infection for HAV RNA quantification.

• HAV RNA levels were measured via real-time reverse transcription PCR (RT-PCR) and analyzed using the ddCt method or standard curve quantification.

• Cell viability was assessed using the dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay.

Gene Expression Shifts Induced by Miso Extracts in Huh7 Cells

• Kurasaigetsuusujiomiso and Igoumiso extracts influenced gene expression in Huh7 cells, with significant upregulation and downregulation of key genes. Kurasaigetsuusujiomiso upregulated genes like REL, GPR82 and MT1A while downregulating RPS28 and HIST1H3C.

• Igoumiso demonstrated even broader effects, boosting genes such as NPIPA5, TLR9, and MT1A while suppressing RN7SL1 and MT1G. These findings point to diverse molecular impacts tied to miso extracts.

Zinc Acetate’s Role in HAV Suppression

• Zinc acetate significantly inhibited HAV replication in both low (1000 mg/dL) and high glucose (4500 mg/dL) conditions.

• In Huh7 cells, replication dropped to 69.1% and 51.0% at 10 and 20 μM in low glucose and 57.4% and 52.8% in high glucose. In GL37 cells, inhibition was even stronger with rates reaching 87.0% and 17.5% in low glucose and 64.3% and 32.5% in high glucose.

Safety of Zinc Acetate on Cell Viability

Zinc acetate exhibited no adverse effects on Huh7 or GL37 cell viability at concentrations up to 100 μM for 48 hours, highlighting its therapeutic potential without cytotoxicity.

Key Insight

Japanese rice-koji miso extracts were found to modulate the expression of zinc-related genes in human hepatocytes. Additionally, zinc acetate exhibited significant in vitro inhibition of HAV replication, highlighting its potential as a therapeutic option for hepatitis A, especially in patients with diabetes.

By - Eeshan Aggarwal

Reference: Hepatology. Volume 80, Issue S1. Abstract Supplement for The Liver Meeting by the American Association for the Study of Liver Diseases (AASLD), November 15-19, 2024, San Diego, CA.