Research on Traditional Chinese Medicine for Treating GJB2 Gene Mutation-Related Diseases

Background and Potential

GJB2 gene mutations (e.g., 35delG) encode defective Connexin 26 protein, a major cause of hereditary deafness linked to gap junction dysfunction. In TCM, deafness is often attributed to “kidney deficiency,” “qi and blood insufficiency,” or “stasis in ear orifices.” Herbs like Rehmannia and Cornus (in Liuwei Dihuang Pill) and blood-activating formulas like Taohong Siwu Decoction may improve microcirculation, regulate intercellular communication, and protect auditory nerves. Single herbs like Astragalus, Angelica, and Ligusticum have antioxidant and microcirculation-enhancing effects, potentially beneficial for GJB2 mutation-related hearing loss.

Pharmacological studies show that Astragalus polysaccharides protect neurons via antioxidant effects, and ligustrazine improves microcirculation, potentially aiding auditory blood supply. Liuwei Dihuang Pill has demonstrated neuroprotective and anti-inflammatory effects. Thus, TCM intervention for GJB2 mutation-related deafness is feasible.

Research Methods

1. Herb Screening and Formula Design

• Single Herb Screening: Select kidney-tonifying (Rehmannia, Cornus), blood-activating (Ligusticum, Angelica), and antioxidant (Astragalus, Goji) herbs. Analyze active components (e.g., ligustrazine, Astragalus polysaccharides) and their effects on GJB2 mutation-related pathways (e.g., oxidative stress, gap junction function).
• Compound Formula Design: Modify Liuwei Dihuang Pill by adding Ligusticum and Angelica to address “kidney deficiency and blood stasis.”
• Pharmacological Basis: Use HPLC-MS to identify key active components in formulas, clarifying their chemical basis and mechanisms.

2. Cell Model Construction

• Cell Line Selection: Use HEK293 cells for gene editing or HEI-OC1 cells (cochlear cell line) to model the cochlear environment.
• Precise Gene Editing:
  1. Design gRNA targeting GJB2 gene (e.g., 35delG mutation).
  2. Construct CRISPR/Cas9 plasmids and transfect into HEK293 or HEI-OC1 cells.
  3. Screen for stable GJB2 mutant cell lines via single-cell cloning.
• Mutation Validation: Verify GJB2 mutations with Sanger sequencing; assess Connexin 26 expression with Western blot; evaluate gap junction function using fluorescence dye transfer assays.

3. TCM Intervention Experiments

• In Vitro Experiments:
  1. Divide GJB2 mutant cells into control, single herb (e.g., Astragalus polysaccharides, ligustrazine), and compound formula groups (e.g., modified Liuwei Dihuang Pill).
  2. Measure cell viability (CCK-8 assay), oxidative stress (ROS detection), and gap junction function (fluorescence transfer assay).
  3. Analyze related pathways (e.g., Nrf2/HO-1 antioxidant pathway).
• Dose Optimization: Determine optimal intervention concentrations and assess cytotoxicity.

4. Validation Methods

• Functional Validation: Assess gap junction function improvement via fluorescence dye transfer assays; measure oxidative stress markers (e.g., SOD, MDA).
• Molecular Mechanism Validation: Use qPCR and Western blot to examine GJB2 and other Connexin gene (e.g., GJA1) expression, identifying TCM’s targets.
• In Vivo Validation: Construct GJB2 mutant mouse models, administer TCM, and assess hearing (ABR testing), cochlear pathology, and Connexin 26 expression.

Expected Outcomes and Significance

TCM may alleviate GJB2 mutation-related hearing loss by reducing oxidative stress, improving microcirculation, and restoring gap junction function. These findings could offer new insights for TCM in treating hereditary deafness and promote its application in precision medicine.