# Identification of mitochondria-related key genes in type 2 diabetes mellitus and elucidation of the Zhimu-Huangbai herb Pair’s mechanism: an integrated approach of bioinformatics, machine learning, and experimental validation

## TL;DR

This study identifies key mitochondrial genes in type 2 diabetes and explains how a traditional Chinese herb pair improves mitochondrial function and reduces disease effects.

## Contribution

The study integrates bioinformatics, machine learning, and experiments to reveal the mechanism of a herb pair in treating type 2 diabetes.

## Key findings

- Eight mitochondria-related genes were identified as key in T2DM and ZB treatment.
- ZB improved mitochondrial function and reduced oxidative stress in INS-1 cells.
- Machine learning models using BCAT2, CASP8, and EPHX2 achieved high diagnostic accuracy for T2DM.

## Abstract

Mitochondrial injury plays a critical role in type 2 diabetes mellitus (T2DM) pathogenesis by impairing cellular energy metabolism and insulin sensitivity. The Zhimu-Huangbai herb pair (ZB), a classic Traditional Chinese Medicine formulation composed of Anemarrhena asphodeloides and Phellodendron chinense, has shown efficacy in T2DM, but its molecular mechanisms remain unclear. In this study, we aimed to identify crucial mitochondrial related genes of type 2 diabetes and the potential mechanism of ZB.

Gene expression datasets for T2DM (GSE76894, GSE25724, and GSE38642) were retrieved from the GEO database. Intersection targets of ZB herb pair and T2DM were identified by screening multiple databases, including the TCMSP and HERB. Mitochondrial function-related genes were obtained from human mitochondria-associated databases. WGCNA was employed to identify differentially expressed genes, which were then intersected with bioactive compound–target genes and mitochondrial-related genes to construct a PPI network. GO and KEGG enrichment analyses were subsequently performed. Four machine learning algorithms—SVM-RFE, RF, GLM, and XGB—were applied to screen feature genes and establish diagnostic models. Furthermore, the correlations between feature targets and immune cell infiltration were analyzed, single-gene GSEA was conducted, and molecular docking was performed to investigate the interactions between feature targets and bioactive constituents of ZB. For experimental validation, INS-1 cells were divided into six groups: the control group, model group, metformin group, and low-, medium-, and high-dose ZB groups. Cell viability, apoptosis, ROS levels, mitochondrial membrane potential, and mitochondrial morphology and function were assessed. Western blot analysis was performed to evaluate the expression of mitochondria-related genes (BCAT2, CASP8, EPHX2, and UCP2) and components of the AMPK–SIRT1–PGC-1α signaling pathway.

A total of eight mitochondria-related differentially expressed genes associated with ZB treatment of T2DM were identified. GO analysis revealed enrichment in multiple biological processes, including response to nutrient levels; cellular components, such as pore complex; and molecular functions, including toxic substance binding. KEGG pathway analysis indicated significant enrichment in pathways including apoptosis, p53 signaling pathway, and necroptosis. Three key genes—BCAT2, CASP8, and EPHX2—were screened through machine learning algorithms, and the constructed T2DM diagnostic models all exhibited area under the curve (AUC) values greater than 0.7, indicating satisfactory discriminative performance. Immune infiltration analysis revealed that all three key genes were significantly correlated with immune cell populations. Molecular docking results demonstrated that the three key genes exhibited strong binding affinities (≤−5.0 kcal/mol) for their corresponding bioactive compounds derived from ZB, with the exception of the CASP8-nicotinamide combination. Experimental validation showed that ZB significantly enhanced the viability of INS-1 cells subjected to high-glucose and high-lipid conditions, inhibited apoptosis, reduced intracellular ROS generation, and ameliorated mitochondrial membrane potential, mitochondrial morphology, and respiratory function. Concurrently, the protein expression levels of UCP2 and BCAT2 were markedly upregulated, whereas those of CASP8 and EPHX2 were significantly downregulated. Additionally, ZB treatment upregulated the p-AMPK/AMPK ratio as well as the expression of SIRT1 and PGC-1α.

The diagnostic model featuring genes BCAT2, CASP8, and EPHX2 provides new insights for T2DM diagnosis and treatment. ZB’s therapeutic mechanism involves regulating mitochondrial-related genes (BCAT2, CASP8, EPHX2, UCP2) and activating the AMPK-SIRT1-PGC-1α pathway, thereby improving mitochondrial morphology and function, reducing oxidative damage, and enhancing energy metabolism.