Tetrandrine alleviates oxaliplatin-induced mechanical allodynia via modulation of inflammation-related genes

Abstract

Oxaliplatin, a platinum-based chemotherapy drug, causes neuropathic pain, yet effective pharmacological treatments are lacking. Previously, we showed that tetrandrine (TET), with anti-inflammatory properties, reduces mechanical allodynia in nerve-injured mice. This study explores the effect of TET on oxaliplatin-induced mechanical allodynia and gene changes in mice. Male C57BL/6J mice received oxaliplatin intraperitoneally to induce mechanical allodynia. Post-treatment with TET or vehicle, the mechanical withdrawal threshold (WMT) was assessed using von Frey filaments. TET alleviated oxaliplatin-induced mechanical allodynia. RNA sequencing identified 365 differentially expressed genes (DEGs) in the Control vs. Oxaliplatin group and 229 DEGs in the Oxaliplatin vs. TET group. Pearson correlation analysis of co-regulated DEGs and inflammation-related genes (IRGs) revealed 104 co-regulated inflammation-related genes (Co-IRGs) (|cor| > 0.8, P < 0.01). The top 30 genes in the PPI network were identified. Arg2, Cxcl12, H₂-Q6, Kdr, and Nfkbia were highlighted based on ROC analysis. Subsequently, Arg2, Cxcl12, Kdr, and Nfkbia were further verified by qRCR. Immune infiltration analysis indicated increased follicular CD4 T cell infiltration in oxaliplatin-treated mice, reduced by TET. Molecular docking showed strong binding affinity between TET and proteins encoded by Arg2, Cxcl12, Kdr, and Nfkbia. In summary, TET may alleviate oxaliplatin-induced peripheral neuropathy in clinical conditions.

Keywords: RNA-Seq; inflammation; mechanical allodynia; molecular docking; oxaliplatin; tetrandrine.

FIGURE 1

Comprehensive flow chart illustration of the experimental and data processing procedures. (A) Experimental design for model conduction: on day 0, a single intraperitoneal injection of oxaliplatin is administered, while the control group receives an equivalent volume of 5% glucose solution. Behavioral tests are conducted on days -1, 2, 5, 7, 10, 14, and 21. (B) Experimental design for testing tetrandrine (TET) in oxaliplatin-induced neuropathy Pain (OINP) mice: administration of TET or pregabalin via oral gavage begins one day prior to the oxaliplatin injection. Behavioral tests are performed every other day. (C) Data processing workflow in the study: following the completion of behavioral assessments, spinal cord tissues are collected for transcriptomic sequencing and qPCR validation. Bioinformatics analysis is then conducted as illustrated.

FIGURE 2

Establishment of an oxaliplatin-induced neuropathic pain mouse model. (A) The MWT data were examined 0, 2, 5, 7, 10, 14, and 21 days after oxaliplatin injection (i.p.), two-way ANOVA followed by Bonferroni’s post-hoc test. ***P < 0.001 compared to the vehicle at the same time point. (B) The area under the curve (AUC) of the data from graph B (2–21 days). One-way ANOVA followed by Newman-Keuls tests. ***P < 0.001 compared to the vehicle group. Data are presented as mean ± SEM, n = 6.

FIGURE 3

Tetrandrine (TET) alleviates oxaliplatin-induced mechanical allodynia. (A) The MWT data within each group were analyzed using two-way ANOVA followed by Bonferroni’s post-hoc test. **P < 0.01, ***P < 0.001 compared to Oxaliplatin at the same time point. (B) The area under the curve (AUC) of the data from graph B (2–10 days). ***P < 0.001 compared to Control group, ^###P < 0.001 compared to the Oxaliplatin group. One-way ANOVA followed by Newman-Keuls tests. (C) Motor function was performed by rotarod test. Data are expressed as mean ± SEM, n = 6. TET and pregabalin use the dose of 45 mg/kg.

FIGURE 4

Identification of DEGs in Control-Oxaliplatin and Oxaliplatin-TET groups using DEseq2, with criteria of |log2FC| > 0.58 and P < 0.05. (A) PCA comparing Control and Oxaliplatin groups based on standardized gene expression. (B) PCA comparing Oxaliplatin and TET groups based on standardized gene expression. (C) Heatmap displaying top 10 DEGs between Control and Oxaliplatin groups. (D) Volcano plot illustrating DEGs distribution between Control and Oxaliplatin groups. (E) Heatmap displaying top 10 DEGs between Oxaliplatin and TET groups. (F) Volcano plot illustrating DEGs distribution between Oxaliplatin and TET groups. Red indicates upregulated genes, and green indicates downregulated genes.

FIGURE 5

Functional enrichment analysis of DEGs compare the Oxaliplatin and TET groups using clusterProfiler. The bubble charts showed BP (A), CC (B), MF (C), and KEGG (D).

FIGURE 6

Identification and functional annotation of Co-regulated IRGs. (A) The Venn diagram displays 99 Co-regulated DEGs between the Control-Oxaliplatin and Oxaliplatin-TET groups. (B) The Venn diagrams indicate that the expression profiles of 95 Co-regulated DEGs were reversed following TET treatment. (C) A bubble chart shows the enriched GO terms for Co-regulated IRGs. (D) A bubble chart presents the enriched KEGG terms for Co-regulated IRGs.

FIGURE 7

Protein-protein interaction (PPI) network and correlation analysis of co-regulated IRGs. (A) PPI network of Co-regulated IRGs established using the STRING database. (B) Venn diagram of candidate genes identified through MCC and Degree algorithms. (C) Correlation of the 30 candidate genes.

FIGURE 8

Receiver operating characteristic analysis. (A) Venn diagram of 13 genes with AUC values greater than 0.7 for both Control-Oxaliplatin and Oxaliplatin-TET groups. (B) ROC analysis of Arg2, Cxcl12, Kdr, H₂-Q6, and Nfkbia in the Control-Oxaliplatin group with AUC values greater than 0.8. (C) Effects of TET on Arg2, Cxcl12, Kdr, and Nfkbia expressions in oxaliplatin induced NP. One-way ANOVA followed by Newman-Keuls tests. *P < 0.05, ***P < 0.001. Data are presented as mean ± SEM, n = 5.

FIGURE 9

Immune infiltration analysis. (A) Heatmap displaying infiltration abundances of 20 immune cells across Control, Oxaliplatin, and TET groups. (B) Pearson correlation heatmap of 20 immune cells. (C) Boxplot comparing immune cell infiltration between Control and Oxaliplatin groups. (D) Boxplot comparing immune cell infiltration between Oxaliplatin and TET groups.

FIGURE 10

Molecular docking results of TET with Arg2 (A), Cxcl12 (B), Kdr (C), and Nfkbia (D).