Clinical Efficacy and Mechanism of Vitamin D2 in Treating Hashimoto's Thyroiditis

Abstract

Objective: Hashimoto's thyroiditis (HT) is one of the most common autoimmune diseases, with the highest incidence rate among autoimmune thyroid disorders. Vitamin D2 may have therapeutic effects on HT. This study aimed to elucidate the molecular mechanisms underlying vitamin D2 therapy for HT.

Methods: Differentially expressed genes (DEGs) associated with vitamin D2-treated HT were identified, and the DEG-associated gene enrichment pathway was explored using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The correlation between the hub genes and infiltrating immune cells was investigated, and the interactions among the hub genes and target drug and competing endogenous RNA (ceRNA; long non-coding RNA [lncRNA]-microRNA [miRNA]-messenger RNA [mRNA]) regulatory networks were determined.

Results: GO and KEGG enrichment analyses identified a total of 102 DEGs (6 upregulated and 96 downregulated) in the vitamin D2-treated group samples. The area under the curve values of the identified 10 hub genes was as follows: CCR1(0.920), CXCL1 (0.960), CXCL8 (0.960), EGR1 (0.960), FCGR3B (0.920), FOS (1.000), FPR1 (0.840), MMP9 (0.720), PTGS2 (0.960), and TREM1 (1.000). The immune enrichment scores of the mast cell (P = 0.008), neutrophil (P = 0.016), and plasmacytoid dendritic cell (P = 0.016) were significantly decreased in the vitamin D2-treated group (P < 0.05). The hub gene/drug regulatory network included 8 hub genes, 108 molecular drugs, and 114 interaction relationship pairs. The ceRNA regulatory network included 129 lncRNAs, 145 miRNAs, mRNAs (hub genes), and 324 interaction relationship pairs.

Conclusion: Vitamin D2 may play an immunomodulatory role by regulating the aforementioned immune-related molecules and immune cells, thereby improving its therapeutic effects on HT.

Keywords: Hashimoto’s thyroiditis; RNA sequencing; differentially expressed genes; immune infiltration; vitamin D2.

Figure 1

(a) Volcano Plot and (b) Heatmap analysis of differentially expressed mRNAs between samples from the Hashimoto’s basic treatment group (Before group) versus Hashimoto’s basic treatment combined with vitamin D2 treatment group (After group). They were differentially expressed with the cutoff fold changes ≥1 or ≤ −1 along with p < 0.05 and false discovery rate (FDR) < 0.05.

Figure 2

(a) The bubble plot of the top 10 enrichment pathways with DEGs by GO analysis; (b) The bubble plot of the top 8 enrichment pathways with DEGs by KEGG analysis.

Figure 3

(a) DEGs with a combined score ≥0.4 was set as the cut-off criterion to construct the PPI network; (b) Top 10 gene by degree method in the network ranked.

Figure 4

(a) Heatmap and (b) Violin plot of the difference in immune cell enrichment scores between samples from the Hashimoto’s basic treatment group (Before group) versus Hashimoto’s basic treatment combined with vitamin D2 treatment group (After group). Red in the boxes represents the After group and blue represents the Before group.

Figure 5

RT-qPCR was used to detect the mRNA expression level of the 10 hub genes CXCL1 (a), TREM1 (b), PTGS2 (c), FOS(d), EGR1 (e), CXCL8 (f), MMP9 (g), PCGR3B (h), FPR1 (i), CCR1 (j) in the Hashimoto’s basic treatment group (Before group) versus Hashimoto’s basic treatment combined with vitamin D2 treatment group (After group). **p < 0.01,***p < 0.001.

Figure 6

Western blot was used to detect the protein expression level of the 10 hub genes CXCL1 (a), TREM1 (b), PTGS2 (c), FOS(d), EGR1 (e), CXCL8 (f), MMP9 (g), PCGR3B (h), FPR1 (i), CCR1 (j) in the Hashimoto’s basic treatment group (Before group) versus Hashimoto’s basic treatment combined with vitamin D2 treatment group (After group). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

ROC curves for CCR1 (a), CXCL1 (b), CXCL8 (c), EGR1 (d), FCGR3B (e), FOS (f), FPR1 (g), MMP9 (h), PTGS2 (i), and TREM1 (j).

Figure 8

(a-o) Scatter plots of correlation between (CCR1, CXCL1, CXCL8, EGR1, FCGR3B, FOS, FPR1, MMP9, PTGS2, and TREM1) expression and different immune cell contents, neutrophil, M2 macrophage, T cells CD8, monocyte, NK cell, naive B cell.

Figure 9

Green nodes indicate Hub genes, blue nodes indicate target drugs and the arrows connecting them indicate interaction pairs.

Figure 10

Red oval nodes indicate mRNAs (hub genes), blue diamond nodes indicate lncRNAs, green triangle nodes indicate miRNAs, and the lines between them indicate interaction pairs.