Cytokine Changes With Effective Drug Therapy for Juvenile Myasthenia Gravis

In order to investigate the therapeutic mechanism of Jianpiyiqi Granule in juvenile myasthenia gravis (JMG) and find the immune-associated cytokines or signaling pathways targeted by this intervention.

(1) We extracted JMG serum samples from the sample bank of the Hebei Provincial Key Laboratory of Myasthenia Gravis before and after treatment. (2) Using Olink Immune Response Panel detects serum cytokines. Data were collated and analyzed using statistical software. Differentially expressed proteins (DEPs) were visualized using heat maps, volcano plots, and other tools. Differentially expressed cytokines were analyzed using KEGG enrichment pathway analysis to identify the relevant signaling pathways they participate in.

(1) Twelve of 92 cytokines were found to be differentially expressed from 20 patients before and after treatment, namely AXIN1, CCL11, CCL13, CCL20, CCL25, CCL3, CD40, IL12B, S100A12, SIRT2, SLAMF1, and STAMBP (|log₂FC| > 0.263; false discovery rate (FDR)-adjusted p  < 0.05). IL12B was upregulated while AXIN1, CCL11, CCL13, CCL20, CCL25, CCL3, CD40, S100A12, SIRT2, SLAMF1 and STAMBP were downregulated after treatment. Notably, three of these-CCL11, IL12B, and STAMBP-showed stronger statistical evidence, with FDR-adjusted p  < 0.01 and |log₂FC| > 0.263. (2) The following pathways were obtained by KEGG enrichment analysis of 12 cytokines: cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, chemokine signaling pathway, Toll-like receptor (TLR) signaling pathway, intestinal immune network for IgA production, IL-17 signaling pathway, NF-kappa B signaling pathway. (3) CCL11 and CCL20 were enriched in the IL-17 signaling pathway. We used enzyme-linked immunosorbent assay (ELISA) to retest additional serum samples before and after treatment from 12 JMG patients before and after treatment. The results showed a significant decrease in CCL11 and CCL20 in after-treatment serum (p  < 0.05).

Drug therapy for JMG revealed 12 differentially expressed cytokines. Due to the exploratory nature and sample volume constraints, only two candidate cytokines were validated by ELISA. We found that the cytokines CCL11 and CCL20-associated with the IL-17 signaling pathway-were downregulated after treatment. The drug therapy may inhibit MG progression by reducing the expression of IL-17-associated cytokines. At the same time, the drug reduced the expression of CCL3, CCL13, CCL25, and CD40, which may play a role in the abnormal proliferation of thymocytes and chronic inflammatory response at the neuromuscular junction. Future studies with multiplex assays are needed to confirm the involvement of other differentially expressed cytokines, such as SIRT2 and AXIN1, which may contribute to disease mechanisms outside the identified pathways.