Longdan Xiegan Decoction Inhibits Notch Signaling Activation by Elevating miR-30b-5p Level in Uveitis: An In Vitro Study Based on Network Pharmacology

Ethnopharmacological relevance: Uveitis is an inflammatory eye disease that seriously threatens human health. Longdan Xiegan Decoction (LXD), a traditional Chinese medicine formula, has commonly been applied in treating uveitis. However, the underlying mechanisms are not fully addressed.Aim of the study To investigate the effect of LXD on the inflammatory process and to address the underlying mechanism of LXD treating experimental autoimmune uveitis (EAU) via inhibiting the Notch signaling activation and T helper (Th) cell differentiation.Materials and methods: Based on network pharmacology, we predicted the potential protein, gene interactions, hub genes, corresponding miRNAs, and the bioactive substances from the LXD formula involved in ameliorating uveitis. Further, to validate the regulatory role of LXD on Th cell differentiation in uveitis based on Notch signaling pathway, the spleen, lymph nodes and eye tissues of the rats in normal control (NC) and EAU groups were harvested at the peak of inflammation. Subsequently, the lymphocytes from the NC and EAU groups were treated with 2% control serum (serum from NC rats), while those from the LXD group were treated with different concentrations of LXD medicated serum. Finally, the levels of Th cell differentiation in the spleen, lymph nodes and ocular tissues of the rats were measured by flow cytometry, and the expression of RORγt, Foxp3, Notch1, DLL4, IL-17A, IL-10 and miR-30b-5p was assessed by Q-PCR and ELISA, respectively.Results: Based on network pharmacology analysis, we noted that T cell lineage commitment and the Notch signaling pathway may be the main inflammatory process and key pathway related to treat uveitis with LXD, and further prediction revealed the miRNA-mRNA interaction between miR-30b-5p and the mRNAs is involved in Notch1 and DLL4.In the in vitro experiment, the pathogenesis of uveitis is involved in the elevated Notch1, DLL4, IL-17A, RORγt and the decreased miR-30b-5p together with the imbalanced Th1/Th2 and Th17/Treg ratios. Alternatively, LXD medicated serum can efficiently elevate miR-30b-5p expression, inhibit the Notch signaling activation and uveitogenic T cell lineage commitment, downregulate the pro-inflammatory cytokine and upregulate the anti-inflammatory cytokine levels, leading to the expansion of Treg and Th2 cell lineages and restoring Th1/Th2, Th17/Treg and immune microenvironment balance.Conclusion: Decreased miR-30b-5p facilitates the pathogenesis of uveitis. LXD formula has a significant inhibitory effect on Notch signaling activation through upregulating miR-30b-5p to regulate the Th cell differentiation to restore the Th1/Th2, Th17/Treg balance. Our findings preliminarily reveal the mechanism of LXD ameliorating the inflammatory response of uveitis .