miR-140-5p Inhibition alleviates LPS-induced cholangitis in rats and is associated with modulation of the NF-κB signaling pathway.

Cholangitis is characterized by persistent inflammatory injury of biliary epithelial cells (BDECs), which is a key driver of biliary fibrosis, cholestatic cirrhosis, and cholangiocarcinoma. Nuclear factor-κB (NF-κB) is the central mediator of the inflammatory cascade in cholangitis. miR-140-5p has been reported to regulate inflammatory diseases via modulating NF-κB signaling, but its role and regulatory mechanism in cholangitis remain completely unelucidated. This study aimed to investigate the functional role of miR-140-5p in lipopolysaccharide (LPS)-induced cholangitis in rats, and its potential interplay with the NF-κB signaling pathway. Thirty specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were randomly divided into 5 groups (n = 6 per group): Control group, LPS-induced cholangitis model group, LPS + negative control (NC) adenovirus group, LPS + miR-140-5p inhibitor group, and LPS + pyrrolidine dithiocarbamate (PDTC, NF-κB specific inhibitor) group. The cholangitis model was established via retrograde injection of LPS (5 μg/mL, 0.9 mL) into the common bile duct. Interventions were administered before modeling. Biliary tissues were harvested for transmission electron microscopy (TEM) to evaluate BDEC ultrastructural injury, and immunohistochemistry (IHC) to detect NF-κB protein expression. Serum levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of Toll-like receptor 4 (TLR4), NF-κB, tumor necrosis factor-α (TNF-α), and miR-140-5p was quantified by quantitative real-time PCR (qPCR). Protein levels of TLR4, NF-κB, and TNF-α were determined by Western blot. The rat cholangitis model was successfully established. Compared with the Control group, the LPS model group showed significant BDEC ultrastructural damage, severe inflammatory infiltration, elevated serum IL-1β and IL-18 levels, and upregulated mRNA and protein expression of TLR4, NF-κB, and TNF-α (all P < 0.01), accompanied by a significant downregulation of miR-140-5p expression (P < 0.01). No significant differences in all indicators were observed between the LPS + NC adenovirus group and the LPS model group (*P* > 0.05). Compared with the LPS model group, the LPS + miR-140-5p inhibitor group exhibited markedly alleviated BDEC injury, reduced serum pro-inflammatory cytokine levels, and downregulated TLR4/NF-κB/TNF-α signaling activation (all P < 0.05). The LPS + PDTC group showed almost complete reversal of LPS-induced inflammatory injury, and a significant upregulation of miR-140-5p expression compared with the LPS model group (P < 0.01). This preliminary in vivo study demonstrates that miR-140-5p expression is downregulated in LPS-induced cholangitis in rats, and that pharmacological inhibition of miR-140-5p significantly alleviates biliary epithelial cell injury and inflammatory response, concomitant with suppression of the TLR4/NF-κB/TNF-α signaling pathway. Additionally, NF-κB inhibition by PDTC upregulated miR-140-5p expression, suggesting a potential negative regulatory relationship between NF-κB activation and miR-140-5p transcription. While these findings identify miR-140-5p as a potential therapeutic target for cholangitis and provide a testable hypothesis for the miR-140-5p/NF-κB regulatory interplay in biliary inflammation, direct mechanistic proof remains to be established in subsequent studies.