miR-140-5p is Associated with the NF-κB Signaling Pathway and Exerts an Interventional Effect on Cholangitis in Rats

Background Cholangitis represents an anti-injury response of cholangiocytes to endogenous and exogenous stimuli. Under chronic inflammatory stimulation, bile duct fibrosis and pathological bile duct remodeling are common pathological alterations in biliary duct tissue, which simultaneously serve as high-risk factors for the development of cholestatic liver cirrhosis and cholangiocarcinoma. NF-κB is a core factor in inflammatory responses. Recent studies have revealed that the small non-coding RNA molecule miR-140-5p may regulate the progression of various inflammatory diseases by influencing NF-κB activity; however, the regulatory role of this molecular pathway in cholangitis remains unclear. Therefore, this study aims to investigate the roles of miR-140-5p and NF-κB in a rat cholangitis model. Methods Thirty healthy SD rats were randomly divided into five groups using a random number table: control group, LPS group, LPS + NC adenoviral vector group, LPS + miR-140-5p inhibitors group, and LPS + PDTC group, with 6 rats in each group. A rat cholangitis model was established by single injection of LPS (5µg/mL) into the common bile duct, with different inhibitors administered prior to modeling. Biliary duct tissues were collected from each group of rats, and ultrastructural changes in cholangiocytes were observed under electron microscopy; NF-κB protein expression levels in biliary duct tissues of rats from each group were measured using immunohistochemical method; Expression levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in serum were determined by ELISA; qPCR was performed to detect mRNA expression levels of Toll-like receptor 4 (TLR4), NF-κB, tumor necrosis factor-α (TNF-α), and miRNA-140-5p in each group; TLR4, NF-κB, and TNF-α protein expression levels were examined by Western Blot. Results A rat cholangitis model was established via common bile duct injection of lipopolysaccharide (LPS). Compared with the control group, the LPS group exhibited significant inflammatory infiltration in biliary tissues. Transmission electron microscopy (TEM) revealed typical pathological manifestations in cholangiocytes (BDECs), including widened intercellular spaces, disrupted membrane structures, and mitochondrial and endoplasmic reticulum damage. Immunohistochemistry and molecular detection revealed that the LPS group exhibited enhanced positive expression of NF-κB protein, with significantly elevated serum levels of IL-1β and IL-18, as well as increased mRNA and protein expression of TLR4, NF-κB, and TNF-α ( p <  0.01). Using adenoviral vector transfection technology to regulate the transcription of miR-140-5p in rats, the LPS + NC adenoviral vector group and LPS + miR-140-5p inhibitors group were established. The results of all indicators in the LPS + NC adenoviral vector group showed no statistically significant difference compared to the LPS group (p > 0.05). Compared with the LPS group, the LPS + miR-140-5p inhibitors group exhibited significantly reduced serum IL-1β and IL-18 levels ( p <  0.01), markedly alleviated BDEC injury, and significantly down-regulated mRNA expression levels of TNF-α and TLR4 ( p <  0.05). Notably, relative to the LPS group, the LPS + miR-140-5p inhibitors group showed a significant decrease in NF-κB mRNA levels, although the reduction in NF-κB protein expression did not reach statistical significance (p > 0.05). In the LPS + PDTC group compared to the LPS group, serum IL-1β and IL-18 levels were significantly reduced ( p <  0.01), BDEC injury was markedly alleviated, and both mRNA and protein expression levels of TNF-α and TLR4 were significantly down-regulated ( p <  0.01). However, mRNA expression of miR-140-5p was significantly elevated ( p <  0.01). Conclusion miR-140-5p exhibits a positive correlation with the severity of cholangitis and the activation of the NF-κB pathway, and it may promote inflammation by positively regulating the NF-κB pathway. Following intervention with NF-κB inhibitors, the expression levels of the aforementioned inflammatory factors and pathway molecules significantly decreased compared to the LPS group ( p <  0.01 ). Moreover, NF-κB activation may transcriptionally inhibit miR-140-5p. When NF-κB activity is suppressed, it induces high expression of miR-140-5p. This feedback loop may participate in the self-regulatory process of inflammation, potentially providing a new target for blocking NF-κB-mediated inflammatory cascade amplification.