Salvianolic Acid B Decreases Interleukin-1b-Induced Colitis Recurrence in Mice
Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal inflammatory condition that includes ulcerative colitis (UC) and Crohn’s disease (CD). The incidence of IBD varies globally, with rates ranging from 0.1 to 11 per 100,000 people for CD and 0.5 to 24.5 per 100,000 people for UC. Despite advances in understanding the disease, the pathological mechanisms of IBD and its recurrence remain poorly understood. This study explores the role of tight junction (TJ) barrier dysfunction in colitis recurrence and evaluates the therapeutic potential of salvianolic acid B (Sal B), a traditional Chinese medicine, in mitigating this condition.
Background and Significance
The intestinal epithelial barrier plays a crucial role in maintaining gut homeostasis by preventing the invasion of luminal pathogens. This barrier is composed of epithelial cells, mucus, antimicrobial proteins, and cell junctions such as tight junctions (TJs) and adherens junctions. TJs are particularly important as they regulate the paracellular permeability of the intestinal epithelium. Dysfunction of TJs has been implicated in the pathogenesis of IBD, leading to increased intestinal permeability and inflammation.
One of the key players in TJ dysfunction is myosin light chain kinase (MLCK), which phosphorylates myosin light chains, leading to cytoskeletal contraction and TJ disruption. Increased expression of MLCK has been observed in IBD patients, contributing to barrier dysfunction. Additionally, epithelial cell apoptosis, another form of barrier dysfunction, is also elevated in IBD, further compromising the intestinal barrier.
The recurrence of IBD is a significant clinical challenge, with a 10-year recurrence rate in UC ranging from 70% to nearly 100%. Current therapeutic strategies, such as non-steroidal anti-inflammatory drugs, often fail to prevent recurrence. Therefore, there is a pressing need for novel treatments that target the underlying mechanisms of IBD recurrence, particularly those related to intestinal barrier dysfunction.
Hypothesis and Objectives
This study hypothesizes that IBD recurrence is closely associated with intestinal barrier dysfunction, particularly TJ barrier dysfunction. The primary objective was to investigate whether enhancing TJ function could reduce colitis recurrence. Salvianolic acid B (Sal B), a compound derived from the traditional Chinese herb Radix Salvia miltiorrhiza (Danshen), was chosen for its known anti-inflammatory, antioxidant, and epithelial barrier-protective effects. The study aimed to compare the efficacy of Sal B with sulfasalazine (SASP), a standard treatment for IBD, in preventing colitis recurrence.
Methods
Ethical Approval and Animal Models
The study was conducted in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the Dalian Medical University Animal Care and Ethics Committee. Eighty C57BL/6 mice were randomly divided into four groups: a normal control group, a colitis group, an SASP-treated group, and a Sal B-treated group. Colitis was induced in the mice by providing them with drinking water containing 4% dextran sulfate sodium (DSS) for seven days. On day two, the SASP and Sal B groups began receiving daily intragastric doses of SASP (80 mg/kg) and Sal B (100 mg/kg), respectively. On day seven, colitis recurrence was induced by intraperitoneal injection of recombinant human interleukin-1b (IL-1b).
Assessment of Colitis Symptoms
Colitis symptoms, including body weight, diarrhea incidence, and food intake, were monitored daily. On day 10, the mice were sacrificed, and colon tissues were collected for analysis. The severity of colitis was assessed using the disease activity index (DAI), which includes parameters such as weight loss, stool consistency, and rectal bleeding. Colon macroscopically visible damage was scored on a scale from 0 to 10, and the colon weight-to-length ratio was measured. Myeloperoxidase (MPO) activity and pro-inflammatory cytokine levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits.
Western Blotting and Flow Cytometry
Colonic segments were analyzed for the expression of apoptosis-related proteins (Bax and Bcl-2) and TJ proteins (MLCK and occludin) using western blotting. Apoptosis was quantified using flow cytometry with Annexin V-FITC and propidium iodide staining. The integrity of the intestinal epithelium was assessed using transmission electron microscopy (TEM).
Results
Colitis Symptoms and TJ Barrier Dysfunction
The induction of colitis with DSS resulted in significant weight loss, diarrhea, and rectal bleeding in the colitis group, peaking on day 3 and improving by day 7. Both SASP and Sal B treatments reduced the DAI and macroscopically visible damage scores, with Sal B showing a more pronounced effect. In the recurrence phase induced by IL-1b, Sal B significantly reduced the DAI and macroscopically visible damage compared to SASP.
Apoptosis and TJ Protein Expression
Apoptosis in the colitis group peaked on day 3 and remained elevated on day 7. Both SASP and Sal B reduced apoptosis, but Sal B was more effective in reversing the increased expression of Bax and the decreased expression of Bcl-2. Sal B also significantly reduced the elevated expression of MLCK and increased the expression of occludin, while SASP had no effect on these TJ proteins.
TEM Analysis
TEM analysis revealed that Sal B restored the integrity of the intestinal epithelium, with clear and intact TJs, while the colitis group showed widened cell gaps and disrupted TJs. SASP did not reverse these changes.
Colitis Recurrence
Sal B significantly reduced the recurrence rate and severity of colitis symptoms induced by IL-1b. The colon weight-to-length ratio, MPO activity, and TNF-a levels were also lower in the Sal B-treated group compared to the SASP-treated group.
Discussion
The findings of this study suggest that TJ barrier dysfunction plays a critical role in the recurrence of colitis. Sal B, by enhancing TJ function through the downregulation of MLCK and upregulation of occludin, effectively reduced colitis recurrence. In contrast, SASP, while reducing apoptosis, did not influence TJ barrier dysfunction, highlighting the distinct mechanisms of action of these two treatments.
The study also underscores the importance of targeting multiple aspects of intestinal barrier dysfunction in IBD treatment. While both apoptosis and TJ dysfunction contribute to barrier loss, TJ dysfunction appears to be a key factor in colitis recurrence. This suggests that therapies aimed at stabilizing TJs, such as Sal B, may be particularly effective in preventing IBD recurrence.
Conclusion
This study provides compelling evidence that enhancing TJ barrier function can reduce colitis recurrence. Sal B, through its ability to modulate MLCK and occludin expression, offers a promising therapeutic strategy for preventing IBD recurrence. The findings also highlight the potential of targeting TJ dysfunction as a biomarker for IBD recovery and a focus for future therapeutic interventions.
doi.org/10.1097/CM9.0000000000000773
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