Sphingosine-1-phosphate Signal Transducer and Activator of Transcription 3 Signaling Pathway Contributes to Baicalein-mediated Inhibition of Dextran Sulfate Sodium-induced Experimental Colitis in Mice
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, characterized by periods of remission and relapse. The most common forms of IBD are Crohn’s disease and ulcerative colitis, which can affect various parts of the gastrointestinal tract, including the oral cavity, esophagus, stomach, intestine, and anus. The etiology of IBD is complex, involving immune-related, environmental, and genetic factors, and its symptoms include diarrhea, abdominal pain, intestinal bleeding, and weight loss. Due to the diverse causes and symptoms, establishing the precise etiology and treatment of IBD has been challenging.
Baicalein, a major bioactive flavonoid isolated from the root of the plant Astragalus membranaceus, has been shown to possess anti-inflammatory, anti-bacterial, anti-hypertensive, and anti-tumor activities. It has also been reported to be beneficial in the treatment of colitis. However, the mechanisms underlying its anti-inflammatory effects on colitis remain unclear. This study aimed to investigate whether baicalein exerts its anti-colitic activity through effects on the S1P-STAT3 signaling pathway.
To explore the effects of baicalein on colitis, a dextran sodium sulfate (DSS)-induced model of acute colitis was established in BALB/c mice. The mice were divided into six groups: (1) control group receiving water for 10 days; (2) DSS group receiving 4% DSS solution in drinking water for 7 days, followed by normal water for 3 days; (3), (4), and (5) DSS plus baicalein (10, 20, 40 mg/kg) administered once daily starting on day 1; and (6) DSS plus 5-aminosalicylic acid (50 mg/kg) administered once daily starting on day 1. Body weights, stool consistency, and hematochezia were recorded, and the severity of colitis was evaluated using a disease activity index (DAI). On day 11, the mice were euthanized, and organs and blood were collected for analysis.
The results showed that baicalein treatment dose-dependently reduced DSS-induced weight loss, splenomegaly, and colonic damage, as reflected by amelioration of diarrhea, rectal bleeding, and colonic ulceration, congestion, and edema. Baicalein also significantly decreased the levels of inflammatory mediators in the serum and colon, and significantly inhibited the expression of NOD2, SPHK1, S1PR1, and p-STAT3 in the colon. These findings suggest that baicalein ameliorates colitis in mice by inhibiting the S1P-STAT3 signaling pathway.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in the regulation of several cellular processes and signal transduction. S1P is mainly produced via phosphorylation of sphingosine by the sphingosine kinases SPHK1 and SPHK2 and activates intracellular signal transduction by binding to one of five cell surface sphingosine-1-phosphate receptors (S1PR1–5). Several studies have identified a close relationship between S1P and the development of IBD. For example, expression of SPHK1, SPHK2, S1PR1, S1PR2, and S1PR4 are significantly up-regulated in children with IBD, and inhibition of SPHK1 reduces the expression of inflammatory markers and the infiltration of neutrophils in colonic tissue of mice with IBD.
Signal transducer and activator of transcription 3 (STAT3) is also over-expressed in the intestinal mucosa of patients with active and inactive IBD. STAT3 mRNA and protein levels are abnormally high in patients with colorectal cancer, and STAT3 is thought to contribute to this cancer via an interleukin (IL)-22-STAT3 signaling pathway. Inhibition of STAT3 and its associated pathways prevents the occurrence and progression of cancer and inflammatory diseases. S1P can maintain STAT3 in an activated state, which contributes to the development of colitis-associated colon cancer.
In this study, immunohistochemistry (IHC) analysis of colon sections from DSS-treated mice revealed large numbers of SPHK1- and p-STAT3-positive cells, but their abundance was significantly reduced by baicalein treatment at 40 mg/kg. Western blot analysis confirmed that baicalein at 40 mg/kg significantly inhibited DSS-induced phosphorylation of STAT3 and reversed the over-expression of SPHK1 and S1PR1. However, the expression of total STAT3 was not changed by baicalein treatment. These results indicate that baicalein exerts its anti-colitic effects through the inhibition of SPHK1 expression and STAT3 activation.
Baicalein also reduced the DSS-induced infiltration of inflammatory cells into the colon. Immunofluorescence microscopy of colon sections for CD11b+ cells, which are expressed on many leukocytes, including monocytes, neutrophils, natural killer cells, granulocytes, and macrophages, showed a large number of CD11b+ cells in the lesional mucosa of colons from DSS/water-treated mice, but the cell abundance was dramatically reduced by baicalein treatment. Moreover, baicalein attenuated the expression of the transcription factor RORgt, which is pivotal in the development of proinflammatory Th17 cells.
Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor expressed in both hematopoietic and non-hematopoietic cells in the intestinal epithelium and plays a key role in stimulating the host immune response. Western blot analysis of colon extracts showed that NOD2 expression was increased by DSS treatment, while baicalein significantly inhibited its expression compared with the DSS/water group.
The results of this study demonstrated that mice treated with DSS exhibited weight loss, colon shortening, and colon tissue damage accompanied by extensive inflammatory cell infiltration. However, these effects were significantly reduced by treatment with baicalein in a dose-dependent manner. Importantly, the DSS-induced increase in serum IL-6 and IL-1b and colonic IL-6, IL-1b, and TNF-a were reduced by baicalein treatment, as was the expression of the Th17 cell transcription factor RORgt in the colon.
In conclusion, baicalein ameliorates colitis in mice by inhibiting the S1P-STAT3 signaling pathway. This study provides a potential mechanism for the protective effect of baicalein in DSS-induced colitis and suggests that this flavonoid might be beneficial in the treatment of colitis in humans. Further studies will be necessary to evaluate the safety of baicalein in clinical applications and to optimize its beneficial effects in patients.
doi.org/10.1097/CM9.0000000000000627
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