The effect of total alkaloids from Sophora alopecuroides on the protein expression of FXR, TGR5, and CYP7A1 in colon and liver tissues of mice with ulcerative colitis
Sophora alopecuroides L. is a perennial herbaceous plant of the Sophora genus in the legume family, mainly distributed in arid and semi-arid desert grasslands, saline alkali land, and sand dunes in northwest China. It is a salt alkali tolerant sand plant. It has the functions of clearing heat and detoxifying, dehumidifying, and has a good therapeutic effect on gastrointestinal diseases. Alkaloids are one of the main active ingredients in Sophora alopecuroides. Studies have shown that Sophora alopecuroides alkaloids have good therapeutic effects on myocarditis, hepatitis, and ulcerative colitis (UC). Our previous research has also shown that total alkaloids from Sophora alopecuroides have good therapeutic effects on UC.

UC is a non-specific colitis with an unclear etiology, mainly affecting the colonic mucosa and submucosa, and is related to various factors such as genetics, environment, intestinal microbiota, and immune imbalance. These factors lead to disturbances in the intestinal environment, dysfunction of intestinal mucosal epithelium, abnormal mucosal immune response, and ultimately recurrent inflammation of the intestine, causing the occurrence and development of UC. In recent years, studies have confirmed a close relationship between bile acids and intestinal diseases. The causes of UC are imbalanced gut microbiota, epithelial dysfunction, and abnormal immune responses, and bile acids participate in these processes through multiple pathways. Our previous research also found that total alkaloids of Sophora alopecuroides can reduce the levels of total bile acids (TBA) and total cholesterol (T-CHO) in the serum of UC mice. Bile acid targeted metabolomics results showed that serum levels of α – muri cholic acid (α – MCA), β – muri cholic acid (β – MCA), ω – muri cholic acid (ω – MCA), and cholic acid (CA) were significantly increased in the model group, while total alkaloids of Sophora alopecuroides decreased significantly after treatment. Bile acids mainly affect their function in the gastrointestinal tract through two receptors, namely farne soy X receptor (FXR) and G protein coupled bile acid receptor 5 (TGR5). FXR and TGR5 have been found to be highly expressed in the liver and colon and play a very important role in inflammation regulation, making them valuable targets for the treatment of UC. The main sign of UC is mucosal inflammation, and FXR can regulate the production of pro-inflammatory cytokines in the colonic mucosa, which can effectively reflect the severity of ulcerative colitis. There is a close connection between the liver and the intestine. Cholesterol is synthesized in liver cells through the neutral (or classical) pathway initiated by the cholesterol 7 α – hydroxylase CYP7A1 on the smooth endoplasmic reticulum. Bile acids act as signaling molecules in the body, regulating metabolism by activating FXR and TGR5 receptors. Previous studies have found that although total alkaloids of Sophora alopecuroides can affect changes in serum bile acids, the bile acid synthesis pathway is regulated by FXR/TGR5. It is not clear how total alkaloids of Sophora alopecuroides affect changes in serum bile acids through the FXR/TGR5 pathway. Therefore, this study focused on the total alkaloids of Sophora alopecuroides and used dextran sulfate sodium (DSS) to induce an acute ulcerative colitis model in mice. The effects of total alkaloids of Sophora alopecuroides on the expression of FXR, TGR5, and CYP7A1 proteins in the colon and liver tissues of mice were analyzed to explore the effects and mechanisms of total alkaloids of Sophora alopecuroides on the FXR/TGR5 pathway in DSS induced acute UC mice.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the colon and colonic mucosa. Due to its complex pathogenesis, it spreads widely in the intestine, is prone to recurrent attacks, has no specific drugs, and is difficult to cure completely. It is listed as one of the modern difficult to treat diseases by the World Health Organization.

This study used a 3.5% DSS induced acute UC mouse model. Compared with other modeling methods, this method is simple to operate, easy to replicate, and suitable for studying drug efficacy and mechanism of action. The results showed that the DSS induced model group of mice had severe rectal bleeding, decreased body weight, significantly increased DAI score, significantly shortened colon length, mucosal detachment and disappearance of crypts observed by pathological observation of colon tissue. The above indicators indicate successful modeling in this experiment. After treatment with total alkaloids of Sophora alopecuroides, the above indicators were improved to varying degrees, indicating that total alkaloids of Sophora alopecuroides have a certain therapeutic effect on DSS induced acute UC mouse model.

Previous studies have shown that the disruption of mucosal barrier function in many intestinal inflammatory diseases is related to the loss of goblet cells and mucus layer caused by the release of a large number of cytokines. In this experiment, PAS and Alizarin blue special staining methods were used to observe the goblet cells in the colon of each group of mice. It was found that the DSS induced model group had a significant decrease in the number of goblet cells, while the total alkali treatment group of Sophora alopecuroides had a significant increase in the number. There are literature reports that inflammatory reactions have a promoting effect on the occurrence and development of UC. The levels of pro-inflammatory factors such as IL-17, IL-23, and tumor necrosis factor – α (TNF – α) are significantly increased, while the levels of anti-inflammatory factors such as IL-10 are significantly decreased, leading to an imbalance between pro-inflammatory and anti-inflammatory factors and causing intestinal inflammatory reactions. In this experiment, ELISA was used to detect the content of cytokines, and it was found that the levels of pro-inflammatory factors IL-23, IL-1 β, and IL-17 in the DSS induced model group mice were significantly increased, while the total alkali treatment group of Sophora alopecuroides was significantly reduced; In addition, it was found that the anti-inflammatory factor IL-10 content in the DSS induced model group mice was significantly reduced, while the total alkaloid treatment group of Sophora alopecuroides showed an increase. The above results indicate that total alkaloids of Sophora alopecuroides can inhibit inflammation and further activate the anti-inflammatory mechanism in mice by regulating the levels of inflammatory factors and goblet cells, thereby improving colitis and achieving therapeutic effects. To provide certain theoretical basis and methods for clinical treatment of UC, but further research is needed on how inflammatory factors and goblet cells improve intestinal mucosal barrier function.

FXR and TGR5 are highly expressed in liver and colon tissues and participate in various physiological activities in the body. They play an important role in regulating inflammation, bile acid metabolism, improving intestinal mucosal mechanical barrier, and maintaining homeostasis. Li Yanxi et al. found that the protective mechanism of Tibetan medicine 25 Wei Songshi Wan against liver injury may be mediated by the FXR signaling pathway, regulating downstream genes and proteins, inhibiting excessive secretion and synthesis of bile acids, and improving bile stasis by stabilizing bile acid metabolism, without affecting the conversion process of bile acids. Wei et al.’s study showed that the expression of FXR was significantly reduced in the colonic mucosa of UC patients, while there was no significant difference in TGR5 expression. These research results are consistent with the findings of this study. Studies have shown that the peroxisome proliferator activated receptor (PPAR α) – UDP glucuronosyltransferase (UGTs) signal is an important determinant of bile acid homeostasis. In patients with enteritis, the peroxisome proliferator activated receptor (PPAR α) is activated, and the decrease in intracellular bile acids leads to the inhibition of the FXR-FGF15 pathway, further resulting in the upregulation of liver CYP7A1, thereby promoting the continuous increase of bile acid synthesis from scratch and breaking the original bile acid homeostasis. FXR can inhibit CYP7A1 negative feedback regulation of bile acid synthesis by inducing small molecule heterodimeric chaperone (SHP). This experiment detected the levels of total cholesterol and total bile acids in the serum of mice. It was found that the DSS induced model group showed a significant increase in total bile acid content and a decrease in total cholesterol. The total bile acid content was significantly reduced and the total cholesterol content was significantly increased in the Sophora alopecuroides alkaloids treatment group. The WB results showed that FXR, TGR5, and CYP7A1 were significantly downregulated in the liver and colon tissues of DSS induced model group mice, while FXR, TGR5, and CYP7A1 were upregulated in the liver tissues of the Sophora alopecuroides total alkaloids treatment group but not significantly; FXR in colon tissue was significantly upregulated, while TGR5 and CYP7A1 were upregulated. The above results suggest that the downregulation of FXR, TGR5, and CYP7A1 in UC mice significantly exacerbates the occurrence and development of inflammation, while total alkaloids of Sophora alopecuroides can activate FXR expression in the colon, thereby reducing the content of total bile acids in serum, alleviating inflammation caused by bile acid accumulation, and reducing its inflammatory response, playing a certain therapeutic and protective role in UC. The detection results of inflammatory factors suggest that FXR may also alleviate the inflammatory response by inhibiting the expression of pro-inflammatory factors.

The correlation analysis between the protein expression of FXR, TGR5, and CYP7A1 and total bile acids and inflammatory factors found that FXR, CYP7A1, and total bile acids in the colon tissue of UC mice were negatively correlated, while they were positively correlated with IL-10. TGR5 was negatively correlated with pro-inflammatory factors IL-23, IL-1 β, and IL-17, indicating that the total alkaloids of Sophora alopecuroides treated UC mice by activating colon FXR, TGR5, and CYP7A1 resulted in a decrease in serum total bile acid content and pro-inflammatory factor content, while the content of anti-inflammatory factor IL-10 increased. It is speculated that this may alleviate inflammatory response by reducing pro-inflammatory factors and increasing anti-inflammatory factor IL-10. Consistent with previous results, this further confirms that the treatment of ulcerative colitis with total alkaloids of Sophora alopecuroides may improve inflammation by regulating the bile acid metabolism pathway. In addition, although the total alkaloids of Sophora alopecuroides have a certain upregulation effect on the activation of TGR5, it is not significant, and it may also have an anti-inflammatory effect, which needs further research.

This study established an acute UC model in BALB/c mice by providing them with 3.5% DSS water for 7 consecutive days of free drinking. The study evaluated and confirmed that total alkaloids of Sophora alopecuroides can reduce tissue damage and inflammatory symptoms of DSS induced colitis in mice. After treatment with total alkaloids from Sophora alopecuroides, the serum total bile acid content decreased, and the colon FXR, CYP7A1, and TGR5 proteins were upregulated to varying degrees. The total alkaloids of Sophora alopecuroides may treat ulcerative colitis in mice by increasing the levels of colonic goblet cells and anti-inflammatory factor IL-10, reducing the levels of pro-inflammatory factors IL-23, IL-1 β, IL-17, and activating the expression of FXR/TGR5 pathway related proteins.