Study on the therapeutic effect of hydroxybenzaldehyde on TNBS induced Crohn’s disease in mice based on the PPAR γ/AMPK/NF – κ B signaling pathway
Crohn’s disease (CD) is a chronic recurrent gastrointestinal disease and one of the main diseases of inflammatory bowel disease (IBD). Its onset is usually characterized by common symptoms such as diarrhea, abdominal pain, rectal bleeding, fever, weight loss, and fatigue. In CD, inflammation extends from the mucosa to the serosa through the intestinal wall, and this disease continues to recur and alleviate. With multiple relapses, CD can progress from mild to moderate inflammation to severe penetrating (fistula formation) or stenotic diseases, and more severe cases can lead to cancer, seriously affecting the patient’s quality of life. In addition, CD patients also exhibit many extraintestinal inflammations, such as inflammatory reactions in the eyes, liver, skin, and joints, reflecting the systemic nature of this disease. The treatment of CD includes first-line therapy and other treatments, among which first-line therapy includes steroids and anti-tumor necrosis factor alpha (TNF – α), which can quickly relieve symptoms. Other treatments may include monoclonal antibodies targeting IL-12/23 or integrin α 4 β 7, immunomodulators, combination therapies, or surgery. However, the unified mechanism of these drugs is immunosuppression, and long-term use can increase the risk of infection in patients. In addition, it is accompanied by other serious side effects, including bone marrow suppression, osteoporosis, and liver damage. Therefore, there is an urgent need to develop alternative drugs from natural resources and functional foods to treat CD.

Nostoc commune is a blue-green algae organism that is often used for the prevention and treatment of diseases due to its comprehensive and rich nutrition. In recent years, its extract has been proven effective in treating intestinal diseases. In our preliminary research, we found that the main active ingredient of thunder fungus, p-hydroxybenzaldehyde (HD), can improve the symptoms of ulcerative colitis (UC) by inhibiting intestinal inflammatory response. Based on the many similarities between UC and CD, we speculate that HD may have anti CD effects. To verify this hypothesis, we induced a mouse CD model and a TNF – α – induced Caco-2 cell inflammation model using 2,4,6-trinitrobenzenesulfonic acid (TNBS) to investigate the protective effect and mechanism of HD on CD, providing scientific evidence for HD rich foods as a supplementary alternative therapy for the prevention and treatment of IBD.

 

CD is an idiopathic chronic inflammatory disease with unknown etiology, which is prone to recurrence and affects the entire digestive tract from the mouth to the anus. The incidence rate is the highest in Europe and North America, but the incidence rate of Asian population has risen sharply in recent years. Research has found that the pathogenesis of CD is closely related to intestinal inflammation caused by dysregulation of intestinal mucosal immune response. Multiple studies have shown that improving intestinal inflammatory response can significantly alleviate CD. For example, thalidomide improves CD in mice by restoring the imbalance of TH17/Treg cells and altering the balance of pro-inflammatory and anti-inflammatory cytokines to alleviate inflammation. Maki polyphenol extract inhibits intestinal inflammation and improves CD in mice by downregulating the expression of COX-2 and iNOS. The above indicates that inhibiting intestinal inflammatory response is a major means of treating CD. Consistently, we found that HD can improve TNBS induced CD by inhibiting inflammatory response.

Inflammation is the main cause of IBD, leading to intestinal tissue damage. As important inflammatory biomarkers, related cytokines include TNF – α, IL-1 β, IL-6, and IL-17, which play important roles in the inflammatory response. Inhibiting their overexpression can improve the intestinal inflammatory response of CD. For example, LB can inhibit the overexpression of IL-6, IL-1 β, and TNF – α in the gut of mice induced by TNBS and improve Crohn’s disease. Sodium butyrate inhibits inflammation and maintains epithelial barrier integrity in TNBS induced inflammatory bowel disease mouse model. The above studies all indicate that inhibiting the production of pro-inflammatory cytokines has a therapeutic effect on CD. In this study, TNBS induced severe intestinal inflammation in mice. However, HD treatment reduced the infiltration of colitis cells and significantly downregulated the levels of inflammatory cytokines at both animal and cellular levels, indicating that HD can inhibit inflammatory response and improve CD in mice.

NF – κ B is an important signaling pathway for the transcription of inflammatory cytokines in IBD, responsible for regulating the transcription of pro-inflammatory cytokines (TNF – α and IL-6). As a member of the NF – κ B family, once activated, p65 can be translocated to the nucleus to upregulate the transcriptional expression of pro-inflammatory cytokines. Research has found that inhibiting the NF – κ B signaling pathway can alleviate inflammation in the TNBS model. In this study, induction of TNBS increased the expression of phosphorylated protein NF – κ B p65 in the colon of CD mice, while intervention with HD led to a decrease in the expression of phosphorylated protein NF – κ B p65. In addition, HD can also inhibit the upregulation of NF – κ B p65 phosphorylation protein expression induced by TNF – α in Caco-2 cells. HD improves colonic inflammatory response by inhibiting the NF – κ B signaling pathway.

AMPK is a serine/threonine kinase that plays important roles in glucose and lipid metabolism, cell growth and apoptosis, autophagy, and inflammation. Research has found that AMPK achieves anti-inflammatory effects by inhibiting the activation of NF – κ B. Xu et al. found that activation of AMPK can inhibit the NF – κ B pathway, thereby suppressing inflammatory responses during hypoxia and reoxygenation processes. Hu et al. found that the use of compound C (AMPK inhibitor) can prevent salidroside from inhibiting the NF – κ B signaling pathway and improving AGEs induced endothelial inflammation and oxidative stress. Similarly, this study found that HD has a dose-dependent effect on promoting AMPK phosphorylation. The above indicates that HD improves TNBS induced inflammatory response by activating AMPK to inhibit the NF – κ B pathway.

PPAR γ is a transcription factor that plays an important role in anti-inflammatory, antioxidant, and phagocytic cell mediated clearance processes, and is highly expressed in colonic epithelium. Many studies have shown that PPAR γ is an activator of AMPK, and the anti-inflammatory effect of PPAR γ is achieved by activating AMPK. For example, PPAR γ agonists (thiazolidinedione drugs) can activate AMPK through phosphorylation of AMPK α to inhibit intestinal pathological inflammation. GW9662, T0070907, and PPAR γ siRNA can prevent the activation of AMPK by hydroxyasiatic acid to restore Th17/Treg balance and improve colitis. This study found that HD has a PPAR γ agonist effect, indicating that HD inhibits the inflammatory response of CD by activating PPAR γ.

In summary, HD can effectively slow down the TNBS induced CD inflammatory response in mice, and its mechanism may be related to the inflammatory response mediated by the PPAR γ/AMPK/NF – κ B signaling pathway. Our research shows that supplementation of T. gondii in daily life can not only prevent and cure UC, but also have a certain preventive and therapeutic effect on CD. In the context of the increasing incidence rate of IBD year by year and the limitations of current therapeutic drugs, our research provides scientific basis for food rich in HD as a complementary alternative treatment for prevention and treatment of IBD.