Demethyleneberberine alleviated the inflammatory response by targeting MD-2 to inhibit the TLR4 signaling.

Introduction: The colitis induced by trinitrobenzenesulfonic acid (TNBS) is a chronic and systemic inflammatory disease that leads to intestinal barrier dysfunction and autoimmunedisorders. However, the existing treatments of colitis are associated with poor outcomes, and the current strategies remain deep and long-time remission and the prevention of complications. Recently, demethyleneberberine (DMB) has been reported to be a potential candidate for the treatment of inflammatory response that relied on multiple pharmacological activities, including anti-oxidation and antiinflammation. However, the target and potential mechanism of DMB in inflammatory response have not been fully elucidated. Methods: This study employed a TNBS-induced colitis model and acute sepsis mice to screen and identify the potential targets and molecular mechanisms of DMB in vitro and in vivo. The purity and structure of DMB were quantitatively analyzed by high-performance liquid chromatography (HPLC), mass spectrometry (MS), Hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), and infrared spectroscopy (IR), respectively. The rats were induced by a rubber hose inserted approximately 8 cm through their anus to be injected with TNBS. Acute sepsis was induced by injection with LPS via the tail vein for 60 h. These animals with inflammation were orally administrated with DMB, berberine (BBR), or curcumin (Curc), respectively. The eukaryotic and prokaryotic expression system of myeloid differentiation protein-2 (MD-2) and its mutants were used to evaluate the target of DMB in inflammatory response. Resluts: DMB had two free phenolic hydroxyl groups, and the purity exceeded 99% in HPLC. DMB alleviated colitis and suppressed the activation of TLR4 signaling in TNBS-induced colitis rats and LPS-induced RAW264.7 cells. DMB significantly blocked TLR4 signaling in both an MyD88-dependent and an MyD88-independent manner by embedding into the hydrophobic pocket of the MD-2 protein with non-covalent bonding to phenylalanine at position 76 in a pi-pi T-shaped interaction. DMB rescued mice from sepsis shock induced by LPS through targeting the TLR4-MD-2 complex. Conclusion: Taken together, DMB is a promising inhibitor of the MD-2 protein to suppress the hyperactivated TLR4 signaling in inflammatory response.

Demethyleneberberine blocked the maturation of IL-1ß in inflammation by inhibiting TLR4-mitochondria signaling.

Demethyleneberberine (DMB) is a natural product from traditional Chinese medicinal herb the rhizome of Coptis chinensis Franch., which has been reported to possess multiple pharmacological activities, especially anti-inflammation and immunoregulation. However, the potential mechanism of DMB in inflammation is still a mystery. In this study, a mouse model of ulcerative colitis (UC) was induced by Dextran sulfate sodium salt (DSS), and in vitro experiments were performed in RAW264.7 macrophages and the primary intestinal macrophages which obtained from Toll-Like receptor 4 (TLR4) and NOD-Like receptor protein 3 (NLRP3) knockout fetal mouse. Mitochondrial was increased by overexpression of peroxlsome proliferator-activated receptor-γ coactlvator-1α (PGC-1α) and exhausted by adding Ethidium Bromide (EtBr) in RAW264.7 to evaluate the function of mitochondria in the maturation of IL-1ß. Additionally, the safety of DMB (50 mg/kg/d) in mice was assessed by orally administrating for 98 days. DMB siginificantly improved colon atrophy, colonic tissue mass score, neutrophil infiltration and histological damage, which was mainly attributed to the anti-inflammatory effect of DMB. Further in vitro analysis showed that DMB blocked the excessive mitochondrial biosynthesis and maintained the homeostasis of mitochondria in inflammatory response. Moreover, the maturation of IL-1ß was suppressed by DMB in a mitochondria dependent manner. Crucially, DMB was a candidate agent for UC with free of toxicity and side effects. These findings demonstrated that DMB ameliorated inflammatory response by inhibiting TLR4-mitochondria signaling, and revealed the effectiveness and mechanism of DMB for alleviation of UC and provided an additional strategy for UC intervention.

Gegen Qinlian decoction relieved DSS-induced ulcerative colitis in mice by modulating Th17/Treg cell homeostasis via suppressing IL-6/JAK2/STAT3 signaling.

BACKGROUND: Gegen Qinlian decoction (GQ) is a traditional Chinese herbal prescription that has been widely used for the treatment of bacterial dysentery and enteric typhoid fever. Recently, GQ has been clinically reported to be a potential candidate for the treatment of ulcerative colitis (UC). However, the immunoregulatory function of GQ in the treatment of UC has not been fully elucidated. PURPOSE: This study focused on the role of immune imbalance in the pathogenesis of UC and the immunomodulatory effect of GQ in the treatment of UC. METHODS: The UC model was established by treating female mice with 3.0% dextran sulfate sodium (DSS) for 7 days, and GQ was orally administered at dosages of 1.5 and 7.5 g/kg/day. Inflammatory factors were detected by ELISA and qRT-PCR. Treg and Th17 cell dysregulation was analyzed by qRT-PCR, immunohistochemistry and flow cytometry. Proteins related to IL-6/JAK2/STAT3 signaling were detected by western blotting. RESULTS: GQ significantly alleviated the symptoms of UC mice and suppressed the activity of myeloperoxidase (MPO). Furthermore, the production of proinflammatory factors, such as IL-1ß, TNF-α and IL-6, was dramatically reduced after GQ administration. Furthermore, GQ improved the infiltration of Treg and Th17 cells into the colons and decreased the expression of inflammatory factors, such as TGF-ß1 and IL-17. The frequencies of Treg and Th17 cells in the Peyer's patches and spleen were reduced by GQ administration; however, GQ had no significant regulatory effect on normal mice. The western blotting results showed that GQ markedly suppressed the phosphorylation of JAK2 and STAT3 and decreased the transcription function of phosphorylated STAT3. CONCLUSIONS: Taken together, these results indicated that GQ alleviated DSS-induced UC by suppressing IL-6/JAK2/STAT3 signaling to restore Treg and Th17 cell homeostasis in colonic tissue.

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling.

BACKGROUND: Gegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium. PURPOSE: This study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models. METHODS: Acute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4-/-) acute UC mice were also used in this study. RESULTS: GQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4-/- UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells. CONCLUSIONS: GQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.