Background: Despite the increased morbidity of ulcerative colitis (UC) in the developing countries, available treatments remain unsatisfactory. Therefore, it is urgent to discover more effective therapeutic strategies. Pulsatilla chinensis was widely used for the treatment of inflamed intestinal diseases including UC for thousands of years in China. Anemoside B4, the most abundant triterpenoid saponin isolated from P. chinensis, exerts anti-inflammatory and antioxidant effects and may be the most active compounds, which is responsible for the therapeutic effects. However, the mechanism how anemoside B4 executes its biological functions is still elusive. Methods: Here, we used the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model to evaluate the therapeutic effect of anemoside B4. Blood samples of colitis rats were collected for hematology analysis. The inflammation-associated factors were investigated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis was determined with EdU cell proliferation assay and TUNEL assay. The proteins regulated by anemoside B4 were identified by label-free quantitative proteomics. The significantly down-regulated proteins were verified by Western blotting analysis. mRNA expression was analyzed by quantitative real-time RT-PCR. Results: The results showed that anemoside B4 ameliorated TNBS-induced colitis symptoms, including tissue damage, inflammatory cell infiltration, and pro-inflammatory cytokine production, apoptosis and slowed proliferation in colon. Quantitative proteomic analyses discovered that 56 proteins were significantly altered by anemoside B4 in the TNBS-induced rats. These proteins mainly clustered in tricarboxylic acid (TCA) cycle and respiratory electron transport chain. Among the altered proteins, S100A9 is one of the most significantly down-regulated proteins and associated with NF-κB and MAPK signaling pathways in the pathogenesis of UC. Further experiments revealed that anemoside B4 suppressed the expression of S100A9 and its downstream genes including TLR4 and NF-κB in colon. In vitro, anemoside B4 could inhibit the NF-κB signaling pathway induced by recombinant S100A9 protein in human intestinal epithelial Caco-2 cells. Moreover, anemoside B4 inhibits neutrophils recruitment and activation in colon induced by TNBS. Conclusions: Our results demonstrate that anemoside B4 prevents TNBS-induced colitis by inhibiting the NF-κB signaling pathway through deactivating S100A9, suggesting that anemoside B4 is a promising therapeutic candidate for colitis.

Background and Purpose: Isoacteoside (is a phenylethanoid isolated from Monochasma savatieri Franch. ex Maxim., which is an anti‐inflammatory herb widely used in traditional Chinese medicine. However, the exact mechanism of the anti‐inflammatory activity of isoacteoside is not completely understood. In this study, its anti‐inflammatory mechanism was elucidated in mouse macrophages.Experimental Approach: The expression of the NF‐κB pathway, MAPK pathway, iNOS, TNF‐α, IL‐6 and IL‐1β was evaluated using Western blotting, quantitative real‐time PCR or ELISA. TLR4 dimerization was determined by transfecting HEK293T cells with TLR4 plasmids. The in vivo anti‐inflammatory effect of isoacteoside was determined using mouse models of xylene‐induced ear oedema, LPS‐induced endotoxic shock and LPS‐induced endotoxaemia‐associated acute kidney injury (AKI).Key Results: Isoacteoside suppressed COX‐2, iNOS, TNF‐α, IL‐6 and IL‐1β expression. Furthermore, isoacteoside attenuated the LPS‐induced transcriptional activity of NF‐κB by decreasing the levels of phosphorylated IκB‐α and IKK and NF‐κB/p65 nuclear translocation. In addition, isoacteoside inhibited LPS‐induced transcriptional activity of AP‐1 by reducing the levels of phosphorylated JNK1/2 and p38MAPK. Isoacteoside blocked LPS‐induced TLR4 dimerization, resulting in a reduction in the recruitment of MyD88 and TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF) and the phosphorylation of TGF‐β‐activated kinase‐1 (TAK1). Pretreatment of mice with isoacteoside effectively inhibited xylene‐induced ear oedema and LPS‐induced endotoxic death and protected against LPS‐induced AKI.Conclusions and Implications: Isoacteoside blocked TLR4 dimerization, which activates the MyD88–TAK1–NF‐κB/MAPK signalling cascades and TRIF pathway. Our data indicate that isoacteoside is a potential lead compound for the treatment of inflammatory diseases.

Tanshinones belong to a group of lipophilic constituents of Salvia miltiorrhiza Bunge (Danshen), which is widely used in traditional Chinese medicine. A deluge of studies demonstrated that tanshinones exert anti-inflammatory effects, but the underlying mechanisms remain unclear to date. This study investigated the anti-inflammatory effects and mechanisms of total tanshinones (TTN). TTN suppressed the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the secretion of TNF-α, IL-6, and IL-1β in RAW264.7 cells, bone marrow-derived macrophages, and THP-1 cells. TTN attenuated the LPS-induced transcriptional activity of NF-κB and decreased IκB-α and IKK phosphorylation and NF-κB/p65 nuclear translocation. Furthermore, TTN inhibited the LPS-induced transcriptional activity of AP-1, which was induced by the reduction of JNK1/2, ERK1/2, and p38MAPK phosphorylation. TTN blocked LPS-induced Toll-like receptor 4 (TLR4) dimerization, which consequently decreased MyD88 recruitment and TAK1 phosphorylation. In addition, TTN pretreatment effectively inhibited xylene-induced ear edema and LPS-induced septic death and improved LPS-induced acute kidney injury in mice. TTN exerts anti-inflammatory effects in vitro and in vivo by blocking TLR4 dimerization to activate MyD88–TAK1–NF-κB/MAPK signaling cascades, which provide the molecular basis of the anti-inflammatory effect of Danshen and suggest that TTN is a potential agent for the treatment of inflammatory diseases.

Four novel compounds (1–4) as well as fourteen reported compounds (5–18) were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound 4 demonstrated the best anti-inflammatory activity and was chosen for further research. Compound 4 greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound 4in vitro. Compound 4 was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound 4 suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.