Ranunculus bulumei Methanol Extract Exerts Anti-Inflammatory Activity by Targeting Src/Syk in NF-κB Signaling

Abstract

1) Background: Ranunculus bulumei is a flowering plant that belongs to the Ranunculus species. Several Ranunculus species, such as R. aquatilis and R. muricatus, have traditionally been used to treat fever and rheumatism throughout Asia, suggesting that plants belonging to the Ranunculus species may have anti-inflammatory effects. To our knowledge, the pharmacological activity of R. bulumei has not been reported. Therefore, in this study, we aim to assess the anti-inflammatory activity of a methanol extract that was derived from R. bulumei (Rb-ME) in macrophage-mediated inflammatory responses and to identify the molecular mechanism that underlies any anti-inflammatory action.(2) Methods: The anti-inflammatory efficacy of Rb-ME was evaluated while using in vitro and in vivo experiments. The RAW264.7 cells and peritoneal macrophages were stimulated by lipopolysaccharide (LPS). In addition, LPS-induced peritonitis and HCl/EtOH-triggered gastritis models were produced. A nitric oxide (NO) assay, real-time PCR, luciferase reporter gene assay, western blot analysis, plasmid overexpression strategy, and in vitro kinase assay were used to determine the molecular mechanisms and target molecules of Rb-ME. The phytochemical active ingredients of Rb-ME were also identified by high performance liquid chromatograph (HPLC). (3) Results: Rb-ME reduced the production of NO and mRNA expression of iNOS, COX-2, IL-1β, and IL-6 without cytotoxicity. The protein secretion of TNF-α and IL-6 was also decreased by Rb-ME. HPLC analysis indicates that quercetin, luteolin, and kaempferol are the main active ingredients in the anti-inflammatory efficacy of Rb-ME. Rb-ME also blocked MyD88-induced NF-κB promoter activity and nuclear translocation of NF-κB subunits (p65 and p50). Moreover, Rb-ME reduced the phosphorylation of IκBα, Akt, p85, Src, and Syk, which are NF-κB upstream signaling molecules in LPS-activated RAW264.7 cells. According to the in vitro kinase assay, Rb-ME directly inhibits Syk kinase activity. The oral administration of Rb-ME alleviated inflammatory responses and the levels of p-IκBα in mice with LPS-induced peritonitis and HCl/EtOH-induced gastritis. (4) Conclusions Rb-ME has anti-inflammatory capacity by suppressing NF-κB signaling and it has been found to target Src and Syk in the NF-κB pathway. Based on this efficacy, Rb-ME could be developed as an anti-inflammatory herbal medicine.

Cissus subtetragona Planch. Ameliorates Inflammatory Responses in LPS-induced Macrophages, HCl/EtOH-induced Gastritis, and LPS-induced Lung Injury via Attenuation of Src and TAK1

“…Besides NF-κB and AP-1 subunits, other upstream signaling proteins have been identified that are involved in the NF-κB and AP-1 pathways, including IκBα and PI3K p85 or MAPKs and MAPKKs, respectively. In agreement with another report [ 21 ], the IκBα and p-IκBα bands were not regularly detected at 15 min due to the degradation, while rapid recovery of the protein at 30 min after LPS stimulation was observed [ 22 ]. Since Cs-EE regulated IκBα at 5 min, we examined whole-cell lysates from LPS-stimulated RAW264.7 cells or primary peritoneal macrophages at earlier time points.…”

“…Numerous studies have demonstrated that Src has a crucial role in activating immune cells and is part of upstream signaling enzymes that mediate transcriptional activation of NF-κB via phosphorylation of PI3K p85 then IKK to degrade IκBα. Indeed, anti-inflammatory remedies derived from compounds or plant extracts that target Src have been shown to diminish inflammatory responses via a blockade of NF-κB pathways [ 10 , 11 , 22 ]. Furthermore, Cs-EE inhibited activation of two MAPKs, JNK and ERK.…”

“…For the gastritis and acute lung injury mouse model experiments, the stock of Cs-EE was prepared in 0.5% CMC and PBS, respectively, at doses of 100 mg/kg and 150 mg/kg. The doses and experimental scheme of Cs-EE in this study were determined, according to previously published papers showing similar in vitro and in vivo activities [ 10 , 11 , 22 , 24 , 35 ].…”

“…In acute lung injury model, C57BL/6 mouse strain was used since this mouse strain is more sensitive to pulmonary edema and was well demonstrated in the pathogenesis of acute lung injury, according to previous papers [ 36 , 37 , 38 ]. In contrast, ICR mice have been utilized to conduct gastric ulcer experiments [ 10 , 11 , 22 , 24 ]. The in vivo experiments were performed in agreement with the guidelines of the Institutional Animal Care and Use Committee Sungkyunkwan University (Suwon, Korea; approval ID: SKKUIACUC2021-03-07-2 and SKKUIACUC2021-03-56-1).…”

“…Cells or stomach and lung tissues of mice were lysed in lysis buffer as previously described [ 22 ]. Around 20–40 μg of protein were separated on SDS/polyacrylamide electrophoresis and then transferred onto an immune-blot PVDF membrane.…”