PMID- 34883218 OWN - NLM STAT- MEDLINE DCOM- 20220224 LR - 20220224 IS - 1872-7573 (Electronic) IS - 0378-8741 (Linking) VI - 286 DP - 2022 Mar 25 TI - Anti-inflammatory activity of Anchusa italica Retz. in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-kappaB signaling pathways. PG - 114899 LID - S0378-8741(21)01129-6 [pii] LID - 10.1016/j.jep.2021.114899 [doi] AB - ETHNOPHARMACOLOGICAL RELEVANCE: Anchusa italica Retz. (Boraginaceae) is an important medicinal plant for the treatment of meningitis and pneumonia in traditional Uygur medicines. AIM OF THE STUDY: To clarify the anti-inflammatory activity of A. italica, to reveal its molecular mechanisms, and to discover the anti-inflammatory active ingredients. MATERIALS AND METHODS: Dried and crushed aerial parts of A. italica were extracted with 75% ethanol to yield crude extract (AICE) and AICE was fractionated to obtain petroleum ether extract (AIPE), dichloromethane extract (AIDE), ethyl acetate extract (AIEE), n-butanol extract (AIBE) and residues (AIW). By measuring the effects of AIPE, AIDE, AIEE, AIBE and AIW on cell viability and nitric oxide (NO) in Lipopolysaccharide (LPS) stimulated RAW264.7 cell lines, AIDE with the lowest cytotoxicity and NO contents was finally selected for further chemical and anti-inflammatory investigations. LC-MS/MS experiment was applied to analyze the chemical composition of AIDE. MTT and Griess methods were used to detect the cell viability and to quantify the nitrite levels in culture supernatants, respectively. Prostaglandin E(2) (PGE(2)), interleukin-6 (IL-6), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) production was examined by ELISA assays. Real-time quantitative PCR was used to detect the expression of hemeoxygenase-1 (HO-1), Nrf2-mediated quinone oxidoreductase 1 (NQO-1), glutathione S-transferase A 1 (GSTA1) and glutathione S-transferase M 1 (GSTM1) mRNA. Western blot analysis was employed to examine the protein expression and enzymatic activities. RESULTS: In preliminary anti-inflammatory screening, AIDE showed the lowest cytotoxicity and the most significant inhibitory effect on the production of NO (the inhibitory is 89%) induced by LPS among the tested five extracts. Thirty-three compounds including twenty-five triterpenoids were identified by LC-MS/MS analysis. AIDE could inhibit LPS-induced the over-expression of NO, IL-6, PGE(2), IL-1beta and TNF-alpha and down-regulate the levels of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38-MAPK (P38) and nuclear transcription factors kappaB-P65 (P65) phosphorylation. It promoted the mRNA expression level of HO-1, NQO-1, GSTA1 and GSTM1 and the protein expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. After the treatment of AIDE, P65 nuclear translocation was inhibited and Nrf2 nuclear translocation was increased. In addition, the protein expression of pyrolytic relevant protein nod-like receptor family pyrin domain-containing 3 (NLRP3) and IL-1beta were decreased after the AIDE treatment. CONCLUSIONS: Anchusa italica Retz. exerted its anti-inflammatory activity by inhibiting the mitogen-activated protein kinase (MAPK), nuclear transcription factors kappaB (NF-kappaB) and pyrolytic relevant proteins, down-regulating inflammatory factor levels, and activating the Nrf2/HO-1 pathway. Triterpenoids might be its major active anti-inflammatory ingredients. CI - Copyright (c) 2021 Elsevier B.V. All rights reserved. FAU - Duan, Xiaomei AU - Duan X AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. FAU - Li, Jun AU - Li J AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. FAU - Cui, Jingxue AU - Cui J AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. FAU - Dong, Yuwei AU - Dong Y AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. FAU - Xin, Xuelei AU - Xin X AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address: xinxl@ms.xjb.ac.cn. FAU - Aisa, Haji Akber AU - Aisa HA AD - Key Laboratory of Chemistry of Plant Resources in Arid Regions, State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, 830011, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address: haji@ms.xjb.ac.cn. LA - eng PT - Journal Article DEP - 20211206 PL - Ireland TA - J Ethnopharmacol JT - Journal of ethnopharmacology JID - 7903310 RN - 0 (Anti-Inflammatory Agents) RN - 0 (Lipopolysaccharides) RN - 0 (Membrane Proteins) RN - 0 (NF-E2-Related Factor 2) RN - 0 (NF-kappa B) RN - 0 (Nfe2l2 protein, mouse) RN - 0 (Plant Extracts) RN - 31C4KY9ESH (Nitric Oxide) RN - EC 1.14.14.18 (Heme Oxygenase-1) RN - EC 1.14.14.18 (Hmox1 protein, mouse) SB - IM MH - Animals MH - Anti-Inflammatory Agents/isolation & purification/*pharmacology MH - Boraginaceae/*chemistry MH - Cell Survival/drug effects MH - Chromatography, Liquid MH - Heme Oxygenase-1/metabolism MH - Inflammation/drug therapy/pathology MH - Lipopolysaccharides MH - MAP Kinase Signaling System/drug effects MH - Membrane Proteins/metabolism MH - Mice MH - NF-E2-Related Factor 2/metabolism MH - NF-kappa B/metabolism MH - Nitric Oxide/metabolism MH - Plant Extracts/*pharmacology MH - RAW 264.7 Cells MH - Signal Transduction/drug effects MH - Tandem Mass Spectrometry OTO - NOTNLM OT - Anchusa italica Retz. OT - Anti-inflammatory OT - Boraginaceae OT - MAPK pathway OT - NF-kappaB pathway OT - NLRP3 inflammasome EDAT- 2021/12/10 06:00 MHDA- 2022/02/25 06:00 CRDT- 2021/12/09 20:15 PHST- 2021/10/20 00:00 [received] PHST- 2021/11/24 00:00 [revised] PHST- 2021/12/05 00:00 [accepted] PHST- 2021/12/10 06:00 [pubmed] PHST- 2022/02/25 06:00 [medline] PHST- 2021/12/09 20:15 [entrez] AID - S0378-8741(21)01129-6 [pii] AID - 10.1016/j.jep.2021.114899 [doi] PST - ppublish SO - J Ethnopharmacol. 2022 Mar 25;286:114899. doi: 10.1016/j.jep.2021.114899. Epub 2021 Dec 6.