A network pharmacology strategy combined with in vitro experiments to investigate the potential anti-inflammatory mechanism of Prunus cerasifera Ehrhart.

This study aimed to investigate the anti-inflammatory activity of Prunus cerasifera Ehrhart (EHP). LC-MS/MS, network pharmacology, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis methods were used to investigate the chemical composition and the anti-inflammatory mechanism of EHP. The LC-MS/MS results showed that flavonoids and phenolic acids were the major compounds in EHP. The network pharmacology analysis results indicated that EHP was related to TNF, inflammatory cytokine, and MAPK signaling pathway. ELISA and Western blot results showed that EHP impeded the increase in inflammatory factors, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), nuclear transcription factors κB (p65), MAPK pathway, pyrolytic relevant proteins nod-like receptor family pyrin domain-containing 3 (NLRP3), and interleukin-1ß (IL-1ß) induced by lipopolysaccharide (LPS) and activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) pathway. Therefore, this research highlighted the potential application of P. cerasifera in the development of anti-inflammatory foods that prevented inflammatory diseases. PRACTICAL APPLICATIONS: In recent years, many synthetic drugs with anti-inflammatory effect have the disadvantages of high price and side effects. Thus, the development of anti-inflammatory drugs from natural resources has its application value. In this study, LPS-stimulated RAW264.7 cells were used to establish inflammatory model to verify the anti-inflammatory effect of Prunus cerasifera (EHP). The results showed that P. cerasifera possessed anti-inflammatory activity through inhibiting pro-inflammatory cytokines secretion, NF-κB, MAPK pathway, and NLRP3 inflammasome activation. Therefore, P. cerasifera has the potential to develop into functional food to prevent the progress of various inflammatory-related diseases.

Chemical component and in vitro protective effects of Matricaria chamomilla (L.) against lipopolysaccharide insult.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamomile (Matricaria chamomilla L.) is a popular herbal tea for the treatment of hepatitis and cholecystitis in traditional Uygur medicines. AIM OF THE STUDY: To investigate the anti-inflammatory activity and chemical composition of M. chamomilla, and clarify its molecular mechanism. MATERIALS AND METHODS: M. chamomilla was extracted with 75% ethanol and then extracted with different solvents to obtain five fractions, namely petroleum ether fraction (EOPE), dichloromethane fraction (EOD), ethyl acetate fraction (EOEA), n-butanol fraction (EOB), and water fraction (EOW). Cytotoxicity and the effect on the nitric oxide (NO) production of RAW264.7 cells induced by LPS of the five fractions were screened, and the most active one (EOD) was selected for further investigations. The components of EOD were identified by LC-MS/MS analysis in combination with comparison of retention time and UV absorption with authentic compounds by HPLC. In addition, five most abundant compounds of EOD were isolation by column chromatography and semi-preparative HPLC and their structures were further confirmed by HRMS and NMR data analysis and comparison with data in literatures. Then the underlying anti-inflammatory mechanism of EOD were predicted through Network pharmacology using the identified compounds from EOD, and further verified by Western Blot and ELISA experiments. RESULTS: EOD showed the most significant inhibition ratio against NO in RAW264.7 cells without toxicity among the tested five fractions. Thirty-seven compounds including flavonoid-O-glycoside, flavonoid aglycone, methylated flavonoid aglycone, phenolic acid, coumarin, sesquiterpene, and triterpene were identified from EOD by LC-MS/MS and comparison with authentic compounds. The five most abundant compounds in EOD were isolated and determined to be axillarin (26), tricin (30), chrysoeriol (31), centaureidin (33) and chrysosplenetin (35). IL-6, NF-κB, ERK1 and ERK2 cascade, TNF were the most important anti-inflammatory targets of EOD predicted by Network pharmacology. Western Blot and ELISA experiments revealed that EOD significantly decreased the protein expression levels of inflammatory factors (PGE2, MCP-1, IL-6, TNF-α), iNOS, COX-2, NF-κB (p-P65 and p-IκBα), MAPKs (p-p38, p-ERK and p-JNK), and increased the protein expression levels of Nrf2, HO-1 and CYP2E1. In addition, EOD blocked the p65 protein into the nucleus and promoted the nuclear translocation of Nrf2 in RAW264.7 cells induced by LPS. CONCLUSION: M. chamomilla exerted anti-inflammatory effect via NF-κB, MAPK and Nrf2/HO-1 pathways. It could be further applied as a safe anti-inflammatory agent from natural source.

Anti-inflammatory activity of Anchusa italica Retz. in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-κB signaling pathways.

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 E2 (PGE2), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) 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, PGE2, IL-1ß and TNF-α 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 κB-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-1ß 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 κB (NF-κB) 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.

Fluorescent Nanoparticles for Targeted Tumor Imaging and DNA Tracking Gene Delivery In Vitro/In Vivo.

Fluorescence detection is desirable to track the gene transfer process in order to explain the mechanism. Here, a fluorescent nanoparticle, diketopyrrolopyrrole-based liposome (DPL), was prepared for DNA delivery and tumor imaging in vitro and in vivo. The process to deliver DNA into cells was detected in real time by DPL according to the fluorescent property. The transfection efficacies (TEs) for luciferase and enhanced green fluorescent protein (EGFP) analysis of DPL were 1.5 times those of the commercial transfection agent Lipo 2000. Importantly, the DPL/DNA system has high EGFP TE in vivo with tumor targeting ability. This work provided an effective strategy for monitoring transfection processes.

Imaging nucleus viscosity and G-quadruplex DNA in living cells using a nucleus-targeting two-photon fluorescent probe.

A two-photon fluorescent probe, TP-2Bz, was designed and synthesized and it exhibited good targeting capabilities toward cell nuclei. In particular, TP-2Bz demonstrated a high selectivity to both G-quadruplex DNA and viscosity inside the nucleus with significant ratiometric enhancement in fluorescence, providing a specific, versatile imaging tool for analyzing the viscosity and G-quadruplex DNA in living cells.