Shuping Sun1, Jiahao Zhang2, Hongxing Li2, Yunyan Du2, Shengli Li3, Anqi Li4, Xiaoguo Suo2, Yang Wang2, Qi Sun4. 1. College of Pharmacy, Wannan Medical College, Wuhu, 241002, Anhui, China; Institute of Natural Daily Chemistry, Wannan Medical College, Wuhu, 241002, Anhui, China. Electronic address: 20060024@wnmc.edu.cn. 2. College of Pharmacy, Wannan Medical College, Wuhu, 241002, Anhui, China. 3. The Fifth People's Hospital of Wuhu, Wuhu, 241000, Anhui, China. Electronic address: bzht@163.com. 4. College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, 150040, China.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: Chloranthus serratus is a traditional Chinese medicine for treating arthritis and bruises. AIM OF THE STUDY: To investigate the dose-effect relationship and molecular mechanisms of the water extract of C. serratus roots (WECR) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. MATERIALS AND METHODS: The cell viability was detected by CCK-8 method. One-step method, DCFH-DA fluorescence probe method and immunofluorescence method were used to detect nitric oxide (NO), reactive oxygen species (ROS) and p65 nuclear transcription, respectively. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) were detected by enzyme linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA were detected by quantitative real-time PCR. Western blotting was taken to determine the contents of the relevant proteins in the nuclear transcription factor E2 related factor 2/heme oxygenase-1 (Nrf2/HO-1), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) pathways. RESULTS: The concentrations of 3, 30 and 300 μg/mL were optimized as low, medium and high concentrations of the WECR, respectively, and 1 μg/mL was selected as the optimal concentration of LPS to activate macrophages. The dose of the positive drug dexamethasone was 0.13 mg/mL. The WECR could not only inhibit LPS-induced cell differentiation and the overexpression of NO, IL-6, TNF-α, PGE2 and ROS but also promote the expression of Nrf2 and HO-1, and down-regulate the phosphorylation levels of ERK, JNK, p38 and p65. After the WECR treatment, the expression levels of iNOS and COX-2 mRNA and nuclear translocation of p65 were all inhibited. CONCLUSIONS: The WECR exerts its anti-inflammatory activity by inhibiting the MAPK and NF-κB pathways, activating the Nrf2/HO-1 pathway and down-regulating inflammatory factor levels in a dose-dependent manner.
ETHNOPHARMACOLOGICAL RELEVANCE: Chloranthus serratus is a traditional Chinese medicine for treating arthritis and bruises. AIM OF THE STUDY: To investigate the dose-effect relationship and molecular mechanisms of the water extract of C. serratus roots (WECR) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. MATERIALS AND METHODS: The cell viability was detected by CCK-8 method. One-step method, DCFH-DA fluorescence probe method and immunofluorescence method were used to detect nitric oxide (NO), reactive oxygen species (ROS) and p65 nuclear transcription, respectively. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) were detected by enzyme linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA were detected by quantitative real-time PCR. Western blotting was taken to determine the contents of the relevant proteins in the nuclear transcription factor E2 related factor 2/heme oxygenase-1 (Nrf2/HO-1), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) pathways. RESULTS: The concentrations of 3, 30 and 300 μg/mL were optimized as low, medium and high concentrations of the WECR, respectively, and 1 μg/mL was selected as the optimal concentration of LPS to activate macrophages. The dose of the positive drug dexamethasone was 0.13 mg/mL. The WECR could not only inhibit LPS-induced cell differentiation and the overexpression of NO, IL-6, TNF-α, PGE2 and ROS but also promote the expression of Nrf2 and HO-1, and down-regulate the phosphorylation levels of ERK, JNK, p38 and p65. After the WECR treatment, the expression levels of iNOS and COX-2 mRNA and nuclear translocation of p65 were all inhibited. CONCLUSIONS: The WECR exerts its anti-inflammatory activity by inhibiting the MAPK and NF-κB pathways, activating the Nrf2/HO-1 pathway and down-regulating inflammatory factor levels in a dose-dependent manner.