| Inflammation is a very common and important pathological process.Moderate inflammation is beneficial to the body,but excessive or lack of reaction will cause some adverse reactions and even lead to death.LPS can stimulate acute inflammatory response in RAW264.7 macrophages,which can be used to constructe in-vitro inflammation research model.NO,TNF-?,IL-1?,IL-6 and IL-10,et al.,are participate in the regulation of inflammation,and the content of them can indirectly reflect the degree of inflammation.MAPK and NF-?B signaling pathways are important signal cascade pathways in inflammatory response.Once they are activated,they will regulate the expression of mRNA and protein of various inflammatory factors.Therefor,MAPK and NF-?B signaling pathways are considered to be the key aims of many anti-inflammatory drugs.Exocarpium Citri Grandis(ECG)has been used as a popular traditional medicine for anti-inflammatory.Whereas,its research has been focused on the extraction and analysis of its main components,and the mechanism of its anti-inflammatory effect has not been demonstrated scientifically.Flavonoids,polysaccharides,volatile oil,etc.are the main components in ECG,and naringin in FECG has been regarded as an indicator of the effectiveness of ECG.In this study,the content of FECG and Naringin were determined,and the main components in FECG were analyzed by UPLC-Q-TOF-MS.RAW264.7macrophages were stimulated with lipopolysaccharide(LPS)to build a model in vitro.NO,TNF-? and IL-1 were detected by Griess reagent,ELISA methods and qRT-PCR to analysis the anti-inflammatory effect of FECG.Western blot was applied to research the effect of FECG on MAPK and NF-?B signaling in RAW264.7 cells.Confocal immunofluorescence was used to detecte the nuclear transfer of NF-?B / p65 protein in RAW264.7 cells.The main results are as follows:(1)Samples were powdered by a mill and 5 g extracted with 70% ethanol of 300 mL for 5.5 h at 50?C.After purificated by AB-8 macroporous resin,the content of FECG was up to 88.37%,and the content of naringin was 72.45% in FECG by HPLC determination.The UPLC gradient elution conditions were optimized as follows: 1.5min,90%A;2.0min,75%A;6.0min,60%A;12min,45%A;15min,20%A;19min,0%A;20min,95%A.The mobile phase A was 0.1% aqueous solution of formic acid and the mobile phase B was acetonitrile.The two main peaks with retention times at 12.008 min and 13.611 min showed in UPLC were determined by UPLC-Q-TOF-MS.The molecular weights of them were determined to be 580 and 578 respectively.According to the parent ion and fragment ion,the main components in FECG were naringin and Rhoifolin.(2)MTT assay was used to determine the cytotoxicity of FECG to RAW264.7macrophages,and 5,20 and 80?g / mL were selected as low,medium and high dose.The cytotoxicity of LPS to RAW264.7 macrophages and effects of LPS on NO secretion were studied.1?g/mL of LPS was used to establish an inflammatory model in RAW264.7 cells.5?g/mL,20?g/mL,80?g/mL of FECG had different inhibitory effects on the secretion of NO in RAW264.7 cells in the prevention mode,the treatment mode and the co-mode.Besides,inhibitory effects on the secretion of NO were showed in cells after treated by FECG for 4h,12 h,24 and 48 h.Compared with LPS-treated cells,high dose of FECG could significantly decrease the activity of NOS(P <0.05).In addition,FECG of 5?g/mL,20?g/mL,80?g/mL had different effects on cytokines such as TNF-?,IL-1?,IL-6 and IL-10.(3)The results in qRT-PCR showed that 5,20 and 80?g/mL of FECG had different effects on the mRNA expression of TNF-?,IL-1?,IL-6 and IL-10.Western blot was used to detect the possible anti-inflammatory mechanism of FECG on MAPK signaling pathway and NF-?B signaling pathway.Compared with the control group,the phosphorylation of p38,ERK1/2 and JNK1/2 in RAW264.7 cells were significantly improved by LPS treated.Compared with LPS-treated cells,the levels of p-p38,p-ERK and p-JNK in the cells were inhibited by FECG.Compared with the control group,the content of I?B in RAW264.7 cells was significantly decreased after LPS stimulation(P<0.01).Compared with LPS-treated group,the content of I?B in RAW264.7 cells was significantly increased by FECG of 20?g/mL and 80?g/mL(P <0.01).Confocal immunofluorescence was used to investigate the effect of 80?g/mL FECG on the activation of NF-?B signaling pathway.The staining of NF-?B / p65 in RAW264.7 cells was observed by immunofluorescence confocal microscopy.The results showed that LPS stimulation promoted the transfer of p65 protein from cytoplasm into the nucleus,and FECG of 80?g/mL could effectively inhibit the nuclear transfer of p65. |
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