Effects Of β-aasarone On The Expression Of HMGB1and NF-κB In LPS-stimulated RAW264.7Cells

ObjectiveLipopolysaccharide (Lipopolysaccharide, LPS) via toll-like receptor on the cell surface to activiate the phlogistic signaling pathway with nuclear transcription factor kappa B (NF-κ B) at the core, producing a large number of inflammatory factors. These inflammatory factor can cause serious inflammatory cascade reactions through the corresponding receptors to activate the intracellular inflammatory signal pathway.Among them, the high mobility group protein-1(HMGB1) is the important inflammatory mediators of endotoxin shock late lethal effects.(β-asarone is one of the six characteristic components in volatile oil of Acorus tatarinowii Schott, and it has many pharmacological activities. The stone calamus is the dry roots of perennial herb Acorus tatarinowii Schott, pungent, warm in nature, fragrant,is a fine good to diffuse Qi and tongqiao. It is mainly used clinically for fever, delirium, dementia, madness, phlegm reversal forgetfulness, deafness, tinnitus, a bored, traumatic injury and so on, may be associated with the inflammatory reaction in the nervous system or cardiovascular system diseases. But the research of it’s role in the inflammatory response is blank.In order to fully tap the β-asarone medicinal value, this study by LPS stimulation of mouse monocyte macrophage cell line RAW264.7to establish the cell model, using Western-blot and fluorescence immunohistochomistry method, to research the β-asarone on late inflammatory factor IIMGB1and inflammatory cytokine transcription related NF-κ B expression influence.Methods (I) β-aasarone on LPS stimulation of RAW264.7cells on the expression of HMGB11. To determine the LPS stimulation of RAW264.7cells:experimental models were divided into control group,0.1u g/mlLPS stimulation of24h、36h and48h groups,0.2u g/mlLPS stimulation of24h. Using Cy3(red fluorescence) to mark HMGB1, Honchest33258(blue fluorescence) to mark nuclei, using fluorescence microscopy and confocal microscopy to observe HMGB1in the nucleus or cytoplasm expression sites, to determine the amount of LPS stimulation time according to HMGBltranslocation to the nucleus and cell activity.2. Fluorescence immunohistochemistry method to detect the expression of HMGBlsite:using0.2u g/ml LPS stimulation of RAW264.7cells from24h model, and the cells were divided into control group, model group, P-asarone (15μM) and P-asarone (150μM) as a total of four groups, the control group without any treatment, the model group and medication group with the use of0.2μ g/ml LPS stimulation, drug groups with different concentrations of3-asarone. Using the same method above to observe HMGB1expression sites, analyze the β-asarone on HMGB1expression of the impact site.3. Western blot method for the detect ion of nuclear HMGB1expression: ditto the establishment of experimental group, by stepwise cracking extraction method of nuclear protein, Western blot HMGB1expression, with LaminB as the internal control.(II）P-asarone on LPS stimulation of RAW264.7cells on the expression of NF-κ B1. Western blot method for the detection of NF-κ B expression:ditto the establishment of the experimental group, lyse cells to extrael the cell total protein, Western blot, for NF kappa B expression, with GAPD as the internal control.2. Dual luciferase report method to detect the NF-κ B promoter activity: the NF-κ B promoter reporter gene plasmi d Luc2P/NF-κ B-Rli/Hygro transfectcd RAW264.7cells, using Renilla luciferase control pi asmid PRL-SV40as the contrast, establishing the experimental group as above. Using the dual luciferase report system to delect β-asarone the NF-kB promoter activity in vitro.Results(I) β-aasarone on LPS stimulation of RAW264.7cells on the expression of HMGB11. The results of Immunofluorescence showed that, after LPS (0.1μ g/ml) stimulation48h of RAW264.7cells and LPS (0.2μ g/ml) stimulation24h of RAW264.7cells, cytoplasmic is bright red, indicating that the cytoplasmic has more HMGBlexpression. To determine the LPS (0.2μ g/ml) stimulation of RAW264.7cells from24h as a subsequent experimental cell model.2. The results of Immunofluorescence showed that,15μ M and150μ M β-asarone group of cytoplasmic are red fluorescence; group15u M cytoplasmic expression of strong red fluorescence, the nuclei were blue fluorescence;150U M β-asarone group within the cytoplasm of red fluorescent weaker expression, the nuclei are orange (blue and red fluorescence overlay); show15μM and150uM β-asarone group can reduce the HMGB1nucleus cytoplasm increased expression of HMGB1,150μM β-asarone group than the15μ M group was more remarkable.3. The results of Western blot showed that, taking LaminB as the internal control, model group HMGB1bands tapering, and blank control group had significant difference (P<0.05);15μ Mβ-asarone group compared with the model group had no statistical difference;150μ M β-asarone group HMGB1with thicker, after statistics treatment compared with the model group had significant difference (P<0.05), indicating that150μM Mβ-asarone group of nuclei within the HMGB1protein expression levels are higher than those in the model group.(II) β-asarone on LPS stimulation of RAW264.7cells on the expression of NF-κ B1. The results of Western blot showed that, after stimulating24h of0.2μ g/ml LPS, NF-κβ protein expression levels were significantly higher than those in blank control group, with significant difference (P<0.01).15μM and150u Mβ-asarone group NF-κ B expression than the LPS group decreased significantly, with especially significant difference (P<0.01);150μ M β-asarone group decreased more significantly. The results show that, β-asarone has inhibition of NF-κ B protein expression function, and the inhibition enhance with the increase of dose.2. The results of Dual luciferase report gene detection system showed that, the NF-κB promoter luciferase activity of RLA of LPS group compared with blank group was significantly enhanced, have significant difference, NF -kB report gone transfecfed successfully.15U M β-asarone group NF-kβ luciforase activity RLA than the LPS group decreased, there was significant difference;150μ M β-asarone group decreased more obviously, have significant difference. The results show that,(β-asarone inhibit NF-κ B promoler activity, and inhibition of the enhancement of increasing dose.Conclusion1.150μ M (β-asarone significantly increased HMGB1expression in nucleus.2.15μ M and150μ M3-asarone can significantly decrease the NF-κB expression, could significantly inhibit NF-κ B promoter activity, and inhibition of the enhancement with the increase of dose.