Anti-inflammatory Effects Of Pentamethylquercetin In LPS-induced Mouse RAW264.7 Macrophages

Flavonoids are rich in diet, vegetables, fruit and are known for a variety of biological activities, including anti-oxidation, anti-inflammation, anti-cancer, improving hyperglyc -emia and decreasing blood pressure et al. We have synthesized a new flavonoid compound through methylation of quercetin, called pentamethylquercetin, with higher metabolic stability. Our previous study showed that PMQ could activate PPARγreceptor. It was also reported that PPARγreceptor may be closely related to inhibit inflammatory responses. On the basis of previous work, we wonder whether PMQ could suppress inflammatory response.To demonstrate these anti-inflammatory effects and the possible mechanism of PMQ, the following two sections were investigated in this thesis:①Anti-inflammatory effects of PMQ on the production of NO in LPS-stimulated mouse RAW264.7 cells.②Anti- Inflammatory mechanism research of PMQ on the expression of iNOS, COX-2, TNF-α, IL-6 mRNA in LPS-stimulated mouse RAW264.7 cells. It provides experiment foundations for further studies.PartⅠEffects of PMQ on LPS-induced cell viability in RAW264.7 macrophage cellsObjective: To study the effects of PMQ on cell viability in RAW264.7 and LPS-induced RAW264.7 macrophage cells. Methods:①MTT assay was adopted to determine the effects of PMQ on cell viability in RAW264.7 macrophage cells. Cells were treated with drugs for 24 h. Experiments were divided into two groups: vehicle group and drug groups, including PMQ 0.1～1μM.②MTT was adopted to determine the effects of PMQ on cell viability in RAW264.7 macrophage cells induced by LPS. Cells were treated with drugs for 24 h. Experiments were divided into three groups: vehicle group, LPS model group and drug groups (PMQ 0.1～1μM). Results:①After treatment with PMQ 0.1μM～1μM for 24 h, compared with vehicle group, no obvious effect was found (P>0.05).②After treatment with 1μg/ml LPS for 24 h, cell viability was increased by 117.6±5.6%. Compared with vehicle group, cell viability was significantly increased in LPS group(P<0.01). After treatment with 1μg/ml LPS and PMQ 0.1～1μM together for 24 h, compared with LPS group, cell viability in 0.3, 1μM PMQ groups decreased from 117.6±5.6% to 97.7±2.2%, 101.2±2.3%, respectively (P< 0.01).PartⅡEffects of PMQ on NO production in LPS-induced RAW264.7Objective: To study the effects of PMQ on NO production in LPS-induced RAW264.7 macrophage cells. Methods: The culture medium was collected and nitrite was measured by the Griess reaction (nitrite reductase assay). Measurement of nitrite accumulation into the culture medium was used to determine NO production. RAW264.7 macrophage cells were treated with drugs for 24h. Experiments were divided into three groups: vehicle control group, LPS model group and drug groups, including PMQ 0.1～1μM. Results: After treatment with 1μg/ml LPS for 24 h,compared with vehicle group, NO production was significantly increased from 3.53±0.50 to 55.84±1.72(P<0.01). After treatment with 1μg/ml LPS and PMQ 0.1～1μM for 24 h, NO production were 51.56±2.34, 42.10±3.4, 40.05±3.45 in PMQ 0.1, 0.3, 1μM groups, respectively. Compared with LPS group, PMQ 0.3, 1μM decreased LPS-induced NO production (P<0.01). PMQ 0.3, 1μM decreased NO production by 25.42±4.99%,29.00±6.05%, respectively.PartⅢEffects of PMQ on iNOS, COX-2, TNF-α, IL-6 mRNA expression in LPS-induced RAW264.7Objective: To study the effects of PMQ on iNOS, COX-2, TNF-α, IL-6 mRNA expressions in LPS-induced RAW264.7 cells. Methods: Semi-quantitative RT-PCR method was used. iNOS/β-actin, COX-2/β-actin, TNF-α/β-actin, IL-6/β-actin density ratio represented mRNA expression level. RAW264.7 cells were treated with drugs for 6h. Experiments were divided into three groups: vehicle control group, LPS model group and drug groups, including PMQ 0.1, 0.3, 1μM. Results:①Compared with vehicle group, LPS 1μg/ml for RAW264.7 cells for 6 h clearly up-regulated the mRNA expressions of iNOS, COX-2, TNF-α, IL-6 (P<0.01).②After treatment with LPS 1μg/ml and PMQ 0.1～1μM for 6 h, compared with LPS group, PMQ 0.1, 0.3, 1μM down-regulated iNOS mRNA expression. The rate of inhibition was 44%(P<0.05), 37%(P<0.05), respectively.③PMQ 0.3, 1μM suppressed COX-2 mRNA expression. The rate of inhibition was 42%(P<0.01), 29%(P<0.05), respectively.④PMQ 0.1, 0.3μM inhibited IL-6 mRNA expression. The rate of inhibition was 42%(P<0.05), 45%(P<0.05), respectively.⑤PMQ 0.1, 0.3, 1μM repressed TNF-αmRNA expression. The rate of inhibition was 48%(P<0.01), 40%(P<0.05), respectively.Conclusion①MTT assey showed that only PMQ 0.1～1μM had no effects on cell viability in RAW264.7. It is concluded that 0.1～1μM PMQ are safe to RAW264.7 cells. 0.3, 1μM PMQ decreased cell viability in LPS-induced RAW264.7 cells, indicating that 0.3, 1μM PMQ could suppress cell growth and cell proliferation in LPS-induced RAW264.7.②Nitrite reductase assay showed that 0.3, 1μM PMQ decreased significantly LPS-induced NO production, indicating that PMQ could directly inhibit NO production in LPS-induced RAW264.7. Semi-quantitative RT-PCR showed that this effect may be related to the inhibition of iNOS gene expression.③Semi-quantitative RT-PCR showed that PMQ inhibited iNOS, COX-2, TNF-α,IL-6 mRNA expression in LPS-induced RAW264.7, indicating that the anti-inflammatory effects of PMQ may be related to the inhibition of iNOS, COX-2, TNF-α,IL-6 gene expression.