Inhibitory Effect Of Catalpol On LPS-induced Inflammation In RAW264.7 Cells And Its Mechanism

Objective: Rehmanniae Radix is a traditional herbal medicine widely used in East Asia that has anti-inflammatory and anti-allergic effects.Catalpol is the primary active component of Rehmanniae Radix.However,it′s effect on RAW264.7 cells and the underlying molecular mechanisms remained poorly understood.This study aimed to assess the anti-inflammation effect of catalpol on lipopolysaccharide-induced RAW264.7 cells and explore the potential mechanisms.Its significance is to provide theoretical basis and ideas for the treatment of inflammatory diseases such as systemic inflammatory response or chronic inflammation of metabolic syndrome.Methods: Mouse mononuclear macrophage RAW264.7 cells were cultured.(1)RAW264.7 cells were divided into six groups: Control,LPS(10ng/ml),LPS + catapol(25μmol/L),LPS + catapol(50μmol/L),LPS + catapol(100μmol/L),catapol(100μmol/L).(2)MTT assay was used to detect cell viability.(3)The messenger RNA(m RNA)expression of IL-1β、IL-6、TNFα was measured by real-time PCR.(4)The expression of IL-1β、IL-6、TNFα、PGE2 in cell supernatant was detected by ELISE kits.(5)The protein expression of i NOS、COX-2、ERK、p-ERK、JNK、p-JNK、p38、p-p38、p65、p-p65、IκBα、p-IкBα was detected by western blot.(6)The expression and distribution of p65 in RAW264.7 cells were detected by cell immunofluorescence.Result:(1)The MTT assay showed that catalpol had no adverse effect on the viability of RAW264.7 cells.(2)LPS significantly stimulated the m RNA expression of IL-1β、IL-6、TNFα in RAW264.7.Catalpol significantly inhibited the LPS-induced m RNA expression of IL-1β、IL-6 but inhibited TNFα m RNA expression only when the drug concentration reached 100 μM.(3)The results of ELISE showed that LPS evidently induced secretion of IL-1β、IL-6、PGE2,which was significantly inhibited by catalpol.However,the inhibitory effect on TNFα appeared when the drug concentration reached 100 μM.The expression of NO in cell supernatant detected by griss reagent was significantly increased in LPS group while decreased in drug group in a concentration dependence.(4)Catalpol significantly decreased LPS-induced protein expression of i NOS 、 COX-2.Furthermore,catalpol pretreatment restored LPS-induced increase of p-p65、p-IκBα、p-p38 、p-ERK dose-dependently,but influenced the JNK activation when the drug concentration reached 100 μM.(5)Cell immunofluorescence assay showed that the expression and nuclear translocation of p65 increased in LPS group compared with control group in RAW264.7 cells.However,the expression and nuclear translocation of p65 decreased in drug group compared with LPS group.Conclusions: Results above indicated that catalpol alleviated LPS-induced inflammatory response by inhibiting the activation of NF-κB pathways and partial MAPK pathways.