Study On The NF-?B Signal Pathway Anti-inflammatory Mechanism Of N-butanol Soluble Fraction From Jingfang Powder

Objective:This study was designed to observe the protective effect of n-butanol soluble fraction from Jingfang Powder on lipopolysaccharide(LPS)-induced acute lung injury(ALI)mice and RAW264.7 cell with inflammation,and clarified the underlying anti-inflammatory mechanism via NF-?B signaling pathway,explored the main material basis of its anti-inflammatory effects to supplement the anti-inflammatory effect of the n-butanol soluble fraction from Jingfang Powder and provide pharmacological basis for its clinical application.Methods:(1)Determination of the main chemical components in n-butanol soluble fraction from Jingfang Powder: the content of prim-o-glucosylcimifugin,hesperidin,4'-O-glucopyranosyl-5-O-methylvisamminol,and rosmarinic acid in the n-butanol soluble fraction from Jingfang Powder were determined using high performance liquid chromatography(HPLC)method;(2)the anti-inflammatory effect of n-butanol soluble fraction from Jingfang Powder was studied: ?1 The mice with acute inflammation induced by acetic acid was used to verify the anti-inflammatory effect of n-butanol soluble fraction from Jingfang Powder;?2 ALI mice established by intratracheal instillation of LPS were used to observe the effect of drugs on lung wet to dry weight ratio(W/D),lung index,lung MPO activity,the total protein content of bronchoalveolar lavage fluid(BALF)and pathological morphology of lung tissue.(3)the mechanism of anti-inflammatory effects of n-butanol soluble fraction from Jingfang Powder was explored:?1 in vivo: the level of inflammatory cytokines(IL-6,IL-1?,and TNF-?)and chemokine(MIP-1? and MIP-1?)in BALF was detected by ELISA;the expression of NF-?B p65,NF-?B p50,I?B? and VEGFa m RNA in lung tissue was determined by Real-Time PCR;and the expression of NF-?B p50 protein in lung tissue was determined by Western-Blot.?2 in vitro: the level of IL-6 and TNF-? in supernatant of RAW264.7 cell after LPS stimulation was detected by ELISA,and the content of NO in RAW264.7 cell supernatant after LPS stimulation was tested by Griess method;the effects of drugs on the expression of NF-?B p65,NF-?B p50,and I?B? m RNA of RAW264.7 cell was determined by Real-Time PCR.(4)the active components of anti-inflammatory effect of n-butanol soluble fraction from Jingfang Powder was studied: the effects of prim-o-glucosylcimifugin,hesperidin,4'-O-glucopyranosyl-5-O-methylvisamminol,and rosmarinic acid on the level of IL-6,TNF-?,and NO in supernatant of RAW264.7 cell after LPS stimulation was detected by ELISA;and the expression of NF-?B p65,NF-?B p50,and I?B? m RNA in RAW264.7 cell was determined by Real-Time PCR.Results:(1)The n-butanol soluble fraction from Jingfang Powder which this study used contains prim-o-glucosylcimifugin 496.08?g/g crude drug,hesperidin 302.02?g/g crude drug,4'-O-glucopyranosyl-5-O-methylvisamminol 271.13?g/g crude drug,and rosmarinic acid 67.70?g/g crude drug.(2)The n-butanol soluble fraction from Jingfang Powder could reduce the total protein content,lung W/D,lung index,and MPO activity in lung tissue of ALI mice,as well as improvement of the inflammation in lung.(3)The n-butanol soluble fraction from Jingfang Powder could reduce the level of IL-6,IL-1?,TNF-?,MIP-1?,and MIP-1? in BALF;inhibited the expression of NF-?B p65 and NF-?B p50 m RNA,as well as the expression of NF-?B p50 protein in lung;the n-butanol soluble fraction from Jingfang Powder decreased the level of IL-6,TNF-?,and NO in supernatant of RAW264.7 cell after LPS stimulation,inhibited the expression of NF-?B p65,NF-?Bp50,and I?B? m RNA in RAW264.7 cell.(4)Hesperidin,4'-O-glucopyranosyl-5-O-methylvisamminol,and rosmarinic acid could reduce IL-6 level in supernatant of RAW264.7 cell after LPS stimulation,4'-O-glucopyranosyl-5-O-methylvisamminol could decrease the NO level,In addition,hesperidin and 4'-O-glucopyranosyl-5-O-methylvisamminol inhibited the expression of NF-?B p50 m RNA in RAW264.7cell,and rosmarinic acid inhibited the expression of NF-?B p65,NF-?B p50,and I?B? m RNA in RAW264.7 cell.Conclusion: The n-butanol soluble fraction from Jingfang Powder showed protective effect against the inflammation in ALI mice and RAW264.7 cell induced by LPS through down-regulation of NF-?B p65,NF-?B p50,and I?B? m RNA expression,as well as the expression of NF-?B p50 protein,and then inhibited the inflammatory mediators including IL-6,IL-1?,TNF-?,MIP-1?,MIP-1?,and NO.(3)Hesperidin,4'-O-glucopyranosyl-5-O-methylvisamminol,and rosmarinic acid may be one of the main material basis of anti-inflammatory effect of the n-butanol soluble fraction from Jingfang Powder.