Sargentgloryvine Stem Extracts Regulate Inflammatory Reaction Through NRF2/ARE Signaling Pathway

Objective:To establish a Sargentgloryvine Stem(SSE)water extraction protocol,analyze the chemical components of Sargentglorjrvine Stem extract by HPLC and set up quality control for aqueous extract of SSE.To observe the effects of SSE intervention on Lipopolysaccharides(LPS)activated human monocytic U937 cell line and explore the molecular mechanisms of anti-inflammation on SSE.To provide experimental data of SSE on clinical application for inflammation diseases.Methods:1.The content of salidroside and rutin in SSE were detected by an Aglient 1200 HPLC system,to establish a protocol of HPLC analysis for quality control of aqueous extract of SSE.2.LPS activated monocyte inflammatory response.We detected the mRNA expression of NRF2 and TNF-α,IL-1β and IL-10 by Real-time PCR,protein expression and activation of NRF2,Keapl and HO-1 in NRF2/ARE pathway by western blot.We also investigated activation of NF-κB p65,IKK,IκB,and the phosphorylation of Akt in Akt/NF-KB,along with the phosphorylation of JNK,P38 MAPK,ERK1/2 in MAPK signaling pathway.NRF2 translocation into the nucleus was also illustrated by immunofluorescent assay.Results:1.According to the optimal process conditions,we detected the content of salidroside and rutin in SSE were 1.059mg/g and 0.5611mg/g,respectively,by HPLC.2.The results of Real-time PCR showed that the expression of TNF-α,IL-1β and IL-10 in LPS model group increased compared to the control group,while treatment with SSE inhibited the mRNA transcription of TNF-α,IL-1β and IL-10 in a dose dependent manner.3.Western blotting and Real-time PCR were performed to determine the effect of SSE on LPS-induced NRF2/ARE activation.At the concentration of 1mg/ml and 2mg/ml,SSE caused a dose dependent increase in the expression of NRF2 and HO-1 protein after LPS stimulate.However,the protein of Keap1 was down-regulated in a dose dependent manner.In order to study the activation of NRF2/ARE pathway,we investigate the protein of NRF2,HO-land Keapl at lmg/ml on SSE in a times dependent manner.The expression of NRF2 protein was down-regulated at 1h and 3h in LPS 1μg/ml group,while 1mg/ml SSE inhibited the NRF2 expession.After treatment with SSE lmg/ml for 6h,12h and 24h,an overproduction of NRF2 by U937 cells was observed.Moreover,the up-regulation of HO-1 on 12h and 24h was observed.Expression of Keapl protein at lh,3h,6h,12h,24h was reduced in a times dependent manner.Immunofluorescent observation indicated LPS treatment caused a decrease of NRF2 translocation into the nucleus.4.We next investigated the activation of Akt/NF-κB pathway in LPS activated U937 cells under SSE interventio.Compare with the unstimulated U937 cells,NF-κB p65,IKK and IκB increased after 3h,6h of 1μg/ml stimulation LPS,while lmg/ml SSE treatment significantly reduced the up-lifted NF-μB p65,IKK and IkB expression by 1μg/ml LPS stimulation.Then,we examined the Akt activation.lmg/ml SSE treatment significantly reduced phosphorylation of Akt which were up regulated by 1μg/ml LPS stimulation in a time dependent manner.5.To elucidate the effects of SSE on the MAPK pathway,the phosphorylation of ERK,JNK,and p38 were examined.lmg/ml SSE treatment significantly reduce of phosphorylation of JNK and P38 at 5min,15min,30min,60min,120 min in a time dependent manner.However,the expression of phosphorylation of ERK1/2 was increased.Conclusions:1.We established the method of HPLC analysis of water extract of Sargentgloryvine Stem,and detected the content of salidroside and rutin in SSE were 1.059mg/g and 0.561 lmg/g,respectively,by HPLC.2.Inhibitory effects of the SSE on LPS-induced production of pro-inflammatory cytokines including TNF-α,IL-1β and IL-10.3.SSE can active the NRF2/ARE,AKT/NF-κB and MAPK signaling pathways to delay the damage of inflammation response.