Study The Inflammatory Mechanism Of Pinellia Ternata And Pinella Pedatisecta And The Detoxification Mechanism Of Ginger Based On ROS-MAPK/NLRP3-IL-1? Signaling Pathway

Pinellia ternata(Thunb.)Breit.and Pinellia pedatisecta Schott.are belong to Araceae family and are important toxic Chinese medicines.It has been reported that these two plants possess similar toxic properties,which have seriously restricted clinical applications of them.A previous study of the research group found that Pinellia ternata and Pinellia pedatisecta contain common toxic substances:the raphides and lectins,which are contained in the poison raphides.Lectins can increase the inflammatory stimulatory toxicity of severe raphides.We have previously verified that macrophages were induced to release large amounts of reactive oxygen species(ROS)and IL-1?after stimulation by PTL(Pinellia ternate lectin)or PPL(Pinellia pedatisecta lectin).This showed that lectins from Araceae have a significant pro-inflammatory effect,and the mechanism remains to be further studied.In ancient literature,ginger was able to antagonize the toxicity of the poisonous family of Araceae.Further study found that gingerol is an active ingredient of anti-inflammatory.Previous studies have shown that components of gingerol can reduce the over-production of ROS and IL-1?,TNF-a stimulated by PTL or PPL,but its anti-inflammatory mechanisms were still little known.ROS is thought to have the role of a second messenger,which regulates MAPK and NLRP3 signaling pathways.The purpose of this paper is to investigate the mechanisms of inflammations caused by PTL or PPL,and whether gingerols could weaken the inflammation of PTL or PPL.The main experimental contents and results are summarized as follows:1.Research on the effect and mechanism of inflammation induced by PTL or PPLDCFH-DA and Elisa kit were used to investigate the effect of PTL and PPL on macrophage-induced ROS overproduction.The results showed that macrophages were generating a large amount of ROS and L-1?after stimulated by PTL or PPL for one hour.The study found that the phosphorylation of p38,JNK and ERK and the expression of pro-IL-1? were increased,and the different concentrations of PTL and PPL could increase expressions of NLRP3,caspase-1 p20 and ASC in a dose-dependent manner.It showed that PTL and PPL could activate MAPK and NLRP3 signaling pathways and promoted the production IL-1?.According to the literature,the maturation of IL-1 ? is dependent on the cleavage of caspase-1.Pretreatment with Ac-YVAD-cmk for 0.5 h followed by addition of lectin protein was designed to further investigate the key role of caspase-1 in activating IL-1?.The results showed that Ac-YVAD-cmk could inhibit the production of IL-1(3 in a dose-dependent manner,compared with the untreated lectin group.It indicates that the maturation of IL-1? is dependent on the activation of caspase-1.The co-immunoprecipitation method was used to investigate the relationship between TXNIP,TRX and NLRP3.After treatment with PTL or PPL for one hour,we detected the dissociation of TRX from TXNIP.In the meantime,we detected ROS-dependent TXNIP-NLRP3 binding.Immunofluorescence assay exhibited that ASC protein was indeed expressed,and the expression and co-localization were augmented with increasing dose.Caspase-1 was expressed intracellularly,and the expression and co-localization were also elevated.2.ROS Mediates PTL and PPL-induced InflammationMacrophages were pretreated with NAC for 0.5 h,and then the lectin protein was added to further investigate the relationship between the inflammatory proteins of lectin and reactive oxygen species ROS.The results showed that the levels of ROS and IL-1? were decreased.It is suggested that the lectin-induced inflammation was associated with over-production of ROS.Meanwhile,the results showed that the phosphorylation levels of p38,JNK,and ERk were decreased,the expression of caspase-1 p20 was decreased,while the expression of TXNIP was increased.The results showed that NAC could inhibit the activation of PTL or PPL on MAPK signaling pathway and NLRP3 inflammasome signaling pathway.The above results indicated that ROS are key signaling molecules that induce inflammation by PTL or PPL.Above results indicated that PTL and PPL could activate ROS-MAPK/NLRP3-IL-1? signaling pathwawy.3.Study on Antagonism of Ginger Compounds in Ginger to Inflammatory Toxicity Induced by PTL or PPLGinger was extracted by refluxing with ethyl acetate to obtain the ethyl acetate extract,the volatile oil and water extractions.Elisa kit was used to study the antagonism of three sites.It was found that the anti-inflammatory effect of ethyl acetate extract site was the strongest.But,the LC-MS MS analysis of the ethyl acetate extracts revealed that the peak percentage of gingerols was only 50.73%,indicating that non-Gingerol components were still present.In order to further enrich the components of gingerols in ginger,petroleum ether and ethyl acetate were used to re-extract the ethyl acetate extracts in sequence to obtain petroleum ether re-extracted parts,ethyl acetate re-extracted parts and water re-extracted parts.HPLC analysis of gingerol species in petroleum ether re-extraction sites.The content of gingerols was the highest.And the anti-inflammatory effect of petroleum ether re-extraction sites was the strongest.It was found that the peak percentage of the gingerol components therein was 89.87%,and it was considered that this part was effective part.The result of the experiment showed that the gingerol group can significantly inhibit the over-production of ROS and the release of the inflammatory cytokine IL-1?.It has been shown that gingerol can inhibit the intensive oxidative stress and inhibit the release of inflammatory factors.4.Study on the Antagonism Mechanism of Gingerin Compounds on MAPK and NLRP3 Signaling Pathway Activated by PTL and PPLThe results of Westren Blotting showed that gingerol can antagonize lectin-stimulated macrophages by inhibiting the activation of MAPK signaling pathway,inhibiting the production of pro-IL-l?,and inhibiting the activation of NLRP3 signaling pathway,thereby inhibiting the activation of caspase-1 and the inflammation stimulated by PTL or PPL.Summary:1.This paper found that PTL and PPL could lead to severe inflammatory reactions.ROS is a key signal molecule that induces inflammation of these two lectins,and the mechanism is the activation of ROS-MAPK/NLRP3-IL-1? signaling pathway.This leads to the maturation of IL-1? and further development of inflammation.2.Ginger is a potent component of anti-inflammatory effects of gingerols.Its mechanism is to block MAPK and NLRP3 signaling pathways by blocking the oxidative stress induced by PTL or PPL.Finally,the inflammatory cascade was blocked.