Study On The Pro-inflammatory Mechanism Of Four Lectins In Araceae And Its Effect By Processing

Pinellia ternata(Thunb.)Breit.,Pinellia pedatisecta Schott.,Arisaema heterophyllum Maxim.,and Typhonium giganteum Engl.,all belong to the Araceae family,have the effect of reducing vomiting,eliminating phlegm and dispersing.However,these four kinds of traditional Chinese medicines have been recorded as "toxic" by the previous Chinese herbal literature,and the clinical misuse of raw or fresh products has strong irritating toxicity.Our previous research has showed that the toxic components of this four medicines were all the "raphides",and the toxic proteins in the raphides were the common lectins.Lectin has strong pro-inflammatory toxicity,which is closely related to macrophages,it can cause neutrophil migration and activation of macrophages,but its further pro-inflammatory mechanism is still unclear.Since the four Chinese medicines of Araceae have strong inflammatory toxicity,it is necessary to be processed for reducing toxicity.The Chinese Pharmacopoeia and the National Chinese Herbal Decoction Processing Regulations describe the processing methods of these toxic Chinese medicines by using ginger,Alunite or heating to reduce its toxicity.Our previous research have found that alunite has an exact role in destroying the needle crystal structure,but its effect on lectin remains to be further studied.In order to further elucidate the pro-inflammatory mechanism of four toxic Chinese medicine lectins and the effect of processing,this paper studied the pro-inflammatory mechanism of this four lectins from the perspective of cell membrane receptors;using adjuvant ginger,Alunite and heating methods respectively,to study the effects of processing.It aims to clearly reveal the pro-inflammatory mechanism of four toxic Chinese medicine lectin proteins in Araceae,and the key scientific connotation of detoxification.What's more,it could provide scientific basis and theoretical support for this four medicines' clinical safety application,processing technology reform.Also,it could provide demonstration and reference for other research on toxic Chinese medicine processing in other family.The main research work is as follows:1.Study on the binding of four toxic Chinese medicine lectins in Araceae to macrophagesIsolation and purification of Pinellia ternata(PTL),Pinellia pedatisecta lectin(PPL),Arisaema heterophyllum lectin(AEL)and Typhonium giganteum lecin(TGL)from the tuber of Pinellia ternata,Pinellia pedatisecta,Arisaema heterophyllum and Typhonium giganteum;four lectins were then labeled with fluorescein isothiocyanate(FITC),and the binding of four lectins to macrophages was detected by flow cytometry and laser scanning confocal microscopy.Flow cytometry results showed that after FITC-labeled four lectin(6.25,12.5,25,50?g/mL)stimulated macrophages for 1 h,there is a significant increase in fluorescence intensity.It is indicated that this four lectins can bind to macrophages,and the binding rate increases significantly with the dose of lectins.According to confocal microscopy,it showed that the four lectins(6.25,12.5,25,50?g/mL)could localize to the macrophage membrane after stimulation for 1h,and with the increase of the dose and the prolongation of the stimulation time,these four lectins gradually enter the cytoplasm.This result indicates that these four lectins of Araceae can bind to macrophages and are mainly localized on the cell membrane.2.Pro-inflammatory mechanism of four toxic Chinese medicine lectins in AraceaeRAW264.7 macrophages were cultured in vitro and stimulated with different concentrations of four lectins for 1 h.Western blot was used to detect the key proteins of the MAPK pathway including p-p38,p38,p-ERK,ERK,p-JNK and JNK,and also the key proteins of the NF-?B pathway including p-p65,p65,p-IK?B and I?B.The release of macrophage inflammatory factors TNF-?,IL-1? and IL-6 were detected by ELISA.The results showed that,after stimulating RAW264.7 macrophages for 1h,these four lectins could activate MAPKs and NF-?B inflammatory signaling pathways,which showed the increase levels of p-p38,p-ERK,p-JNK,p-I?B,p-p65.At the same time,the four lectins can cause a large release of macrophage inflammatory factors TNF-?,IL-1?,and IL-6.This result indicates that these four lectins have strong pro-inflammatory toxicity,which could activate the MAPK and NF-?B inflammatory signaling pathways,and lead to the release of inflammatory factors.The activation of inflammatory pathways and the release of inflammatory factors are regulated by upstream cell membrane receptors.Fluorescent labeling experiments show that the four lectin proteins can be localized on the macrophage membrane,and whether the lectin protein localized on the cell membrane binds to the receptor?What type of receptor is it associated with?This paper intends to further study the pro-inflammatory mechanisms of four lectins from the perspective of cell membrane receptors.By using gene silencing technology,macrophages were transfected with shRNA plasmid,and six important cell membrane inflammation-related receptors SR-A,DC-SIGN,TLR4,TNFR1,TNFR2 and IL-1R1 receptors were silenced,respectively.Silencing efficiency was determined by microscopy,PCR,and Western Blot,respectively.After gene silencing,Western Blot and ELISA were used to detect the effects of four lectins on MAPK,NF-?B signaling pathways and the release of inflammatory factors TNF-?,IL-1? and IL-6.The key molecules were then screened and obtained,and the key receptor molecules and their downstream adaptor proteins were determined by Western Blot to further verify the role of the receptor.The results showed that after transfection of macrophages with shSR-A,shDC-SIGN,shTLR4,shTNFR1,shTNFR2 and shIL-1R1 plasmids for 4 h,the silencing efficiency of each gene reached 70%,indicating the gene silencing effect is suitable.Silencing of the above six receptors,respectively,can inhibit the activation of MAPK and NF-?B pathways and the release of inflammatory factors TNF-?,IL-1?,IL-6 to a certain extent,and the effect of silencing TNFR1 and TLR4 receptors on their inhibition pathway activation and inflammatory factor release was most pronounced,indicating that TNFR1 and TLR4 receptors play important roles in the pro-inflammatory processes of four lectins.Western Blot results showed that all four lectins couldincrease the levels of TNFR1 and its downstream adaptor proteins TRADD,TRAF2 together with TLR4 and its downstream adaptor proteins MyD88,IRAK4 and TRAF6,further indicating both TNFR1 and TLR4 play important roles in the pro-inflammatory process of these four lectins.Based on the above results,we speculate that the four lectins have direct binding effect to TNFR1 or TLR4.For this,we will use protein-protein interaction methods for further research and validation.Since the four lectins have no three-dimensional structure,this paper uses homology modeling to simulate the three-dimensional structure of these four lectins,respectively,and then dock with TNFR1,TLR4 receptors,and determine four lectins'binding capacity to TNFR1,TLR4 receptors.At the same time,the affinity of four lectins to TNFR1 and TLR4 was studied by biofilm interferometry(BLI),and their affinity was determined.The protein-protein docking results indicated that the binding ability of the four lectins to TNFR1 was much greater than that of the TNFR1-specific protein ligand and the TNFR1 receptor,and the binding ability of the PTL to the TNFR1 receptor was its twice the binding capacity of the TNFR1-specific protein ligand to the TNFR1 receptor;and the binding ability of the four lectins to TLR4 is similar to that of the TLR4-specific protein ligand and the TLR4 receptor.The results of BLI showed that the four lectins have similar affinity to TNFR1,and the affinity constant is between 5 and 8×10-8mol/L.The affinity of four lectins to TLR4 is between 2×10-6?4×10-5mol/L.In general,the smaller the affinity constant,the stronger the binding between molecules.Therefore,compared with the TLR4 receptor,the TNFR1 receptor has a stronger affinity with the four lectins.The above results indicate that the four lectins of Araceae have a strong affinity with the TNFR1 receptor,and the TNFR1 receptor may be the main molecular target of the pro-inflammatory effects of the four lectins,and TLR4 may be the second.3.Transcriptome sequencing of macrophage after stimulated by Pinellia ternata lectinIn order to further study the effect of Pinellia ternata lectin on RAW264.7 cells,the high-throughput sequencing was used.After stimulation of macrophages by Pinellia ternata lectin(50?g/mL)for 1 h,high-throughput sequencing was performed using Illumina HiSeq sequencing platform to construct a phage library of macrophages,and using sequencing,gene function annotation and other methods for analysis.The results showed that after the stimulation of macrophages by Pinellia ternata lectin,a total of 652 differentially expressed genes of macrophages were obtained,including 616 up-regulated genes with 43 genes greatly associated with inflammation,and 36 down-regulated genes.GO functional analysis showed that Pinellia ternata lectin mainly affects the molecular function,biological processes and cell composition of macrophages.KEGG pathway analysis showed that TNF signaling pathway and MAPK signaling pathway were significantly enriched in cells after stimulation by Pinellia ternata lectin.This result is consistent with the results above,this is the lectin could bind to TNFR1 and then activate the MAPK,NF-?B signaling pathway to lead to massive release of inflammatory factors.4.Effects of processing on four lectins of Araceae familyThis four Chinese medicines of Araceae have strong inflammatory toxicity,it is necessary to be processed for reducing toxicity,and the methods for processing are usually using ginger,alunite or heating.In order to further study the effect of processing on lectins,the ginger,alunite and heating was used.First,the content of lectins in Araceae and their processed products was determined by Western Blot.Then,the four medicines and lectins were prepared by ginger,alunite and heating.SDS-PAGE was used to investigate the protein precipitates of four lectins after processing.MALDI-TOF was used to detect the protein sequence.The results showed that the content of each lectin protein in Pinellia pedatisecta,Pinellia ternata,Arisaema heterophyllum,and Typhonium giganteum was 7.3%,4.9%,2.7%,2.3%,and the content of lectin in Pinelliae Rhizoma praeparatum cum alumine was 0.027%.While lectins were not detected in the Pinelliae Rhizoma praeparatum cum zingibere et alumine,Arisaema heterophyllum praeparatum and Typhonium giganteum praeparatum.The effects of ginger,alunite and heating on the contents of four lectin proteins were investigated.The results showed that heating can greatly down-regulate the contents of lectins,followed by alunite,while ginger had no effect on lectin protein content.SDS-PAGE and MALDI-TOF results showed that the after processed by alunite or heating alone can cause denaturation and degradation of lectin protein,resulting in a decrease in active lectin content.The results reveal that after processed by alunite or heating could reduce the toxicity,rather than ginger.This also shows that the process of using alunite or heating can always be used,and the separate ginger process has been eliminated.Based on our group's previous research,this paper continues to study the pro-inflammatory mechanism of four toxic Chinese medicine lectins of Araceae family and its detoxification.After thoughtful research,we revealed that the needles in the four toxic Chinese medicine tubers can penetrate into the body tissues,and the lectin proteins in the needle crystals and tubers can bind to the TNFR1 and TLR4 receptors on the cell membrane,with the large generation of ROS,thereby activating the downstream MAPK,NF-?B and NLRP3 signaling pathways,leading to the release of inflammatory factors,resulting in strong inflammatory irritating toxicity.Alunite can destroy the structure of needle crystals in four kinds of toxic Chinese medicines,and the alunite and heating can cause denaturation or degradation of lectin proteins,thereby significantly reducing the irritating toxicity;seperate ginger can not destroy lectin protein.This research more clearly revealed the pro-inflammatory mechanism of four toxic Chinese medicine lectins of Araceae family,as well as the key scientific connotation of detoxification.Also,it provides scientific basis and theoretical support for other research on toxic Chinese medicine processing in other family.