| Croton Tiglium,dry and mature fruit of Euphorbiaceae,was first published in Shennong Herbal Classic and was listed as the second grade,because it’s property-Spicy,hot and poisonous.Clinically,it can be used to treat cough,asthma,gastrointestinal diseases(swelling syndrome,deficiency cold abdominal pain in children),intestinal obstruction and other diseases.However,because of its toxicity,the clinical application needs to be processed to reduce toxicity.The main processing method is to press oil after heating to make frost.The processed product is called Crotonis Seme Pulveratum.The toxicity of Croton Tiglium can be manifested as follows:in the process of processing and steaming,volatile vapor can easily cause skin and mucosa swelling,bubbles and acute inflammation in front-line workers;mistakenly taking Croton Tiglium can cause mouth,throat burning,tingling,vomiting,and severe abdominal pain and diarrhea in clinical poisoning cases.The main toxic component of Croton Tiglium is fatty oil,which has strong inflammation effect on skin and intestine.Therefore,all the processing methods in the past dynasties have been degreasing and frosting to reduce the content of fatty oil.There are two different methods of Croton frosting in the Chinese Pharmacopoeia 2015 Edition-dilution method and heat pressing method(dilution method is adding appropriate starch,heat pressing method is degreasing after heating),which stipulates that the content of fatty oil is 18%-20%.To further study the difference of intestinal toxicity of Crotonis seme pulveratum prepared by different methods and the preliminary mechanism of intestinal inflammation caused by croton protein.In this paper,three different processed products of croton frost:diluted crotonis seme pulveratum,heat crotonis seme pulveratum and non-heated crotonis seme pulveratum(without heating),were prepared.The effects of Croton Tiglium and three Crotonis seme pulveratum maked by different processing methods on intestinal inflammation toxicity and intestinal barrier function were studied at the animal level as a whole.The mechanism of Croton protein causing intestinal inflammation was preliminarily studied at the cellular and molecular level.The main work and research results are as follows:1.Study on the Intestinal Toxicity of Croton tiglium and Different Croton CreamsThree Croton frosts were prepared by heat-pressing deoiling,nonheated-pressing deoiling and starch dilution.The mice were given intragastric administration(40 mg/kg,160 mg/kg)for 7 consecutive days.Real-time PCR was used to determine the effects of Croton Tiglium and different Crotonis seme pulveratum on the expression of TNF-alpha and IL-lbeta in duodenum,jejunum,ileum and colon.The results showed that Croton Tiglium could significantly increase the expression of inflammatory cytokines in duodenum,jejunum and ileum of mice,and the expression of inflammatory cytokines in intestine of mice in three Crotonis seme pulveratum groups was significantly decreased,which was significantly different from Croton Tiglium group There were differences in the expression of inflammatory factors in different Crotonis seme pulveratum groups.The order of intestinal inflammatory toxicity was non-heated Croton cream>diluted Croton cream>heated Crotonis seme pulveratum.To further study the difference of impairment of intestinal barrier function between Croton Tiglium and different Crotonis seme pulveratum.The contents of diamine oxidase(DAO)and D-lactic acid in serum of mice were determined by ELISA,and the expression of tight junction protein(occluding,claudin-1)in different intestinal segments was determined by Western blot.The results showed that Croton Tiglim could significantly increase the levels of diamine oxidase(DAO)and D-lactic acid in serum,and decrease the expression of tight junction protein in duodenum and jejunum.Compared with Croton Tiglium group,the levels of DAO and D-lactic acid in serum and the expression of tight junction protein in intestine of three Crotonis seme pulveratum groups were significantly decreased.The intestinal barrier damage of different Croton frost was different,and the order of damage was non-heated Croton cream>diluted Croton cream>heated Crotonis seme pulveratum.It indicated that Croton Tiglium could destroy intestinal epithelial barrier function and reduce the damage after frosting.Combining different methods of Crotonis seme pulveratum preparation,it shows that dilution and heating can reduce the toxicity of croton frost,but the attenuation effect of heating process is more significant,indicating that heating is the key step in Croton frost preparation.2.Study on the Difference of Fatty Oil and Protein in three Crotonis seme pulveratumThe composition of fat oil in different samples was analyzed by GC-MS.The results showed that the content of fat oil decreased after frosting.The content and composition of fatty oil in different Crotonis seme pulveratum were basically the same.The results showed that different methods of Croton frosting had no effect on the content and composition of fatty oil.BCA method was used to determine the protein content in PBS aqueous solution of each sample.The results showed that there were significant differences in protein content among different croton frosts.The order of protein content was Croton Tiglium=non-heated Crotonis seme pulveratum=diluted Crotonis seme pulveratum>heated Crotonis seme pulveratum.The results showed that the hot pressing frost method had a significant effect on the protein content in the frost.When preparing Croton frost by dilution method,starch was added to dilute,and protein was enriched by deoiling method(heated pressing frost method and non-heated pressing frost method).The order of protein content in Crotonis seme pulveratum with the same quality was non-heated Crotonis seme pulveratum>heated Crotonis seme pulveratum>diluted Crotonis seme pulveratum.SDS-PAGE was used to analyze the changes of protein composition in different samples.The protein molecular weight bands in PBS supernatant of non-heated Crotonis seme pulveratum and diluted Crotonis seme pulveratum were the same as those of Croton Tiglium,while the protein molecular weight bands in heated Crotonis seme pulveratum were significantly reduced.The Croton total protein(CTP)was extracted by saturated ammonium sulfate precipitation method.,then simulated steaming for 1 hour.SDS-PAGE showed that the reduced protein molecular weight bands in the supernatant of CTP PBS solution existed in the precipitation.The results showed that the steaming process of heat pressing frost could reduce the protein content and change the protein composition of Crotonis seme pulveratum by denaturing some proteins.According to the results of the first chapter,and the order of toxicity of Croton seme pulveratum was non-heated Crotonis seme pulveratum>diluted Crotonis seme pulveratum>heated Crotonis seme pulveratum.The results showed that the toxicity difference of Crotonis seme pulveratum prepared by different processing methods might be due to the different protein content and composition.3.Simulated Steaming of CTPTo further study the effect of different steaming time on CTP,this paper simulated the processing of Croton total protein.The results showed that the solubility of protein in PBS decreased gradually and the content of protein in supernatant decreased with the steaming time prolonged(0,1,2,5,10,20,40 min).This indicated that Croton protein denatured during the steaming process and was no longer soluble in PBS.SDS-PAGE method showed that the molecular weight bands of CTP decreased gradually with the steaming time prolonging.The results showed that steaming had a significant effect on the solubility(water solubility)and composition of Croton total protein.The supernatant and precipitation of CTP after steaming were further studied.SDS-PAGE experiments showed that the reduced molecular weight bands of CTP in PBS supernatant existed in the precipitation,indicating that CTP denatured during steaming.Small molecule peptides in the supernatant of CTP before and after steaming were detected by 5800 MALDI-TOF/TOF.The results showed that small molecule peptides appeared in the supernatant of CTP after steaming,indicating that CTP degraded during steaming.In summary,it is indicated that the steaming process of heated Crotonis seme pulveratum can reduce the protein content and change the protein composition of Crotonis seme pulveratum by denaturing and degrading some proteins.4.Toxicity of Croton total protein(CTP)and effects of steaming on itWestern blot was used to detect the effect of CTP in pre-and pro-steaming on tight junction protein expression in IEC-6 cells.The results showed that CTP(10,20,40 μg/mL)could significantly reduce the expression of tight junction proteins occludin and claudin-1 compared with the blank group,while CTP steamed for 30 minutes did not significantly reduce the effect of tight junction proteins,which was significantly different from that of unsteamed CTP group.To further study the effect of CTP steaming on cell barrier permeability.In this study,IEC-6 cells were cultured in Transwell chamber for 9 days to establish a single barrier of IEC-6 cells.FD4 transmittance was used to evaluate the effect of CTP on the permeability of single barrier.The results showed that the CTP(10,20,40 μg/mL)significantly increased the transmission of FD4 compared with the blank group.Compared with unsteamed CTP group,FD4 transmittance decreased significantly after 30 minutes of steaming.The results showed that CTP could increase the permeability of cell monolayer barrier and decrease the permeability damage significantly after steaming.ELISA and Real-time PCR were used to detect the effect of CTP in pre-and pro-steaming on the release of inflammatory factors in RAW264.7 macrophages.The results showed that CTP(5,10,20 μg/mL)could significantly increase the levels of TNF-alpha and IL-lbeta in macrophages and promote the release of inflammatory factors compared with the blank group.Compared with unsteamed CTP group,30 minutes after steaming,the level and release of inflammatory factors mRNA decreased,and the inflammatory toxicity decreased.Intestinal inflammation caused by macromolecule substances often needs to destroy intestinal epithelial barrier function before it can enter the lamina propria and stimulate immune cells to release inflammatory factors.In this study,we simulated the intestinal structure in vivo,established a co-culture model of IEC-6 cell monolayer barrier and macrophage Transwell,labeled CTP with FITC,and studied whether CTP could enter the lower chamber of Transwell to stimulate macrophages to release inflammatory factors through IEC-6 cell monolayer barrier.The results showed that with the increase of CTP(10,20,40 μg/mL),the fluorescence value of the lower chamber increased,indicating that the transmittance of FITC-CTP increased,indicating that CTP could destroy the single barrier of IEC-6 cells.At the same time,with the increase of CTP entering the lower chamber,the level of macrophage inflammatory factor mRNA increased significantly in a dose-dependent manner.Compared with the unsteamed CTP group,after 30 minutes of steaming,the content of CTPentering the lower chamber decreased significantly,and the inflammatory cytokine gene level of macrophages decreased significantly.These results suggest that CTP can stimulate macrophages to release inflammatory factors by reducing the expression of tight junction protein in IEC-6 cells and increasing the permeability of single barrier.Steaming reduced the damage of IEC-6 cell monolayer barrier function and macrophage inflammation toxicity,indicating that steaming could reduce the toxicity of CTP.5.Isolation and Purification of Croton Toxicity Protein and Identification of Toxic ProteinIn order to further study the toxic protein in Croton Tiglium,four kinds of proteins were isolated by hydrophobic chromatography column and gel column chromatography:protein F2,protein F3-F1,protein F3-F2 and protein F4-F3.The relative molecular weight of F2(39.8 kDa),F3-F1(39.2 kDa)，F3-F2(14.2 kDa)and F4-F3(13.3 kDa)were determined by SDS-PAGE.The toxicity of four proteins was compared.The results showed that F3-F1 had the strongest toxicity when the release of macrophages inflammatory factors TNF-alpha,IL-lbeta and the expression of tight indirect proteins occludin and claudin-1 were chosen as index.Protein F2 has the strongest toxicity in terms of the permeability of FD4 and the level of macrophage inflammatory factor mRNA in co-culture model.Preliminary identification of protein F2 and F3-F1 by LC-MS/MS showed that protein F2 and F3-F1 contained a variety of proteins,of which heat shock protein 70 was a common protein sequence,and literature studies found that heat shock protein had inflammatory toxicity.To further determine whether heat shock protein 70 exists in the two proteins,Western blot was used to verify the identification results.The results showed that HSP70 was a common protein.Summary1.This study found that Croton Tiglium can cause intestinal inflammation and damage of intestinal epithelial barrier function.After frosting,the toxicity decreases.Comparison of intestinal toxicity of three kinds of Crotonis seme pulveratum:non-heated Croton cream>diluted Croton cream>heated Crotonis seme pulveratum.The protein content in Croton Tiglium can be diluted and the toxicity of Crotonis seme pulveratum can be reduced in the process of dilution frosting.Hot-pressing method can denaturate and degrade protein,reduce the content of water-soluble protein,thus reducing the toxicity of heated Croton cream is more significant,and the toxicity of heated Croton cream is the lowest.2.The co-culture model of IEC-6 cell monolayer barrier and macrophage Transwell was established.Studies have shown that CTP can reduce the expression of IEC-6 tight junction protein,increase the permeability of cell monolayer barrier and damage the barrier function,thus entering the lower chamber to stimulate macrophages to release inflammatory factors.After 30 minutes of steaming,the barrier function was restored and the inflammatory stimulation of macrophages was weakened.It is suggested that the intestinal inflammation caused by CTP stimulating macrophages to release inflammatory factors is related to the destruction of barrier function.Steaming can reduce the toxicity of CTP.Therefore,steaming is needed to reduce the toxicity of protein,so as to reduce the toxicity of Crotonis seme pulveratum.It is indicated that heating is the key step in croton frosting.3.Four proteins were isolated from Croton total protein,protein F2(39.8kDa),protein F3-F1(39.2kDa),protein F3-F2(14.2kDa),protein F4-F3(13.3kDa).The four proteins can stimulate macrophages to release inflammatory factors and reduce the expression of tight junction protein,increase the permeability of cell monolayer barrier and the level of macrophage inflammatory factor mRNA in co-culture model,among which F2 and F3-F1 are the most toxic.Both of them have inflammatory toxicity of heat shock protein 70. |
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