Investigation Of The Toxic Constituents And Toxicological Mechanism Of Macleaya Cordata (Willd.) R. Br.

Macleaya cordata(Willd.)R.Br.(Papaveraceae family)is a well-known traditional Chinese medicine has been used for years to treat muscle pain,arthritis,rheumatism and joint pain.It has been developed as an anti-diarrhea drug for clinical use,and is also widely used as the first second-class Chinese medicine feed additive in China.Although it is listed as a highly toxic traditional Chinese medicine,few studies have been reported on its toxicity,especially the study on its toxic substance basis and toxic mechanism,which makes the hidden danger of its toxicity always exist.In view of this,toxicity tracing method was used in this study to clarify the basis of toxic substances and evaluate the key toxic components.On this basis,transcriptomic methods were used to explore the mechanism of neurotoxicity.This topic mainly covers the following aspects:1 Screening and toxicity evaluation of chlorophenolIn this study,acute toxicity test of mice was used for toxicity screening.On the basis of confirming the toxicity of the alcohol extract of Chloroform,toxicity tracing mode was adopted.First,n-butanol extract of chloroform ethanol extract was identified as the toxic part,and then repeated silica gel column chromatography and HPLC were used to trace the two main toxic components.They were identified by HR-MS,1H-NMR and 13C-NMR as proto-opioids and allocryptoopioids with synergistic toxicity.Proto-opioid is the main toxic component.The effects of acute toxicity on clinical symptoms,autopsy and histopathological sections of mice were observed,and the target organs of toxic injury of the extract of Brogue gyro were determined including heart,lung,liver,kidney,brain and gastrointestinal tract.2 General toxicity evaluation of protopine in vivo.First of all,the classical Kohl’s method was used to measure the median lethal dose(LD50)of proopiate in mice was 313.10 mg/kg,which was a highly toxic drug.According to LD50,the dosages of acute and chronic toxicity test mice for 7 and 60 days were determined.The changes of body weight,organ coefficient,general behavior,conventional biochemical indexes,pathological damage(optical pathology and ultrastructural observation)and oxidationantioxidant indexes were observed in mice exposed to protopine,and the acute and chronic toxicity characteristics of protopine in vivo were evaluated.(1)Acute toxicity evaluation of proto-opioid in vivo.(1)Compared with control group(A),the mice exposed to proopiate(62.6 mg/Kg/d,H)for one week had significantly decreased body weight,increased cerebral visceral coefficient,increased heat pain threshold,gait instability,ataxia,cerebral hemorrhage edema,nerve cell degeneration and necrosis and other neurotoxic damage.(2)Compared with group A,the levels of ALT,AST,BUN,Cre and LDH in serum of group H were significantly increased on the 3rd to 5th day of proopiate exposure.H&E staining showed that mice in different doses showed different degrees of liver and kidney injury,including liver cell bubble degeneration,inflammatory cell infiltration and liver cell necrosis.Kidney renal tubule congestion,dilation and other acute liver,kidney injury.(3)Flatulence appeared in mice exposed to protopine for one week.H&E staining showed hyperemia,inflammatory cell infiltration,mucosal edema,epithelial cell injury and other pathological changes.The results of 16SR-RNA and ITS high-throughput sequencing showed that protopine exposure for one week mainly affected the changes of intestinal bacteria,decreased the abundance of bacteria,and significantly changed the composition of bacterial community,especially the abundance of Lactobacillus significantly increased.(4)Ultrastructural observation showed that mitochondria,endoplasmic reticulum,golgi apparatus,apoptosis and autophagy were involved in the damage of brain,liver and kidney induced by protopine.(5)Compared with group A,the contents of T-SOD,GSH and CAT in serum,brain and colon of proopiate(62.6 mg/Kg/d,H)were significantly decreased and the content of MDA was significantly increased.Oxidative stress is involved in nerve cell degeneration and necrosis,colon inflammation and neurotoxicity induced by protopine.(2)Evaluation of chronic toxicity of protopine in vivo.Protopine(7.3 mg/Kg/d H)was administered continuously for 60 days,and learning and memory disorders appeared in mice.Myocardial hypertrophy and myocarditis;Mild fatty liver disease;Pneumonia and other chronic toxicity.Oxidative stress and mitochondrial lesions are involved in proopiate-induced chronic neurotoxicity and cardiotoxicity.It is suggested that cardiac and neurotoxicity of protopine should be paid attention to after long-term administration.3 To investigate the mechanism of acute neurotoxicity induced by protopine based on transcriptomeHigh-throughput transcriptome sequencing was performed on the brain tissues of the d H group and control group(A)mice exposed to protopine(62.6 mg/Kg/d)for one week.A total of 156 differentially expressed genes(DEGs)were obtained from the transcriptome.After GO enrichment analysis,these differentially expressed genes mainly focused on the biological process of animal organ morphogenesis.KEGG enrichment analysis showed that cholinergic synapses,dopaminergic synapses,synaptic vesicles circulation pathways,relaxins signaling pathways,tight junctions related to blood brain barrier and cellular adhesion molecules(CAMs)signaling pathways were significantly enriched.In this experiment,13 DEGs were randomly selected,There were 6 cholinergic synaptic genes(CHRM5,SLC18A3,SLC5a7,Creb3i1,CHRnb4,Kcnj14),5 tightly linked genes(CLDN1,CLDN2,CLDN9,Rab13,Plcz1)and 2 significantly altered genes(Plcz1,Lefty1))was verified by QRT-PCR.4 The cholinergic system is part of the autonomic nervous system and regulates higher neurological functions related to learning,memory,attention,and movement.Tight junctions are essential for maintaining the blood-brain barrier as well as the intestinal mucosal barrier.To investigate the role of cholinergic synapses and tight junctions in proopiate-induced neurotoxicity in vivo(acute and chronic)mice and in vitro based on the results of omics.In this section,q RT-PCR and Western blot were used to detect the expression changes of cholinergic synapses and tightly connected genes and proteins in mouse acute chronic brain and colon tissues and PC12 cells.The Ach E content in tissues,blood and cells was detected by the kit.The results showed that CLDN 1,CLDN2,CLDN9,SLC18A3,SLC5a7,CHRnb4 and CHRM5 genes and proteins were significantly decreased,while the content of Ach E in brain and blood was significantly decreased,but the content of Ach E in colon was significantly increased after protopine exposure.The cytotoxicity of PC12 cells increased in a dosedependent manner with different concentrations of protopine(2.28,1.14,0.56,0.28,0.14,0.07,0.04,0 m M).Slc18a3,SLC5A7 and CHRnb4 genes and proteins were significantly decreased in PC2 cells exposed to protopine(1.14 m M),while CHRM5 genes and proteins were significantly increased,and Ach E content was significantly decreased in PC2 cells exposed to protopine(1.14 m M).In summary,the main toxic substance base of brogues is proto-opioid,LD50 is 313.10 mg/Kg,causing liver,kidney,brain,heart,colon and other organs damage.Original opioid alkali short-term high-dose exposure cause weight loss,sensory function and motor dysfunction such as nerve toxicity symptoms,pathological changes in the cerebral cortex,hippocampus,mitochondria,endoplasmic reticulum and golgi expansion,apoptosis mediated neural cell edema,degeneration,necrosis,the transcriptome analysis,cholinergic synapses,closely connected pathway enrichment significantly.Based on the results of omics,further in vivo and in vitro studies revealed that cholinergic synapses and tightly connected pathways were involved in proopiate-induced neurotoxic injury.