The Study On "Toxicity-effect" Correlation Evaluation And Biosynthesis Of Bufadienolides In Bufonis Venenum

Background:Bufonis venenum(BV),also known as "toad eyebrow oil",is the dried secretion from the skin glands and parotid venom glands of Bufo gargarizans Cantor and Bufo melanosticus Schneider.It is a traditional Chinese animal medicine that can treat malignant swelling.Studies have shown that BV has therapeutic effects on various cancer,and its effective components,bufadienolides,is widely used,such as Huachansu,Liushen Pill,Shexiang Baoxin Pill,and so on.However,with changes in external factors,the available resources of BV is becoming increasingly scarce.Objective:Screening the key "toxicity-effect" components in BV,analyzing the correlation between "toxicity-effect" of BV,providing theoretical basis for establishing a reasonable quality evaluation system for BV,and promoting the rational and full use of BV resources.Analyzing the metamorphosis process of toads,providing a research basis for artificial breeding and resource expansion of toads;exploring the biosynthesis regulation pathway of bufadienolides,providing a theoretical basis for the renewable resources of BV.Methods:1.Normal zebrafish were used as the toxicity evaluation animal and the zebrafish HepG2 tumor xenograft model was used as the activity evaluation model to evaluate the toxicity and anti-tumor activity of eight batches of BV from different regions.Meanwhile,UHPLC-Orbitrap-MS was used to detect the eight batches of BV,and theorthogonal partial least squares discriminant analysis was used to screen the key"toxicity-effect" components of BV based on the toxicity and activity evaluation results.Representative toxic and effective components were selected for regulation research to elucidate the "toxicity-effect" correlation of BV.2.Using zebrafish as a toxicity evaluation model animal,and zebrafish HepG2 xenograft model as an activity evaluation model,the key "toxicity-effect" components selected were ranked,and the top ranked bufadienolides were used as representative substances for the toxicity of BV to carry out research on the toxicity mechanism.3.Using UHPLC-Orbitrap-MS and Illumina NovaSeq for conducting lipomic and transcriptomic analyses on zebrafish samples,to screen differential lipids and genes related to the toxic effects of BV,and to ascertain the mechanism of said toxic effects based on test results.Furthermore,verifying the gene expression levels of key proteins in relevant pathways by using RT-qPCR.4.Using UHPLC-Orbitrap-MS to detect bufadienolides in various tissues of toads,determine the synthetic sites of bufadienolides in the toads body,and validate them using mass spectrometry imaging and homogenate incubation experiments to determine the key organs for the biosynthesis of bufadienolides.5.The livers of 14 adult toads were tested,using weighted gene co-expression network analysis and other bioinformatics analysis methods to identify potential regulatory genes for the biosynthesis of bufadienolides.Based on the enrichment analysis results,potential regulatory genes of key concern were identified,and RT-qPCR detection was conducted in tadpole and juvenile toad samples to verify.Results:1.According to the evaluation results of zebrafish models,BV has clear cardiovascular toxicity and anti-tumor activity.A total of 1365 features were detected by UHPLC-Orbitrap-MS,and combined with the results of the BV toxicity and activity experiment,8 key "toxic effective" components of toad venom were selected using orthogonal partial least squares discriminant analysis method,namely,resibufogenin,cinobufagin,arenobufagin,bufotalin,bufalin,gamabufatalin,desacetylcinobufagin,and telocinobufagin.2.Assessment and ranking of BV’s key components via the zebrafish model.Toxicity:bufalin>resibufogenin>telocinobufagin>cinobufagin>arenobufagin>bufotalin>gamabufatalin>desacetylcinobufagin.Activity:desacetylcinobufagin>resibufogenin>bufotalin>arenobufagin>cinobufagin>telocinobufagin>gamabufatalin(under the treatment concentration of 0.1 μg·ML-1).3.6.BV can exert a "toxic-effect" by regulating calcium ion signaling pathways and lipid metabolism.At an intervention concentration of 5 μg·mL-1,bufalin and cinobufagin upregulate Caveolin-2(CaV2)to interfere with the ion homeostasis of cells,causing abnormal heart rate and inducing cell apoptosis.Bufalin and cinobufagin toxin upregulate LPC and other differential lipids to promote the release of anti-inflammatory mediators,causing blood flow reduction and even thrombus formation.At treatment concentration(0.01～0.1 μg·mL-1),the intervention of 3 BDs can have a callback effect on energy metabolism,amino acid metabolism,and differential lipids.4.Tadpoles have undergone significant changes in diet,energy metabolism,and lipid metabolism,including ketogenesis,adipogenesis,cholesterol metabolism,and fatty acid oxidation during metamorphosis,which could reshape the body to adapt to terrestrial life.It was also found that bufadienolides tended to accumulate during the metamorphosis.Transcriptome data suggest that the "pentose phosphate pathway"and "aromatase activity" may be the key pathways in the biosynthesis of bufadienolides.The CYP2s have a regulatory role in the biosynthesis of bufadienolides.5.The results of tissue distribution in toads indicate that bufadienolides were mainly enriched in the gall bladder,except for the parotid venom gland and skin tissues.Therefore,it is speculated that the liver can biosynthesis bufadienolides.The detection results of DESI-MSI also showed that bufadienolides were mainly concentrated in the toad liver rather than in the immature parotid venom gland.Incubation experiment demonstrates that the liver can independently synthesize bufadienolides.6.Transcriptome sequencing was performed on the livers of toads to screen differential genes that potentially regulate the biosynthesis of bufadienolides.The enrichment results and RT-qPCR detection in tadpoles samples indicate that there is an intersection between the biosynthetic pathways of bile acids and bufadienolides,and that CYP2C23,CYP2F4 may be involved in the process of converting the side chain of cholesterol C-17 into a six-membered unsaturated lactone ring of bufadienolides.Conclusion:BV has clear cardiovascular toxicity and anti-tumor activity.The bufadienolides including bufalin and cinobufagin,in BV can exert a "toxic-effect" relationship by regulating calcium ion signaling pathways and lipid metabolism.Based on the evaluation model of zebrafish,it was found that bufalin,cinobufagin,and resibufogenin ranked high in toxicity.As the Chinese Pharmacopoeia’s quality control indicators for BV,their use should be limited.In addition,bufalin,cinobufagin,and deacetylcinobufagin have weak toxicity but higher anti-tumor activity than cinobufagin,proving the value orientation of the "only content theory" to be unreasonable.A more comprehensive quality evaluation method can help to ensure the full,effective,and safe use of existing BV.The toads gradually establishes chemical defense during its metamorphosis,and the liver is a crucial organ for bufadienolides biosynthesis.Cholesterol serves as an important precursor for bufadienolides,and the "pentose phosphate pathway"provides NADPH for this process.Regulatory enzymes in the bile acid synthesis pathway,such as HSD3B7,are involved in the biosynthesis and regulation.P450 epoxidase enzymes(CYP2C23,CYP2F4)may be involved in the process of cholesterol C-17 side chain cyclization to six membered unsaturated lactone ring of bufadienolides.