| Venenum bufonis is a precious Chinese herbal medicine produced by processing and drying the white serous fluid secreted by the gland behind the ear and the skin of the toad such as the Chinese big toad or the black socket toad.It has high medicinal value.However,the toxicity of Venenum bufonis is so high that its application is limited.Microbial transformation,as a classical technique used in the industrialization of food and medicine,has the characteristics of increasing efficacy,reducing toxicity and increasing indications.In this paper,the microbial transformation of Venenum bufonis was studied to reveal the mechanism of microbial transformation and anti-tumor effect.At the cellular level and at the animal level,MTT method found that Venenum bufonis at a concentration of 0.001-1.0 mg/ml could effectively inhibit the prolifer ation of lung cancer cell A549,and flow cytometry found that the concentration was0.00001,0.005 and 0.01 mg/ml Venenum bufonis could induce the early apoptosis of lung cancer cells A549,while 0.05 and 0.1 mg/ml toad venom could induce the late apoptosis and necrosis of lung cancer cells A549,and the expression of pro-apoptotic protein Bax and Bad protein were significantly up-regulated.Under scanning electron microscope,lung cancer cells A549 after Venenum bufonis intervention showed obvious irregular shape.It was found that Venenum bufonis effectively inhibit tumor growth at week 7 by using nude mice model and intervention.By collecting soil samples from different environments and soil suspension was diluted 100 times with sterile water and mixed with 10 times of sterile water,the strains resistant to Venenum bufonis toxicity could be well screened on the transformation medium containing 0.5 mg/ml Venenum bufonis.More than 150 microbial strains were obtained according to the morphology,color and texture of the colonies,which were numbered 1-150.According to the transformation ability of microorganisms to Venenum bufonis,a microbial strain that can fully transform Resibufogenin was selected,and was numbered BV-77.The strain BV-77 DNA was extracted,the 16 Sr DNA was amplified and sequenced.The species classificationstatus was confirmed by BLAST comparison and phylogenetic tree analysis.The strain bv-77 was Bacillus marisflavi strain TF-11,belonging to Firmicutes,Bacilli,Bacillales and Bacillus.TLC method was used to determine the Rf value of each components.Among them,the Rf value of Resibufogenin is 0.81,the Rf value of Cinobufogenin is 0.71,the Rf value of Bufalin is 0.62,the Rf value of Bufotalin is 0.50,the Rf value of Telocinobufagin is 0.42,and the Rf value of Arenobufagin is 0.31.Identifying Venenum bufonis and its transformation products,and it was found that strain BV-77 could effectively transform the widely abundant Resibufogenin into Telocinobufagin.The mobile phase was 0.1 % formic acid water(A)-acetonitrile(B)and gradient elution(0-15 min:70 %A?45 %A;15-40 min:55% A),velocity of 0.7ml/min,column temperature 40 ?,sample quantity 10 ?l,detection wavelength of296 nm.Using external standard method,the substrate in the Resibufogenin and Telocinobufagin accounted for 23.25 %,1.25 %.Respectively,the transformation product in Resibufogenin and Telocinobufagin accounted for0.26 %,22.57 %.The conversion efficiency of the system reached 82.20 %.To compare the antitumor effect of Venenum bufonis and transformation product on lung cancer cell A549.MTT assay showed that the inhibitory effect of 0.001-1.0mg/ml transformation product on proliferation was better than that of the substrate Venenum bufonis,and the toxicity of 0.001-0.1mg /ml transformation product on normal cells was reduced compared with that of the substrate Venenum bufonis at the same concentration.Flow cytometry showed that compared with Venenum bufonis,the transformation product could promote the production of large amount of ROS in cells at a concentration of 0.1 mg/ml and 0.5 mg/ml.It was found that the transformation products could induce high expression of Bad and Bax of apoptotic proteins,and the effect was better than that of Venenum bufonis.Combined with molecular biology method,explore the molecular mechanism of Venenum bufonis and its transformation products to inhibit tumor.It was found that AMPK,the regulatory center of energy metabolism was activated.They had significant effects on glycolysis level and mitochondrial function.By inhibiting the expression of the key rate-limiting protein like HK2 and PLM2 in the glycolysis pathway,reducing the P-PFK-2 / PFK-2 glcolysis rate,damaging the mitochondrial structure,changing mitochondrial function,and up-regulating the expression of mitochondrial fission protein Drp1 and autophagy protein Beclin,it achieves the killing effect on lungcancer cells A549. |
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