| Objective:Rats pathological model was established with an arsenic trioxide. Explored the mechanism of liver injury induced by arsenic trioxide and observed the intervention effect of different doses of Radix Saposhnikoviae on the toxicity model. Explored death protective effect of Radix Saposhnikoviae extracts on mice. Explored the effect arsenic trioxide casts on cell and impacts of four Radix Saposhnikoviae compounds on the effect caused by arsenic trioxide.Methods:1. Rats were divided into five groups or normal control group, ATO poisoning model group (arsenic suspension,5mg·kg-1·d-1), Radix Saposhnikoviae high-dose group (12g·kg-1·d-1), Radix Saposhnikoviae middle dose group (6g·kg-1·d-1), Radix Saposhnikoviae low dose group (3g·kg-1·d-1). Normal control group was orally administrated with0.5%sodium carboxymethyl cellulose solution, the rest of groups were orally administrated with arsenic suspension,8hours later, the control group was orally administrated with distilled water, the rest of groups were orally administrated with different doses of Radix Saposhnikoviae solution accordingly, for23consecutive days. The rat liver histopathology was observated by optical microscopy and electron microscopy.2. Mice were divided into13groups (n=20, half male and half female) or ATO poisoned model group (ATO injected by tail vein,100mg·kg-1), Radix Saposhnikoviae A high-dose group (ATO+Radix Saposhnikoviae A (10mg-kg-1)), middle-dose group (ATO+Radix Saposhnikoviae A (5mg·kg-1)), low-dose group (ATO+Radix Saposhnikoviae A (2.5mg·kg-1)), Radix Saposhnikoviae B high-dose group (ATO+Radix Saposhnikoviae B (50mg·kg-1)), middle-dose group (ATO+Radix Saposhnikoviae B (25mg·kg-1)), low-dose group (ATO+Radix Saposhnikoviae B (12.5mg·kg-1)), Radix Saposhnikoviae C high-dose group (ATO+Radix Saposhnikoviae C (50mg·kg-1)), middle-dose group (ATO+Radix Saposhnikoviae C (25mg·kg-1)), low-dose group (ATO+Radix Saposhnikoviae C (12.5mg·kg-1)), Radix Saposhnikoviae D high-dose group (ATO+Radix Saposhnikoviae D(50mg·kg-1)), middle-dose group (ATO+Radix Saposhnikoviae D(25mg·kg-1)), low-dose group (ATO+Radix Saposhnikoviae D(12.5mg·kg-1)). Corresponding drugs were orally administrated by lml/100g weight. The mice were injected by tail intravenous with ATO injection accordingly by1ml/100g weight at30min later. Survival time and mortality of the mice were observed.3. Effects of Radix Saposhnikoviae compounds on ATO-treated HL-60/H9c2cells:Adding different concentrations of ATO (0.5,1.0,2.0,4.0,8.0,16.0μg/ml), prim-o-glucosylcimifugin (12.5,25,50,100,200,400μg/ml),5-o-methylvisammi-oside (12.5,25,50,100,200,400μg/ml), Imperatorin (2,4,8,16,32,64μg/ml), hamaudolglucoside (2,4,8,16,32,64μg/ml) to HL-60/H9c2cells and treated for48h. Cells proliferation was measured by MTT colorimetric method. Selected the appropriate drug concentration to the next experiment. The cells’proliferation condition was observed by MTT assary with the four compounds and Different concentrations (0.5,1.0,2.0,4.0,8.0,16.0μg/ml) of ATO after48h.4. Annexin V/PI double staining detected the effects of drugs on apoptosis:cell suspension0.5ml,0.5ml test sample/well, treated in37℃incubator for48h. Order and dose of the each test substance①solvent control②prim-o-glucosylcimifugin (50.100.200μg/ml)③5-o-methylvisammioside (50、100.200μg/ml)④Imperatorin (50.100.200μg/ml)⑤hamaudolglucoside (50.100.200μg/ml)⑥ATO (50.100.200μg/ml)Results:1. Pathological effects of Radix Saposhnikoviae extract on rat liver tissue toxicity induced by ATO1.1Optical microscopy results showed: the normal control group:no animal liver tissue swelling, no sinusoidal congestion, no central vein dilation, no increased perportal bile duct, no hyperplasia of biliary epithelial cells, no pathological changes in liver tissue, normal structure. Model group:Part of the liver shows various degrees of karyopyknosis and nuclear hyperchromatism, accompanied with pathologies like dikaryon and karyorrhexis. Cells in certain region of the liver distal end shows significant swelling, assuming a ballooning degeneration with karyopyknosis and emptier cytoplasm, which just resembles apoptotic cells.Most of the animals show both signifivant hyperplasia of biliary epithelial cells and increase of periportal bile duct, we can also observe a severe hyperplasia and fiber extension of some periportal bile ducts, which is a progression to fibrosis. High dose groups, middle dose groups and small dose groups of Radix Saposhnikoviae:Compared with model groups, these3groups show various degrees of remission in pathologic changes such as cell swelling, hyperplasia of periportal bile duct.1.2Electron microscopic observation shows: Normal control group:we can observe a complete structure of mitochondrial both outer and inner membrane, a clear structure of mitochondria cristae, no swelling in mitochondria. Model group:we observe that most of mitochondria is swollen, part of outer membrane shows rupture and cristae disorder with its structure disappeared, in matrix, stromatolysis and cavitation is observed.High dose groups, middle dose groups and small dose groups of Radix Saposhnikoviae:These3groups show various degrees of remission in mitochondrial swelling, integrity of outer and inner membrane and inner structure.2. Effects of Radix Saposhnikoviae extract on survival time and rate of ATO-poisoned miceIn female mice survival experiment, compared to the model group, high-dose, middle-dose group of Radix Saposhnikoviae extraction A and high-dose group of extraction C showed significant difference (P<0.05); in male mice survival experiment, compared to the model group, high-dose group of Radix Saposhnikoviae extraction A, high-dose, middle-dose of extraction B, middle-dose group of extraction C showed significant difference (P<0.05);3. Effects of Radix Saposhnikoviae compounds on ATO-treated HL-60cell IC50Different concentrations of ATO (0.5、l.0、2.0、4.0、8.0、16.0μg/ml) added in HL-60cells culture fluid, after adding four Radix Saposhnikoviae monomers with the concentration of100μg/ml, IC50values are2.626μg/ml (prim-o-glucosylcimifugin),2.802μg/ml (5-o-methylvisammioside),2.661μg/ml (Imperatorin) and2.503μg/ml (hamaudolglucoside), the difference with2.695μg/ml (ATO only, no monomer added) is insignificant, hinting that four Radix Saposhnikoviae monomers have no effect on ATO’s inhibition to HL-60cells proliferation;4. Effect of Radix Saposhnikoviae compounds on ATO-induced HL-60cell apoptosisIn the experiment of detecting apotosis cells with Annexin V/PI double staining, we found that compared to the normal group, apoptosis cells significantly increased in3ATO-treated groups (p <0.01), compared with the ATO treated group, apoptosis cells reduced in the3groups:ATO+prim-o-glucosylcimifugin (3+100μg/ml) and ATO+5-o-methylvisammioside(5-.10+100μg/ml)(p<0.05);5. Effects of Radix Saposhnikoviae compounds on ATO-treated H9c2cell IC50Different concentrations of ATO (0.5,1.0,2.0,4.0,8.0,16.0μg/ml) were added to rat cardiomyocytes H9c2cells, after adding100ug/ml of four Radix Saposhnikoviae monomers, IC50values are3.236μg/ml (prim-o-glucosylcimifugin),3.467μg/ml (5-o-methylvisammioside),3.311μg/ml (Imperatorin),2.691μg/ml (hamaudolglucoside), and showed insignificant difference with3.155μg/ml (ATO only, no monomer added), hinting that four Radix Saposhnikoviae monomers exerted no effect on ATO’s inhibition to H9c2cell proliferation;6. Effect of Radix Saposhnikoviae compounds on ATO-induced H9c2cell apoptosisIn the experiment of detecting apotosis cells with Annexin V/PI double staining, we found that compared to the normal group, apoptosis cells significantly increased in3ATO-treated groups (p<0.01), compared with the ATO treated group, apoptosis cells reduced in the4groups:ATO+prim-o-glucosylcimifugin (10+100μg/ml), ATO+5-o-methylvisammioside (10+100μg/ml), ATO+Imperatorin (10+100μg/ml), ATO+hamaudolglucoside (10+100ug/ml)(p<0.05), and2groups: ATO+Imperatorin (10+100μg/ml), ATO+hamaudolglucoside (10+100ug/ml) showed significant improved survival (p<0.01), hinting that ATO can strongly inhibit H9c2cells survival, adding four Radix Saposhnikoviae monomers can alleviate apoptosis to some extent, Imperatorin and hamaudolglucoside may increase H9ce cells survival. Conclusion:1. Rat liver histopathological examination showed that Radix Saposhnikoviae has a certain degree of protection on pathological tissue damage induced by ATO;2. Radix Saposhnikoviae extract can prolong the survival time of ATO-poisoned mice and improve survival rate to a certain extent.3. ATO can induce apoptosis of HL-60cells. Radix Saposhnikoviae compounds: prim-o-glucosylcimifugin and5-o-methylvisammioside showed no inhibition on ATO-induced HL-60cell apoptosis.4. ATO can strongly inhibit H9c2cell survival.The four compounds of Radix Saposhnikoviae can alleviate apoptosis, in which imperatorin and hamaudolglucoside may improve H9c2cells survival rate. |
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