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Objective@# To explore whether RhoA plays a role in the migration and invasion of the salivary adenoid cystic carcinoma cell lines SACC-LM and SACC-83.@*Methods@#Total RNA and total protein were extracted from 20 salivary adenoid cystic carcinoma (SACC) and normal adjacent tissues frozen in liquid nitrogen to detect RhoA expression. RhoA-siRNA was constructed to transfect two cell lines (SACC-LM and SACC-83) for cytological experiments. The research included an experimental group (RhoA-siRNA transfection), negative control group (siRNA-NC transfection) and blank group by transient transfection with liposomes. Expression of RhoA mRNA and protein as well as the protein expression of biomarkers of epithelial-mesenchymal transition (EMT) were analyzed, including E-cadherin, N-cadherin, and Vimentin. Furthermore, the changes in invasion and migration of cells in each group were analyzed by comparing the number of transmembrane cells in the Transwell assay and the results of the scratch test.@*Results@#Compared with normal adjacent tissues, RhoA protein and mRNA levels increased in SACC tissues. Compared with the control group, the relative expression levels of RhoA mRNA and protein decreased (P < 0.01), the relative expression levels of E-cadherin protein increased, and the relative expression levels of N-cadherin and vimentin protein increased in the experimental group (P < 0.01). Additionally, the trial results revealed that RhoA knockdown restrained cell migration and invasion (P < 0.01).@*Conclusion @#RhoA expression increased in SACC tissue. Silencing RhoA in vitro could effectively restrain cell migration and invasion in SACC-LM and SACC-83 cells through the regulation of EMT signaling pathways.
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@# Objective: To investigate the effect of salivary adenoid cystic carcinoma (SACC) derived exosomes on PD-L1 expression in fibroblasts. Methods: Exosomes of SACC-83 cell line were extracted by exosome isolation kit, and its particle size, density and phenotypes were identified by electron microscope.After being labeled with PKH67 fluorescence, exosomes were co-incubated with HPLF to observe whether the exosomes could be ingested by fibroblasts under confocal microscope. After co-incubation with the exosomes, the differentially expressed genes (DEGs) in HPLF cells were detected by whole transcriptome sequencing. GO analysis together with KEGG enrichment analysis was used to clarify the biological functions and related signaling pathways related with the DEGs. qPCR, Western blotting and Flow cytometry were used to detect the effect of tumor exosomes on the mRNAand protein expressions of PD-L1, LAG3 and IDO1 in HPLF fibroblasts. Results: SACC exosomes specifically expressed CD63, CD81 and TSG101 molecules, and could be ingested by fibroblasts. After the treatment of fibroblasts by exosomes, the expression of PD-L1 molecule was significantly up-regulated (Fold change=10.19), and the DEGs were significantly enriched in the immune response signaling pathways, such as TNF, NF-κB and cGAS-String pathway, etc. In vitro experiments showed that exosomes could significantly promote the expression of PD-L1 in HPLF cells at both mRNA and protein levels (24.7±4.75 vs 1.03±0.11,P<0.05). Conclusion: Exosomes of SACC can promote the expression of immunocheckpoint ligand PD-L1 in fibroblasts.
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OBJECTIVE: To research for the identification of Dongchongxiacao [Cordyceps sinensis( Berk.) Sacc.] and its counterfeits-processed materials from Cordyceps taii Liang et Liu. METHODSE: On the basis of the specimens of Dongchongxiacao and four types of counterfeits, the macroscopic and microscopic identification methods were studied, including the comparison of the macroscopic features, such as insertion position of stroma, colour of caterpillar part, abdominal leg, and the microscopic features such as the transverse section of the stroma, the caterpillar's body wall and the planta of abdominal leg. RESULTS: The differences between Dongchongxiacao and four types of counterfeits were obvious in macroscopic and microscopic features. CONCLUSION: Dongchongxiacao and its counterfeits-processed materials from Cordyceps taii Liang et Liu. can be identified by the differernces of macroscopical and microscopical fetures.
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Objective To investigate the effects of cultivated Cordyceps sinensis on inhibiting the proliferation and migration of B16 melanoma cells. Methods MTT assay and clone formation assay were used to examine the inhibitory effect of cultivated C. sinensis on the proliferation of B16 cells; Scratching test and transwell assay were used to detect its effect on migration; Cell adhesion assay was used to detect its effect on adhesion. Flow cytometry was used to detect its effect on cell cycle arrest; Western blotting was used to detect its effect on the expression of the proteins of MMP-2, MMP-9, Bax, Bcl-2, CyclinD1, CDK2, CDK4, P21, and p-Akt. Results The results showed that cultivated C. sinensis can significantly inhibit the proliferation and clone formation of B16 cells via arresting cells at G1/S phase in a dose-dependent manner. Moreover, cell migration was also substantially inhibited in a dose-dependent manner. Western blotting analysis showed that cultivated C. sinensis obviously increased the levels of Bax and P21, and meanwhile, decreased the expression of Bcl-2, MMP-2, MMP-9, CDK2, CDK4, CyclinD1, and p-Akt. Conclusion The inhibitory effects of cultivated C. Sinensis on the proliferation and migration of B16 cells probably associated with the expression of related regulating proteins, MMPs family proteins and p-Akt protein in cell cycle.
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Objective To investigate the metabolites related to the course of anthracnosis of Dendrobium officinale and its possible mechanism based on the technique of metabonomics, so as to provide theoretical support for the creation or breeding of disease resistant varieties of D. officinale. Methods The normal and infected by Colletotrichum gloeosporioides leaves of D. officinale were collected respectively, and to find out the differential metabolites through the sample pretreatment, GC-MS analysis, and bioinformatics analysis. Then the differential metabolites and analysis of metabolic pathways involved in the course of disease were carried on the preliminary discussion. Results The multidimensional statistical models of each analysis group were successfully established. The dispersion points of VIP > 1.0 were selected as the potential differential materials, combined with P < 0.05 in the analysis of single dimensional Statistics (test) as the standard to verify. A total of 84 differential metabolites screened from 305 identified metabolites were considered to be pathogenesis related metabolites. A total of 34 differential metabolites were found out to be involved in the metabolic pathways through the pathway enrichment analysis, and the obtained ZC-GB metabolic pathways were of significance. Conclusion Based on GC-MS technology, the metabolomics analysis of D. officinale samples (normal/infected) was carried out and the metabolites related to the course of anthracnosis of D. officinale were found out. It could lay the foundation for studying the disease of D. officinale and cultivating resistant varieties on molecular level.
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Objective Try to find the new biological compounds, the research on the chemical constituents in the fermentation products of an endophytic fungus Phomopsis fukushii had been carried out. Methods The chemical constituents in this fermentation products were isolated by silica gel, Sephadex LH-20 column chromatographies and RP-HPLC methods. Their structures were elucidated by using various spectroscopic techniques. Results Four pentylated diphenyl ethers (1-4) were isolated from this fermentation products, and the new compound (1) was evaluated for its anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. Compounds 2-4 were identified as diorcinol C, diorcinol D, and diorcinol E. Conclusion Compounds 2-4 are isolated from the fermentation products of endophytic fungus Phomopsis fukushii for the first time. Compound 1 is a new compound named phomodiphenyl ether A and given the system name of 1-[4-(3-hydroxy-5-methylphenoxy)-2-methoxy-6-methylphenyl]-3-methylbut- 3-en-2-one. Compound 1 also shows strong anti-MRSA activity with MIC90 value of (54 ± 4) μg/mL. This valve is close to that of positive control, levofloxacin with MIC90 value of [≥ (56 ± 6) μg/mL].
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Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.
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Objective To establish an HPLC method for simultaneous determination of uridine, vernine, adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine in Cordyceps (Cordyceps militaris, Cordyceps Fungus Powder CS-4, Hirsutella sinensis, and C. sinensis), and determine the characteristic components of C. militaris to provide the scientific basis for quality control of C. militaris and its extract. Methods HPLC was performed on Inertsil ODS-3 column (250 mm × 4.6 mm, 5 μm) with mobile phase A (acetonitrile) and B (water) for gradient elution. The flow rate was 0.8 mL/min, detection wavelength was 260 nm, and column temperature was 30 ℃. Results All mentioned five nucleosides can be detected from C. militaris. However, cordycepin and N6-(2-hydroxyethyl)-adenosine were undetected in the other three Cordyceps species. The sample preparation method of C. militaris has a great effect on the content of nucleosides. The content of uridine, vernine and adenosine was the highest in samples prepared by ultrasonic extraction 180 min. Cordycepin was stable form six sample preparation methods, and N6-(2-hydroxyethyl)-adenosine was unbearable to heat and acid. The contents of cordycepin and N6-(2-hydroxyethyl)-adenosine were the same in four preparation methods. Conclusion This experiment provides a basis for the quality analysis of C. militaris and its extracts. Cordycepin and N6-(2-hydroxyethyl)-adenosine can be used as markers for the quality control of C. militaris and its extracts.
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Objective: To establish an HPLC fingerprint method for water-soluble components in Cordyceps sinensis which could provide the basis for authenticity identification and quality evaluation of C. sinensis. Methods: HPLC method was adopted to construct the fingerprint of water soluble components in C. sinensis and its main confused species (C. liangshanensis, C. gunnii and artificial C. militaris). Total 16 common peaks were marked, and 12 common peaks were confirmed by the method with tandem high performance liquid chromatography (HPLC) and quadrupole time-of-flight mass spectrometry (Q-TOF MS). Results: The common mode of HPLC fingerprint for C. sinensis was developed. The method could distinguish between C. sinensis and its confused species, and 12 common peaks were identified, including cytosine, uracil, cytidine, hypoxanthine + guanine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, and cordycepin respectively. Conclusion: This method is simple and easy, providing scientific basis for identification of C. sinensis.
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Objective Recent years, some studies have been studied on the biosynthesis of cordycepin, but it is not clear. To sequence the transcriptomes of the Ophiocordyceps sinensis which could provide the basis for revealing the bio-synthesis mechanism of cordycepin. Methods In this study, by Illumina/Solexa HiSeq 2500 technology, the transcriptomes of the O. sinensis fungus (anamorph) and the fruiting body (teleomorph) was sequenced, assembled and analyzed. By RT-PCR, the full lengths of RNRL (RNR large subunit) and RNRM (RNR small subunit) cDNA were cloned from the fresh O. sinensis fruit body. Results The pathway and the genes involved in cordycepin biosynthesis were predicted. Among of them, RNR was the critical enzyme in the metabolism of adenosine, also predicted to play an important role in the biosynthesis of cordycepin. From the transcriptome data, one large, one small subunits, and four similar sequences of RNR were found. RNRL mRNA was 2 733 bp, encoding 910 aa and RNRM mRNA 1 257 bp, encoding 418 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in RNRL, RNRM contained a ferritin-like conserved sequence. Conclusion This study would be established for revealing the bio-synthesis mechanism of cordycepin.
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Objective: To investigate the effects of three active components in Cordyceps sinensis (cordycepin, adenosine, and ergosterol) on angiogenesis and the function of hepatic endothelial cells. Methods: Chick chorioallantoic membrane was used to record angiogenesis area, and transgenic zebrafish was used to evaluate the changes of intersegmental vascular and alkaline phosphatase. The toxicity of three active components was assayed in SK-HEP-1 cells by MTT. The proliferation of SK-HEP-1 cells was induced by vascular endothelial growth factor (VEGF), with sorafenib as the positive control. Cell proliferation was analyzed by MTT assay. Cell migration and tube formation were observed by Matrigel and Transwell assay, respectively. Fluorescence probe method was used to detect the levels of intracellular nitric oxide (NO) and nitric oxide synthase (NOS). Results: Adenosine and ergosterol inhibited angiogenesis of chick chorioallantoic membrane, and decreased the number of transgenic zebrafish intersegmental vascular. However, cordycepin can promote angiogenesis of chick chorioallantoic membrane. Compared with the blank control group, VEGF induced SK-HEP-1 cells proliferation, promoted cells migration and tube formation, further significantly increased the levels of NO and NOS in SK-HEP-1. All three active components inhibited the proliferation, tube formation of SK-HEP-1 and decreased the levels of NO and NOS in a dose-dependent manner. Moreover, adenosine and ergosterol inhibited SK-HEP-1 cells migration significantly. Conclusion: Three active components, especially adenosine, could inhibit SK-HEP-1 cells proliferation, migration and tube formation in different degrees. And the mechanism is related to the inhibition of intracellular NO levels and NOS activity.
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PA-824 is a novel bicyclic nitroimidazole anti-tuberculosis (TB) drug. Cordyceps sinensis (Berk.) Sacc. (CS) was proven to be a good immunomodulatory compound. This research aimed to investigate the effect of CS on PA-824 in Mycobacterium tuberculosis (M.tb) infected mice (female CBA/J mice, 6 to 8 weeks of age and 20±2 g of weight). Mice were randomly assigned to 4 groups: PA-824, CS, PA-824+CS, and control. To verify the effect of PA-824 and CS on M.tb, after drug administration, mice lungs were harvested and bacterial colony formations were measured. Cells were isolated from infected lungs and spleens to analyze the percentage of CD4+ T cells (CD11a positive). Lung cells were cultured to detect the secretion of interferon-γ (IFN-γ) and interleukin-10 (IL-10) by ELISA. IFN-γ and IL-10 double-positive CD4+ cells in peripheral blood were measured by flow cytometry. The expression levels of IL-2 and IL-10 in mice lungs were analyzed by real-time PCR and western blot. Results showed that PA-824 combined with CS led to the lowest lung colony-forming units (CFU) counts among treated groups. Furthermore, this beneficial outcome might be associated with the decreased CD11a on CD4+ cells in mice lungs and spleens. Moreover, the suppressed secretion of IFN-γ and IL-10, and IL-10 expressions, as well as the decreased IFN-γ and IL-10 double-positive CD4+ cells in blood, could also be associated with the positive effect. However, no significant effect on IL-2 production was found. The combination of PA-824 and CS had more effective bacteriostatic and immunomodulatory effects on M.tb infected mice than PA-824 alone. In conclusion, CS has the potential to be an effective adjuvant in TB treatment.
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Animals , Male , Mice , Anti-Bacterial Agents/pharmacology , Cordyceps/chemistry , Interleukin-10/immunology , Mycobacterium tuberculosis/immunology , Nitroimidazoles/pharmacology , Blotting, Western , Disease Models, Animal , Flow Cytometry , Immunomodulation/drug effects , Immunomodulation/immunology , Mice, Inbred CBA , Mycobacterium tuberculosis/drug effects , Real-Time Polymerase Chain Reaction , Tuberculosis, Pulmonary/immunologyABSTRACT
Objective:To investigate the clinical significance of epidermal growth factor receptor(EGFR),proliferating cell nuclear antigen(PCNA),laminin(LN)and type IV collagen expression in salivary adenoid cystic carcinoma(SACC).Methods:EGFR gene in 78 cases of SACC with complete clinical data was detected by fluorescence in situ hybridization(FISH)technique,the expression of EGFR,PCNA,LN and type IV collagen protein was detected by immunohistochemistry technique(IHC),their correlation with the clin-icopathological parameters was analysed by SPSS 13.00 software.Results:EGFR gene amplification levels(69.2%)was positively related to the ratio of EGFR protein positive expression(7 1 .8%),the expression of EGFR,PCNA,LN and type IV collagen was posi-tively related to the clinical pathological parameters(P<0.05).There was a positive correlation between EGFR and PCNA expression (P<0.05),a negative correlation between LN protein and type IV collagen protein expression(P<0.05).Conclusion:EGFR gene is amplified in SACC.EGFR,PCNA,LN and type IV collagen take part in the occurrence and development of SACC.
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Objective: To study the optimum particle size of Cordyceps sinensis for liver fibrosis in combination with in vitro dissolution experiment from serum pharmacology. Methods: To prepare the powder samples with different grinding degrees, Cordyceps sinensis was crushed through 100-, 150-, 200-, and 300-mesh sieves. The in vitro dissolution of adenosine was measured at different time points. Meanwhile, the powder sample was ig administered to rats, and pharmacodynamic approach was adopted to study the inhibition of medicated serum on HSC-T6 proliferation. Results: The accumulative in vitro dissolution of C. sinensis by 200-300 meshes was higher than that of other meshes. Medicated serum could significantly inhibit HSC-T6 cell proliferation. The AUC of HSC-T6 inhibition kinetics of medicated serum crushed to 200-300 meshes was significantly higher than that in other groups. Conclusion: The in vitro dissolution and pharmacodynamic method could be used for the study on different particle sizes of C. sinensis for anti-hepatic fibrosis, and 200-300 meshes are the optimal particle size.
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Objective: The method of loop-mediated isothermal amplification (LAMP) was employed to detect and identify Cordyceps sinensis rapidly. Methods: Specific LAMP primers were designed according to the CS2 serine protease (csp2) gene of Cordyceps sinensis. C. sinensis DNA was extracted using CTAB method. The reaction conditions of LAMP were optimized. Specificity of LAMP reaction was validated by six different strains and using restriction enzyme Taq1 digested the LAMP products. Sensitivity of LAMP was tested with diluted C. sinensis solution with 10-fold gradient. LAMP products were shown by gel electrophoresis or adding SYBR Green I. Results: The method of LAMP for detecting C. sinensis was effective and specific. The detection limit of LAMP assay was up to 6 pg/mL. Conclusion: LAMP protocol is a promising method for the identification and detection of C. sinensis and Chinese materia medica as well.
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Objective To study the effect of plant Coleus forskohlii active elements Isoforskolin(ISOF)and CT-E(analogs mixture of Isoforskolin)on human neutrophill(PMN)in vitro in order to uncover the mechanism of their properties of mitigating acute lung injury(ALI).Method The effects of ISOF and CF-E on PMN aggregation induced by N-formyl-methiony-leucyl-phenylalanine(fMLP)was performed by using a 4-channel platelet aggregometer.Cytometry Was applied to analyze the effect of tested samples on adhension between PMN and endothelial cells(ECV-304)activated by using lipopolysaccharides(LPS).Expression of LPS-induced PMN adhension molecules was determined with flow cytometry.Radioimmunoassay Was applied to detect the level of TNT-α liberated bv PMN and intracellular cyclic adenosine monophosphate(cAMP)level of PMN.Results It was found that ISOF(25,50,100 μmnol/L)and CF-E(1.25,2.5,5 mg/ml)inhibitted PMN aggregation induced by fMLP,PMN adhemion to ECV-304 indeed by LPS,expression of PMN adhesion molecules,and TNF-α level released by PMN.ISOF and CF-E also increased intracellular cAMP level of PMN.Condusions ISOF and CF-E inhibit PMN aggregation,adhension and adhension molecules,and TNF-α released by PMN,while they increase intracellular cAniP level of PMN.It suggests that their specific alleviating the ALI by the mechanism of the modulation of PMN function.
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Objective To observe inhibition rate of mouse mammary adenocarcinoma(EMT6) and inhibition effect on tumor angiogenesis of Panax notoginseng F.H. Chen and Cordyecps sinensis Sacc. powder. Methods BALB/C mice as bearing cancer animals with subcultured mouse-transplantable EMT6 cell train had accepted intervention treatment by Panax notoginseng F.H. Chen and Cordyceps sinensis Sacc. powder. In this way,inhibition rate of the mouse-transplanted EMT6 by the powder was recorded and the expression of microvessel density(MVD),vascular endothelial growth factor(VEGF) and tissue inhibitor of metalloproteinase-2(TIMP-2) in the tumor tissue after paraffin imbedding and section were detected through immunohistochemistry technique. Results The tumor inhibition rates of two designed dose groups were separately 48.95% and 37.64%. MVD and VEGF expressions of tumor tissue in the two group were significantly suppressed while the TIMP-2 expression increased. Conclusions Panax notoginseng F.H.Chen and Cordyceps sinensis Sacc. powder can restrain the growth of EMT6,and the pathway of this function is likely related to inhibiting tumor angiogenesis.
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Object To study the growth speed and dynamic spore production of CY-8202, which is an asexual strain of entomopathogenic fungi isloated from Cordyceps sinensis (Berk.) Sacc., to explore the method of artificial culture of CY-8208 strain, to assay its antibiotic activity and spectrem and to provide the experimental basis for studies of its active components. Methods Variations in colony diameter of the cordyceps hypha cultured on the fungi media were measured. The spore-count was used to determine the dynamic colony spore outputs of the hypha on several fungi media and water agar. The agar-piece method was used to test its antibiotic activity.Results There were linear relationships between the colony extensions and the culture times on common fungi media such as PDA medium, etc.. The amounts of spore produced by the single colony of the fungi were more than 10 7 and gradually increased, but the rates of increase tended to be gentle after 200 h. Antimicrobial tests against 22 microbial strains showed strong inhibition of gram positve bacteria including Bacillus subtilis, Micrococcus tetragenus and Staphylococcus albus, and to gram negative bacteria, including Proteus vulgaris, Salmonella typhi, Aerobacter aerogenes and Salmonella sp., as well as weak inhibition of three mold strains and one actinomycetes strain, but no inhibition was observed in four yeast tested.Conclusion The growth activity and the spore-production ability of Cordyceps hypha are two important factors to infect validly its insect-host. For the growth activity, its strong penetration seems more important than its fast growth. The ability to product numerous spores of Cordyceps hypha may be an important mark of its strong infectivity to insect-host. The antimicrobial tests show that CY-8202 may secrete some metabolites which have a more broad-spectrum antibacterial activity than cordycepin isolated initially from Cordyceps militaris.
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Objective To compare the antitumor effects and chemical components of the extracts in cultivated Cordyceps sinensis (Cs) fungus HK-1 and natural Cs. Methods The cultivated Cs fungus HK-1 and natural Cs were extracted with petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH), and water. The cytotoxicity of all the extracts was observed with MTT assay on breast cancer cell line MCF-7, mouse melanoma cell line B16, human premyelocytic leukemia cell line HL-60, and human hepatocellular carcinoma cell line Hep G2. The antitumor activity in vivo of the extract was further studied with animal model. The chemical constituents in the extracts were analyzed by HPLC. Results All of the extracts from the cultivated Cs fungus HK-1 had much stronger cytotoxicity on B16 than those from natural Cs, and the EtOAc extract had the most potent activity. The activity of EtOAc extract from the cultivated Cs fungus HK-1 was more potent than that from natural Cs on all the four cancer cell lines. EtOAc extract from the cultivated Cs fungus HK-1 also can inhibit the growth of tumor in vivo. Chemical components of all extracts were analyzed and ergosterol and adenosine were found to be potential compounds. Conclusion The cultivated Cs fungus HK-1 has stronger antitumor activity than natural Cs and is a potential source of antitumor compounds.
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Objective To investigate the protective effect of the extractions of synthetic Cordyceps Sinensia Sacc powder on cold preserved injured tissues of rat livers. Methods The extractions of synthetic Cordyceps Sinensia Sacc powder were added into LR at certain proportion. Having been preserved for 6 or 12 hours, the liver grafts of rats were reperfused for 30 minutes. And then, ATP, ADP, AMP in livers were measured and added up to total adenine nucleotide (TAN). Besides, AST, ALT, MDA, and ET in the effluent of the reperfusion were assayed. Pathological sections were studied and apoptosis index was detected by TUNEL method. Results The values of ATP, TAN in the experimental group preserved for 12 hours were significantly higher than the control group (P0.05), but the mean of the former was higher than the latter. There was obvious increase of apoptosis index (P