| ObjectiveThis study was conducted to investigate the effects of Si-wu decoction on hepatocyte and T lymphocyte apoptosis, expression of Bax, Bcl-2, Fas, Caspase3and Caspase8in mice with blood deficiency.Methods60C57BL/6J mice were randomly divided into6groups, which were negative control group(healthy mice), model control group, low concentration Si-wu decoction group, middle concentration Si-wu decoction group, high concentration Si-wu decoction group and carnine(positive control) group, respectively. The blood deficiency model was made by intraperitoneal injection of cyclophosphamide. At the first day, model control group, low concentration Si-wu decoction group, middle concentration Si-wu decoction group, high concentration Si-wu decoction group and carnine group were injected intraperitoneally with0. lg/kg dose cyclophosphamide1times according to the weight of mice. The negative control group was injected intraperitdneally with isometric saline. Si-wu decoction was intragastric administrated to low concentration group with5g·kg-1·d-1dose, middle concentration group with10g·kg-1·d-1dose and high concentration group with20g·kg-1·d-1dose, respectively. Carnine was intragastric administrated to carnine group with0.06g·kg-1·d-1dose. Isometric saline was administrated to the negative control group and model control group. All of mice were intragastric administrated once a day for7consecutive days. At the seventh day, a volume of about0.1mL blood sample was collected from post-orbital venous plexus of each mouse after administration of1h. The peripheral hemogram was tested by full-automatic blood analyzer. At the eighth day, the mice were killed by cervical dislocation and celiotomized to take the liver, spleen and thymus. The spleen and thymus of mice were weighed to calculate the spleen index and thymus index. The liver tissue block, the size of which was about1. Ocm×1.0cm×1.0cm, was excised from right hepatic lobe. After that, the tissue was fixed with10%formaldehyde, embedded in paraffin and cut into slices successively. The hepatocyte apoptosis was tested by Tunel assay. The spleen was taken to prepare lymphocyte suspension. Then T lymphocyte was separated and purified by immunomagnetic beads. T lymphocyte was made into cells smear.Tunel assay was carried out to detect T lymphocyte apoptosis. The left hepatic lobe was stored at-80℃in order to extract total RNA and protein. Real-time fluorescence quantitative PCR technology was applied to assess mRNA expression of Bax, Bcl-2and Fas. The total RNA was turn into cDNA after reverse transcription. The reaction conditions are as follows:incubation for10min at25℃, incubation for50min at42℃, and heating95℃for5min. Then PCR was performed with cDNA. The reaction conditions were as follows:pre-denaturation for2min at95℃, denaturation for30s at95℃, annealing for30s at58℃, extension for lmin at72℃,and reaction for35cycles. After all cycles, extension for5min was added at72℃. Ct (Cycle threshold) of each sample was collected to compare the expression level of mRNA. Western blot technology was applied to assess the protein expression of Caspase3and Caspase8. Firstly, SDS-PAGE gel was prepared, including10%separation gel8mL and5%stacking gel4mL, and placed at room temperature for40min until the gel solidified. Secondly, the protein sample and electrophoresis loading buffer was mixed and denatured for10min at95℃. Then protein electrophoresis was carried out. Thirdly, the protein was transferred to PVDF membrane under65V voltage for2h. After that, the electrotransfer membrane was removed to blocking buffer and blocked for1h. Fourthly, the blocked membrane was placed into primary antibody working solution and incubated at4℃for one night. The incubated membrane was washed in1XTBST three times. Each time the membrane was washed for10min. Fifthly, the washed membrane was placed into secondary antibody working solution and incubated at room temperature for1h. The incubated membrane was washed in1×TBST three times again. Finally, the washed membrane was developed by electrochemiluminescence. After exposure and processing, the film was scanned by gel imaging system and band grayscale was detected as semi-quantitative analysis of protein. Results(1) The peripheral hemogram:Compared with model group mice, the counts of erythrocyte, leukocyte and hemoglobin in peripheral blood of middle, high concentration Si-wu decoction group and carnine group increased (P<0.05) Moreover, the counts of erythrocyte and hemoglobin in peripheral blood of low concentration Si-wu decoction group were significantly higher than that of model control group. The counts of leukocyte in peripheral blood of low concentration Si-wu decoction group did not increase significantly (P>0.05). The counts of platelet in peripheral blood of all of Si-wu decoction group were not significantly higher than that of model control group(P>0.05). The counts of platelet in peripheral blood of carnine group were even lower than that of model control group, but the difference was not significantly (P>0.05).(2) The viscera index:Compared with model control group, the spleen indices of all of Si-wu decoction group and carnine group increased significantly (P<0.05). The thymus indices of low concentration Si-wu decoction group were not significantly higher than that of model control group(P>0.05). The thymus indices of middle-dose, high-dose Si-wu decoction group and carnine group were even lower than that of model control group, but the difference was not significantly (P>0.05)(3) The percents of apoptosis:Compared with model control group, the percents of hepatocyte apoptosis of low-dose, middle-dose and high-dose Si-wu decoction group decreased significantly (P<0.05). Besides, the percents of hepatocyte apoptosis of carnine group did not decrease significantly (P>0.05).The percents of T lymphocyte apoptosis of middle-dose and high-dose Si-wu decoction group were significantly lower than that of model control group (P<0.05). Moreover, the percents of T lymphocyte apoptosis of low-dose Si-wu decoction group and carnine group did not decrease significantly (P>0.05).(4) The mRNA expression of apoptosis genes:Compared with model control group, the mRNA expression of Bax and Fas, both of genes that promote apoptosis, of high-dose Si-wu decoction group and carnine group reduced significantly (P<0.05).The mRNA expression of Bax and Fas of low-dose and middle-dose Si-wu decoction group was not significantly lower than that of model control group (P>0.05). The mRNA expression of Bcl-2, one of anti-apoptotic genes, of middle-dose, high-dose Si-wu decoction group and carnine group was significantly higher than that of model control group (P<0.05). Moreover, the mRNA expression of Bcl-2of low-dose Si-wu decoction group was not significantly higher than that of model control group (P>0.05).(5) The expression of initiator and executioner of apoptosis:Compared with model control group, the expression of Caspase3, the final executioner, and Caspase8, the key initiator, of all of Si-wu decoction group and carnine group decreased significantly (P<0.05) ConclusionSi-wu decoction increased the count of erythrocyte, leukocyte and hemoglobin in peripheral blood of blood deficiency model mice. It showed that Si-wu decoction could improve the peripheral hemogram of blood deficiency model mice. Si-wu decoction raised the spleen indices of blood deficiency model mice. It showed that Si-wu decoction could increase the quality of immune organ and enhance the immune function. Si-wu decoction decreased the percents of hepatocyte and T lymphocyte apoptosis of blood deficiency model mice. The mechanism is Si-wu decoction could down-regulate the expression of Bax and Fas, up-regulate the expression of Bcl-2and down-regulate the expression of Caspase3and Caspase8.In conclusion, the mechanism of Si-wu decoction for reinforcing liver and nourishing blood may be associated with its reduction of apoptosis. |
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