| Tripterygium wilfordii Hook. F. (TWH) is toxic to digestive system, reproductive system, urinary system, cardiovascular system, haematopoietic system, which relatively more incidents of human poisoning have been reported in recent years. Experiment on toxicity to haematopoietic system of TWH was few, and the mechanism is not very clear. So BMCs in vitro were chosen as research object, and effect and mechanism of TWH on cell growth and apoptosis were observed. And effect and mechanism of serum containing Chinese medical formulas on BMCs co-cultured with ethanol extraction of TWH were observed, using serum pharmacological test. Thinking of there was no national standard for TWH, contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were tested. And in order to study the toxicity, LD50 was tested.Part One Study on Contents of Tripterygium Total Lactones and Triptolide in TWH and Acute Toxicity of Ethanol Extraction of TWHMethods:A colorimetric method was applied for the assay of Tripterygium total lactones, and HPLC applied for triptolide. According to the results of preliminary experiments, mice were divided into six groups, administered ethanol extraction of TWH once by oral gavage. Toxic reactions were observed in continuous 14 days. Results:The contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were 0.0839%o-0.0859%o and 0.0507%o-0.0525%o in the experiment. The LD50 (95%confidence limit) of ethanol extraction of TWH in mice was 5.6 g/kg(4.8-6.4 g/kg).Part Two Study on Effects of Ethanol Extraction of TWH on Cell Growth and Apoptosis in Bone Marrow CellsMethods:BMCs were co-cultured in vitro with different concentrations of ethanol extraction of TWH. MTT assay was used to evaluate the growth inhibition of BMCs. The cell cycle and apoptosis rate were detertmined by FCM with PI staining and AnnexinⅤ/PI staining. The activities of caspase3, caspase8 and caspase9 were detected by spectrophotometry. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were measured by ELISA. Results:After applied with different concentrations of ethanol extraction of TWH, viability of BMCs were significantly reduced in a time-and dose-dependent manner. The IC50 were 8.742μg/mL for 24 h,5.106μg/mL for 48 h and 4.684μg/mL for 72 h. Cell cycle flow cytometry demonstrated that BMCs treated with different concentrations of ethanol extraction of TWH resulted in G1 phase arrest. Apoptosis rate were increased in BMCs treated with different concentrations of ethanol extraction of TWH tested by FCM with AnnexinⅤ/PI staining. The activities of caspase3 and caspase9, but not caspase8, were up-regulated in the experimental group compared with the control group (P<0.01), indicating ethanol extraction of TWH may induce apoptosis of BMCs through mitochondrion pathway. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were decreased in the experimental group compared with the control group (P<0.01).Part Three Study on Protective Effects of Serum Containing Chinese Medical Formulas on BMCs Co-cultured with Ethanol Extraction of TWHMethods:BMCs were co-cultured in vitro with ethanol extraction of TWH and different concentrations of serum containing LWDH, JGSQ and GP. MTT assay was used to evaluate the growth proliferation of BMCs. The cell cycle and apoptosis rate were detertmined by FCM with PI staining and AnnexinⅤ/PI staining. The activities of caspase3, caspase8 and caspase9 were detected by spectrophotometry. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were measured by ELISA. Results:After applied with different concentrations of serum containing LWDH, JGSQ and GP, viability of BMCs were significantly increased. The best was serum containing JGSQ. Cell cycle flow cytometry demonstrated that BMCs treated with different concentrations of serum containing JGSQ resulted in BMCs of Go/G1 phase decreased and BMCs of S and G2/M phase increased. Apoptosis rate were decreased in BMCs treated with different concentrations of serum containing JGSQ. The activities of caspase3 and caspase9, but not caspase8, were decreased in JGSQ group, indicating serum containing JGSQ might inhibit apoptosis of BMCs through mitochondrion pathway. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were increased in LWDH group.Conclusion:1. The contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were 0.0839‰-0.0859‰and 0.0507‰-0.0525‰in the experiment.2. The LD50 (95%confidence limit) of ethanol extraction of TWH in mice was 5.6 g/kg(4.8-6.4 g/kg). Hepatotoxic effect of ethanol extraction of TWH is a major cause of mobility in mice.3. After applied with different concentrations of ethanol extraction of TWH, viability of BMCs were significantly reduced in a time-and dose-dependent manner, resulted in G1 phase arrest. Ethanol extraction of TWH could induce the apoptosis of BMCs. It is probably mediated by activating mitochondrion pathway. Decrease of contents of cytokines in bone marrow micro-enviroment might be relevant to inhibition.4. After applied with different concentrations of serum containing LWDH, JGSQ and GP, viability of BMCs were significantly increased. The best one was serum containing JGSQ, the second serum containing LWDH, the third serum containing GP.5. Serum containing JGSQ could significantly increase viability of BMCs, promoted BMCs of G0/G1 phase entering proliferation cycle. It might inhibit apoptosis of BMCs through mitochondrion pathway. It also could increase the contents of cytokines in bone marrow micro-enviroment. |
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