588 HepG2 2 2 53002 E-mail huan_chaos@ hotmail. com 2 3602 HepG2 MTT PI HepG2 Western Blot HepG2 Bid Bcl-2 HepG2 HepG2 G0 /G Bid Bcl-2 Bax Bid Bcl-2 Bax HepG2 HepG2 R 735. 7 A 0253-4304 204-588-05 DOI 0. 675 /j. issn. 0253-4304. 204.. 24 Matrine Induces Apoptosis of HepG2 Cells via Mitochondrial Apoptotic Pathway ZHOU Huan XU Ming-ying 2 LEI Yu 2 LIANG Kai GUO Qing HU Tian-hui 2 Department of Pharmacy Affiliated Tumor Hospital of Guangxi Medical University Nanning 53002 China 2 Cancer Research Center School of Medicine Xiamen University Xiamen 3602 China Abstract Objective To explore the mechanism of matrine inducing apoptosis of HepG2 cells via mitochondrial apoptotic pathway. Methods MTT assay PI staining flow cytometry were used to evaluate the anti-proliferation cell cycle regulation and inducing apoptosis effects of matrine on HepG2 cells. Western Blot assay was used to detect the expression levels of anti-apoptotic protein Bid and Bcl-2 in HepG2 cells. Results Matrine could inhibit the proliferation and induce the apoptosis of HepG2 cells in a time- and dose-dependent manner and the cell cycle of HepG2 was arrested in G0/G phase. Apoptosis of HepG2 cells induced by matrine was associated with collapse of mitochondrial membrane potential which made the Bid and Bcl-2 expression levels down-regulated but the Bax expression level up-regulated. Conclusion Matrine induces the apoptosis of human hepatoma cell line HepG2 via the mitochondrial apoptotic pathway by regulating the expression levels of Bid Bcl-2 and Bax proteins. Key words Matrine HepG2 Mitochondrial apoptotic pathway 04. 9 /0 3-4 2 HepG2 872284 200J065 988 ~ 973 ~ E-mail thu@ xmu. edu. cn
204 36 589 24 h. 0 mg /ml 0 2 24 36 48 h EDTA. HepG2 Annexin V-FITC /PI Bid Bcl-2 Bcl-xl Bax 3 0 5 Santa Cruz Biotech DMEM 500 μl Binding buffer Gibco PAA 5 μl Annexin V-FITC 5 μl PI MTT Sigma Chemical Annexin-FITC 5 min h 488 nm 50 mg /ml 0. 25 g 50 ml ddh 2 O - 20. 6 24 h 0 0. 25 0. 5. 2 HepG2 DMEM. 0. 5 2. 0 mg /ml 24 h 37 5% CO 2 DMEM 0 μg /ml 0% 00 U /ml 00 U /ml 23 37 5 min 37 80% ~ 90% PBS 60 min 0. 5 ml EDTA 0. 25%. 7 Western Blot 4x Loading buffer 00 5 min. 3 MTT 0 5 /ml 96 marker 4 24 h TBS-T 0 min 0. 25 0. 5. 0. 5 0. 25% 60 min 2. 0 mg /ml 2 24 48 h 20 μl ECL 3 min 5 mg /ml MTT 4 h MTT 50 μl DMSO 0 min x ± s 570 nm P < 0. 05 OD 630 nm 2. 4 PI 2. MTT HepG2 24 h HepG2 0. 25 0. 5. 0 mg /ml 24 h P < 0. 05 EDTA HepG2 70% - 20 2 h - DNase RNase HepG2 x ± s % 37 30 min 5 μl PI 30 min 488 nm. 5 Annexin V-FITC /PI 24 h 0 0. 25 0. 5. 0. 5 2. 0 mg /ml. 8 GraphPad Prism 5. 0 mol /L 2 h 24 h 48 h 0. 25 8. 23 ±. 57 29. 3 ± 0. 87 * 3. 79 ± 2. 67 * 0. 5 27. 78 ± 2. 6 * 36. 27 ± 2. 38 * 4. 65 ± 2. 42 *. 0 33. 47 ± 2. 49 * 53. 9 ± 3. 53 * 52. 98 ± 3. 5 *. 5 42. 23 ± 2. 03 * 59. 25 ± 3. 26 * 63. 5 ± 4. 0 * 2. 0 58. 38 ± 5. 6 * 70. 69 ± 4. 33 * 80. 2 ± 4. 57 * * P < 0. 05
590 2. 2 HepG2 G0 /G 0. 25 0. 5. 0 mol /L HepG2 G0 /G 7. 97 ± 2. 54 % 74. 487 ± 0. 64 % 77. 223 ± 0. 4 % 66. 772 ± 0. 65 % G0 /G F = 45. 579 P = 0. 000 HepG2 G0 /G P < 0. 05 2A HepG2 Western Blot G0 /G p27 p27 2B 2 2. 3 HepG2 0. 5. 0 HepG2. 5 2. 0 mol /l 24 h HepG2 26. 20 ±. 8 % 34. 063 ± 2. 43 % 22. 47 ± 3. 42 % 33. 654 ±. 33 % 43. 833 ± 2. 63 % 63. 026 ± 5. 6 % F = 4. 665 P < 0. 00 HepG2. 67 ± 0. 57 % P < 0. 05 F = 22. 28 P < 0. 003B P < 0. 05 3A. 0 mol /l 24 36 48 h HepG2 45. 453 ± 2. 92 % 5. 59 ± 0. 75 % 3 HepG2 2. 4 HepG2 2. 233 ± 3. 9 30. 074 ± 4. 82 38. 573 ± 5. 89. 0 mg /L 2. 267 ± 0. 72 HepG2 4. 0 F = 36. 529 P < 0. 00. 5 2. 0 mol /l P < 0. 05
204 36 59 4 4 HepG2 2. 5 HepG2 HepG2 HepG2 p27 Western Blot G S p27 Bid Bcl-2 3 Bax HepG2 G0 /G p27 PARP Bcl-xl AIF 5 5 HepG2 HepG2 ψm 3 HepG2 MMP MMP Bcl-2 MMP BxPC-3 6 - PANC- SMMC-772 Bcl-2 Bcl-XL Bax Bak 8 7 Bik Bad Bid Akt ERK /2 9 BH3 0 c-myc p53 Bcl-2 7 AIF - 2 Western Blot HepG2 24 h p27 p27 G0 /G ψm MMP ψm ATP 4-5 Rhodamine 23 HepG2 - DNA 8 G0 /G S HepG2 G0 /G Bid
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