378 216 5 1 41 5 LO2 [ ] (CCl 4 ) LO2 LO2 CCl 4 (1 5 1mg/L) E(Vit E) (5mmol/L) 24h CCl 4 ( 1mmol/L 6h) LO2 (ALT) (AST) MTT CCl 4 (1mg/L) Vit E (5mmol/L) (HCA) LO2 C DNACCl 4 Vit E ALT AST (P<.5 P<.1) 1mg/L Vit E Vit E CCl 4 (P<.5 P<.1) HCA 1mg/L Vit E (P<.1) C (P<.1) (P<.1) (P<.1) (P<.1) (P<.5) CCl 4 [ ] [ ] R285.5 R575.1 [ ] A [ ] 577-742(216)5-378-6 [DOI] 1.11855/j.issn.577-742.216.5.6 Effect of Paeonia lactiflora formula granule against the damage of LO2 cells induced by carbon tetrachloride REN Yan-qing, NIU Li-ying, TIAN Yu-rou, WANG Xin-guo * Pharmacological Lab of Traditional Chinese Medicine, Hebei University of Chinese Medicine, Hebei TCM Formula Granule Engineering Technology Research Center, TCM Formula Granule Research Center of Hebei Province University, Shijiazhuang 591, China * Corresponding author, E-mail: wangxinguozy@163.com This work was supported by the Natural Science Foundation of Hebei Province (H21542326), and the Science and Technology Research Project in Colleges and Universities of Hebei Province (ZD2151) [Abstract] Objective To investigate the effect of Paeonia lactiflora formula granule (PLFG) against the damage of LO2 cells induced by carbon tetrachloride (CCl 4 ) and explore its mechanism. Methods LO2 cells were divided into control group, CCl 4 model group, PLFG (1, 5, 1mg/L) groups and Vit E (5mmol/L) group. LO2 cells of PLFG groups and Vit E group were pretreated by drugs for 24 hours, then except control group, other groups were treated by 1mmol/L CCl 4 for 6 hours, which induced hepatic cell damage, the alanine aminotransferase (ALT) and aspartate transaminase (AST) content of the cell culture supernatant were measured, the survival rates of LO2 cells were analyzed by MTT method. LO2 cells were divided into control group, CCl 4 model group, PLFG (1mg/L) group and Vit E (5mmol/L) group, cell loss, changes in nuclear size and morphology, DNA content, mitochondrial membrane potential (MMP), cell permeability changes, and cytochrome C release were measured simultaneously by high content analysis (HCA). Results Compared with CCl 4 model group, PLFG and Vit E pretreatment could obviously decrease thelevelofaltandast(p<.5 or P<.1), especially in the 1mg/L PLFG group and Vit E group. PLFG (1, 5, 1mg/L) and Vit E could also significantly weaken the decrease of LO2 cell viability, which were induced by CCl 4 treatment, the results showed in dose-dependent to some extents (P<.5 or P<.1). Meanwhile, treatment with 1mg/L PLFG and Vit E could significantly [ ] (H21542326) (ZD2151) [ ] [ ] 591 ( ) [ ] E-mail wangxinguozy@163.com
Med J Chin PLA, Vol. 41, No. 5, May 1, 216 379 increase the level of MMP (P<.1), prevent cytochrome C release (P<.1), decrease the membrane permeability (P<.1), increase nuclear size (P<.1), decrease total nuclear intensity (P<.1) and increase the cell count (P<.5). Conclusion PLFG may act an obvious protective effect against the liver injury induced by CCl 4, and it might be due to protecting the integrity of mitochondria membrane, decreasing cell membrane permeability and inhibiting the apoptosis of LO2 cells. [Key words] Paeonia lactiflora formula granule; drug-induced liver injury; membrane potential, mitochondrial; cell membrane permeability; apoptosis; high content analysis [1-2] Paeonia lactiflora Pall. [3-4] UPLC-Q-TOF-MS [5] CCl 4 [6] [7] [8-9] LO2 CCl 4 (high content analysis HCA) 1 1.1 ( 1g 1g HPLC 1g 25.8mg 1411511 1) DMEM.22 m ( 11736-21438) E(Vit E) Sigma CCl 4 ( ) DMEM Gibco ( 1148777) (FBS) Gibco ( 142768) (MTT) Amresco (ALT) ( 21517) (AST) ( 2156) (Mito Tracker QC217368) Hoechst 33342( 62249) (UU1 QC217369) Cytochrome C ( PI1912662A) DyLight 649 IgG ( PB195113) ( PH26495) ( PF19225) ( PF248) Thermo Fisher Scientific 2 4 6 8 1 t/min A 2 4 6 8 1 t/min 1 (A) (B)HPLC Fig. 1 HPLC chromatogram of Paeonia lactiflora formula granule (PLFG) (A) and paeoniflorin (B) B 1.2 ( ) MCO-15AC CO 2 ( SANYO ) TGL-16 ( ) DMI 3 B ( Leica ) Multiskan FC ( Thermo Fisher Scientific ) VTI7 ( Thermo Fisher Scientific ) 1.3 (LO2) 1% FBS DMEM 37 5% CO 2 2~3d.25% 1:3 1.4 MTT
38 216 5 1 41 5 1.4.1 LO2 LO2 1 1 5 /ml 96 (.1 1 1 mg/l) 24h 24h DMEM l/ MTT 2 l/ 4h DMSO 57nm (A) =A /A % 1.4.2 CCl 4 LO2 CCl 4 (1mmol/L) (1 5 1mg/L +1mmol/L CCl 4 ) Vit E (5mmol/L Vit E [1] +1mmol/L CCl 4 ) 1 5 1mg/L Vit E( 5mmol/L) 24h 1mmol/L CCl 4 (.1%) 6h 1.4.1 A 1.5 ALT AST 1.4.2 CCl 4 6h ALT AST 1.6 HCA C DNA CCl 4 (1mmol/L) (1mg/L +1mmol/L CCl 4 ) Vit E (5mmol/L Vit E+1mmol/L CCl 4 ) 96 5 1 4 /ml 3 / 1mg/L 5mmol/L Vit E 24h CCl 4 5 l ( ) 37 3min 4% ( l/ ) 2min ( l/ ) 3 C (1:4)5 l/ 6min Hoechst 33342 DyLight TM 649 IgG(1:5) 5 l/ 6min 1 16 16 / 552/576nm C / 646/674nm / 491/59nm Hoechst 33342 / 35/461nm 1.7 SPSS 19. x±s SNK-q P<.5 2 2.1 CCl 4 LO2.1~mg/L LO2 (P>.5) LO2 ( 2A) CCl 4 LO2 (P<.1) 63.9% Vit E (P<.5 P<.1) 1 5 1mg/L 69.4% 77.8% 8.6% ( 2B) 2.2 CCl 4 LO2 ALT AST CCl 4 ALT AST Cell viability (%) 8 6 4 2 Control.1 1 1 PLFG/(mg/L) A Control Model 1 5 1 Vit E PLFG/(mg/L) 2 (A) CCl 4 (B)LO2 (x±s, n=6) Fig. 2 Effects of Paeonia lactiflora formula granule (PLFG) on survival rates of LO2 cells (A) and of damaged LO2 cells induced by CCl 4 (B) (x±s, n=6) P<.1 compared with control group; (2)P<.5, P<.1 compared with model group Cell viability (%) 8 6 4 2 (2) B
Med J Chin PLA, Vol. 41, No. 5, May 1, 216 381 (P<.1) CCl 4 Vit E ALT (P<.5 P<.1) 1mg/L AST (P<.5 P<.1 1) 1mg/L Vit E ALT AST 1 CCl 4 LO2 ALT AST (U/L x±s, n=6) Tab.1 Effects of PLFG on levels of ALT and AST in damaged LO2 cells induced by CCl 4 (U/L, x±s, n=6) Group ALT ASTr Control 15.5±1.87 13.3±1.86 CCl 4 model 33.8±3.31 23.7±4.18 PLFG (mg/l) 1 29.3±2.8 (2) 22.7±5.32 5 24.2±4.36 18.7±3.27 (2) 1 22.3±2.66 16.2±2.64 Vit E (5mmol/L) 21.7±3.72 16.±3.52 PLFG. Paeonia lactiflora formula granule; P<.1 compared with control group; (2)P<.5, P<.1 compared with CCl 4 model group 2.3 CCl 4 LO2 C DNA HCA C CCl 4 (P<.1) C (P<.1) (P<.1) (P<.1) CCl 4 Vit E (P<.1) C (P<.1) (P<.1) Vit E (P<.1) (P<.1) (P<.5) 1mg/L (P<.5 P<.1) C (P<.1) 1mg/L 5mmol/L Vit E ( 3) 3 [11] CCl 4 [12-13] [14] CCl 4 P45 CCl 3 [12,15] ALT AST CCl 4 LO2 ALT AST CCl 4 ALT AST LO2 CCl 4 HCA HCA [16] HCA [17] HCA [18] [19] [2] [21-22] C C C - caspase [23] C
382 216 5 1 41 5 Membrane potential Cytochrome C Permeability Nuclei Merge Control Model PLFG Vit E 4 Mitochondria membrane potential 4 Cytochrome C 3 2 3 2 3 25 2 15 5 Membrane permeability 4 3 2 Total nuclear intensity 2 Nuclear size 2 Cell loss 18 18 (2) 16 (2) 16 14 14 12 12 1 8 8 6 3 CCl 4 LO2 C DNA (n=3) Fig.3 Effects of PLFG on mitochondria membrane potential, cytochrome C, membrane permeability, DNA content, nuclear size, andcelllossinccl 4 -induced LO2 cell damage by HCA ( ) (n=3) P<.1 compared with control group; (2)P<.5, P<.1 compared with model group Cell count
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