| Objective: To explore the safe dose range of TanshinoneIIA in hepatocyte and to investigate protective effect of TanshinoneIIA on TNFαand H2O2 damage models, and to search for inhibitory action of TanshinoneIIA against actived hepatic stellate cell.Methods: 1. Human hepatocyte line HL-7702 was cultured in vitro and established respectively the groups treated with different concentration of TanshinoneIIA(1㎎/L,2㎎/L,4㎎/L,8㎎/L,10㎎/L,20㎎/L,40㎎/L) and the normal control group (incubated with equal PBS), then cell survival of hepatocyte was detected by MTT assay. 2. Human hepatocyte line HL-7702 was cultured in vitro and established respectively the groups treated with different concentration of TanshinoneIIA(1㎎/L,2㎎/L,4㎎/L) and the normal control group (incubated with equal PBS),observe supernate ALT,LDH. 3. Human hepatocyte line HL-7702 was cultured in vitro and established respectively the normal control group (incubated with equal PBS) and the groups treated with different concentration of TanshinoneIIA and TNFα(TanshinoneIIA 2㎎/L group,TNFα20ug/L group and TanshinoneIIA2㎎/L+TNFα20ug/L group), then cell survival of hepatocyte was detected by MTT assay and observe supernate ALT,LDH. 4. Human hepatocyte line HL-7702 was cultured in vitro and established respectively the groups treated with different concentration of H2O2(7.5mmol/L,15mmol/L,30mmol/L,50mmol/L,100mmol/L) and the groups protected with TanshinoneIIA(2mg/L group) and the normal control group (incubated with equal PBS), then cell survival of hepatocyte was detected by MTT assay. 5. Activated rat HSC-T6 was cultured in vitro and established respectively the groups treated with different concentration of TanshinoneIIA(12.5㎎/L,25㎎/L,50㎎/L,100㎎/L) and the normal control group (incubated with equal PBS), then inhibition ratio of HSC was detected by MTT assay.Results: 1. TanshinoneIIA have no cytotoxicity to hepatocyte in certern dose range(1-4㎎/L). This dose range is safe to hepatocyte. (TanshinoneIIA 1㎎/L: 0.599±0.028; TanshinoneIIA 2㎎/L: 0.594±0.082; TanshinoneIIA 4㎎/L: 0.576±0.064), compared with the normal control group(0.602±0.080), (P>0.05); exceeding this dose rang, the characterization of cytotoxicity is lower cell survival rate(TanshinoneIIA 8㎎/L: 0.563±0.113; TanshinoneIIA 10㎎/L: 0.554±0.014; TanshinoneIIA 20㎎/L: 0.526±0.010; TanshinoneIIA 40㎎/L: 0.492±0.013), compared with the normal control group(0.602±0.080), (P<0.05). 2. Supernate ALT,LDH of the model groups of TanshinoneIIA was advanced. Compared with the normal control group(ALT: 53.02±6.27; LDH: 1158.27±340.00), TanshinoneIIA 1mg/L(ALT: 54.14±6.85; LDH: 1183.88±412.06), TanshinoneIIA 2mg/L(ALT: 53.07±6.31; LDH: 1194.24±423.69), (P>0.05); TanshinoneIIA 4mg/L(ALT: 57.84±6.97; LDH: 1316.55±457.83), (P<0.05). So, the safe dose range of TanshinoneIIA was 1-2mg/L, the concentration of TanshinoneIIA (4mg/L) was doubtfully unsafe dose. 3. Compared with the groups treated with TanshinoneIIA(2mg/L)+TNFα(20ug/L), the groups treated with TNFα(20ug/L), cell survival of hepatocyte was lower than in that(0.718±0.047 vs 0.821±034, P<0.05), Meanwhile, the results of supernate ALT,LDH was identical(ALT: 53.02±5.48 vs 49.40±5.10; LDH: 1648.43±412.57 vs 1489.15±405.73, P<0.05). 4. Compared with the normal control group(0.796±0.066), the groups treated with different concentration of H2O2(7.5mmol/L,15mmol/L,30mmol/L,50mmol/L,100mmol/L), cell survival of hepatocyte was lower(H2O27.5mmol/L: 0.521±0.043; H2O215mmol/L: 0.449±0.020; H2O230mmol/L: 0.349±0.018; H2O250mmol/L: 0.330±0.054; H2O2100mmol/L: 0.164±0.025, P<0.05), and there was a dose-dependent relationship in a certain range. Meanwhile, compared with the groups treated with TanshinoneIIA(2mg/L)+ H2O2(7.5mmol/L-15mmol/L), cell survival of hepatocyte of the groups treated with different concentration of H2O2 (H2O27.5mmol/L: 0.521±0.043 vs 0.593±0.024; H2O215mmol/L: 0.449±0.020 vs 0.455±0.014, P<0.05) was lower than in that. The descent of cell survival induced H2O2 (30-100mmol/L) could not be improved by TanshinoneIIA(H2O230mmol/L: 0.349±0.018 vs 0.358±0.017; H2O250mmol/L: 0.330±0.054 vs 0.344±0.031; H2O2100mmol/L: 0.164±0.025 vs 0.178±0.024, P>0.05). 5. TanshinoneIIA(12.5㎎/L) have no cytotoxicity to activated HSC (0.256±0.028 vs 0.258±0.029, P>0.05), proliferation of activated HSC was not inhibited. Compared with the normal control group(0.258±0.029), with the concentration increase of TanshinoneIIA(25-100㎎/L), cell proliferation of activated HSC was inhibited(TanshinoneIIA 25mg/L: 0.246±0.027; TanshinoneIIA 50mg/L: 0.166±0.029; TanshinoneIIA 100mg/L: 0.148±0.015, P<0.05).Conclusions: 1. The safe dose range of TanshinoneIIA in cultured hepatocyte is 1-2㎎/L. 2. The damage induced TNFα(20ug/L) and H2O2(7.5-15mmol/L) could be improved by TanshinoneIIA in the safe dose range. 3. Activated HSC could be inhibited by TanshinoneIIA in 25-100㎎/L. Objective: To identify the effect of anti-IGFBP7(rP1) antibody on inhibitory ratio and apoptosis in activated hepatic stellate cell.Methods: Rat activated HSC-T6 was cultured in vitro and established respectively the groups treated with different concentration of anti-IGFBPrP1 antibody(0.25mg/L,0.50mg/L,1.00mg/L) and the normal control group (incubated with equal PBS), cell-coated dishes were attained or the monoplast suspension was prepared after 14 hours, then the inhibitory ratio of activated hepatic stellate cell was detected by MTT assay. Apoptosis was detected by flow cytometry with Annexin/PI.Results: 1. The results of MTT assay showed that the experimental groups treated with anti-IGFBPrP antibody significant difference compared with the normal control group. Inhibitory ratio(anti-IGFBPrP1 antibody0.5mg/L:12.30; anti-IGFBPrP1 antibody1.0mg/L:26.90, P<0.05). 2. Apoptosis detected flow cytometry with Annexin/PI showed that compared with normal control group, the anti-IGFBPrP1 antibody(0.5mg/L) group markedly increased in the percentage of apoptosis(20.25±1.41 vs 8.84±0.78, P<0.05).Conclusions: Activated HSC could be inhibited by anti-IGFBPrP1 antibody. Apoptosis of activated HSC could be induced by anti-IGFBPrp1 antibody. So anti-IGFBPrP1 antibody could be used a new therapeutics. |
PDF Full Text Request