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Tanshinone IIA and anti- IGFBP7 ( RP1 ) antibody in vitro study of liver protection mechanism

Author: ZhangDongMei
Tutor: LiuLiXin
School: Shanxi Medical
Course: Internal Medicine
Keywords: Tanshinone IIA Tumor necrosis factor alpha ( TNF - alpha ) H2O2 Hepatocytes Hepatic stellate cells ( HSC ) Anti - insulin-like growth factor binding protein 7 ( associated protein 1 ) antibody Hepatic stellate cells Apoptosis
CLC: R285.5
Type: Master's thesis
Year: 2008
Downloads: 92
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Abstract


Objective: To explore Tanshinone IIA on cultured liver cell line HL-7702 safe use of dose and observation of Tanshinone IIA on tumor necrosis factor-α (tumor necrosis factorα, TNFα) and hydrogen peroxide (H2O2) in vitro through liver cell injury role; explore tanshinone IIA on proliferation of activated hepatic stellate cells. Method: 1. Choose vitro cultured human liver cell line (HL-7702) as the research object, set up tanshinone IIA (1 mg / L, 2 mg / L, 4 mg / L, 8 mg / L, 10 mg / L, 20 mg / L, 40 mg / L) treatment group and the control group (add equal amount of phosphate-buffered saline (PBS)), liver cell viability by MTT assay. 2 Select vitro cultured human liver cell line (HL-7702) as the research object, the establishment of tanshinone IIA (1 mg / L, 2 mg / L, 4 mg / L) treatment group and normal control group (add equal amount of PBS) , ALT and LDH in the detection of cell culture supernatant. 3 Select vitro cultured human liver cell line (HL-7702) as the research object, set up the TNFα (20ug / L) treated control group of tanshinone IIA (2 mg / L), tanshinone IIA (2 mg / L) TNF-alpha ( 20ug / L) treatment group and normal control group (adding an equal amount of PBS), liver cell viability, ALT and LDH in the cell culture supernatant was measured by MTT assay. Select vitro cultured human liver cell line (HL-7702) as the research object, namely the establishment of H2O2 (7.5 mmol / L and 15 mmol / L, 30 mmol / L, 50 mmol / L, 100 mmol / L) treatment groups, tanshinone IIA (2 mg / L) control group, H2O2 (7.5 mmol / L, 15 mmol / L, 30 mmol / L, 50 mmol / L, 100 mmol / L) (mg / L), tanshinone IIA treatment group and normal control group (add equal amount of PBS) liver cell viability by MTT assay. 5 Select vitro cultured rat activated hepatic stellate cells (HSC-T6) as the research object, the establishment of tanshinone IIA (12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L) treatment and normal control group (add equal amount of PBS), hepatic stellate cell viability by MTT assay. Results: 1.MTT assay results showed that the absorbance of the normal control group (0.602 ± 0.080), Tan IIA group (tanshinone IIA1mg / L group: 0.599 ± 0.028; tanshinone IIA2mg / L group: 0.594 ± 0.082; tanshinone IIA4mg / L group: 0.576 ± 0.064) compared with the control group (P gt; 0.05), Tanshinone IIA of liver cells in a dose range of 1-4 mg / L non-toxic, safe dose range; With Tanshinone IIA concentration increased (8 -40mg / L) significant inhibition of cell proliferation (tanshinone IIA8mg, / L group: 0.563 ± 0.113; groups of tanshinone IIA10mg, / L: 0.554 ± 0.014; groups of tanshinone IIA20mg, / L: 0.526 ± 0.010; groups of tanshinone IIA40mg, / L: 0.492 ± 0.013) compared with the control group (0.602 ± 0.080) (P lt; 0.05), and the higher the concentration, the inhibition became more apparent, there is a dose-response relationship, the safe range of more than dose toxic to cells. Lai's method Determination of cell culture supernatant ALT and LDH content was increased, of Tan IIA group ALT and LDH levels than normal control group, which tanshinone IIA1mg / L group (ALT: 54.14 ± 6.85; LDH: 1183.88 ± 412.06), 2 mg / L group (ALT: 53.07 ± 6.31; LDH: 1194.24 ± 423.69) and normal control group (ALT: 53.02 ± 6.27; LDH: 1158.27 ± 340.00) no significant difference (P gt; 0.05); tanshinone IIA4mg / L group (ALT: 57.84 ± 6.97; LDH: 1316.55 ± 457.83) and normal control group (ALT: 53.02 ± 6.27; LDH: 1158.27 ± 340.00) were significantly different (P lt; 0.05) Tanshinone IIA in 1-2mg / L as a safe dose of 4 mg / L suspicious as unsafe dose. Determined by 3.MTT method results show that TNF-alpha (20ug / L) treatment group (0.718 ± 0.047) and tanshinone IIA (2 mg / L) TNF-alpha (20ug / L) compared to the treatment group (0.821 ± 0.034) survival rate of liver cells ( P lt; 0.05); while Lai's method for the determination of ALT and LDH content of cell culture supernatant, TNFa (20ug / L) treated with tanshinone IIA (2 mg / L) TNF-alpha (20ug / L) compared to the treatment group ( ALT: 53.02 ± 5.48 vs 49.40 ± 5.10; LDH: 1648.43 ± 412.57 vs 1489.15 ± 405.73, P lt; 0.05) increased. The assay results display 4.MTT H2O2 (750-100 mmol / L) treatment group and the normal control group (0.796 ± 0.066) compared to liver cell viability decreased significantly (H2O27.5mmol / L group: 0.521 ± 0.043; H2O215mmol / L group: 0.449 ± 0.020; H2O230mmol / L group: 0.349 ± 0.018; H2O250mmol / L group: 0.330 ± 0.054; H2O2100mmol / L group: 0.164 ± 0.025, P lt; 0.05), and a dose-dependent effect. MTT assay results showed that H2O2 (7.5-15 mmol / L) treated with tanshinone IIA (2 mg / L) H 2 O 2 (7.5-15 mmol / L) compared to the treatment group, Tanshinone IIA can improve liver cell damage caused by H2O2 ( H2O27.5mmol / L group: 0.521 ± 0.043 vs 0.593 ± 0.024; H2O215mmol / L group: 0.449 ± 0.020 vs 0.455 ± 0.014, P lt; 0.05); H2O2 (30-100 mmol / L) treated with tanshinone IIA (2 mg / L) H 2 O 2 (30-100 mmol / L) treatment group compared Tanshinone IIA not improve liver cell injury caused by H2O2 (H2O230mmol / L group: 0.349 ± 0.018 vs 0.358 ± 0.017; H2O250mmol / L group: 0.330 ± 0.054 vs 0.344 ± 0.031; H2O2100mmol / L group: 0.164 ± 0.025 vs 0.178 ± 0.024, P gt; 0.05) 5.MTT assay results show tanshinone IIA12.5mg / L treatment group compared with the control group no significant difference in cell viability (0.256 ± 0.028 vs 0.258 ± 0.029, P gt; 0.05), Tanshinone IIA activation at this dose no inhibitory effect on hepatic stellate cells; With the increase of the concentration (25-100mg / L), with the normal control group (0.258 ± 0.029) compared to a significant inhibition of cell proliferation (tanshinone IIA25mg / L group: 0.246 ± 0.027; tanshinone IIA50mg / L Group: 0.166 ± 0.029; tanshinone IIA100mg / L group: 0.148 ± 0.015, P lt; 0.05), and the higher the concentration, the inhibition of the more obvious, there is a dose-response relationship, in this dose range, the cell significantly inhibition. Conclusions: 1. Tanshinone IIA on cultured liver cell line (HL-7702) the safe use of dose range of 1-2 mg / L. Tanshinone IIA safe dose range can reduce TNF-alpha (20ug / L) and hydrogen peroxide (7.5 - 15 mmol / L) in vitro through damage to the liver cells. Tanshinone IIA in the dose range of 25-100 mg / L can effectively inhibit the proliferation of activated hepatic stellate cells. Objective: To investigate the anti-insulin-like growth factor binding protein 7 (associated protein 1) (IGFBP7/IGFBPrP1) antibody activated hepatic stellate cell proliferation inhibition and apoptosis. Methods: cultured rat activated hepatic stellate cell line (HSC-T6) as the research object, set up anti-IGFBP7 (RP1) antibody-treated group (0.25 mg / L, 0.50mg / L, 1.00mg / L) normal control group (adding an equal amount of PBS), intervention factors deal with 14h, MTT assay was used to detect anti-IGFBP7 (RP1) antibody on the activation of hepatic stellate cell proliferation inhibition; flow cytometry instrument Annexin-at the same time under the same processing factors Ⅴ / PI double staining to detect the activation of hepatic stellate cell apoptosis incidence. Results: 1.MTT results showed: no significant difference in cell activity of the antibody anti-IGFBP7 (RP1) 0.25 mg / L group and the normal control group, at this dose no inhibitory effect on hepatic stellate cells; anti-IGFBP7 (RP1) antibody 0.50 (RP1) mg / L group and anti-IGFBP7 antibody 1.00mg / L group, can significantly inhibit the activation of hepatic stellate cell proliferation inhibition rate (RP1) antibody (anti-IGFBP7 0.50mg / L Group: 12.30%; anti-IGFBP7 (rP1 ) antibody 1.0mg / L group: 26.90%, P lt; 0.05). Flow cytometry instrument Annexin Ⅴ / PI double staining: anti-IGFBP7 (RP1) antibody 0.5mg / L group activated hepatic stellate cells apoptosis was significantly increased compared with the normal control group (20.25 ± 1.41 vs 8.84 ± 0.78, P lt; 0.05). Conclusion: The anti-IGFBP7 (RP1) antibody can inhibit the activation of hepatic stellate cell proliferation and induce apoptosis, suggesting that anti-IGFBP7 (RP1) antibody may serve as a new drug for treating liver fibrosis.

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