HfPS1 protects the ethanolinduced injury to IEC6 cells

MTS assays of IEC-6 cells after exposure to 100-1000 mg/ml Hf-PS or Hf-PS-1 showed no cytotoxicity (Fig. 11.9). Furthermore, Hf-PS-1, but not Hf-PS, appeared to protect against ethanol-induced cytotoxicity (Fig. 11.10A). When cell viability assays were performed on cells after exposure to 0-500 mg/ml Hf-PS-1 for 3-24 h followed by exposure to 5% ethanol for 1 h, Hf-PS-1 was found to have a concentration- and time-dependent protective effect (Fig. 11.10B). Results of MTS assays showed that ethanol treatment induced the death of the IEC-6 cells and that pretreatment with Hf-PS-1 for 6-24 h counteracted this effect. Morphological studies also confirmed protection against ethanol toxicity by Hf-PS-1. Ethanol treatment induced cell shrinkage and decreased the number of viable cells (Fig. 11.11), but Hf-PS-1 pretreatment inhibited this damage. These results indicate that Hf-PS-1 pretreatment is protective against ethanol-induced cell death.

Control 100 200 300 500 1000

FIGURE 11.9 Effect of Hf-PS and Hf-PS-1 on cell viability. IEC-6 cells were incubated with Hf-PS or Hf-PS-1 at the indicated concentrations for 24 h. The results indicated mean ± SD in three independent experiments. Different alphabets are significant values among the group by Duncan's multiple range test.

Control 100 200 300 500 1000

FIGURE 11.9 Effect of Hf-PS and Hf-PS-1 on cell viability. IEC-6 cells were incubated with Hf-PS or Hf-PS-1 at the indicated concentrations for 24 h. The results indicated mean ± SD in three independent experiments. Different alphabets are significant values among the group by Duncan's multiple range test.

Si 100

■ Treated 3 h 0 Treated 12 h □ Treated 6 h H Treated 24 h aaa

a

FIGURE 11.10 Protective effect of Hf-PS-1 on ethanol-induced damage. (A, B) Cells were incubated with SFM or Hf-PS-1 at the indicated concentrations and times and treated ethanol for 1 h. Cell viability was measured with MTS assay kit as manufacturer's instruction. Data were represented % of control (100%). Values are the mean ± SD. Different alphabets are significant values among the group by Duncan's multiple range test. (C) Cell morphology (400x). 'a' control; 'b' treated only ethanol; 'c' treated ethanol (5%, 1 h) after Hf-PS-1 (500 mg/ml) pretreatment.

IP:IGF-IR

IB:PY99

IB:IGF-IR

IB:IGFIR

IB:IGFIR

I IB:PY99

I IB:PY99

IP:Shc

IP:Shc c

FIGURE 11.11 Effect of ethanol and Hf-PS-1 on IGF-IR and Shc phosphorylation in IEC-6 cells. Cells were pretreated with Hf-PS-1 (500 mg/ml) and ethanol (5%) or with ethanol only (5%) after incubation with SFM for 12 h. Cell lysates were immunoprecipitated with anti-IGF-IR, Shc antibody and the immune complexes were then analyzed by electrophoresis and immunoblotting with anti-phosphotyrosine, or anti-IGF-IR, Shc, Grb2 antibody. This gel photograph is representative of three experiments. Values are the mean ± SD. Different alphabets are significant values among the group by Duncan's multiple range test.

IB:IGF-IR

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