| Objective: To identify the effect of IGFBP7(rP1) on both the activating of hepatic stellate cells (HSC) and the synthesis and secretion of extracellular matrix (ECM).Methods: HSC-T6 was cultured in vitro and established respectively the groups treated with IGFBP7(rP1) 10μg/L, 20μg/L, 30μg/L and the blank control group (incubated with equal phosphate buffer saline(PBS)), cell-coated dishes were attained or cytoplasmic proteins were extracted after 24 hours, then the expression of alpha- smooth muscle actin (α-SMA), Collagen I and fibronectin(FN) were detected by immunocytochemical staining and analyzed by Image- Pro image analysis system. The synthesis of Collagen I in HSC was confirmed by Western Blot again and analyzed by Bandscan 5.0 image analysis system. Simultaneously, the supernatant were collected and the content of FN was measured by enzyme-linked immunosorbent assay (ELISA).Results: (1) The results of immunocyochemical staining: Each group had positive staining in cytoplasm showing some buffy or brown particles. The results of image analysis showed that the positive staining ofα-SMA, Collagen I and FN in IGFBP7(rP1) treatment with 10μg/L group (α-SMA 11.57±0.50,Collagen I 9.74±0.34,FN 10.54±0.67), 20μg/L group (α-SMA 11.84±0.50,Collagen I 10.69±0.65,FN 13.99±0.72), 30μg/L group(α-SMA 12.90±0.37,Collagen I 11.15±0.41,FN 13.32±0.70) were significantly higher than that in the blank control group(α-SMA 7.67±0.41,Collagen I 6.82±0.47,FN 4.34±0.46)(P<0.05), and there was a dose-dependent relationship in a certain range. The positive staining ofα-SMA and Collagen I in IGFBP7(rP1) treatment with 30μg/L group were the most strong among the total groups and the positive staining of FN in IGFBP7(rP1) treatment with 20μg/L group. (2) The results of Western Blot analysis: The place of 95kD, 43kD represented Collagen I andβ-actin respectively. The groups of IGFBP7(rP1) all had obvious expression of protein straps, and the blank control group in Collagen I saw minute strap. After corrected byβ-actin, Collagen I in IGFBP7(rP1) treatment with 10μg/L group (0.7663±0.0412), 20μg/L group(0.7439±0.0720) were higher than that in the blank control group(0.6402±0.0475) (P<0.05). (3) The results of ELISA: The content of FN in IGFBP7(rP1) treatment with 10μg/L group (3.92±0.33), 20μg/L group(5.16±0.49), 30μg/L group (4.99±0.50) were significantly higher than that in the blank control group(3.40±0.19) (P<0.05).Conclusions: (1) HSC could be activated by IGFBP7(rP1) in vitro and the level of HSC activation gradually enhanced in a certain dose range of IGFBP7(rP1). (2) The synthesis and secretion of both collagen I and FN which are the principal component of ECM, were increasing by IGFBP7(rP1). Objective: To identify the effect of IGFBP7(rP1) on the DNA binding activity of nuclear factor-κB p65(NF-κB p65).Method: HSC-T6 was cultured in vitro and established respectively IGFBP7(rP1) treatment with 10μg/L group, 20μg/L group, 30μg/L group and the blank control group (incubated with equal PBS), the single cell suspension was prepared after 24 hours, The DNA binding activity of nuclear factor-κB p65(NF-κB p65) was detected by flow cytometry(FCM).Result: Flow cytometry analysis showed that there was the distinct activity of nuclear factor-κB in different group, and the percentage of positive cells of NF-κB p65 in IGFBP7(rP1) treatment with 10μg/L group(72.48±7.87), 20μg/L group(81.27±7.50), 30μg/L group (68.83±7.82) markedly increased compared with the blank control group(48.69±2.56)(P<0.05).Conclusion: IGFBP7(rP1) can enhance the DNA binding activity of NF-κB p65 in HSC. |
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