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Protection effect of kallistatin on carbon tetrachloride-induced liver fibrosis in rats via antioxidative stress.

Huang X, Wang X, Lv Y, Xu L, Lin J, Diao Y - PLoS ONE (2014)

Bottom Line: Furthermore, residual hepatic functional reserve was improved by kallistatin treatment.CCl4 induced elevation of malondialdehyde level and reduced superoxide dismutase activity in the liver, while kallistatin reduced these oxidative parameters.We also investigated the effects of kallistatin on rat primary HSC and LX-2, the human HSC cell line.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Huaqiao University, Quanzhou, China.

ABSTRACT
Prolonged inflammation and oxidative stress are emerging as key causes of pathological wound healing and the development of liver fibrosis. We have investigated the effects of recombinant human kallistatin, produced in Pichia. pastoris, on preventing carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Daily administration of kallistatin prevented development of CCl4-induced liver fibrosis, which was evidenced by histological study. In all kallistatin treated rats, activation of hepatic stellate cells (HSC) as assessed by s-smooth muscle actin staining was attenuated, TGF- β1 expression was inhibited, class I serum biomarkers associated with the process of fibrogenesis, such as hyaluronic acid, laminin, and procollagen III, were lowered, compared with that in the model control group. Furthermore, residual hepatic functional reserve was improved by kallistatin treatment. CCl4 induced elevation of malondialdehyde level and reduced superoxide dismutase activity in the liver, while kallistatin reduced these oxidative parameters. We also investigated the effects of kallistatin on rat primary HSC and LX-2, the human HSC cell line. Kallistatin scavenged H2O2-induced ROS in the LX-2 cells, and suppressed the activation of primary HSC. These results suggest recombinant human kallistatin might be a promising drug candidate for therapeutic intervention of liver fibrosis.

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Kallistatin prevents CCl4–induced liver fibrogenesis in rats.(A) Representative images of immunohistochemical staining for TGF-β1 (brown in color, arrowheaded) and α-SMA (brown in color, arrowheaded) are shown (original magnifications ×10) respectively. Expression of α-SMA around the periportal fibrotic band areas, central vein and fibrous septa were arrowed. Scale bars = 100 µm. (B) Deposition of α-SMA and TGF- β1 was quantitated by image analysis based on the immunohistochemistry results. Data are expressed as mean±SD (n = 8). ## p<0.01 vs. negative control; ** p<0.01 vs. model control group. (C) Immunoblotting analysis of α-SMA and TGF- β1 in livers from CCl4 alone or plus kallistatin treated rats. Data from immunoblotting were confirmed, showing kallistatin-dependent abrogation of α-SMA and TGF-β1 expression. Immunoblotting and immunohistochemistry results were consistent. Housekeeping proteins GAPDH are useful as loading controls for western blot and protein normalization.
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pone-0088498-g003: Kallistatin prevents CCl4–induced liver fibrogenesis in rats.(A) Representative images of immunohistochemical staining for TGF-β1 (brown in color, arrowheaded) and α-SMA (brown in color, arrowheaded) are shown (original magnifications ×10) respectively. Expression of α-SMA around the periportal fibrotic band areas, central vein and fibrous septa were arrowed. Scale bars = 100 µm. (B) Deposition of α-SMA and TGF- β1 was quantitated by image analysis based on the immunohistochemistry results. Data are expressed as mean±SD (n = 8). ## p<0.01 vs. negative control; ** p<0.01 vs. model control group. (C) Immunoblotting analysis of α-SMA and TGF- β1 in livers from CCl4 alone or plus kallistatin treated rats. Data from immunoblotting were confirmed, showing kallistatin-dependent abrogation of α-SMA and TGF-β1 expression. Immunoblotting and immunohistochemistry results were consistent. Housekeeping proteins GAPDH are useful as loading controls for western blot and protein normalization.

Mentions: Histological assessment of liver tissue has been the bedrock for diagnosis and staging of fibrosis. However, as a relatively static process of liver injury, fibrosis content alone is not enough to convey the information about the fibrogenic activity. Accordingly, tests that reflect fibrogenesis are important complements to the tests that assess only fibrosis content. As a marker of HSCs activation α-SMA is one of the sensitive indicators of the rate of fibrogenesis [2]. Although several potential non-HSC sources of myofibroblast have been identified, the concept that major myofibroblasts in hepatic fibrosis can be recognized as activated HSCs is still generally accepted [2]. Our results showed that there were hardly any α-SMA positive cells in the negative control (Figure 3). In contrast, considerable expression of α-SMA was detected in the model control group, around the periportal fibrotic band areas, central vein and fibrous septa (Figure 3). Expression of α-SMA in all the CCl4 plus kallistatin-treated groups showed a remarkable reduction, compared with the model control group, although higher than that in the negative control.


Protection effect of kallistatin on carbon tetrachloride-induced liver fibrosis in rats via antioxidative stress.

Huang X, Wang X, Lv Y, Xu L, Lin J, Diao Y - PLoS ONE (2014)

Kallistatin prevents CCl4–induced liver fibrogenesis in rats.(A) Representative images of immunohistochemical staining for TGF-β1 (brown in color, arrowheaded) and α-SMA (brown in color, arrowheaded) are shown (original magnifications ×10) respectively. Expression of α-SMA around the periportal fibrotic band areas, central vein and fibrous septa were arrowed. Scale bars = 100 µm. (B) Deposition of α-SMA and TGF- β1 was quantitated by image analysis based on the immunohistochemistry results. Data are expressed as mean±SD (n = 8). ## p<0.01 vs. negative control; ** p<0.01 vs. model control group. (C) Immunoblotting analysis of α-SMA and TGF- β1 in livers from CCl4 alone or plus kallistatin treated rats. Data from immunoblotting were confirmed, showing kallistatin-dependent abrogation of α-SMA and TGF-β1 expression. Immunoblotting and immunohistochemistry results were consistent. Housekeeping proteins GAPDH are useful as loading controls for western blot and protein normalization.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928242&req=5

pone-0088498-g003: Kallistatin prevents CCl4–induced liver fibrogenesis in rats.(A) Representative images of immunohistochemical staining for TGF-β1 (brown in color, arrowheaded) and α-SMA (brown in color, arrowheaded) are shown (original magnifications ×10) respectively. Expression of α-SMA around the periportal fibrotic band areas, central vein and fibrous septa were arrowed. Scale bars = 100 µm. (B) Deposition of α-SMA and TGF- β1 was quantitated by image analysis based on the immunohistochemistry results. Data are expressed as mean±SD (n = 8). ## p<0.01 vs. negative control; ** p<0.01 vs. model control group. (C) Immunoblotting analysis of α-SMA and TGF- β1 in livers from CCl4 alone or plus kallistatin treated rats. Data from immunoblotting were confirmed, showing kallistatin-dependent abrogation of α-SMA and TGF-β1 expression. Immunoblotting and immunohistochemistry results were consistent. Housekeeping proteins GAPDH are useful as loading controls for western blot and protein normalization.
Mentions: Histological assessment of liver tissue has been the bedrock for diagnosis and staging of fibrosis. However, as a relatively static process of liver injury, fibrosis content alone is not enough to convey the information about the fibrogenic activity. Accordingly, tests that reflect fibrogenesis are important complements to the tests that assess only fibrosis content. As a marker of HSCs activation α-SMA is one of the sensitive indicators of the rate of fibrogenesis [2]. Although several potential non-HSC sources of myofibroblast have been identified, the concept that major myofibroblasts in hepatic fibrosis can be recognized as activated HSCs is still generally accepted [2]. Our results showed that there were hardly any α-SMA positive cells in the negative control (Figure 3). In contrast, considerable expression of α-SMA was detected in the model control group, around the periportal fibrotic band areas, central vein and fibrous septa (Figure 3). Expression of α-SMA in all the CCl4 plus kallistatin-treated groups showed a remarkable reduction, compared with the model control group, although higher than that in the negative control.

Bottom Line: Furthermore, residual hepatic functional reserve was improved by kallistatin treatment.CCl4 induced elevation of malondialdehyde level and reduced superoxide dismutase activity in the liver, while kallistatin reduced these oxidative parameters.We also investigated the effects of kallistatin on rat primary HSC and LX-2, the human HSC cell line.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, Huaqiao University, Quanzhou, China.

ABSTRACT
Prolonged inflammation and oxidative stress are emerging as key causes of pathological wound healing and the development of liver fibrosis. We have investigated the effects of recombinant human kallistatin, produced in Pichia. pastoris, on preventing carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Daily administration of kallistatin prevented development of CCl4-induced liver fibrosis, which was evidenced by histological study. In all kallistatin treated rats, activation of hepatic stellate cells (HSC) as assessed by s-smooth muscle actin staining was attenuated, TGF- β1 expression was inhibited, class I serum biomarkers associated with the process of fibrogenesis, such as hyaluronic acid, laminin, and procollagen III, were lowered, compared with that in the model control group. Furthermore, residual hepatic functional reserve was improved by kallistatin treatment. CCl4 induced elevation of malondialdehyde level and reduced superoxide dismutase activity in the liver, while kallistatin reduced these oxidative parameters. We also investigated the effects of kallistatin on rat primary HSC and LX-2, the human HSC cell line. Kallistatin scavenged H2O2-induced ROS in the LX-2 cells, and suppressed the activation of primary HSC. These results suggest recombinant human kallistatin might be a promising drug candidate for therapeutic intervention of liver fibrosis.

Show MeSH
Related in: MedlinePlus