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Up-regulation of nerve growth factor in cholestatic livers and its hepatoprotective role against oxidative stress.

Tsai MS, Lin YC, Sun CK, Huang SC, Lee PH, Kao YH - PLoS ONE (2014)

Bottom Line: Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice.Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG.In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, E-DA Hospital, Kaohsiung, Taiwan; The School of Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan.

ABSTRACT
The role of nerve growth factor (NGF) in liver injury induced by bile duct ligation (BDL) remains elusive. This study aimed to investigate the relationship between inflammation and hepatic NGF expression, to explore the possible upstream molecules up-regulating NGF, and to determine whether NGF could protect hepatocytes from oxidative liver injury. Biochemical and molecular detection showed that NGF was up-regulated in cholestatic livers and plasma, and well correlated with systemic and hepatic inflammation. Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice. Immunohistochemistry showed that the up-regulated NGF was mainly localized in parenchymal hepatocytes. In vitro mechanistic study further demonstrated that TGF-β1 up-regulated NGF expression in clone-9 and primary rat hepatocytes. Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG. TUNEL staining confirmed the involvement of anti-apoptosis in the NGF-exhibited hepatoprotection. Moreover, NGF potently induced Akt phosphorylation and increased Bcl-2 to Bax ratios, whereas these molecular alterations by NGF were only seen in the H2O2-, but not TGF-β1-treated hepatocytes. In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases.

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Anti-apoptotic effect of exogenous NGF pretreatment on TGF-β1-induced and oxidative cell death of primary hepatocytes.Primary rat hepatocytes grown on collagen-coated chamber slides were treated with recombinant NGF at 20 ng/mL for 24 hrs and exposed to either TGF-β1 at 50 ng/mL or H2O2 at 500 µM for another 24 hrs. After treatments, cells were fixed with paraformaldehyde and subjected to in situ TUNEL staining for cellular apoptotic events. (A) Representative TUNEL staining images are shown (Bars  = 10 µm). Positive control (PC) was performed by treating cells with DNase I. (B) Nuclear TUNEL-positive signals were quantified by measuring 20 high-power fields per group and the positivity are shown in mean±SEM. * indicates P<0.05 as compared with negative control (NC) or between groups.
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pone-0112113-g007: Anti-apoptotic effect of exogenous NGF pretreatment on TGF-β1-induced and oxidative cell death of primary hepatocytes.Primary rat hepatocytes grown on collagen-coated chamber slides were treated with recombinant NGF at 20 ng/mL for 24 hrs and exposed to either TGF-β1 at 50 ng/mL or H2O2 at 500 µM for another 24 hrs. After treatments, cells were fixed with paraformaldehyde and subjected to in situ TUNEL staining for cellular apoptotic events. (A) Representative TUNEL staining images are shown (Bars  = 10 µm). Positive control (PC) was performed by treating cells with DNase I. (B) Nuclear TUNEL-positive signals were quantified by measuring 20 high-power fields per group and the positivity are shown in mean±SEM. * indicates P<0.05 as compared with negative control (NC) or between groups.

Mentions: To confirm the involvement of anti-apoptogenesis in the NGF-exhibited hepatoprotection against oxidative stress, in situ TUNEL detection was used to quantify the cellular apoptotic events under in vitro hepatotoxic injury. The TUNEL staining (Figure 7A) and quantitative results (Figure 7B) clearly indicated that both TGF-β1 and H2O2 insults significantly increased nuclear apoptotic signals in treated primary rat hepatocytes, while NGF pretreatment effectively prevented the increased hepatocytic apoptosis induced by both agents, supporting that NGF may functionally protect hepatocytes against TGF-β1- and oxidation-induced hepatocellular apoptosis.


Up-regulation of nerve growth factor in cholestatic livers and its hepatoprotective role against oxidative stress.

Tsai MS, Lin YC, Sun CK, Huang SC, Lee PH, Kao YH - PLoS ONE (2014)

Anti-apoptotic effect of exogenous NGF pretreatment on TGF-β1-induced and oxidative cell death of primary hepatocytes.Primary rat hepatocytes grown on collagen-coated chamber slides were treated with recombinant NGF at 20 ng/mL for 24 hrs and exposed to either TGF-β1 at 50 ng/mL or H2O2 at 500 µM for another 24 hrs. After treatments, cells were fixed with paraformaldehyde and subjected to in situ TUNEL staining for cellular apoptotic events. (A) Representative TUNEL staining images are shown (Bars  = 10 µm). Positive control (PC) was performed by treating cells with DNase I. (B) Nuclear TUNEL-positive signals were quantified by measuring 20 high-power fields per group and the positivity are shown in mean±SEM. * indicates P<0.05 as compared with negative control (NC) or between groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232375&req=5

pone-0112113-g007: Anti-apoptotic effect of exogenous NGF pretreatment on TGF-β1-induced and oxidative cell death of primary hepatocytes.Primary rat hepatocytes grown on collagen-coated chamber slides were treated with recombinant NGF at 20 ng/mL for 24 hrs and exposed to either TGF-β1 at 50 ng/mL or H2O2 at 500 µM for another 24 hrs. After treatments, cells were fixed with paraformaldehyde and subjected to in situ TUNEL staining for cellular apoptotic events. (A) Representative TUNEL staining images are shown (Bars  = 10 µm). Positive control (PC) was performed by treating cells with DNase I. (B) Nuclear TUNEL-positive signals were quantified by measuring 20 high-power fields per group and the positivity are shown in mean±SEM. * indicates P<0.05 as compared with negative control (NC) or between groups.
Mentions: To confirm the involvement of anti-apoptogenesis in the NGF-exhibited hepatoprotection against oxidative stress, in situ TUNEL detection was used to quantify the cellular apoptotic events under in vitro hepatotoxic injury. The TUNEL staining (Figure 7A) and quantitative results (Figure 7B) clearly indicated that both TGF-β1 and H2O2 insults significantly increased nuclear apoptotic signals in treated primary rat hepatocytes, while NGF pretreatment effectively prevented the increased hepatocytic apoptosis induced by both agents, supporting that NGF may functionally protect hepatocytes against TGF-β1- and oxidation-induced hepatocellular apoptosis.

Bottom Line: Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice.Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG.In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, E-DA Hospital, Kaohsiung, Taiwan; The School of Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan.

ABSTRACT
The role of nerve growth factor (NGF) in liver injury induced by bile duct ligation (BDL) remains elusive. This study aimed to investigate the relationship between inflammation and hepatic NGF expression, to explore the possible upstream molecules up-regulating NGF, and to determine whether NGF could protect hepatocytes from oxidative liver injury. Biochemical and molecular detection showed that NGF was up-regulated in cholestatic livers and plasma, and well correlated with systemic and hepatic inflammation. Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice. Immunohistochemistry showed that the up-regulated NGF was mainly localized in parenchymal hepatocytes. In vitro mechanistic study further demonstrated that TGF-β1 up-regulated NGF expression in clone-9 and primary rat hepatocytes. Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG. TUNEL staining confirmed the involvement of anti-apoptosis in the NGF-exhibited hepatoprotection. Moreover, NGF potently induced Akt phosphorylation and increased Bcl-2 to Bax ratios, whereas these molecular alterations by NGF were only seen in the H2O2-, but not TGF-β1-treated hepatocytes. In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases.

Show MeSH
Related in: MedlinePlus