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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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Related in: MedlinePlus

Increased serum NGF levels and intrahepatic NGF up-regulation in mice receiving bile duct ligation (BDL) surgery.Normal mice (n = 6) and those receiving BDL surgery were sacrificed at 7 or 14 post-operative days (POD7, n = 4) or (POD14, n = 4). (A) Serum levels of NGF and inflammatory cytokines, including TNF-α, IL-6 and TGF-β1 were determined by ELISA detection. (B) Liver tissue extracts were collected for mRNA isolation and subjected to RT-qPCR analysis. (C) Contents of NGF and TGF-β1 proteins in pooled liver extracts in experimental groups were measured using Western blot detection. Subsequent densitometrical analysis showed that cholestatic injury increased protein abundance of NGF (D) and TGF-β1 (E). All data are shown in mean±SEM. * indicates P <0.05 as compared to normal controls.
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pone-0112113-g001: Increased serum NGF levels and intrahepatic NGF up-regulation in mice receiving bile duct ligation (BDL) surgery.Normal mice (n = 6) and those receiving BDL surgery were sacrificed at 7 or 14 post-operative days (POD7, n = 4) or (POD14, n = 4). (A) Serum levels of NGF and inflammatory cytokines, including TNF-α, IL-6 and TGF-β1 were determined by ELISA detection. (B) Liver tissue extracts were collected for mRNA isolation and subjected to RT-qPCR analysis. (C) Contents of NGF and TGF-β1 proteins in pooled liver extracts in experimental groups were measured using Western blot detection. Subsequent densitometrical analysis showed that cholestatic injury increased protein abundance of NGF (D) and TGF-β1 (E). All data are shown in mean±SEM. * indicates P <0.05 as compared to normal controls.

Mentions: To elucidate the role played by NGF in cholestatic liver injuries, mice serum and liver tissues were collected at 7 days and 14 days post BDL operation. Elevated plasma levels of AST, ALT, and total bilirubin confirmed the effectiveness of surgery-induced cholestatic injury in mouse livers (Figure S1). ELISA detection showed that the pro-inflammatory cytokine levels including TNF-α, IL-6, and TGF-β1 in those mice with liver injury were significantly elevated. In parallel, the plasma NGF levels also increased along with the progression of liver fibrosis (Figure 1A). To investigate whether the cholestatic insult triggers de novo synthesis of NGF in livers, total RNA and protein extracts were used for further molecular measurements. RT-qPCR analysis indicated that the intrahepatic transcript contents of TNF-α, IL-6, TGF-β1, and NGF genes were remarkably up-regulated at POD14 of BDL surgery (Figure 1B), while gene expression patterns correlated with those of serum peptides. Western blotting demonstrated a similar increasing trend between intrahepatic NGF and TGF-β1 peptides (Figure 1C, 1D, 1E).


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)

Increased serum NGF levels and intrahepatic NGF up-regulation in mice receiving bile duct ligation (BDL) surgery.Normal mice (n = 6) and those receiving BDL surgery were sacrificed at 7 or 14 post-operative days (POD7, n = 4) or (POD14, n = 4). (A) Serum levels of NGF and inflammatory cytokines, including TNF-α, IL-6 and TGF-β1 were determined by ELISA detection. (B) Liver tissue extracts were collected for mRNA isolation and subjected to RT-qPCR analysis. (C) Contents of NGF and TGF-β1 proteins in pooled liver extracts in experimental groups were measured using Western blot detection. Subsequent densitometrical analysis showed that cholestatic injury increased protein abundance of NGF (D) and TGF-β1 (E). All data are shown in mean±SEM. * indicates P <0.05 as compared to normal controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112113-g001: Increased serum NGF levels and intrahepatic NGF up-regulation in mice receiving bile duct ligation (BDL) surgery.Normal mice (n = 6) and those receiving BDL surgery were sacrificed at 7 or 14 post-operative days (POD7, n = 4) or (POD14, n = 4). (A) Serum levels of NGF and inflammatory cytokines, including TNF-α, IL-6 and TGF-β1 were determined by ELISA detection. (B) Liver tissue extracts were collected for mRNA isolation and subjected to RT-qPCR analysis. (C) Contents of NGF and TGF-β1 proteins in pooled liver extracts in experimental groups were measured using Western blot detection. Subsequent densitometrical analysis showed that cholestatic injury increased protein abundance of NGF (D) and TGF-β1 (E). All data are shown in mean±SEM. * indicates P <0.05 as compared to normal controls.
Mentions: To elucidate the role played by NGF in cholestatic liver injuries, mice serum and liver tissues were collected at 7 days and 14 days post BDL operation. Elevated plasma levels of AST, ALT, and total bilirubin confirmed the effectiveness of surgery-induced cholestatic injury in mouse livers (Figure S1). ELISA detection showed that the pro-inflammatory cytokine levels including TNF-α, IL-6, and TGF-β1 in those mice with liver injury were significantly elevated. In parallel, the plasma NGF levels also increased along with the progression of liver fibrosis (Figure 1A). To investigate whether the cholestatic insult triggers de novo synthesis of NGF in livers, total RNA and protein extracts were used for further molecular measurements. RT-qPCR analysis indicated that the intrahepatic transcript contents of TNF-α, IL-6, TGF-β1, and NGF genes were remarkably up-regulated at POD14 of BDL surgery (Figure 1B), while gene expression patterns correlated with those of serum peptides. Western blotting demonstrated a similar increasing trend between intrahepatic NGF and TGF-β1 peptides (Figure 1C, 1D, 1E).

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