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Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis.

Tao YY, Yan XC, Zhou T, Shen L, Liu ZL, Liu CH - BMC Complement Altern Med (2014)

Bottom Line: In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax.Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203, China. chenghailiu@hotmail.com.

ABSTRACT

Background: What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.

Methods: Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.

Results: Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.

Conclusion: These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.

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Related in: MedlinePlus

The DNA of Apoptotic hepatocyte up-regulates α-SMA expression in rat HSCs and FZHY inhibited hepatocyte apoptosis and the expressions of α-SMA and type I collagen in HSCs. (A) Fragmented apoptotic DNA in cultured hepatocytes was induced by 200 ng/ml Act D and 20 ng/ml TNF-α. Lane 1, Marker, Lane 2; phosphate-buffered saline (PBS)-treated hepatocytes; Lane 3, Act D/TNF-α treated hepatocytes. (B) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. 25 ~ 100 μg/ml DNA from apoptotic hepatocyte stimulated α-SMA expression in rat HSCs in a dose dependent manner (Immunofluorescence staining, × 400). (C) Western blot analysis of expressions of α-SMA and type I collagen in rat HSCs. (D) Graphic presentation of the relative expressions of α-SMA and type I collagen. The values were represented as the density of α-SMA and collagen I vs. housekeeping gene GAPDH (%) from 3 samples. (E) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. Then, 100 μg/ml DNA from apoptotic hepatocytes was added into HSCs after culture for 4 days. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. The apoptotic DNA from hepatocytes treated by FZHY or Z-VAD-FMK (FMK) had less stimulating effects on α-SMA expression in HSCs (Immunofluorescence staining, × 400). **P < 0.01 vs. Control; ##P < 0.01 vscpc Act D/TNF-α group.
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Fig5: The DNA of Apoptotic hepatocyte up-regulates α-SMA expression in rat HSCs and FZHY inhibited hepatocyte apoptosis and the expressions of α-SMA and type I collagen in HSCs. (A) Fragmented apoptotic DNA in cultured hepatocytes was induced by 200 ng/ml Act D and 20 ng/ml TNF-α. Lane 1, Marker, Lane 2; phosphate-buffered saline (PBS)-treated hepatocytes; Lane 3, Act D/TNF-α treated hepatocytes. (B) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. 25 ~ 100 μg/ml DNA from apoptotic hepatocyte stimulated α-SMA expression in rat HSCs in a dose dependent manner (Immunofluorescence staining, × 400). (C) Western blot analysis of expressions of α-SMA and type I collagen in rat HSCs. (D) Graphic presentation of the relative expressions of α-SMA and type I collagen. The values were represented as the density of α-SMA and collagen I vs. housekeeping gene GAPDH (%) from 3 samples. (E) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. Then, 100 μg/ml DNA from apoptotic hepatocytes was added into HSCs after culture for 4 days. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. The apoptotic DNA from hepatocytes treated by FZHY or Z-VAD-FMK (FMK) had less stimulating effects on α-SMA expression in HSCs (Immunofluorescence staining, × 400). **P < 0.01 vs. Control; ##P < 0.01 vscpc Act D/TNF-α group.

Mentions: Four experimental protocols were followed. Firstly, to address the influence of FHZY on apoptosis of hepatocyte following CCl4-induced acute hepatic injury. Mice were subcutaneously injected with 100% CCl4 3 ml/kg body weight for one time, starting at 8 weeks of age. Three days before the first CCl4 injection, once-daily treatment with FZHY powder 4.0 g (crude drug)/kg or NAC 0.1 g/kg body weight respectively was started and mice were followed as noted in Figure 1. Eighteen hours after the first CCl4 injection, mice were analyzed for liver function, hematoxylin-eosin (HE) staining and immunohistochemical evaluation of apoptosis (Figure 1). Secondly, to focus on the effect of FHZY on established liver fibrosis, mice were injected at 8 weeks of age with 100% CCl4 3 ml/kg body weight for the first time, then 50% CCl4/olive oil 3 ml/kg body weight, two times per week for 8 weeks. Three days before the first CCl4 injection, once-daily treatment with FZHY powder 4.0 g (crude drug)/kg or NAC 0.1 g/kg body weight respectively was started and mice were followed as noted in Figure 2. At 16 weeks of age, or 8 weeks following the initial exposure to CCl4, mice were sacrificed for histological assessment of liver fibrosis, and immunohistochemical evaluation of apoptosis (Figure 2). Thirdly, to detect the effect of FHZY on hepatocyte apoptosis induced by Act D/TNF-α (Figure 3), After cultured for 24 hour, primary hepatocytes were treated with 10% FZHY-medicated serum or 10% rats’ serum + 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for another 6 h. Immunofluorescence, flow cytometry and DNA ladder for hepatocyte apoptosis and immunoblotting for TNFR1, Bcl-2 and Bax were also analyzed. Finally, to explore whether FZHY attenuated hepatic fibrosis by inhibiting apoptosis of hepatocytes, we performed the subsequently experiments. At day 4 after isolation, primary HSCs were treated with the DNA of apoptotic hepatocytes incubated by Act D/TNF-α and 10% FZHY-medicated serum. Immunofluorescence and immunoblotting for α-SMA and type I collagen in HSCs were analyzed (Figures 4 and 5).Figure 1


Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis.

Tao YY, Yan XC, Zhou T, Shen L, Liu ZL, Liu CH - BMC Complement Altern Med (2014)

The DNA of Apoptotic hepatocyte up-regulates α-SMA expression in rat HSCs and FZHY inhibited hepatocyte apoptosis and the expressions of α-SMA and type I collagen in HSCs. (A) Fragmented apoptotic DNA in cultured hepatocytes was induced by 200 ng/ml Act D and 20 ng/ml TNF-α. Lane 1, Marker, Lane 2; phosphate-buffered saline (PBS)-treated hepatocytes; Lane 3, Act D/TNF-α treated hepatocytes. (B) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. 25 ~ 100 μg/ml DNA from apoptotic hepatocyte stimulated α-SMA expression in rat HSCs in a dose dependent manner (Immunofluorescence staining, × 400). (C) Western blot analysis of expressions of α-SMA and type I collagen in rat HSCs. (D) Graphic presentation of the relative expressions of α-SMA and type I collagen. The values were represented as the density of α-SMA and collagen I vs. housekeeping gene GAPDH (%) from 3 samples. (E) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. Then, 100 μg/ml DNA from apoptotic hepatocytes was added into HSCs after culture for 4 days. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. The apoptotic DNA from hepatocytes treated by FZHY or Z-VAD-FMK (FMK) had less stimulating effects on α-SMA expression in HSCs (Immunofluorescence staining, × 400). **P < 0.01 vs. Control; ##P < 0.01 vscpc Act D/TNF-α group.
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289302&req=5

Fig5: The DNA of Apoptotic hepatocyte up-regulates α-SMA expression in rat HSCs and FZHY inhibited hepatocyte apoptosis and the expressions of α-SMA and type I collagen in HSCs. (A) Fragmented apoptotic DNA in cultured hepatocytes was induced by 200 ng/ml Act D and 20 ng/ml TNF-α. Lane 1, Marker, Lane 2; phosphate-buffered saline (PBS)-treated hepatocytes; Lane 3, Act D/TNF-α treated hepatocytes. (B) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. 25 ~ 100 μg/ml DNA from apoptotic hepatocyte stimulated α-SMA expression in rat HSCs in a dose dependent manner (Immunofluorescence staining, × 400). (C) Western blot analysis of expressions of α-SMA and type I collagen in rat HSCs. (D) Graphic presentation of the relative expressions of α-SMA and type I collagen. The values were represented as the density of α-SMA and collagen I vs. housekeeping gene GAPDH (%) from 3 samples. (E) Primary hepatocytes were incubated with or without 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. Then, 100 μg/ml DNA from apoptotic hepatocytes was added into HSCs after culture for 4 days. 100 μg/ml DNA from hepatocytes incubated with PBS acted as a control. The apoptotic DNA from hepatocytes treated by FZHY or Z-VAD-FMK (FMK) had less stimulating effects on α-SMA expression in HSCs (Immunofluorescence staining, × 400). **P < 0.01 vs. Control; ##P < 0.01 vscpc Act D/TNF-α group.
Mentions: Four experimental protocols were followed. Firstly, to address the influence of FHZY on apoptosis of hepatocyte following CCl4-induced acute hepatic injury. Mice were subcutaneously injected with 100% CCl4 3 ml/kg body weight for one time, starting at 8 weeks of age. Three days before the first CCl4 injection, once-daily treatment with FZHY powder 4.0 g (crude drug)/kg or NAC 0.1 g/kg body weight respectively was started and mice were followed as noted in Figure 1. Eighteen hours after the first CCl4 injection, mice were analyzed for liver function, hematoxylin-eosin (HE) staining and immunohistochemical evaluation of apoptosis (Figure 1). Secondly, to focus on the effect of FHZY on established liver fibrosis, mice were injected at 8 weeks of age with 100% CCl4 3 ml/kg body weight for the first time, then 50% CCl4/olive oil 3 ml/kg body weight, two times per week for 8 weeks. Three days before the first CCl4 injection, once-daily treatment with FZHY powder 4.0 g (crude drug)/kg or NAC 0.1 g/kg body weight respectively was started and mice were followed as noted in Figure 2. At 16 weeks of age, or 8 weeks following the initial exposure to CCl4, mice were sacrificed for histological assessment of liver fibrosis, and immunohistochemical evaluation of apoptosis (Figure 2). Thirdly, to detect the effect of FHZY on hepatocyte apoptosis induced by Act D/TNF-α (Figure 3), After cultured for 24 hour, primary hepatocytes were treated with 10% FZHY-medicated serum or 10% rats’ serum + 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for another 6 h. Immunofluorescence, flow cytometry and DNA ladder for hepatocyte apoptosis and immunoblotting for TNFR1, Bcl-2 and Bax were also analyzed. Finally, to explore whether FZHY attenuated hepatic fibrosis by inhibiting apoptosis of hepatocytes, we performed the subsequently experiments. At day 4 after isolation, primary HSCs were treated with the DNA of apoptotic hepatocytes incubated by Act D/TNF-α and 10% FZHY-medicated serum. Immunofluorescence and immunoblotting for α-SMA and type I collagen in HSCs were analyzed (Figures 4 and 5).Figure 1

Bottom Line: In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax.Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New Area, Shanghai 201203, China. chenghailiu@hotmail.com.

ABSTRACT

Background: What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis.

Methods: Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed.

Results: Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression.

Conclusion: These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.

Show MeSH
Related in: MedlinePlus