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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

Fuzheng Huayu recipe (FZHY) inhibits Act D/TNF-α induced hepatocyte apoptosis. Z-VAD-FMK (FMK) is a synthetic peptide that irreversibly inhibits activity of caspase family proteases and blocks apoptosis, and was used as a positive control in this work. Twenty four hours after plating, primary hepatocytes were pretreated with 10% FZHY medicated serum or 10% rats’ serum plus 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. (A) Confocal microscopy observation of hepatocyte apoptosis stained by FITC labeled Annexin V (green) and propidium iodide (PI) (red) (×400). (B) Cells were trypsinized and stained with Annexin V and PI followed by analysis with flow cytometry. Early apoptotic cells (Annexin V positive and PI negative) were in the right lower quadrant. Late apoptotic or necrotic cells were in the right upper quadrant. (C) The apoptotic index (AI) of five high-power fields at × 400 magnification of confocal microscopic figures in Figure 3A. **P < 0.01 vs. control group; ##P < 0.01 vs. Act D/TNF-α treated group. (D) Primary hepatocytes were pretreated with 10% FZHY medicated resum 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 6 h. Cells were harvested, and DNA was purified by the DNeasy Kit. DNA was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light. Lane 1, marker; lane 2, phosphate-buffered saline (PBS) treated cells; lane 3, PBS and Act D/TNF-α treated cells; lane 4, FZHY and Act D/TNF-α treated cells; and lane 5, Z-VAD-FMK and Act D/TNF-α treated cells. Agarose gel electrophoresis showed that FZHY and Z-VAD-FMK inhibited apoptosis of hepatocytes induced by Act D and TNF-α.
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Fig3: Fuzheng Huayu recipe (FZHY) inhibits Act D/TNF-α induced hepatocyte apoptosis. Z-VAD-FMK (FMK) is a synthetic peptide that irreversibly inhibits activity of caspase family proteases and blocks apoptosis, and was used as a positive control in this work. Twenty four hours after plating, primary hepatocytes were pretreated with 10% FZHY medicated serum or 10% rats’ serum plus 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. (A) Confocal microscopy observation of hepatocyte apoptosis stained by FITC labeled Annexin V (green) and propidium iodide (PI) (red) (×400). (B) Cells were trypsinized and stained with Annexin V and PI followed by analysis with flow cytometry. Early apoptotic cells (Annexin V positive and PI negative) were in the right lower quadrant. Late apoptotic or necrotic cells were in the right upper quadrant. (C) The apoptotic index (AI) of five high-power fields at × 400 magnification of confocal microscopic figures in Figure 3A. **P < 0.01 vs. control group; ##P < 0.01 vs. Act D/TNF-α treated group. (D) Primary hepatocytes were pretreated with 10% FZHY medicated resum 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 6 h. Cells were harvested, and DNA was purified by the DNeasy Kit. DNA was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light. Lane 1, marker; lane 2, phosphate-buffered saline (PBS) treated cells; lane 3, PBS and Act D/TNF-α treated cells; lane 4, FZHY and Act D/TNF-α treated cells; and lane 5, Z-VAD-FMK and Act D/TNF-α treated cells. Agarose gel electrophoresis showed that FZHY and Z-VAD-FMK inhibited apoptosis of hepatocytes induced by Act D and TNF-α.

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)

Fuzheng Huayu recipe (FZHY) inhibits Act D/TNF-α induced hepatocyte apoptosis. Z-VAD-FMK (FMK) is a synthetic peptide that irreversibly inhibits activity of caspase family proteases and blocks apoptosis, and was used as a positive control in this work. Twenty four hours after plating, primary hepatocytes were pretreated with 10% FZHY medicated serum or 10% rats’ serum plus 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. (A) Confocal microscopy observation of hepatocyte apoptosis stained by FITC labeled Annexin V (green) and propidium iodide (PI) (red) (×400). (B) Cells were trypsinized and stained with Annexin V and PI followed by analysis with flow cytometry. Early apoptotic cells (Annexin V positive and PI negative) were in the right lower quadrant. Late apoptotic or necrotic cells were in the right upper quadrant. (C) The apoptotic index (AI) of five high-power fields at × 400 magnification of confocal microscopic figures in Figure 3A. **P < 0.01 vs. control group; ##P < 0.01 vs. Act D/TNF-α treated group. (D) Primary hepatocytes were pretreated with 10% FZHY medicated resum 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 6 h. Cells were harvested, and DNA was purified by the DNeasy Kit. DNA was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light. Lane 1, marker; lane 2, phosphate-buffered saline (PBS) treated cells; lane 3, PBS and Act D/TNF-α treated cells; lane 4, FZHY and Act D/TNF-α treated cells; and lane 5, Z-VAD-FMK and Act D/TNF-α treated cells. Agarose gel electrophoresis showed that FZHY and Z-VAD-FMK inhibited apoptosis of hepatocytes induced by Act D and TNF-α.
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Fig3: Fuzheng Huayu recipe (FZHY) inhibits Act D/TNF-α induced hepatocyte apoptosis. Z-VAD-FMK (FMK) is a synthetic peptide that irreversibly inhibits activity of caspase family proteases and blocks apoptosis, and was used as a positive control in this work. Twenty four hours after plating, primary hepatocytes were pretreated with 10% FZHY medicated serum or 10% rats’ serum plus 50 μM Z-VAD-FMK for 18 h and then incubated with 200 ng/ml ActD and 20 ng/ml TNF-α for 6 h. (A) Confocal microscopy observation of hepatocyte apoptosis stained by FITC labeled Annexin V (green) and propidium iodide (PI) (red) (×400). (B) Cells were trypsinized and stained with Annexin V and PI followed by analysis with flow cytometry. Early apoptotic cells (Annexin V positive and PI negative) were in the right lower quadrant. Late apoptotic or necrotic cells were in the right upper quadrant. (C) The apoptotic index (AI) of five high-power fields at × 400 magnification of confocal microscopic figures in Figure 3A. **P < 0.01 vs. control group; ##P < 0.01 vs. Act D/TNF-α treated group. (D) Primary hepatocytes were pretreated with 10% FZHY medicated resum 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 6 h. Cells were harvested, and DNA was purified by the DNeasy Kit. DNA was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light. Lane 1, marker; lane 2, phosphate-buffered saline (PBS) treated cells; lane 3, PBS and Act D/TNF-α treated cells; lane 4, FZHY and Act D/TNF-α treated cells; and lane 5, Z-VAD-FMK and Act D/TNF-α treated cells. Agarose gel electrophoresis showed that FZHY and Z-VAD-FMK inhibited apoptosis of hepatocytes induced by Act D and TNF-α.
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