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reoxygenation injury of HepG2 cell line
Posters ~International Hepatology Communications 3 Suppl. (1995) $37-S169
S86
P-197
CYTO~OTECTI=
cyclic AMP(DBc-AMP) OF HepG2 CELL LINE
~r~CTS
OF D~EOTYR~L
ON HYPOXIA/REOXUG~L~ATION
TNJL~Y
KUSUO Ayukawa, Hironori Sakai, Hajime Nawata 3rd.Dept.of Internal Medicine,Faculty of Medicine,Kyushu University,Fukuoka,Japan It was proven that Dibutyryl cyclic AMP(DBc-AMP) inhibits ischemia/reperfusion liver injury in vivo by improvement of the microcirculatory disturbance or energy metablism. The aim of this study was to investigate direct effects of DBc-AMP on HepG2 cell line under hypoxia/reoxygenation in vitro. METHODS: HepG2 cells were incubatehd in a atmosphere of air/CO2(95:5). After cells became confluent, subsequent incubation was continued under the same condition or hypoxic condition [O2/N2/CO2 (0.8:94.2:5)] with or without DBc-AMP. After 24hrs of incubation under each condition, LDH activity of the supernatants was measured by UVmethods. RESULTS: LDH activity IU (mean±S.D.) hypoxia/normoxia 10mM 0.1mM control 0h/24h 1.29~1.65 1.29± 1.65 6h/18h 1.72 • 1.99 12.9±3.29 12h/12h 0.0~0.0 28.1 • 4.97 39.1±17.6 18h/6h 117.0~15.1 208 .I ± 98.3 CONCLUSION: DBc-AMP showed a dose-dependent inhibition of the hypoxia/reoxygenation injury of HepG2 in vitro. These data suggest that DBc-AMP has also direct cytoproection for hypoxia/reoxygenation injury. We speculate that the mechanism of directly protective effects were related to enhancement of gap junctional intercellular communication or cell membrane stabilizing action.
P-199
T H E MECHANISM OF THE PRIMARY ISCHEMIA/ REOXYGENATION INJURY FROM T H E ASPECT OF IRON CHELATION M, Ozaki, S . F u c h i n o u e , S. Teraoka, K. Ota D e p a r t m e n t of S u r g e r y III, Tokyo W o m e n ' s Medical College, Tokyo, J a p a n T h e in vivo e f f e c t s o f a s c o r b i c acid(AsA) a n d d e f e r r l o x a m i n e (DFO) on the r e o x y g e n a t e d liver t i s s u e w e r e e x a m i n e d , with r e g a r d to t h e following effects: I) the effects of s c a v e n g i n g radicals a n d / o r r e d u c i n g peroxidative reactions, a n d 2) t h e effects o f t h e c h e l a t i o n with low m o l e c u l a r weight iron a n d affecting its reactivity(radical production). Lipid p e r o x i d a t i o n of t h e r e o x y g e n a t e d liver t i s s u e ( t h e p r o d u c t i o n of t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e a n d lipid h y d r o p e r o x i d e s ) w a s s u p p r e s s e d effectively by the p r e - i s c h e m i c i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of both AsA a n d DFO. They also s h o w e d this a n t i - o x i d a n t effects in a d o s e - d e p e n d e n t m a n n e r . The a n a l y s i s o f t h e levels of AsA a n d g l u t a t h i o n e of the liver t i s s u e r e v e a l e d t h a t AsA w o r k s a s a n a n t i - o x i d a n t by b e i n g oxydized to dehydro-AsA j u s t after the reoxygenation. The latter w a s r e d u c e d to AsA again, coupled with the c o n v e r s i o n of GSH to GSSG in the post-ischemic time course. DFO also successfully s u p p r e s s e d t h e lipid p e r o x i d a t l o n of t h e liver t i s s u e . H e p a t o c e l l u l a r d a m a g e ( s e r u m levels of GOT, GPT a n d LDH) a n d t h e e n e r g y s t a t e of the p o s t - i s c h e m l c liver t i s s u e (tissue ATP level) w e r e well p r e s e r v e d by AsA, b u t not by DFO, The p r e d o m i n a n t effect of AsA on the r e o x y g e n a t e d liver t i s s u e s e e m s to be c a u s e d by t h e s c a v e n g i n g r a d i c a l s a n d r e d u c i n g peroxidative r e a c t i o n s , r a t h e r t h a n by c h e l a t i n g iron a n d i n c r e a s i n g its reactivity. On the o t h e r h a n d , DFO c h e l a t e s iron strongly a n d r u d u c e s its reactivity a n d the lipid peroxidation of t h e liver tissue.
P-198
Cytoprotective effect of hepatocyte growth factor (HGF) for acute liver injury in HGF transgenic mice J. Okano, G. Shiota and H. Kawasaki 2nd Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan
Aims: HGF is a potent mitogen for hepatocytes in vivo as well as in vitro. Recent studies have suggested that HGF can reduce acute liver injury induced by ct-naphtylisothyanate (ANIT). We evaluated cytoprotective effect of HGF for acute liver injury caused by acetoaminophen (AAP) or D-galactosamine (D-GalNI u,;ing transgenic mice expressing HGF in hepatocytes. Methods: Genotyping of HGF transgenic mice (TG) and control mice (WT) was done by using tail DNA. Exp 1. After intraperitoneal injection of AAP (5mmol/kg), serum ALT and liver histology were examined at 2, 4 and 8 hr in 12 TG and 22 WT Exp 2. Serum ALT and liver histology were evaluated at 24 and 48 hr alter D-GaIN (3g/kg) administration in 7 TG and7 WT Exp 3. Survival rate after6g/kg D-GaIN injection was assessed by using 16 TG and 16 WT. Results: Exp 1. Serum ALT levels at 4 hr were significantly lower in TG than in WT (108_+39 IU/L, 231_+66 IU/L, respectively), although no differencewas observed at 2 hr. Exp 2. At 24 hr after 3g/kg D-GaIN administration, TG showed lower levels of ALT than those of WT (131_+69 IU/L, 451+384 IU/L, respectively). At 48 hr aftertreatment, serum levels of ALT in TG and WT were 200--65 IU/L and 500 IU/L, respectively Histological examination showed that D-GaIN caused wide spread necrotic areasin parenchyma and HGF reduced the parenchymal lesions. Exp 3. Survival rates of TG after 6g/kg D-GaIN treatment were 7/8 (89%) at 24 hr and5/8 (63%)at 48 hr, while those of WT were 4/8 150c/~)and I/8 (13c/Q, respectively Conclusion: We concludethat HGF can reduceacute liver injury caused by AAP or D-GaIN. Its responsible mechanisms are underinvestigation.
P-200
INTERACTION BETWEEN HEPATOCYTE AND KUPFFER CELL : .FROM THE VIEWPOINrl" OF BILIARY EXCRETION OF LIPOPOLYSACCHARIDE IN THE LIVER Y.HORI, Y.TAKEY,~MA, T.UEDA, J.NqSHIKAWA, N.UENO, MYAMAMOTO, Y.SAITOH I st Department of Surgery, Kobe University School of Medicine, Kobe, Japan Bacterial lipopolysaccharides (LPS) are cleared through the liver, predominantly by Kupffer cells. However, conflicting results show that parenchymal cells are also involved. Clinically, LPS are detected in bile but little is known about the transport pathway of LPS across the hepatocyte from blood to bile. In the present study, we attempted to detect LPS in bile, using the isolated perfused rat liver (IPRL) model, and examined the effect of colchicine on the biliary excretion of LPS. Moreover, to investigate the interaction between hepatocytes and Kupffer cells, we examined the effect of gadolinium chloride (GdCI3) which ilLhibits the phagocytosis of Kupffer cells. When 5mg/ml LPS were infused as a 1-min pulse load, the biliary excrenon of LPS peaked at approximately 25-30 n~n Pretreatment with colchicine significantly decreased the peak, from 064--+0.05 to 0.36-0.05 pgtmn g liver. In these experiments, colckicine reduced bile flow nonsi~m~_tficanfly,from 1.13--0 16 to 0.95--+0.22 p.I/mm/g liver. On the other hand, pretreatment with GdCI3 elevated the biliary excretion of LPS. These results suggest that LPS, as well as IgA and horseradish peroxadase enter bile via the microtubule-dependent transcytotic vesicle pathway, and this pathway is probably the major one, and that hepatocytes correlate with Kupffer cells in protecting the spill over of LPS into systemic circulation.
S86
P-197
CYTO~OTECTI=
cyclic AMP(DBc-AMP) OF HepG2 CELL LINE
~r~CTS
OF D~EOTYR~L
ON HYPOXIA/REOXUG~L~ATION
TNJL~Y
KUSUO Ayukawa, Hironori Sakai, Hajime Nawata 3rd.Dept.of Internal Medicine,Faculty of Medicine,Kyushu University,Fukuoka,Japan It was proven that Dibutyryl cyclic AMP(DBc-AMP) inhibits ischemia/reperfusion liver injury in vivo by improvement of the microcirculatory disturbance or energy metablism. The aim of this study was to investigate direct effects of DBc-AMP on HepG2 cell line under hypoxia/reoxygenation in vitro. METHODS: HepG2 cells were incubatehd in a atmosphere of air/CO2(95:5). After cells became confluent, subsequent incubation was continued under the same condition or hypoxic condition [O2/N2/CO2 (0.8:94.2:5)] with or without DBc-AMP. After 24hrs of incubation under each condition, LDH activity of the supernatants was measured by UVmethods. RESULTS: LDH activity IU (mean±S.D.) hypoxia/normoxia 10mM 0.1mM control 0h/24h 1.29~1.65 1.29± 1.65 6h/18h 1.72 • 1.99 12.9±3.29 12h/12h 0.0~0.0 28.1 • 4.97 39.1±17.6 18h/6h 117.0~15.1 208 .I ± 98.3 CONCLUSION: DBc-AMP showed a dose-dependent inhibition of the hypoxia/reoxygenation injury of HepG2 in vitro. These data suggest that DBc-AMP has also direct cytoproection for hypoxia/reoxygenation injury. We speculate that the mechanism of directly protective effects were related to enhancement of gap junctional intercellular communication or cell membrane stabilizing action.
P-199
T H E MECHANISM OF THE PRIMARY ISCHEMIA/ REOXYGENATION INJURY FROM T H E ASPECT OF IRON CHELATION M, Ozaki, S . F u c h i n o u e , S. Teraoka, K. Ota D e p a r t m e n t of S u r g e r y III, Tokyo W o m e n ' s Medical College, Tokyo, J a p a n T h e in vivo e f f e c t s o f a s c o r b i c acid(AsA) a n d d e f e r r l o x a m i n e (DFO) on the r e o x y g e n a t e d liver t i s s u e w e r e e x a m i n e d , with r e g a r d to t h e following effects: I) the effects of s c a v e n g i n g radicals a n d / o r r e d u c i n g peroxidative reactions, a n d 2) t h e effects o f t h e c h e l a t i o n with low m o l e c u l a r weight iron a n d affecting its reactivity(radical production). Lipid p e r o x i d a t i o n of t h e r e o x y g e n a t e d liver t i s s u e ( t h e p r o d u c t i o n of t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e a n d lipid h y d r o p e r o x i d e s ) w a s s u p p r e s s e d effectively by the p r e - i s c h e m i c i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of both AsA a n d DFO. They also s h o w e d this a n t i - o x i d a n t effects in a d o s e - d e p e n d e n t m a n n e r . The a n a l y s i s o f t h e levels of AsA a n d g l u t a t h i o n e of the liver t i s s u e r e v e a l e d t h a t AsA w o r k s a s a n a n t i - o x i d a n t by b e i n g oxydized to dehydro-AsA j u s t after the reoxygenation. The latter w a s r e d u c e d to AsA again, coupled with the c o n v e r s i o n of GSH to GSSG in the post-ischemic time course. DFO also successfully s u p p r e s s e d t h e lipid p e r o x i d a t l o n of t h e liver t i s s u e . H e p a t o c e l l u l a r d a m a g e ( s e r u m levels of GOT, GPT a n d LDH) a n d t h e e n e r g y s t a t e of the p o s t - i s c h e m l c liver t i s s u e (tissue ATP level) w e r e well p r e s e r v e d by AsA, b u t not by DFO, The p r e d o m i n a n t effect of AsA on the r e o x y g e n a t e d liver t i s s u e s e e m s to be c a u s e d by t h e s c a v e n g i n g r a d i c a l s a n d r e d u c i n g peroxidative r e a c t i o n s , r a t h e r t h a n by c h e l a t i n g iron a n d i n c r e a s i n g its reactivity. On the o t h e r h a n d , DFO c h e l a t e s iron strongly a n d r u d u c e s its reactivity a n d the lipid peroxidation of t h e liver tissue.
P-198
Cytoprotective effect of hepatocyte growth factor (HGF) for acute liver injury in HGF transgenic mice J. Okano, G. Shiota and H. Kawasaki 2nd Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan
Aims: HGF is a potent mitogen for hepatocytes in vivo as well as in vitro. Recent studies have suggested that HGF can reduce acute liver injury induced by ct-naphtylisothyanate (ANIT). We evaluated cytoprotective effect of HGF for acute liver injury caused by acetoaminophen (AAP) or D-galactosamine (D-GalNI u,;ing transgenic mice expressing HGF in hepatocytes. Methods: Genotyping of HGF transgenic mice (TG) and control mice (WT) was done by using tail DNA. Exp 1. After intraperitoneal injection of AAP (5mmol/kg), serum ALT and liver histology were examined at 2, 4 and 8 hr in 12 TG and 22 WT Exp 2. Serum ALT and liver histology were evaluated at 24 and 48 hr alter D-GaIN (3g/kg) administration in 7 TG and7 WT Exp 3. Survival rate after6g/kg D-GaIN injection was assessed by using 16 TG and 16 WT. Results: Exp 1. Serum ALT levels at 4 hr were significantly lower in TG than in WT (108_+39 IU/L, 231_+66 IU/L, respectively), although no differencewas observed at 2 hr. Exp 2. At 24 hr after 3g/kg D-GaIN administration, TG showed lower levels of ALT than those of WT (131_+69 IU/L, 451+384 IU/L, respectively). At 48 hr aftertreatment, serum levels of ALT in TG and WT were 200--65 IU/L and 500 IU/L, respectively Histological examination showed that D-GaIN caused wide spread necrotic areasin parenchyma and HGF reduced the parenchymal lesions. Exp 3. Survival rates of TG after 6g/kg D-GaIN treatment were 7/8 (89%) at 24 hr and5/8 (63%)at 48 hr, while those of WT were 4/8 150c/~)and I/8 (13c/Q, respectively Conclusion: We concludethat HGF can reduceacute liver injury caused by AAP or D-GaIN. Its responsible mechanisms are underinvestigation.
P-200
INTERACTION BETWEEN HEPATOCYTE AND KUPFFER CELL : .FROM THE VIEWPOINrl" OF BILIARY EXCRETION OF LIPOPOLYSACCHARIDE IN THE LIVER Y.HORI, Y.TAKEY,~MA, T.UEDA, J.NqSHIKAWA, N.UENO, MYAMAMOTO, Y.SAITOH I st Department of Surgery, Kobe University School of Medicine, Kobe, Japan Bacterial lipopolysaccharides (LPS) are cleared through the liver, predominantly by Kupffer cells. However, conflicting results show that parenchymal cells are also involved. Clinically, LPS are detected in bile but little is known about the transport pathway of LPS across the hepatocyte from blood to bile. In the present study, we attempted to detect LPS in bile, using the isolated perfused rat liver (IPRL) model, and examined the effect of colchicine on the biliary excretion of LPS. Moreover, to investigate the interaction between hepatocytes and Kupffer cells, we examined the effect of gadolinium chloride (GdCI3) which ilLhibits the phagocytosis of Kupffer cells. When 5mg/ml LPS were infused as a 1-min pulse load, the biliary excrenon of LPS peaked at approximately 25-30 n~n Pretreatment with colchicine significantly decreased the peak, from 064--+0.05 to 0.36-0.05 pgtmn g liver. In these experiments, colckicine reduced bile flow nonsi~m~_tficanfly,from 1.13--0 16 to 0.95--+0.22 p.I/mm/g liver. On the other hand, pretreatment with GdCI3 elevated the biliary excretion of LPS. These results suggest that LPS, as well as IgA and horseradish peroxadase enter bile via the microtubule-dependent transcytotic vesicle pathway, and this pathway is probably the major one, and that hepatocytes correlate with Kupffer cells in protecting the spill over of LPS into systemic circulation.