- Email: [email protected]
Effects of idoxifene and estradiol on NF-κB activation in cultured rat hepatocytes undergoing oxidative stress
1828
tn hepatocytesand 8ECs. However. the marked bending lines of ZO-1, 7H6, Claudin-1 were observed, mainly during 36 - 96 hr after PH. By electron mtcroscopy, marked bending bile canaliculi were observed. Conclusions: The loss of integral network of bile canalicutus may be one of the possible causativefactors of cholestasis associatedwith regeneratingliver.
Alpha-napthylisothiocyanate-induced CholangiocyteApoptosis is Dependent on Increased Reactive Oxygen Species (ROS) Gene Lesage,Shannon Glaser, Heather Francis, Scott & White Memorial Hosp, Temple, TX; Mikel Ludvik, The Texas A&M Univ System Health Science Ctr, Temple, TX; Jo Lynne Phinizy, Scott & White Memorial Hosp, Temple, IX; Noriatsu Kanno, Gianfranco Aipini, The Texas A&M Univ System Health Science Ctr, Temple, TX
1831
Cholangiocytesrespondto ANITfeeding with cholangiocytedeathand compensatoryproliferation. Cholangiopathiesare associatedwith cholangiocyteapoptosis limited to a range of bile ducts sizes. It remains unknown if ANIT induces cholangiocyteapoptosis and if ANIT-induced apoptosis, like cholangiopathies,is limited to certain sized ducts. The cellular mechanisms of ANIT-inducedcholangiocyteinjury are unknown.Apoptosis is triggered by the accumulation of reactive oxygen species (ROS) in cells. Antioxidants (such as glutathione) protect cells from apoptosis, and hydrogen peroxide by increasing ROS induces apoptosis in cells. The OBJECTIVES of the studies were to determine (i) if ANIT directly induces cholaogiocyte apoptosis in vitro; and (ii) if ANIT-induced apoptosis is due to accumulation of ROS in cholangiocytes.METHODS:Rats were fed 0.1% ANIT for 2 weeks. Small and large cholangiocytes (from small and large bile ducts, respectively)were isolatedfrom rat liver. Cholangiocytes isolated from normal liver were treated with ANIT (10/~), or HA (100 pM) in the presence and absenceof Ethyl ester glutathione (10/~M) for 3 hours at 37°C, Cholangiocyteapoptosis was assessed by the presenceof nuclear fragmentation by DAPI staining. ROS in chotangiocytes was measured by addition of the ROS indicator dye, 2',7'-dichlorofluorascein for 30 minutes and then assessing cellular fluorescence which is proportional to the intracellular ROS concentration. RESULTS:Both apoptosis and ROS generationwere significantly greater in large and small cholangiocytesfrom ANIT-fed comparedto control rats. In vitro, ANIT and H202 induced cholangiocyte apoptosis in both large and small cholangiocytes and this was associatedwith an increasedROSgeneration.Consistentwith increasedROSas the mechanism for ANIT-inducedapoptosis,we found that Ethyl ester glutathionepreventedboth ROS generation and ANIT-induced apoptosis. CONCLUSION:These studies for the first time establish that the hepatotoxin,ANIT induces cholangiocyteapoptosisin both small and large intrahepatic bile ducts. ANIT-inducedapoptosis is due to increasedcholangiocyteROS.Thesedata suggest that the mechanism of drug-induced cholangiopathiesmay be due to ROS and are consistent with the novel idea that cholangiocyteapoptosis in human cholangiopathieasuch as PBC and PSC could alleviated by therapies that reduce ROS in cholangiocytes. 1829
Effects of Idoxifono and Esfradiol on NF-KB Activation in Cultured Rat Hepatocytes Undmleiq OxlaUce Stress Hiroshi Inoue, Ichiro Shimizu, Guaogming Lu, Yajun Zhou, Mina Itonaga, Hirohito Honda, Susumu Ito, Second Dept of Internal Medicine, Tokushima Univ, Tokushima Japan Background: Idoxifeneis one of a series of synthesizedanaloguesof tamoxifen, and a tissuespecific selectiveestrogen receptor modulator. Estradiol is a potent endogenousantioxidant, and nuclear factor kB (NF-kB) is a key transcription factor that induces multiple genes in response to inflammation or oxidative stress. The aim of this study was to explore the inhibitory effects of idoxifena and estradiol on NF-kB activation in hepatocytes in a state of oxidative stress. Methods: Lipid peroxidation was induced in cultured rat hepatocytes by incubation with ferric nitrilotriacetate solution. NF-kBactivity was evaluatedby electrophoretic mobility shift assay. Results:The oxidative stress-inducedactivation of NF-kBand degradation of IkB-alpha are maximal at 3-5 h, with an increase in lactate dehydrogenase(LDH) and malondlaldebyde (MDA) secretion into the culture medium. Treatment with idoxifene and estrediol inhibited the IkB-alphadegradationand NF-k8 activation through the attenuation of hepatocyte oxidative bursts and decreasedextracellular levels of LDH and MDA. In addition, iitoxifena and estradiol inhibited lipid peroxidationin rat liver mitochondria. Whereastamoxiten had no such inhibitory effect. A potent NF-k8 inhibitor, pyrrolidine dithiocarbamate,prevented NF-kB activation by inhibition of IkB-alpha degradation and decreasedLDH and MDA levels, suggesting that NF-kB might be a regulator in agenetic responseto increase oxidative stressinduced hepaticinjury. Conclusion:Thesefindings suggestthat idoxifeneand estradiolfunction as a n t i o x ~ and protect bepatocytesfrom inflammatory cell injury. 18~ Direct Evidence Of Production Of Nitric Oxide In The Lipopolysaccharide-Sensitized Rat HepatonytesAnd Kupfter Coils Tom Kono, Jun Iwamoto, Kazushi Ishikawa, Yosiaki Ebisawa,Takanori Aoki, Nobutosi Ando, Shinichi Kasal, Asahikawa Medical Coil, AsahikawaJapan Backgroundand Aims: We have detectedthe intra-abdominal nitric oxide (NO) releasedfrom the liver surface of the rat sensitized with lipopolysaccharids (LPS) by analyzing the intraabdominal gas by chemiluminescence (Ando et al., Nitric Oxide, 2, 481 1998 and Kamlya et al., Shock, 14, 229, 2000) However, the origin of NO in the liver has not been clarified. In order to identify the production site of NO in the liver, we attemptedto detect NOfluorescence from isolatedhapatocytes(Hc) and Kupffer cells (Kc) by using daiaminofluoresceine-2diacetate (DAF-2DA). Methods: Wistar male rats were injected with 0.1 mg/kg LPS i.p. After 6 hr, rat liver was perfusod with Hanks solution containing collagenasevia portal vein and Hc and Kc were harvested altogether and preserved at 4C. For later identification of Kc, rhodamineconjugated Latex particles (1 pm diameter) were also injected to the LPS-sensitizedrats t hr before perfusion. Kc was isolatedfrom the cluster of harvestedcells with a percoll gradient centrifuge technique immediately after the perfusion. Either Hc or Kc were the transferred to a chamber perfused with Krebs solution equilibrated at pH 7.4 at 33C. Consequently, DAF2DA (10 p.M) was added in the chamber as being excited at 495 nm under a fluorescent micmscopa. The observedHc was later identified using immunohistochemistryfor the intracelMar albumin. Inducible-NOS was also confirmed in the observed Hc and Kc using antiiNOS monoclonal antibody. During observation of NO fluorescence from the cells, Kc was simuRaneouslyidentified with red fluorescence of rhodamine particles, which had beentaken within the cells by phagocytosis. The control experiment was also performed in order to detect NO production under the normal condition. Insteadof LPS administration, the rat only receivedvehicle (saline). The following procedureswere similar to those in the LPS-sensitized groups. Results: Hc and Kc cultured in the chamber gradually developedbright green fluorescence (515 nm wavelength)after excitation in the LPS-sensitizedgroups. However, unsensitized liver did not develop fluorescence in the Hc, whereas the Kc demonstrated similar fluorescence as LPS-sensitizedcells. Conclusions: Theseresu/ts indicatethat intra-abdominal NO accumulationfrom the rat liver surface after sensitizationwith LPS may be releasedfrom Hc and Kc. It seems that Kc keep releasing NO under physiological condition. The present study is the first direct evidenceof NO production in the Hc and Kc.
TNF-a And IFN-~ Affect On Barrier Function Of Tighl Junction In Immorlallzod Mouse CholanoJocytes. Shinichiro Hanada, Masaru Harada, Kurume Univ, Kurume Japan; Shotaro Sakisaka, Fukuoka Univ, FukuokaJapan; Hironori Koga, Takumi Kawaguchi, Eitaro Taniguchi, Shinji Baba, Shoichiro Shishido, Ryukichi Kumashiro, Takato Ueno, Kurume Univ, Kurume Japan; Yoshiyuki Ueno, Motoyasu Ishii, Tohoku Univ, Sendal Japan Background/Objectives: In primary biliary cirrhosis (PBC), it is well known that activated T lymphocytes are present around the destroyed intralobular bile ducts. Cytokinesfrom CD4+ T lymphocytes are thought to contribute to the bile duct damage together with cellular immunity by CDS+ T lymphocytes. We hypothesizedthat the developmentof inflammation and cholestasis in PBC might be partially promoted by disruption of the barrier function of tight junction (TJ) in cholangiocytes. To reveal this hypothesis, we investigated the effects of TNF-a and IFN-',/in barrier function of TJ in immortalizedmouse cholangiocytes.Materials and methods: Immortalizedmouse cholangiocyteswere grown on the porous filters and either TNF-~ or IFN-~,was added to the apical or basolateralcompartment. To examinethe barrier function of TJs, we measuredtransegithelialresistance(TER) using an epithelialvoltohmmetor. Distribution of TJ-associated proteins including ZO-1, claudin-1 and 7H6 was evaluated by immunofluorescent microscopy. Results: Both TNF-~and IFN--y,when addedto the hasolateral compartment but not to the apical compartment, decreasedTER of cholangiocytes in doseand time-dependentmanner. NeitherTNF-~ nor IFN-~ affects the expressionand distribution of TJ-associated proteins. Conclusions: Results indicate that both TNF-a and IFN-.y inhibit the barrier function of TJs without affecting the distributions of TJ-associeted proteins in cholangiocytes. Cytokines around the intralobular bile ducts may affect the barrier function of TJs and may induce the leakageof bile. Furthermore, some bile constituents may influence the inflammation around the bile ducts in patients with PBC. 1830 Distribution of Tight Junction Associated Proteins, ZO-1, 7H6, and Clandin-1, in Regenerating Rat Liver Eitaro Taniguchi, Masaru Hareda,Takumi Kawaguchi,Hiroto Kumemum, Shinichiro Hanada, Shouichiro Shishido, Shinji Baba, Hironori Koga, Ryukichi Kumashiro, Kurume Univ, Kurume Japan; Shotaro Sakisaka, Fukuoka Univ, FukuokaJapan; Michio Bata, Kurume Univ, Kurume Japan
lm
Phagocytosis of Opsonlzed Red Blood Cells by HepatocytesAfter Gone Transfer of Chimeric Fc ~R III Aa and ,/cDNA Masaya Furukawa, Yasushi Magami, Daiju Nakayama,Fuminori Moriyasu, Tokyo Medical Univ, Tokyo Japan; Jong-Gu Park, Alan D. Schreiber, Univ of PennsylvaniaSch of Medicine, Philadelphia,PA
Background: The functional liver cell mass is restored by the process of liver regeneration in responseto various forms of liver injuries including partial hepatectomy(PH). This complex process is accompanied by transient cholestasis, although the mechanisms are not known. Tight junction (TJ) prevents bile regurgitation into the sinusoidal spaces and alterations in the hepatocyteTJs are seen in many types of cholestasis. However,there is no availabledata about the change of hepatocyteTJs in regenerating liver. Aims: The aim of the present study is to clarify the alteration on the distribution of TJ associatedproteins, ZO-1, 7H6, and Claudin1, in regeneratingrat liver. Materialsand methods: SD rats weighing 200 to 250 g underwent 70% partial hepatectomy (PH) and were sacrificed O, 12, 24, 36, 48, 72, 96, or 168 hr after PH. Resecteitlivers were processedfor immunohistochemistryto investigatethe expression of ZO-1, 7H6, Claudin-l. In separateexperiments, resectedlivers were processedfor electron microscopy to observe the alteration of bile canaliculi. Results: In normal liver, all of ZO-1, 7H6, Claudin-1 were expressed in hepatocytesand biliary epithelial cells (BECs), and they were colocalizedeach/[email protected] regeneratingliver, they were also expressedand colocalized
Aims/Background: Fc-/receptorswhich bind with the Fc portion of IgG play an important role in host defenseand in the inflammatory response by inducing the phagocytosisof antibodycoated microorganisms and the release of inflammatory mediators. Clearanceof circulating immune complexes is disturbed in chronic liver diseaseand in cirrhosis, in which enhanced susceptivity to infection is associatedwith a downregulation of Fc~,receptors.Recentlyit has been reported that adenovirus-mediatedgene transfer of Fc-/R II A cDNA in hepatocytesand lung epithelial cells enhanced clearance of immune complexes. This study evaluates the induction of phagocytosis in hepatocytesby transfection of other type Fc~/receptor,Fc~,RIII A. Methods: We constructed the chimeric cDNA Fc'yR III Aot-'y-Tcoding sequence of the extracellulardomain of Fc-tR III A -aand the transmembraneand cytoplasmic domain of Fc~,R Ill A -'y. cDNA were inserted into the expression vector pRC/CMV under control of the CMV promoter. Fc~,RIII Aot-h,-',/pRC/CMVwas introduced into chang liver cells by electropolation. To assessprotein expressionon the surfaceof transfected cells, FACSanalysiswas performed
A-356
tn hepatocytesand 8ECs. However. the marked bending lines of ZO-1, 7H6, Claudin-1 were observed, mainly during 36 - 96 hr after PH. By electron mtcroscopy, marked bending bile canaliculi were observed. Conclusions: The loss of integral network of bile canalicutus may be one of the possible causativefactors of cholestasis associatedwith regeneratingliver.
Alpha-napthylisothiocyanate-induced CholangiocyteApoptosis is Dependent on Increased Reactive Oxygen Species (ROS) Gene Lesage,Shannon Glaser, Heather Francis, Scott & White Memorial Hosp, Temple, TX; Mikel Ludvik, The Texas A&M Univ System Health Science Ctr, Temple, TX; Jo Lynne Phinizy, Scott & White Memorial Hosp, Temple, IX; Noriatsu Kanno, Gianfranco Aipini, The Texas A&M Univ System Health Science Ctr, Temple, TX
1831
Cholangiocytesrespondto ANITfeeding with cholangiocytedeathand compensatoryproliferation. Cholangiopathiesare associatedwith cholangiocyteapoptosis limited to a range of bile ducts sizes. It remains unknown if ANIT induces cholangiocyteapoptosis and if ANIT-induced apoptosis, like cholangiopathies,is limited to certain sized ducts. The cellular mechanisms of ANIT-inducedcholangiocyteinjury are unknown.Apoptosis is triggered by the accumulation of reactive oxygen species (ROS) in cells. Antioxidants (such as glutathione) protect cells from apoptosis, and hydrogen peroxide by increasing ROS induces apoptosis in cells. The OBJECTIVES of the studies were to determine (i) if ANIT directly induces cholaogiocyte apoptosis in vitro; and (ii) if ANIT-induced apoptosis is due to accumulation of ROS in cholangiocytes.METHODS:Rats were fed 0.1% ANIT for 2 weeks. Small and large cholangiocytes (from small and large bile ducts, respectively)were isolatedfrom rat liver. Cholangiocytes isolated from normal liver were treated with ANIT (10/~), or HA (100 pM) in the presence and absenceof Ethyl ester glutathione (10/~M) for 3 hours at 37°C, Cholangiocyteapoptosis was assessed by the presenceof nuclear fragmentation by DAPI staining. ROS in chotangiocytes was measured by addition of the ROS indicator dye, 2',7'-dichlorofluorascein for 30 minutes and then assessing cellular fluorescence which is proportional to the intracellular ROS concentration. RESULTS:Both apoptosis and ROS generationwere significantly greater in large and small cholangiocytesfrom ANIT-fed comparedto control rats. In vitro, ANIT and H202 induced cholangiocyte apoptosis in both large and small cholangiocytes and this was associatedwith an increasedROSgeneration.Consistentwith increasedROSas the mechanism for ANIT-inducedapoptosis,we found that Ethyl ester glutathionepreventedboth ROS generation and ANIT-induced apoptosis. CONCLUSION:These studies for the first time establish that the hepatotoxin,ANIT induces cholangiocyteapoptosisin both small and large intrahepatic bile ducts. ANIT-inducedapoptosis is due to increasedcholangiocyteROS.Thesedata suggest that the mechanism of drug-induced cholangiopathiesmay be due to ROS and are consistent with the novel idea that cholangiocyteapoptosis in human cholangiopathieasuch as PBC and PSC could alleviated by therapies that reduce ROS in cholangiocytes. 1829
Effects of Idoxifono and Esfradiol on NF-KB Activation in Cultured Rat Hepatocytes Undmleiq OxlaUce Stress Hiroshi Inoue, Ichiro Shimizu, Guaogming Lu, Yajun Zhou, Mina Itonaga, Hirohito Honda, Susumu Ito, Second Dept of Internal Medicine, Tokushima Univ, Tokushima Japan Background: Idoxifeneis one of a series of synthesizedanaloguesof tamoxifen, and a tissuespecific selectiveestrogen receptor modulator. Estradiol is a potent endogenousantioxidant, and nuclear factor kB (NF-kB) is a key transcription factor that induces multiple genes in response to inflammation or oxidative stress. The aim of this study was to explore the inhibitory effects of idoxifena and estradiol on NF-kB activation in hepatocytes in a state of oxidative stress. Methods: Lipid peroxidation was induced in cultured rat hepatocytes by incubation with ferric nitrilotriacetate solution. NF-kBactivity was evaluatedby electrophoretic mobility shift assay. Results:The oxidative stress-inducedactivation of NF-kBand degradation of IkB-alpha are maximal at 3-5 h, with an increase in lactate dehydrogenase(LDH) and malondlaldebyde (MDA) secretion into the culture medium. Treatment with idoxifene and estrediol inhibited the IkB-alphadegradationand NF-k8 activation through the attenuation of hepatocyte oxidative bursts and decreasedextracellular levels of LDH and MDA. In addition, iitoxifena and estradiol inhibited lipid peroxidationin rat liver mitochondria. Whereastamoxiten had no such inhibitory effect. A potent NF-k8 inhibitor, pyrrolidine dithiocarbamate,prevented NF-kB activation by inhibition of IkB-alpha degradation and decreasedLDH and MDA levels, suggesting that NF-kB might be a regulator in agenetic responseto increase oxidative stressinduced hepaticinjury. Conclusion:Thesefindings suggestthat idoxifeneand estradiolfunction as a n t i o x ~ and protect bepatocytesfrom inflammatory cell injury. 18~ Direct Evidence Of Production Of Nitric Oxide In The Lipopolysaccharide-Sensitized Rat HepatonytesAnd Kupfter Coils Tom Kono, Jun Iwamoto, Kazushi Ishikawa, Yosiaki Ebisawa,Takanori Aoki, Nobutosi Ando, Shinichi Kasal, Asahikawa Medical Coil, AsahikawaJapan Backgroundand Aims: We have detectedthe intra-abdominal nitric oxide (NO) releasedfrom the liver surface of the rat sensitized with lipopolysaccharids (LPS) by analyzing the intraabdominal gas by chemiluminescence (Ando et al., Nitric Oxide, 2, 481 1998 and Kamlya et al., Shock, 14, 229, 2000) However, the origin of NO in the liver has not been clarified. In order to identify the production site of NO in the liver, we attemptedto detect NOfluorescence from isolatedhapatocytes(Hc) and Kupffer cells (Kc) by using daiaminofluoresceine-2diacetate (DAF-2DA). Methods: Wistar male rats were injected with 0.1 mg/kg LPS i.p. After 6 hr, rat liver was perfusod with Hanks solution containing collagenasevia portal vein and Hc and Kc were harvested altogether and preserved at 4C. For later identification of Kc, rhodamineconjugated Latex particles (1 pm diameter) were also injected to the LPS-sensitizedrats t hr before perfusion. Kc was isolatedfrom the cluster of harvestedcells with a percoll gradient centrifuge technique immediately after the perfusion. Either Hc or Kc were the transferred to a chamber perfused with Krebs solution equilibrated at pH 7.4 at 33C. Consequently, DAF2DA (10 p.M) was added in the chamber as being excited at 495 nm under a fluorescent micmscopa. The observedHc was later identified using immunohistochemistryfor the intracelMar albumin. Inducible-NOS was also confirmed in the observed Hc and Kc using antiiNOS monoclonal antibody. During observation of NO fluorescence from the cells, Kc was simuRaneouslyidentified with red fluorescence of rhodamine particles, which had beentaken within the cells by phagocytosis. The control experiment was also performed in order to detect NO production under the normal condition. Insteadof LPS administration, the rat only receivedvehicle (saline). The following procedureswere similar to those in the LPS-sensitized groups. Results: Hc and Kc cultured in the chamber gradually developedbright green fluorescence (515 nm wavelength)after excitation in the LPS-sensitizedgroups. However, unsensitized liver did not develop fluorescence in the Hc, whereas the Kc demonstrated similar fluorescence as LPS-sensitizedcells. Conclusions: Theseresu/ts indicatethat intra-abdominal NO accumulationfrom the rat liver surface after sensitizationwith LPS may be releasedfrom Hc and Kc. It seems that Kc keep releasing NO under physiological condition. The present study is the first direct evidenceof NO production in the Hc and Kc.
TNF-a And IFN-~ Affect On Barrier Function Of Tighl Junction In Immorlallzod Mouse CholanoJocytes. Shinichiro Hanada, Masaru Harada, Kurume Univ, Kurume Japan; Shotaro Sakisaka, Fukuoka Univ, FukuokaJapan; Hironori Koga, Takumi Kawaguchi, Eitaro Taniguchi, Shinji Baba, Shoichiro Shishido, Ryukichi Kumashiro, Takato Ueno, Kurume Univ, Kurume Japan; Yoshiyuki Ueno, Motoyasu Ishii, Tohoku Univ, Sendal Japan Background/Objectives: In primary biliary cirrhosis (PBC), it is well known that activated T lymphocytes are present around the destroyed intralobular bile ducts. Cytokinesfrom CD4+ T lymphocytes are thought to contribute to the bile duct damage together with cellular immunity by CDS+ T lymphocytes. We hypothesizedthat the developmentof inflammation and cholestasis in PBC might be partially promoted by disruption of the barrier function of tight junction (TJ) in cholangiocytes. To reveal this hypothesis, we investigated the effects of TNF-a and IFN-',/in barrier function of TJ in immortalizedmouse cholangiocytes.Materials and methods: Immortalizedmouse cholangiocyteswere grown on the porous filters and either TNF-~ or IFN-~,was added to the apical or basolateralcompartment. To examinethe barrier function of TJs, we measuredtransegithelialresistance(TER) using an epithelialvoltohmmetor. Distribution of TJ-associated proteins including ZO-1, claudin-1 and 7H6 was evaluated by immunofluorescent microscopy. Results: Both TNF-~and IFN--y,when addedto the hasolateral compartment but not to the apical compartment, decreasedTER of cholangiocytes in doseand time-dependentmanner. NeitherTNF-~ nor IFN-~ affects the expressionand distribution of TJ-associated proteins. Conclusions: Results indicate that both TNF-a and IFN-.y inhibit the barrier function of TJs without affecting the distributions of TJ-associeted proteins in cholangiocytes. Cytokines around the intralobular bile ducts may affect the barrier function of TJs and may induce the leakageof bile. Furthermore, some bile constituents may influence the inflammation around the bile ducts in patients with PBC. 1830 Distribution of Tight Junction Associated Proteins, ZO-1, 7H6, and Clandin-1, in Regenerating Rat Liver Eitaro Taniguchi, Masaru Hareda,Takumi Kawaguchi,Hiroto Kumemum, Shinichiro Hanada, Shouichiro Shishido, Shinji Baba, Hironori Koga, Ryukichi Kumashiro, Kurume Univ, Kurume Japan; Shotaro Sakisaka, Fukuoka Univ, FukuokaJapan; Michio Bata, Kurume Univ, Kurume Japan
lm
Phagocytosis of Opsonlzed Red Blood Cells by HepatocytesAfter Gone Transfer of Chimeric Fc ~R III Aa and ,/cDNA Masaya Furukawa, Yasushi Magami, Daiju Nakayama,Fuminori Moriyasu, Tokyo Medical Univ, Tokyo Japan; Jong-Gu Park, Alan D. Schreiber, Univ of PennsylvaniaSch of Medicine, Philadelphia,PA
Background: The functional liver cell mass is restored by the process of liver regeneration in responseto various forms of liver injuries including partial hepatectomy(PH). This complex process is accompanied by transient cholestasis, although the mechanisms are not known. Tight junction (TJ) prevents bile regurgitation into the sinusoidal spaces and alterations in the hepatocyteTJs are seen in many types of cholestasis. However,there is no availabledata about the change of hepatocyteTJs in regenerating liver. Aims: The aim of the present study is to clarify the alteration on the distribution of TJ associatedproteins, ZO-1, 7H6, and Claudin1, in regeneratingrat liver. Materialsand methods: SD rats weighing 200 to 250 g underwent 70% partial hepatectomy (PH) and were sacrificed O, 12, 24, 36, 48, 72, 96, or 168 hr after PH. Resecteitlivers were processedfor immunohistochemistryto investigatethe expression of ZO-1, 7H6, Claudin-l. In separateexperiments, resectedlivers were processedfor electron microscopy to observe the alteration of bile canaliculi. Results: In normal liver, all of ZO-1, 7H6, Claudin-1 were expressed in hepatocytesand biliary epithelial cells (BECs), and they were colocalizedeach/[email protected] regeneratingliver, they were also expressedand colocalized
Aims/Background: Fc-/receptorswhich bind with the Fc portion of IgG play an important role in host defenseand in the inflammatory response by inducing the phagocytosisof antibodycoated microorganisms and the release of inflammatory mediators. Clearanceof circulating immune complexes is disturbed in chronic liver diseaseand in cirrhosis, in which enhanced susceptivity to infection is associatedwith a downregulation of Fc~,receptors.Recentlyit has been reported that adenovirus-mediatedgene transfer of Fc-/R II A cDNA in hepatocytesand lung epithelial cells enhanced clearance of immune complexes. This study evaluates the induction of phagocytosis in hepatocytesby transfection of other type Fc~/receptor,Fc~,RIII A. Methods: We constructed the chimeric cDNA Fc'yR III Aot-'y-Tcoding sequence of the extracellulardomain of Fc-tR III A -aand the transmembraneand cytoplasmic domain of Fc~,R Ill A -'y. cDNA were inserted into the expression vector pRC/CMV under control of the CMV promoter. Fc~,RIII Aot-h,-',/pRC/CMVwas introduced into chang liver cells by electropolation. To assessprotein expressionon the surfaceof transfected cells, FACSanalysiswas performed
A-356