A combination of Ang Ⅱ and carbon tetrachloride accelerates process of hepatic fibrosis

62.Chinese Medical Journal 2003 ; 116( 1 ):62-65A combination of Ang ]I and carbon tetrachloride accelerates processof hepatic fibrosisZHOU Xin周馨 , LI Dingguo李定国, LI Xuanhai李宣海, LU Hanming陆汉明and ZHANG Wenzhu张文竹Keywords : fibrosis . angiotensin I . carbon tetrachlorideObjective To assess whether Angiotensin I ( Ang I ) and carbon tetrachloride ( CCI4 ) used incombination could accelerate the process of fibrosis and whether Ang I play a role in exaggerating hepaticfibrosis in rats.MethodsAng I was injected into the abdominal cavity of Sprague-Dawley( SD ) rats together withsubcutaneous injection of CC4. Rats were klld after 14 and 28 d. Blood serum and liver specimen werecollected. The extent of fibrosis in the stained liver tissue sections was determined with the KS 400 ImageAnalysis System.Results Rats receiving Ang I and CCl4 for 28 d showed extensive liver fibrosis . Along with the increaseof hepatic fibrosis , the serum concentration of Ang I went up gradually.Conclusions A combination of Ang I and CCl4 would accelerate the process of hepatic fibrosis. Ang Iprobably took part in the ccurrence of heparic fibrosis.Chin Med J 2003 ; 116( l ):62-65Hepatic fibrosis is the common pathway leading to hepaticbought from Shanghai Bi Kai Experimental Animal Co Ltd ,cirrhosis in various chronic liver diseases. Hepatic stellateChina. Powder form Ang II was bought from Callbiochem (cells( HSC ) play a pivotal role in the occurrence of hepaticLtd , Germany. We dissolved the powder in 5% acetic acidfibrosis. Activated by many cytokines and chemokines , HSCand adjusted the solution with 0.5 mol/L PBS to pH 7.2-transform into myofibroblasts and form an excess of7.6. CCl4 was diluted to a concentration of 40% with oliveextracellular matrix ( ECM ) far exceeding its degradationoil. 108 healthy male SD rats were divided into 6 groupsuntil hepatic fibrosis forms .randonly , 15 - 22 each. They were normal control group( 17 rats ), buffer control group( 16 rats ) , olive oil controlAt present，the major way to study experimental hepaticgroup( 18 rats ), CCl4 group( 20 rats), Ang II and CClfibrosis is to make a model by injecting CCl4 to ratscombined group( 22 rats )and Ang II group( 15 rats ). Thesubcutaneously to cause hepatocytic damage and subsequentrats of the CCl4 group were received CCl4 subcutaneouslyliver tissue fibrosis. It takes at least 8 wk. The study of(0.3 mg/100 g) every 3 d and PBS solution by abnominalHafizi et al' and Brown2 revealed that if the myocardiumcavity , equal to the volume in the combined group. Ang Ior kidney interstitial myofibroblasts were incubated in awas injected into the abdominal cavity of the combined groupsolution of Ang II , myofibroblasts would proliferate andrats(0.3 mg/100 g/d , equal to 1 ng/g/min), and to beECM would increase. If activated HSC were incubated irvarious concentrations of Ang I ，they would divide ,divided into 2 doses , given twice daily besides subcutaneousinjection of CCl4. The Ang II group rats also receivedproliferate and contract .These changes were all throughthe action of Ang I typeI receptor. Thus we may supposeAng Il takes part in the generation of hepatic fibrosis. InDepartment of Gastroenterology and Hepatology ，Xin Hua Hospital ,this experiment we combined the use of Ang II and CCI4 toShanghai Sgrnd Mwtign! Ininnit Slonghai 200092 , China ( Zhousee whether they will accelerate the process of hepaticX,Li DC中国煤化工ShanghaiC N M H Gehai 20025 ,China( Li XH)fibrosis in rats and to verify the role of Ang II in hepaticDepartment ol Tatthology ，Ain Hua Hosptal , Shanghai Second Medicalfibrosis.University , Shanghai 200092 , China ( Zhang WZ )METHODSCorrespondence to :Dr. Zhou Xin , Department of Gastroenterology andHepatology , Xin Hua Hospital , Shanghai Second Medical University ,AnimalsShanghai 200092 , China( Tel : 86-21-65790000 ext 5319. Fax :86-21-Male Spragu万振y( SD )rats , weighing 250土10 g ,vere55571294. E-mail : xzhouch@ hotmail . com )Chinese Medical Journal 2003 ; 116( 1 ):62-65injections of olive oil subcutaneously every 3 d( equal to the .level of ALT and AST of the combined group at the end ofvolume in the CCI4 group ) and Ang II ( equal to the volumethe second and the fourth week were significantly higher,in the combined group ). The normal control rats were givenwhile ALB lowered( P <0.01 ). As time went on , liverno treatment and the buffer control group animals werefunctions of these rats deteriorated( Table 1 ).injected equal volume of PBS solution as in the combinedTable 1. 'The infuence of combining CC4 and Ang IIgroup. Rats of the olive oil group were injected olive oil in ato ALT ,AST ,ALB(xts)volume equal to that in the CCL4 group. Animals were killedGroupALT(μ/1)AST(p/1l)ALB( g/)at the end of the second and the fourth week. LiverNormal control 9132.75+11.66 217.00+22.37 19.38土 1.69specimen were preserved at - 20C.Buffer control130.00+ 15.03232.00+27.9618.50+1.51Olive oil control 10 126.50+14.78 212.63+19.11 19.13+1.25Serum concentration of Ang I[CCL48 715.75+97.38* 997 8士104.79* 15.88士1.25*Radioimmunity Test Kit of Ang II was bought from BeijingCombined13 1132.63+113.29↑ 1038.69土126.04* 14.46土0.88*Northerm Biotech Institute , China. 25 μl enzyme inhibitorAng II72.00+32.24*__ 869.00+64.97*_ 16.86 +0.69”was put into 1 ml of rat blood , fllowed by centrifuging in a￥P<0.01 vs control goup,+ P<0.01 vs CCL4 group,O P<0.05 vs CC4groupBeckman GS-6 centrifuge , 4000 r/min ,4C. 200 pl blood .serum was aspirated and then 125I labeled antigen andMarkers of hepatic fibrosis after Ang II and CCl4antibody were added into the serum. 7 standard tubes of Angcombined treatmentII at concentrations of 0 , 25 , 50，100, 200 , 400 , 800Collagen IV ,laminin , hyaluronic acid and PIl P are usuallypg/ml and another T tube were set up. The blood serumused as serum markers of hepatic fibrosis. When Ang I andconcentration of Ang II was determined by Radio immuno-CCl4 were used together , the values of collagen IV , lamininassay.were heightened. This means an enhancement of the processof hepatic fibrosis after the comined treatment( Table 2).Comparison of liver functionAt the end of the second and the fourth week , AlanineTable 2. The infuence of combining CCl4 and Ang IIaminotransferase ( ALT ) , Aspartate aminotransferase( AST )on collagen IV and laninin( 无土s )and Albumin( ALB ) of rats were detected according to theGroup .Collagen IV ( ng/ml )routine biochemical method.Nornal control16.75+0.97 .44.36+3.5018.08土1.0544.663.28Marker of hepatic fibrosisOlive oil17.09土1.63.44.78+3.97cc75.01 +7.85Radio immuno test Kit for collagen IV and laminin were usedCombining1369.90+7.11*95.53士2.20*to detect the two markers of hepatic fibrosis.Ang II __47.81+3.14*58.40+5.45*+* P<0.01 vs contol goup,t P<0.01 vs CCL4 groupThe degree of hepatic fibrosisVG-stained liver sections were observed and analyzed byThe influence of combining Ang I and CCl4 on the .KS400 Image Analysis System of Zeiss Co Ltd , Japan. Theprocess of hepatic fbrosispercentage of the fibrosis area over the whole observed fieldImage analysis showed that processes of hepatic fibrosis inwas assessed to represent the degree of hepatic fibrosis.the CCl4 group , combined group and Ang I group werequite notable compared with the three control groupsStatistics( P<0.05 ). Among them，the hepatic fibrosis of theData were expressed as元土s. One factor analysis olcombined group is the most remarkable( P<0.05 ). Aftervariance was used to analyze the variance among groups and28 d，we could see pseudo-lobules in the liver of theStudent' s t test was used to analyze the variance betweencombined group , whereas in the CCl4 group fibrosis was onlytwo groups. All data were analyzed by Statpal Statisticalmild( Table 3 , Figs.1 and2 ).Package .Table 3. The infuence of combining Ang II and CCI4 on the processRESULTSof hepatic fibrosis at the end of the secondand fourth week( x士s)The influence of Ang I and CCl combined treatment onALT , AST and ALBGrou中国煤化工28d(%)No-0.45+0.13The liver function of rats in normal control group , bufferBuffeYHCNMHG0.44+0.08control group , olive oil control group showed no differenceOlive oil control0.40土0.08.0.46+0.082.83 +0.44*7.53+3.46 'before and after the experiment. Levels of ALT and AST of4.02 +0.87*+212.52+6.14*the CCI4 , Ang II and combined groups went up strikinglyAng2.68+0.40*t4.09+0.66( P<0.01 ). Levels of ALB in these groups lowered greatly* P<0.05 vs the three control group,+ P<0.05 vs the CC4 goup,△P<( P<0.0ared with the CC4 and Ang II goups ,0.05 vs the Ang II group64.Chinese Medical Journal 2003 ; 116( 1 ):62-65DISCUSSIONLocal activation of RAS is the major mechanism of fibrosis inthe heart and kidney. But it' s still unconcemed whether itplays a role in hepatic fibrosis. In this study we combinedthe use of Ang II and CCI4 to set up a new model of hepaticfibrosis, and to see whether Ang I could enhance theprocess of hepatic fibrosis. It was observed that with thecombined treatment hepatic function got worse , blood serummarkers deteriorated and progression of hepatic fibrosisspeeded up. It would take at least 8 wk to set up a model ofhepatic fibrosis with CCI4 alone , whereas in our study , after4 wk , pseudo-lobules were extensively formed in combinedFig. 1. After 28 d , pseudo-lobules can be seen obviously ( HE ,group. Thus, we might suggest that Ang Iwas weloriginal magnificationx 200 ).absorbed by the abdominal cavity and hepatic fibrosisspeeded up. Kohara et al* injected Ang II into vein of ratscontinuously at 6 nmol/h corresponding to 1ng/ 'g weight/min. They found that blood serum level of renin descendedwhile the level of blood angiotensinogen and Ang IIelevated. This is coincided with our study. But we stillcannot say whether it is the result of endogenous 0Iexogenous activation of AngII .Recent studies indicated that RAS is relevant to theformation of fibrosis in myocardium and kidney. Localactivation of RAS is also correlated with the fibrosis of skinand pancreas. Fibrosis induced by Ang II has nothing to dowith alteration of hemodynamics.5A study led by Australiaepidemiologists showed thatHCVpatients withFig. 2. After 28 d , hepatic fibrosis is mild in the CCL4 group ofangiotensinogen and/ or TGF_β genotype were more likely tcrats，and pseudo-lobules cannot be found ( HE，originalresult in hepatic fibrogenesis.' Wei et al7 usedmagnification x 200 ).angiotensin converting enzyme inhibitor and Ang I receptorinhibitor to treat rats with hepatic fibrosis induced by CCl4Serum level of Ang I at the end of second and fourthand discovered that the two could restrict the process ofweekhepatic fibrosis obviously , along with a gradual elevation ofAt the beginning，there was no difference among each groupserum Ang lI level. Besides their studies , many otherin the serum levels of AngII . But after two weeks , serumforeign scholars also found that angiotensin convertinglevels of Ang II in the three treated groups went upenzyme inhibitor and Ang II typeI receptor inbibitor( Table 4 ). The concentration in the combined group was thecould relieve the degree of hepatic fibrosis via the Ang IIhighest among the three groups( P<0.01 ).type I receptor89 and the serum level of Ang II in themodel groups were much higher than the nommal controlTable 4. The variation of serum levels of Ang II at the beginninggroup and the treated groups. It means that serum level ofandtheendofthe2ndand4thweek(元士s)Ang II went up , following the process of hepatic fibrosis.Al theAt the endExogenous Ang II can be used to induce heart and kidneyGroupbeginningof 2nd weekof 4th weekfibrosis. 10.11Fibfosis formed , followed by the increase ofNormal control830.29+193.06 782.13+39.49778.70土26.66Ang中国煤化工ase might be related to770.89士169.35764.95 +49.70753.55+ 18.15fibrosis:MYHCNMHGOlive oil control763.35+82.62 7.58+41.27801.41士48.94CA4758.16+125.79 922.07 土66.211263.57士26.02 *In conclusion ，Ang l may promote hepatic fibrogenesisCombined752.15土110.81 1207.77+91.77* 157.55+22.59*induced by CC4. The blood serum Ang I[ concentrationAng II779.91+45.54 952.65+57.96*1088. 19+25.55** P<0.01智鹦理,十P<0.01 vs CCI4 group,0 P <0.01 vs AnglIrises , as hepatic fibrosis progresses. Ang II might play agroup.role in the process of hepatic fibrosis.Chinese Medical Journal 2003 ; 116( 1 ):62-65REFERENCESHepatology 2000 31 828-833.7. Wei HS ,Lu HM ,Li DG ,et al. The expression of ATI receptor1. Hafizi S , WhartonJ , Morgan K ,et al. Expression of functionalon hepatic sellate cells in rat fibrosis induced by CCl4. Chin Medangiotensin- converting enzyme and AT1 receptors in culturedJ 2001 ;114 583-587.human cardiac fibroblasts. Circulation 1998 98 2553-2559.8. Yoshiji H , KuriyamaS, Yoshii J ,et al. Angiotensin I type I2. BrownL ,Duce B , Miric G ,et al. Reversal of cardiac fibrosis inis a major regulator for liver fibrosisdeoxycorticosterone acetate- salt hypetensive rats by inhibition ofdevelopment in rats. Hepatol 2001 34(4 pt 1 ) .745-750.the rennin- angiotensin system. J Am Soc Nephrol 1999 ;10 S143-. Jonsson JR，Clouston AD，Ando Y，et al. Angiotensin-S148.converting enzyme inhibitor attenuates the progression of rat3. Ramon B , Pere G , Josep MN , et al. Angiotensin I inducedhepatic fibrosis. Gastroenterol 2001 ;121 :148- 155.contraction and proliferation of human hepatic stellate cell.10. Matsusaka T，Katori H，Inagami T，et al. CommunicationGastroenterology 2000 ;118 :1 149-1156.between myocytes and fibroblasts in cardiac remodeling in4. Kohara K , Brosnihan KB , Ferratio CM，et al. Peripheral andangiotensin chimeric mice. J Clin Invest 1999 ;103 :1451- 1458.central angiotensin II regulates expression of genes of the rennin-11. Yoo KH , Thomhill BA , Wolstenhome JT ,et al. Tissue specificangiotensin system. Am J Physiol 1992 262 :E651-E657.regulation of growlb-factors and clusterin by angiotensin II. J5. Gill GN, 1l CR，Simonian MH. Angiotensin stimulation oHypertens 1998 11(6 pt 1)715-722.bovine adrenocortical cell growth. Proc Natl Acad Sci U S A1997 ,74 5569-5573.，Powell EE, Edwards-Smith CJ, Hay JL, et al. Host genetic( Received December 30 , 2001 )factors influence disease progression in chronic hepatitis C.本文编辑:王德罗丹中国煤化工MHCNM HG