| Objective: Hepatic fibrosis is a wound-healing response to liver injury and the result of imbalance of extracellular matrix(ECM)accumulation and degradation.Although significant progress has been achieved in elucidating the mechanisms of fibrogenesis,effective anti-fibrotic strategies are still lacking.However,the treatment of traditional Chinese medicine has the holism in mind and has a superiority in synthetic action,because the treatment based on syndrome differentiation in anti-fibrosis therapy.Astragalus membranaceus,salvia miltiorrhiza,poria,radix curcumae,white cardamom,etc compose the Yiqi Huoxue recipe(YQHX),has the effects of“supplementing qi and activating blood circulation,soothing liver and strengthening spleen”.Studies have found that astragaloside exerts the protective effect from the injured cells through inhibiting the cell damage induced by excessive autophagy.Autophagy is a catabolic intracellular pathway,targeting defective or excessive organelles to the lysosomes for degradation into amino acids,free fatty acids or other small molecules used for material recycling or energy harvest.A recent study identifying the breakdown of intracellular lipids by autophagy to generate fatty acids for energy production in hepatocytes raised the possibility that the loss of LDs by autophagy drives the fibrogenic response in stellate cells.However,the exact relationships between autophagy and hepatic fibrosis,the molecular mechanism of Yiqi Huoxue(YQHX)recipe in modulation of autophagy are not totally clear and need further investigations.This study was demonstrated that the effects of YQHX on the carbon tetrachloride(CCl4)-induced rats liver fibrosis,along with Fuzheng Huayu(FZHY)as the positive control.Secondly analyses the relationship of autophagy and liver fibrosis,and YQHX make a contribution to therapeutic strategy of liver fibrosis.Methods: Male Wistar rats were randomly divided into four groups: i)CCl4 group,rats were administrated with a mixture of CCl4(30%)and olive oil(70%)(2 ml/kg)twice a week for 8 weeks by intraperitoneal injection;ii)CCl4+YQHX group,rats were treated with YQHX(3.4g/10ml/kg·d)by gavage from the beginning of CCl4 injection for 8 weeks;iii)CCl4+FZHY group,rats were treated with FZHY(0.46g/10ml/kg·d)by gavage from the beginning of CCl4 injection for 8 weeks;?)control group,rats were administered with equivoluminal vehicle.All rats were sacrificed at the end of8 weeks,blood and liver samples were obtained.Serums alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were tested by enzymic method.Hepatic inflammation and fibrosis of rats were observed by hematoxylin and eosin(HE)and Masson's trichromatism staining(Masson)stained liver sections.Hepatic mRNA and protein expressions of?-smooth muscle actin(?-SMA)and alpha-1 type I collagen(Col1A1)were detected with immunohistochemistry,quantitative real-time PCR and western blot.The mRNA and protein expressions of autophagy-related gene 7(Atg7),microtubule-associated protein 1 light chain 3(LC3)and sequestosome1(SQSTM1/p62)were analyzed by quantitative real-time PCR and western blot,respectively.Statistical analysis on the data was performed by one-way analysis of variance(ANOVA),with Student-Newman-Keuls analysis for comprison among groups.P-values below 0.05 were considered statistically significant.Results:1 The general situation of experimental animals: Control rats looked spirited,swift;while the rats admistrated with CCl4 for 8 weeks looked exhausted,less dynamic;rats administered with YQHX and FZHY presented better general condition compared to CCl4 group.2 The serum biochemistry of experimental animals: The serum ALT and AST levels were increased in CCl4 group(106.80 ± 18.24 vs 38.17 ± 4.99 and278.40 ± 66.14 vs 121.00 ± 11.87,P<0.001)compared to the control group,and were amelioration by the administration of YQHX and FZHY(66.80 ±10.42,122.60 ± 16.65,and 73.20 ± 10.33,125.4 ± 16.92,P < 0.001).3 The change of liver histopathology in the rats: Rat admistrated with CCl4 for 8 weeks showed severe hepatic injury including necro-inflammation and fibrosis,pseudo lobular formation(S 5.64 ± 0.22),The administration of YQHX and FZHY were found to reverse hepatic fibrosis(S 3.70 ± 0.35 and S3.90 ± 0.34,respectively).4 The hepatic mRNA expression of ?-SMA,Col1A1,Atg7,LC3 B and p62 in the rats: Compared to the control group,the mRNA expression of?-SMA,Col1A1,Atg7 and LC3 B were up-regulated in CCl4 group,p62 was down-regulated(0.88±0.13 vs 0.19±0.07,0.99±0.08 vs 0.05±0.02,1.11±0.12 vs 0.33±0.07,0.92±0.07 vs 0.32±0.02,1.16±0.17 vs 3.18±0.18,respectively,P<0.001);whereas the levels of ?-SMA,Col1A1,Atg7 and LC3 B in rats admistrated with YQHX and FZHY were descended compared with CCl4 group,and p62 was increased(0.34±0.05,0.32±0.09,0.66±0.08,0.66±0.07,1.79±0.09,and 0.48±0.04,0.31±0.11,0.74±0.07,0.58±0.03,1.45±0.03,respectively,P<0.05).5 The hepatic protein expression of ?-SMA,Col1A1,Atg7,LC3 B and p62 in the rats: The integrated optical density(IOD)of ?-SMA and Col1A1 were increased in CCl4 group compared with the control group(34675.09±1200.29 vs 10360.13±1442.25,22480.85±1035.92 vs2915.58±574.99,respectively,P<0.001);The IOD of ?-SMA and Col1A1 were suppressed by YQHX and FZHY treatment compared to the CCl4 group(22051.47±1610.70,12565.8±932.23,and 22556.52±1510.60,12997.9±903.85).Compared to the control group,the protein expression of?-SMA,Col1A1,Atg7 and LC3 B were up-regulated in CCl4 group,p62 was down-regulated(0.83±0.05 vs 0.05±0.006,0.73±0.002 vs 0.29±0.02,0.12±0.002 vs 0.06±0.002,0.83=±0.02= vs 0.53±0.02,0.26±0.02 vs 0.68±0.03,respectively,P<0.001);whereas the levels of ?-SMA,Col1A1,Atg7 and LC3 B in rats administrated with YQHX and FZHY were descended compared with CCl4 group,and p62 was increased(0.19±0.02,0.35±0.003,0.10±0.002,0.67±0.02,0.4467±0.02 and 0.31±0.02,0.47±0.003,0.10±0.003,0.62±0.03,0.35±0.04,respectively,P<0.001).Conclusions:1 Autophagy of liver cell was involved in the inhibitory effects of liver fibrosis,Atg7,LC3 B and p62 played a crucial role in the process of liver fibrosis induced by carbon tetrachloride.2 Both YQHX and FZHY could improve fibrosis through suppressing the expression of autophagy factor in rat with CCl4-induced liver fibrosis.3 Yiqi Huoxue recipe has the effects of “replenishing qi to invigorate the spleen,promoting blood circulation to remove blood stasis”.On tne basis of physical and chemical indicators,YQHX can be multiple targets for resisting hepatic fibrosis,accordingly,YQHX is expected to become new type of traditional Chinese medicine prescription for the treatment of liver fibrosis and cirrhosis of the liver. |
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