The Protective Effect Of The Licorice Flavonids On The Thioacetamide-induced Liver Fibrosis And Related Mechanism Study In Rats

Background:Hepatic fibrosis （HF）is a healing response to liver injury on a variety of damage factors.The pathological feature is the excessive deposition of the extra cellular matrix （ECM）, whichis a common pathological process in a varity of chronic liver disease. The studies showed thatthe hepatic frbrosis is a kind of reversal pathological changes. But there is still no effectiveantifibrotic mdication available in drug based therapy and in clinical methods. So, how tolook for the drugs that can reverse the liver fibrosis are still the hot spots in searching thenew hepatoprotective drug.Anti-liver fibrosis drugs driven from the Chinese herbal medicine have goodpharmacological efficacy. According to our preliminary work, the flavonoids extract fromChinese herbal medicine Licorice has many pharmacological activities in animal and inhuman. Licorice of artificial cultivation technology has been successful in NingXia. Thegrowing area has reached50,000acres; fenced area has reached400,000acres, which is asthe rich resources to protect. Combined with industrialization and after a lot of work had beendown in the chemical composition and quality an alysis, the relevant quality standards and（total） extraction of flavonoids content stan-dards had been drawn up.In the present study, we applied the thioacetamide-induced hepatic fibrosis as an animalmodel in rats, and clearfied the anti-liver fibrosiscal pharmacological activities of the licoriceflavonoids（LF）. In study the molecular mechanisms of its hepato protective effect at the same time, we still hoped to provide a reliable pharmacological studies in the development andutilization of licorice.Aim:The pharmacological effects of licorice flavonoids intervention of the hepatic fibrosis inexperimental animals were studied by employing the thioacetamide-induced rat liver fibrosismodel, the blood biochemical and histopathological method.And the biochemicalindices of liver function and liverfibrosis-related pathological parameters wre investgated inthe study as well. By molecular biological and histopathological methods, the proteinand gene expression levels of transforming growth factor-beta1（TGF-β1） and caspases-3were been observed, and the possible mechanism of licorice flavonoids against liver fibrosiswere been discussed in the licorice flavonoids intervention.Methods:Eighty-four male Sprague-Dawley rats were divied in seven groups. Rats were givenintraperitoneal injection of thioacetamide （TAA）（3%,150mg/kg body weight, solution insaline）, twice a week for twelve weeks. At the same time, some of these rats were givenlicorice flavonoids （LF） and control drug silymarin（30mg/kg） by a intragastric administrationin one time every day. Thus, seven groups each including twelve animals could bedistinguished. Control group, saline water alone; TAA group, TAA with saline water;low-dose LF group, TAA with LF （50mg/kg bodyweight）; middle-dose LF group, TAA withLF （100mg/kg body weight）; plus middle-dose LF group, TAA with LF （200mg/kg bodyweight）; high-dose LF group, TAA with LF （400mg/kg body weight）.At the end of the experiment （after twevle weeks）, the rats were anaesthetized withpentobarbital sodium （45mg/kg, ip）. The blood was centrifuged with3000rmp after fourhours in room temperature for serum markers of hepatic fibrosis and function study. Theserum alanine transaminase （ALT）, aspartate transaminase （AST）, alkaline phosphatase（ALP） and liver hydroxyproline（HYP）contents were assayed by spectrophotometric method. The serum hyaluronic acid （HA） was assessed by radioimmunoassay. Liver samplescollected were stained with hematoxylin and eosin （HE） and Masson’s Trichrome. TheTGF-β₁and Caspase-3protain in liver tissue was examined by immunohistochemistryapproaches. The mRNA expression of the TGF-β₁was examined by Quantitative real-timePCR analysis.Results：1. Effects of LF on liver damage（1） Compated with the control group, TAA caused the serum levels of ALT、AST andALP increases significantly（P<0.01）.（2） Co-administration of LFs and silymarin was associated with a significant reductionin the serum levels of AST, ALT, and ALP（P<0.01）.The data suggested that LFprotects hepatocytes against TAA-induced damage in rats as well as silymarin.2. Serum markers of hepatic fibrosis and function（1） TAA can induce the hepatic fibrosis by increased the levels of HA in the serum andHYP level in liver tissues respectively（P<0.01）. The rat livers treated with TAA fortwelve weeks developed hepatic fibrosis.（2） LF groups and silymarin group significantly attenuated the TAA-induced increase inserum HA and liver HYP levels.3. Histopathological examination（1） Histological analysis showed that TAA cause the rat liver volume increase andweightincrease. It shows that the liver surface is not smooth with sizes granularappearance. And the grains and adhered with surroundings organs can be observedalso.（2） By HE and Masson’s trichrome staining showed that distorted tissue architect-turewith bundles of collagen surrounding the lobules as well as large fibrous septa.（3） Co-administration of LFs and silymarin significantly attenuated the deposition of collagen fibers. in a dose-dependent manner（P<0.01）. The data suggested that LFcan suppress TAA-induced liver fibrosis in rats as well as silymarin.（4） LF groups in400mg/kg dosage could maintain the liver tissue lobule structurecompleted, and reduced the degree of liver cell degeneration, crosis and fibroustissue HYPerplasia significantly （P <0.05, P <0.01）,4. Affect the outcome of liver TGF-β₁protein expression（1） The normal tissue TGF-β₁protein expression was weak.（2） TAAcan attenuate the expression of TGF-β1significantly （P<0.01）.（3） Co-administration of LFs and silymarin was associated with a significant reduction inTGF-β₁protein expression in a dose-dependent manner（P<0.01）.（4） The efficacy of the400mg/kg dose is superior to other groups. LFs and silymarin hasno differenc in TGF-β₁protein expression.5. Affect the outcome of liver Caspase-3protein expression（1） TAAcan attenuate the expression of Caspase-3significantly （P<0.01）.（2） Co-administration of LFs and silymarin was associated with a significant redu-ctionin Caspase-3protein expression in a dose-dependent manne（rP<0.01）.The efficacy ofthe400mg/kg dose is superior to other groups.（3） LFs and silymarin has no differenc in Caspase-3protein expression（P>0.05）.6. Quantitative real-time PCR analysis（1） TAAcan attenuate the TGF-β₁mRNA significantly （P<0.01）.（2） Co-administration of LFs and silymarin was associated with a significant reduction inTGF-β1mRNA expression in a dose-dependent manner（P<0.01）. The efficacy of the400mg/kg dose is superior to other groups.（3） LFs and silymarin has no differenc in TGF-β₁mRNA expression（P>0.05）.Conclusion：1. The licorice flavonoids can suppress the thioacetamide-induced hepatic fibrosis in rats. 2. The mechanisms of anti-hepatic fibrosis may be associated with reduced TGF-β₁and Caspase-3protein expression in liver.