Our aim was to investigate whether the antioxidant quercetin protects against liver injury and ameliorates the systemic oxidative stress in rats with common bile duct ligation. Secondary biliary cirrhosis was induced through 28 days of bile duct obstruction. Animals received quercetin (Q) after 14 days of obstruction. Groups of control (CO) and cirrhotic (CBDL) animals received a daily 50 mg/kg body weight i.p. injection of quercetin (CO + Q; CBDL + Q) or vehicle (CO; CBDL). Quercetin corrected the reduction in superoxide dismutase (SOD), catalase CAT, and glutathione peroxidase GPx activities and prevented the increase of thiobarbituric acid reactive substances (TBARS), aminotransferases, and alkaline phosphatase in cirrhotic animals. Quercetin administration also corrected the reduced total nitrate concentration in the liver and prevented liver fibrosis and necrosis. These effects suggest that quercetin might be a useful agent to preserve liver function and prevent systemic oxidative stress.
Biliary cirrhosis is extrahepatic structure and function and is a potential late complication resulting from prolonged obstruction of extrahepatic biliary pathways. It occurs in less than one tenth of patients with biliary cirrhosis, as described previously [
The cytotoxicity of biliary acids is attributed to several mechanisms, including its detergent properties, alterations in intracellular Ca++ homeostasis, decreases in ATP, and mitochondrial damage. It has been suggested that alterations in antioxidant mechanisms occur with the pathogenesis of cholestatic hepatic damage, resulting in an imbalance of oxidative and antioxidative processes, which stimulates lipoperoxidation and leads to injuries in several systems [
Many experimental studies have reported beneficial effects of antioxidants in cholestasis [
Flavonoids are phenolic phytochemicals that represent essential constituents of the nonenergetic part of the human diet. They are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against reactive oxygen species (ROS) [
By increasing the endogenous antioxidant defenses, flavonoids can modulate the redox state of organisms. The major endogenous antioxidant systems include superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx), which are essential for the detoxification of lipid peroxides [
In cirrhosis patients, the use of blood markers to determine the oxidative stress level and the efficiency of the antioxidant used may qualify the diagnosis and allow for a better survival curve [
All studies were performed in accordance with the Guiding Principles for Research Involving Animals at HCPA [
Plasma was collected from the remaining sample by centrifugation at 2195 ×g for 5 min at 4°C. Erythrocytes were washed with cold saline three times. Hemoglobin content of blood was estimated by the cyanmethemoglobin method described by Drabkin and Austin [
For histological studies, a piece of the liver was trimmed and fixed by immersion in 10% buffered formalin for 24 hr. The obtained blocks were dehydrated in a graded series of ethanol, embedded in paraffin, and stained with hematoxylin and eosin or picrosírius, which mainly stains collagen fibers (performed by the Laboratory of Pathology at HCPA-RS, Brazil).
Frozen liver from each rat was homogenized in ice-cold phosphate buffer (KCl 140 mmol/L and phosphate 20 mmol/L, pH 7.4) and centrifuged at 14,000 ×g for 10 min. Nitric oxide production was measured indirectly using a quantitative colorimetric assay based on the Griess reaction, according to Granger et al. [
Results were expressed as the mean values ± SEM. The data were compared by analysis of variance (ANOVA); when the analysis indicated a significant difference, the means were compared with the Student Newman-Keuls test. Values were considered significant at
The cirrhotic group had higher levels of AST, ALT, and ALP compared to the control group. Treatment with quercetin significantly reduced the higher levels of aminotransferases and ALP induced by biliary obstruction (Table
Effects of biliary obstruction determined by hepatic integrity tests.
Parameters | Experimental groups | |||
CO | CO + Q | CBDL | CBDL + Q | |
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AST (U/L) | 95.67 ± 10.34 | 66.44 ± 2.26 | 510.6 ± 46.78a | 146.9 ± 23.44 |
ALT (U/L) | 65.78 ± 9.02 | 38.44 ± 2.51 | 127.5 ± 13.03a | 52.8 ± 9.36 |
FA (U/L) | 156.00 ± 16.62 | 143.33 ± 9.23 | 386.8 ± 27.71a | 218.9 ± 42.73 |
CO: control; CBDL: common bile duct ligation; CO + Q and CBDL + Q: animals received a daily 50 mg/kg body i.p. injection of quercetin.
aSignificant difference between the CBDL group and groups CO, CO + Q, and CBDL + Q;
The histological analysis of liver tissue from the animals in the control group (CO) showed normal architecture of the parenchyma (Figures
Photomicrographs of liver sections from rats (100x). Liver sections were stained with hematoxylin and eosin (a, c and e) or with picrosírius (b, d and f). (a) and (b): control rats (CO), showing normal architecture of liver parenchyma. (c) and (d): untreated rats with common bile duct obstruction (CBDL), showing regenerative nodules (arrow (c)) and fibrosis (arrow (d)). (e) and (f): Biliary obstructed rats treated with quercetin (CBDL + Q), showing the regeneration of liver parenchyma (arrow (e)) and light fibrosis (arrow (f)).
The TBARS concentration was significantly increased in the CBDL rats, whereas this increase was inhibited by quercetin treatment. Lipid peroxidation was not modified by quercetin in normal rats (Table
Effects of quercetin (Q) on lipid peroxidation, antioxidant enzyme activities, and total nitrates on damage due to biliary obstruction.
Parameters | Experimental groups | |||
CO | CO + Q | CBDL + Q | CBDL + Q | |
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TBARS (nmol/mg Hb) | 3.01 ± 0.22 | 2.46 ± 0.29 | 5.69 ± 0.23a | 3.87 ± 0.06b |
SOD (U-SOD/mg protein) | 9.26 ± 0.55 | 8.26 ± 1.02 | 5.35 ± 0.53a | 9.35 ± 0.46 |
CAT (pmol/mg protein) | 0.1 ± 0.01 | 0.11 ± 0.01 | 0.05 ± 0.01a | 0.09 ± 0.01 |
GPx (mmol/min/mg Hb) | 3.45 ± 0.36 | 4.00 ± 0.61 | 2.15 ± 0.69a | 4.63 ± 0.66 |
Nitrates ( |
125.6 ± 18.1 | 110.5 ± 9.12 | 64.99 ± 5.67a | 92.65 ± 5.75 |
CO: control; CBDL: common bile duct ligation; CO + Q and CBDL + Q: animals received a daily 50 mg/kg body i.p. injection of quercetin
Results represent the mean ± S.E.
aSignificant difference between the CBDL group and the CO, CO + Q, and CBDL + Q groups;
bSignificant difference between the CBDL + Q group and the CO and CO + Q groups;
We measured SOD enzymatic activity in erythrocytes and verified a reduction in enzymatic activity in the CBDL group compared to the CO and CO + Q and to the cirrhotic group treated with quercetin (CBDL + Q) (Table In the evaluation of the antioxidant enzymes GPx and CAT, both exhibited a significant reduction in the cirrhotic group CBDL. When animals were treated with quercetin, significantly increased enzyme activity, restoring values similar to those of the control group (Table
Total liver nitrate concentration was significantly reduced in obstructed rats. Values were significantly increased in groups of quercetin-treated animals (Table
Biliary cirrhosis is a chronic and diffuse hepatic disease that alters the intrahepatic or extrahepatic structure and the function of the biliary tree [
Hepatic damage results in increased concentrations of serum AST, ALT, and ALP. Animals that underwent common bile duct ligation showed a significant increase in serial enzymes AST, ALT, and ALP, which are regarded as sensitive markers of hepatic damage and indicators of parenchymal and biliary tree injuries. Hepatic lesions and increases in these serial enzymes were also reported in several studies on bile duct obstruction [
Animals made cirrhotic by CBDL and treated with the antioxidant quercetin experienced restoration of the evaluated enzymes representing hepatic integrity. These results demonstrate that the flavonoid, due to its antioxidant potential, had a hepatoprotective function. Peres et al. have also reported quercetin treatment to reduce the high AST, ALT, and ALP levels present in bile duct obstruction-induced cirrhosis [
When we evaluated the levels of total nitrates in the homogenized livers of the CBDL group, we observed a significant 52% reduction of these metabolites compared to the CO group (Table
Oxidative stress is defined as an imbalance between pro-oxidants and antioxidants; ROS-induced lipid peroxidation can occur either in overwhelmed scavenging systems (excessive production of ROS) or in impaired antioxidant systems. In cirrhotic animals, there is a considerable increase in liver lipoperoxidation due to the formation of ROS. When lipoperoxidation was evaluated, we observed an increase in TBARS in the erythrocytes of the CBDL animals compared to the CO rats. We suggest that the increase in this process is related to the increase in oxidative stress, which has already been shown in hepatic tissue by other studies [
After quercetin treatment, animals from the CBDL group had TBARS values that were similar to those of the control group, indicating the antioxidant potential of this flavonoid. The reduction of lipoperoxidation in the hepatic tissue of the CBDL rats had already been demonstrated by the use of the antioxidant quercetin in the CBDL model [
Due to its antioxidant action, quercetin seems to protect the liver and ameliorate hepatic function; in a secondary pathway, it diminishes bacterial translocation from the gastrointestinal tract and, consequently, the severity of the disease [
The SOD enzyme is the cell’s first line of defense against oxidative stress [
GPx activity was significantly reduced in animals with CBDL, possibly due to increased superoxide anions and hydrogen peroxide, which presumably bind to the active site of the enzyme. Additionally, rats with CBDL lacked vitamin E, and selenium deficiency is known to cause depletion of tissue GPx activity, which makes tissue more vulnerable to oxidative damage [
In summary, our data indicate that quercetin maintains the antioxidant defense system and reduces systemic oxidative damage, ductular proliferation, and fibrosis in biliary obstructed rats. Evaluation of blood during oxidative stress facilitates the monitoring of treatments and the evolution of disease. In this study, we were able to verify the same oxidative alterations reported previously by our group using blood samples instead of tissue homogenates, which were used in previous studies to evaluate oxidative stress on biliary cirrhosis [