The Liquor Containing Pueraria Ameliorates Hepatic Fibrosis in Alcoholic Liver Disease

Hepatic fibrosis is a pathological process in which extracellular matrix accumulates and forms scar tissue due to chronic liver injury caused by various factors, including but not limited to alcoholism, vital hepatitis, and steatohepatitis [1]. Hepatic fibrosis will progress to irreversible cirrhosis and even liver cancer without timely intervention [2]. Recent studies indicate that hepatic fibrosis is reversible when the causative agent(s) is removed in the early stage [3,4]. Therefore, timely intervention before hepatic fibrosis develops into irreversible lesions is warranted. In recent years, traditional Chinese medicine has attracted much attention in the treatment of hepatic fibrosis due to its multi-component and multi-target characteristics. The traditional Chinese medication Pueraria, contains a bioactive isoflavone glucoside called puerarin (4′,7-dihydroxy-8-β-d-glucosylisoflavone), which is a C-glycoside compound and has been shown to have several anti-inflammatory and anti-fibrosis characteristics [5-7]. According to reports, puerarin has an inhibitory effect on Transforming Growth Factor (TGF)-β expression in cardiac tissues, preventing myocardial fibrosis [8]. Puerarin can alleviate kidney fibrosis in renovascular hypertensive rats [9]. Regarding the liver, puerarin exhibits hepatoprotection against CCl4-induced hepatic fibrosis in mice [10]. However, the anti-alcoholic fibrosis effect of pueraria has not been fully confirmed. This study comprehensively evaluated the effects of puerarin on alcoholic hepatic fibrosis in rats through blood biochemical indicators, pathology and imaging, and proposed a basis for puerarin to improve alcoholic fibrosis..

Male Sprague-Dawley (SD) rats (300 ± 10g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd. All rats were kept at 22 ℃ with 12:12 h light-dark cycle. Animal handling and procedures were carried out in accordance with international guidelines for the use and care of laboratory animals. The experimental protocol was authorized by the local ethics committee. During the experiment, rats were divided into two treatment groups: (1) alcohol (n = 8, 42% ethanol by volume alcohol, Niulanshan Liquor); (2) pueraria plus alcohol (n = 8, 42% ethanol by volume alcohol; Yuzhe Liquor). All treatments were administered by gavage once daily. The initial dose of alcohol was 5mL/kg/d, increased at week 2 as tolerance developed to a maintenance dose of 20mL/kg/d and continued for 4 weeks at all. CT images were collected in axial, coronal and sagittal view using liver window (L: 150HU, W: 30HU). The acquired CT images were transferred to United Imaging Medical Processing Software (uWS-CT, United Imaging). The Region of Interest (ROI) was drawn on the liver and spleen, and the average CT value was measured. After CT examination, the activities of serum Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), γ-Glutamyl Transpeptadase (GGT), Alkaline Phosphatase (ALP), Total Bilirubin (TBIL) and Direct Bilirubin (DBIL) were measured using the commercial assay kits, and liver pathological changes were evaluated by Haematoxylin and Eosin (H&E) staining. Hepatic fibrosis was analyzed by Sirius Red staining according to the manufacturer’s instructions. Statistical analyses were performed using SPSS 23.0 software (SPSS Inc., USA). Data were expressed as the mean ± SD. Student’s t test was used for data comparisons, and p < 0.1 was considered as statistically significant.

Effect of puerarin on liver density of CT imaging

Typical CT plain scans of the two groups of rats are presented in Figure 1. CT images showed diffusely increased hepatic uptake in the two groups of rats. However, the mean CT value of liver of the pueraria plus alcohol group was significantly lower than that of the alcohol-induced group. The mean CT value of the liver in the alcohol-induced group was 76.87 ± 1.81, the mean CT value of the spleen was 72.47 ± 1.72, and the ratio of liver to spleen CT value was 1.06 ± 0.01. In contrast, the mean CT value of the liver in the pueraria plus alcohol group was 64.07 ± 0.31, the mean CT value of the spleen was 71.36 ± 1.64, and the ratio of liver to spleen CT value was 0.90 ± 0.02. There were statistically significant differences in the mean CT value of the liver and the ratio of liver to spleen CT values between the two groups of rats (p< 0.05)

Figure 1: A. Typical cross-sectional CT images of the pueraria plus alcohol. B. Alcohol groups of rats. C. The mean CT values of the liver of the two groups of rats. D. The ratio of liver to spleen CT values in the two groups of rats.

Effects of puerarin on serum ALT, AST, GGT, ALP, TBIL and DBIL

The serum ALT, AST, GGT, ALP, TBIL and DBIL levels were measured at 4 weeks after gavage with alcohol in presence of puerarin. As shown in Table 1, ALT, AST and ALP levels in the puerarin plus alcohol group were higher than those in the alcohol group [(28.59 ± 5.23) U/L vs. (26.94 ± 8.26) U/L, p = 0.70; (23.91 ± 5.63) U/L vs. (22.49 ± 7.94) U/L, p = 0.74; (230.46 ± 26.70) U/L vs. (197.73 ± 33.96 U/L), p = 0.10]. Serum GGT, TBIL and DBIL levels in the puerarin plus alcohol group were lower than those in the alcohol group [(7.73 ± 2.19) U/L vs. (8.24 ± 4.04) U/L, p = 0.80; (35.70 ± 10.19) U/L vs. (48.31 ± 9.48) U/L, p = 0.06; (4.91 ± 0.10) U/L vs. (6.10 ± 4.20) U/L, p = 0.52]. The difference between the two groups in ALT, AST, GGT, ALP and DBIL was non-significant. The difference between the two groups in TBIL was significant (p< 0.1).

Table 1:Effect of puerarin on liver related serum indexes in rats.

*p< 0.1

Effects of puerarin on liver pathology

As shown in Figure 2, after the intervention of alcohol, the morphology of liver tissue cells was irregular. In detail, the hepatic cords were arranged disorderly and the structure of the liver lobules was blurred. In addition, round fat vacuoles (lipid droplets) were scattered in the liver. In the puerarin intervention group, the cell structure was complete and the hepatic cord arrangement was significantly improved. The fat vacuoles were reduced, and no obvious degeneration, necrosis and inflammatory infiltration were observed.

Figure 2: A. Sirius Red staining of liver in alcohol. B. Pueraria plus alcohol group.

Several chronic liver diseases frequently result in hepatic fibrosis. Hepatocellular carcinoma can occur as a result of chronic fibrosis, according to abundant evidence [11]. Interrupting and/or reversing hepatic fibrosis is critical in preventing the development of hepatocellular carcinoma. However, there is no well-validated therapy for reversing liver fibrosis at this time. The potential of puerarin to alleviate alcohol-induced hepatic fibrosis was proven in this study via assessing liver-related biochemical indicators, CT imaging, and pathological changes. Chronic alcohol consumption caused considerable damage to hepatocytes, resulting in pathological elevation of the serum levels of ALT, AST, GGT, ALP, TBIL and DBIL. However, on administering puerarin, we have observed decreased levels of serum GGT, TBIL and DBIL, supporting the hypothesis that puerarin could reduce hepatic fibrosis by metabolizing total bilirubin. In addition, histological analysis showed that the rat model of alcohol-induced fibrosis was successfully established, which was characterized by marked fatty degeneration and peri hepatocyte fibrosis. Furthermore, histological analysis showed that the pathogenic characteristics of liver tissues were significantly reduced in the puerarin therapy group. CT diagnosis of hepatic fibrosis could be preferred because of its repeatability, non-invasiveness, and ability to dynamically assess the lesion. This study noninvasively assessed the effect of puerarin on fibrosis by CT imaging, which was manifested in the decrease of diffuse CT density of the liver compared with the alcohol-induced group. In conclusion, the results of this study suggest that puerarin is effective in the treatment of alcohol-induced liver fibrosis in rats. The main mechanism of this therapeutic effect may be due to its protection against liver injury by metabolizing total bilirubin. Therefore, based on our results, puerarin should be considered as a drug or an additive with potential application in the prevention and treatment of alcoholic liver fibrosis.

Puerarin may serve as a candidate with high potential to manage hepatofibrosis via effectively ameliorating the metabolism of total bilirubin.

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