Studies on the Antiliver Injury of Effective Parts from Saururus chinensis by Rats and Mice Model In Vivo

The aim of this study was to evaluate the pharmacodynamics of the effective parts from Saururus chinensis (EPS) in vivo. The antihepatic fibrosis and injury effects of EPS were investigated with the following four model animals including the effect on Wistar rats with liver fibrosis induced by complex factors, mice with acute liver injury induced, respectively, by carbon tetrachloride and alcohol, and Sprague-Dawley (SD) rats with acute liver injury caused by D-galactosamine hydrochloride. The pharmacodynamics results showed that the rats' oral administration of EPS can significantly inhibit the formation of liver fibrosis in rats caused by complex factors and has significant preventive and therapeutic effects on acute liver injury caused by various factors as shown by decreased levels of serum biochemical indicators and improved pathological grade. Taken all together, our findings showed that EPS exhibits potent activities and should be considered a good option and an additional source of natural compounds for the treatment of hepatic fibrosis and hepatic injury.


Introduction
Saururus chinensis (S. chinensis) is the dry aerial part of Saururus chinensis (Lour.) Baill (saururaceae), and it has been used in chinese folk medicine for the treatment of various diseases such as jaundice, urinary tract infection, edema of nephritis, dysuria, gonorrhea, leucorrhea abnormality, swelling sores, and eczema [1,2]. Modern studies have shown that S. chinensis contains lignans, alkaloids, terpenes, flavonoids, and other components and has a variety of biological activities such as anti-inflammatory, antitumor, and antioxidative [3].
Liver fibrosis is the intermediate link and necessary stage of various causes of liver disease from the progressive development to end-stage liver disease, and it is very important to prevent the progression of liver fibrosis and the reversion of developed liver fibrosis, which can reduce the occurrence of end-stage liver disease (cirrhosis, liver cancer) and thus reduce the mortality related to liver disease. It is urgent to reduce the damage to the liver and to slow down the progress of liver injury to fibrosis and cancer. erefore, on the basis of our previous chemical components and biological studies of S. chinensis [4][5][6][7][8][9], in our present study, effective parts from the herbs of S. chinensis (EPS) (the sum of total lignans, total lactams, and total sterols is greater than 50%) were used to examine its hepatoprotective effects. Its antiliver injury efficacy was studied systematically by four animal models, including the effects on liver fibrosis rats induced by complex factors, acute liver injury mice and rats caused by carbon tetrachloride, alcohol, and D-galactosamine hydrochloride, respectively. e liver inflammation and hepatofibrosis-improving effects were evaluated according to AST, ALT (serum biochemical indicators for liver inflammation), HA, LN, and COLIV, PC III (four serum markers of rats with liver fibrosis), TP, ALB (liver cell regeneration indicators), SOD, T-AOC, and MDA (antioxidant indicators), as well as histopathological examination of rats and mice liver. is is the first study systematically on hepatoprotective effects of S. chinensis in vivo. is is the first time to use four different liver injury models to study antifibrosis and liver injury systematically.

Plant Material.
e whole plant materials were purchased and collected from Chenzhou City, Hunan Province, and identified as S. chinensis by Professor Dacheng Jiang, College of Pharmacy, Changchun University of Chinese Medicine, China.

Preparation of Experimental Medication (EPS).
e dried herbs of S. chinensis (10 kg) were cut into sections, extracted twice with aqueous 70% EtOH under reflux for 2 hours and 8 times the amount of medicinal material each time. e extract was concentrated until the weight ratio (EtOH-dried herb) was 1 : 1, left to stand at room temperature overnight, brown yellow precipitates were obtained after filtration of the solution, the precipitates were further purified and dried at 55°C by vacuum drying, and the effective parts of S. chinensis (EPS) were obtained (232 g).

Medication Composition Analysis.
e content of total lignans and lactams in EPS was determined by UV spectrophotometry, and total sterols were determined by vanillin-perchloric acid method [8][9][10]. e determinations process is as follows.

Content Determinations of Total
Lignans. 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, and 0.7 mL of the reference solution (0.25 mg Sauchinone per mL) were accurately measured, the absorbance was measured at the wavelength of 242 nm by UV-Vis spectrophotometry, the standard curve with absorbance was drawn, and about 25 mg of EPS powder was accurately weighed, put into a conical flask, added 40 mL of chloroform, ultrasonic treated (Power 120 W, frequency 40 KHz) for 20 minutes, filtered, quantitatively transferred the filtrate into a 50 mL measuring flask, added chloroform to the scale, and shaken well. 0.6 mL of the sample solution was accurately measured, put into a 10 mL measuring bottle, added chloroform to the scale, and shaken well. With the corresponding reagent as the blank, the absorbance was measured at the wavelength of 242 nm by UV-Vis spectrophotometry. e concentration of total lignans in the test solution was calculated with the standard curve.

Content
Determinations of Total Lactams. 0.3 mL, 0.6 mL, 0.9 mL, 1.2 mL, 1.5 mL, and 1.8 mL of the sauristolactam reference solution (90 μg/mL) were accurately measured, the absorbance was measured at the wavelength of 277 nm by UV-Vis spectrophotometry, the standard curve with absorbance was drawn, and about 13 mg of EPS powder was weighed accurately, placed in a conical flask with a stopper, accurately added 25 mL of methanol, weighed, ultrasonic treated (Power 120 W, frequency 40 KHz) for 20 minutes, cooled, weighed again, 0.8 mL of supernatant was accurately measured, put into a 10 mL measuring bottle, methanol was added to the scale, and shaken well. With the corresponding reagent as the blank, the absorbance was measured at the wavelength of 277 nm by UV-Vis spectrophotometry. e concentration of total lactams was calculated in the test solution with the standard curve.

Content Determinations of Total Sterols.
e β-sitosterol reference solution of 25 μL, 50 μL, 75 μL, 100 μL, 125 μL, and 150 μL (0.6 mg per mL) was accurately measured, put into the test tube with stopper, respectively, volatilized the solvent, added 0.2 mL of newly prepared 5% vanillin glacial acetic acid solution and 0.8 mL of perchloric acid solution, shaken well, kept warm in a water bath at 60°C for 15 minutes, cooled with running water, added 5 mL of glacial acetic acid, and shaken well. With the corresponding reagent as the blank, the absorbance was measured at the wavelength of 541 nm by UV-Vis spectrophotometry, and about 25 mg of this product powder was weighed accurately, put into a conical flask, added 30 mL of chloroform, ultrasonic treated (power 120 W, frequency 40 KHz) for 20 minutes, filtered, evaporated the filtrate, the residue were dissolved with 2 mL of chloroform, mixed with 1 g of column chromatography silica gel (60-100 mesh), volatilized the solvent, and added to the treated silica gel column (petroleum ether column, 60-100 mesh, 2 cm × 9 cm), eluted with 150 mL of petroleum ether/ethyl acetate (7 : 3), the eluent was collected, evaporated, an appropriate amount of chloroform was added to dissolve the residue, quantitatively put into a 5 mL measuring bottle, accurately measured 150 μL, operated according to the law from "volatilizing the solvent" under the preparation of the standard curve, and the absorbance was measured and the concentration of total sterols in the test solution was calculated with the standard curve. (Benxi, China). e animal feeding laboratory was kept at 23 ± 2°C and 50 ± 5% relative humidity with a 12 h light/dark cycle. Animals were freely accessed to a commercial standard rat/mouse cube diet and water ad libitum, and they were allowed for acclimation under climate-controlled conditions for a week before use. All studies were conducted in compliance with Good Laboratory Practice Regulations from the State Food and Drug Administration of China. All animal protocols were approved by the Animal Welfare Ethics Committee of Jilin Provincial Academy of Chinese Medicine Sciences [11,12].

Effect on Hepatic Fibrosis of Complex Factors in Rats.
Wistar rats were randomly divided into two groups, 15 rats were normal control group and the rest 81 rats were model group. Liver fibrosis was induced in rats of model group by following complex factors: rats were fed with high-lipid and low-protein diets (containing 79.5% corn flour, 20% lard, and 0.5% cholesterol) for two weeks and subsequently with pure corn flour for two weeks, meanwhile, these model group rats were subcutaneously injected with 40% carbon tetrachloride olive oil solution twice a week at 3 mL/kg and gavaged with 30% ethanol every other day (1 mL per rat). Rats of the normal control group were given regular feed and drinking water and injected with corresponding volume of olive oil. Animals are weighed once a week.
One month after the establishment of the model, 10 rats died, and the remaining 71 rats were randomly divided into six groups according to the difference in body weight. Gavage is as follows: namely, the normal control group and the model group rats were given 10 mL/kg 0.5% carboxymethylcellulose sodium water suspension; western medicine positive drug group rats were given 40 mg/kg Silibinin capsules (Tianshili Pharmaceutical Group Co. Ltd., Tianjin, China); Chinese medicine positive drug group rats were given 1.08 g/kg Compound Biejia Ruangan Tablet (CBRT, Inner Mongolia Furui Zhongmeng Pharmaceutical Technology Co. Ltd.); the high-, middle-, and low-dose groups of EPS were given 140, 70, and 35 mg/kg, and the volume of gastric perfusion was 10 mL/kg. During the administration period, except the control group, the other groups' rats were given high-fat and low nutrition feed and gavaged with 30% ethanol every other day (1 mL per rat), and 40% carbon tetrachloride oil solution was injected subcutaneously once a week at 3 mL/kg, the control group was given normal feed and drinking water, and injected with equal volume edible oil; the drug was given once a day for 8 weeks [13][14][15].
After the last administration, the rats were fasted for 16 hours, weighed, blood samples were collected from abdominal aorta, and hyaluronic acid (HA), laminin (LN), procollagen III (PC III), and collagen IV (Col IV) in the serum were determined according to the enzyme-linked immunosorbent assay; total protein (TP) was determined by Bradford method; albumin (ALB) was determined by bromocresol green colorimetry; alanine transaminase (ALT) and aspartate transaminase (AST) activity were determined by the method of Lai's colorimetry; superoxide dismutase (SOD) activity was determined by hydroxylamine method; malondialdehyde (MDA) content was determined by thiobarbituric acid (TBA) method. e liver was fixed in 10% formalin solution for pathological examination [16][17][18][19][20][21][22].

Effect on Carbon Tetrachloride-Induced Acute Liver
Injury in Mice. 66 Kunming mice were randomly divided into six groups, the first group was normal control group and the second group was model group, which all were given 10 mL/kg 0.5% carboxymethylcellulose sodium water suspension by gavage; the third group was positive drug group, which was given 60 mg/kg Silibinin capsule by gavage; the fourth to the sixth groups were the high-, middle-, and lowdose group of EPS, which were, respectively, given 200, 100, and 50 mg/kg by gavage. e drug was given once a day for 10 days.
One hour after the last administration, except for the normal control group, mice of the other groups were intraperitoneally injected with 0.1% carbon tetrachloride olive oil solution 10 mL/kg, fasted for 16 hours later, blood was collected from the ophthalmic vein, centrifuged at 5000 rpm for 5 minutes. e activity of ALT and AST in the serum of mice was determined by the method of Lai's colorimetry.

Effect on Alcohol-Induced Acute Liver Injury in Mice.
68 Kunming mice were randomly divided into six groups, the first group was the normal control group, which was given 10 mL/kg0.5% carboxymethylcellulose sodium water suspension; the second group was the model group, administrated as above; the third group was the positive drug group, which was given 60 mg/kg Silibinin capsules by gavage; the fourth to the sixth groups were the high-, middle-, and low-dose groups of EPS, which were, respectively, given 200, 100, and 50 mg/kg by gavage. e drug was given once a day for 10 days.
After the last administration, except the normal control group, the mice in other groups were fasted for 12 hours, given 15 mL/kg (40°) Beijing Baijiu by gavage. Six hours later, the blood was collected from the ophthalmic vein and the activity of ALT and AST was measured by the method of Lai's colorimetry. e liver was fixed in 10% formalin solution for pathological examination [25][26][27].

Effect on D-Galactosamine Hydrochloride-Induced
Acute Liver Injury in Rats. 60 SD rats were randomly divided into six groups, the first group was normal control group and the second group was model group, which all were given 10 mL/kg 0.5% carboxymethylcellulose sodium water suspension by gavage; the third group was positive drug group, which was given 40 mg/kg Silibinin capsules by gavage; the fourth to the sixth groups were high-, middle-, and low-dose groups of EPS, which were given 140, 70, and 35 mg/kg by gavage. e drug was given once a day for 14 days [28][29][30].
Except for the normal control group, the rats in the other groups were intraperitoneally injected with 400 mg/kg D-galactosamine hydrochloride one hour after the eleventh administration, and again intraperitoneally injected repeatedly with 400 mg/kg D-galactosamine hydrochloride 48 hours later, and blood was collected from the abdominal aorta and centrifuged at 3000 rpm for 10 minutes. ALT, AST, SOD activities, and MDA content in the serum were measured, and the liver was fixed in 10% formalin solution for pathological examination [21][22][23].
Additionally, for liver injury induced by various factors (mice induced by carbon tetrachloride and alcohol, and SD rats with acute liver injury caused by D-galactosamine hydrochloride), hepatocyte necrosis or degeneration severity was also graded as follows: grade 0: no hepatocyte degeneration; grade 1: mild hepatocyte degeneration, with no more than 1/4 of the hepatocytes denatured; grade 2: moderate hepatocyte degeneration, with no more than 1/2 of the hepatocytes denatured; grade 3: severe hepatocyte degeneration, with no more than 3/4 of the hepatocytes denatured; and grade 4: severe hepatocyte degeneration, liver tissue is almost replaced by denatured hepatocytes. e liver scoring examination was performed by a pathologist who was blinded to the rats' treatment assignment. Fibrosis and hepatocyte scores were given after the pathologist had examined throughout three different areas in the tissue slide for each rat, the histopathological changes were observed, and the score data were analyzed.

Effect on General Conditions of Rats.
e rats in the normal control group had increased body weight, tight fur, luster, free movement, and no death. After subcutaneous injection of carbon tetrachloride, the rats in the model group had reduced activity, rough hair, obvious weight loss, and hair removal. From the second week after modeling, some rats had orange urine, some rats had blood in their urine, there were secretions in their eyes, and some rats lost weight and died. e anatomy of the dead rats showed that there was exudate in the abdominal cavity, small granular protrusions in the liver, the section was thick, the color was yellow or white, and the color of the kidney became dark and dull. Compared with the rats in the model group, most rats in the high-, medium-, and low-dose groups of EPS increased activity, increased weight, and reduced hair withering and depilation.

Effect on Serum Index in Rats with Liver Fibrosis.
e contents of LN, HA, COL IV, and PC III in serum of model group were significantly increased compared with the normal control group (P < 0.05 or P < 0.01), the contents of HA and COL IV were significantly reduced by EPS at doses of the high, middle, and low doses (140, 70, and 35 mg/kg) (P < 0.001, P < 0.01 or P < 0.05), and LN content was significantly reduced by the high and middle doses (P < 0.01 or P < 0.05). Meanwhile, the content of HA, LN, and COL IV in the Compound Biejia Ruangan Tablets (CBRT) group and Silibinin capsules group was significantly decreased compared with the model group (P < 0.001, P < 0.01, and P < 0.05).
Compared with the model group, the high and middle doses of EPS can significantly reduce the ALT and AST activities (P < 0.01, P < 0.05) and significantly increase the ALB content (P < 0.05). e activities of ALT and AST in Silibinin capsules group and CBRT group were significantly lower and that of ALB were significantly higher than those in the model group (P < 0.05).
Compared with the model group, the high-dose group of EPS can significantly improve SOD activity (P < 0.05) and significantly reduce MDA content (P < 0.05), and the highand middle-dose group can significantly reduce Hyp content (P < 0.05). e content of Hyp in CBRT group and Silibinin capsule group was decreased significantly compared with the model group (P < 0.05). For the contents of PC III, EPS and positive drug have no effects. e results are shown in Figures 1-3.

Histopathological Evaluation.
According to the prevention and treatment plan for viral hepatitis [10], the histopathological changes were observed and analyzed. e normal control group had normal liver tissue structure, clear portal area and hepatic sinus structure, no fibrous connective tissue hyperplasia, no abnormality, and no inflammatory cell infiltration. No blue-stained collagen fibers separated normal liver tissue to form pseudolobular structure, and the collagen tissue components of blood vessels were dyed blue. In the model group, the proliferation of hepatic fibrous tissue was extensive (especially in the portal area), accompanied by proliferation in the hepatic lobules. In the model group, the hepatic lobules were divided and wrapped by the blue-stained fibrous components of the liver to form pseudolobules of different sizes, degeneration of hepatocytes in pseudolobules, disordered arrangement of hepatocytes, compression of hepatic sinuses, deviation or absence of central veins, and focal lymphocyte infiltration between proliferating fibers. In EPS high-and middle-dose groups and CBRT group, the results showed that the formation of liver fibers and pseudolobules was significantly reduced, which indicated that the formation of liver fibers in rats could be significantly inhibited. e results are shown in Figure 4 and Table 1.

Effect on Liver
Function. e activity of ALT and AST in the model group was significantly higher than that in the normal control group, indicating that the model was 4 Evidence-Based Complementary and Alternative Medicine established (P < 0.001 or P < 0.01). e ALT activity of mice with acute liver injury induced by carbon tetrachloride was significantly inhibited in the high-, middle-, and low-dose group of EPS (P < 0.001 or P < 0.05). e AST activity of mice with acute liver injury induced by carbon tetrachloride was significantly inhibited in the high-and middle-dose groups of EPS (P < 0.01 or P < 0.001). e activities of ALT and AST in Silibinin capsule group were significantly inhibited compared with the model group (P < 0.001). e results are shown in Figure 5.

Pathological Examination Results.
e results showed that the control group liver was reddish brown in appearance, soft and elastic in texture, clear in structure of liver lobule, and intact in structure of liver cell, without necrosis, swelling, and other lesions. In the model group, the structure of liver lobule was damaged, inflammatory cell infiltration, spot necrosis, and cell degeneration were observed. Compared with the model group, EPS administration group was alleviated in varying degrees.
e results are shown in Figure 5 and Table 2.

Effect on Liver Function.
Six hours after intragastric administration of alcohol, the activities of ALT and AST in the serum of the model group were significantly higher than that of the normal control group (P < 0.01 or P < 0.05), indicating that the model was established. ree dose group of EPS had no significant effect on ALT activity in serum of mice with alcoholism; high-dose group could significantly reduce AST activity compared with the model group (P < 0.05). ere was no significant effect of Silibinin capsule on ALT and AST activities in serum of mice with alcoholism. e results are shown in Figure 6.

Pathological Examination Results.
In the normal group, there was no degeneration of hepatocytes in the liver, the structure of hepatic lobules was clear, and the hepatocytes were arranged in a radial manner from the central vein to the periphery of the lobules. Hepatocyte degeneration with different number of cases was observed in the liver of the model group and each administration group. EPS group can alleviate the degeneration process of liver injury induced by alcohol in mice to varying degrees, and the results are shown in Table 3 and Figure 6.   Evidence-Based Complementary and Alternative Medicine reduce the ALT and MDA activities and improve the SOD activity (P < 0.05); the low-dose group of EPS can significantly reduce the ALT and AST activities and MDA content and improve the SOD activity (P < 0.01). Compared with the model group, Silibinin capsule group can significantly reduce ALT, AST, and MDA content and significantly improve SOD activity (P < 0.01 or P < 0.05). e results are shown in Figure 7.

Pathological Examination
Results. Normal control group: hepatocytes are arranged radially with the central vein as the center, the structure of hepatic lobules is complete, and there is no hepatocyte degeneration and inflammatory cell infiltration in intrahepatic portal area. In the model group, the liver showed pathological changes, most of the hepatocytes were swollen, the cytoplasm was loose and ballooned, and extensive hepatocyte degeneration and necrosis could be seen; inflammatory cell infiltration was also observed. 5/10 animals showed severe liver injury and degeneration of a large area of hepatocytes. e number and degree of liver degeneration in the positive western medicine group decreased significantly. e number and degree of liver degeneration of animals in EPS administration groups were significantly reduced. Comparative analysis shows that EPS can effectively alleviate the liver injury of rats caused by D-galactosamine. e results are shown in Figure 7 and Table 4.

Discussion and Conclusion
Liver fibrosis occurs as a result of many chronic liver diseases such as hepatitis viral infection, including hepatitis B and C, in patients. Liver fibrosis plays a pivotal role in liver function impairment. It is characterized by altered hepatic function and the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. e progression of liver fibrosis often develops into irreversible cirrhosis and is associated with liver cancer. erefore, it is urgent to reduce the damage to the liver and to slow down the progress of liver injury to fibrosis and cancer.
Carbon tetrachloride complex liver fibrosis model is a continuous infusion of Chinese Baijiu (liquor) by repeated injection of carbon tetrachloride, feeding high-fat (lard, cholesterol), low-protein (cornmeal) feed, resulting in fatty degeneration, necrosis, and fibrous hyperplasia of liver cells in rats. e model is stable, reliable, and has a high success rate. It is a commonly used animal model of liver fibrosis. Rats were administered EPS by gavage for 8 weeks, which can significantly reduce the levels of hyaluronic acid (HA), laminin (LN), type IV collagen (Col IV) content, and malondialdehyde content and inhibit the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum. e content of serum albumin (ALB) was significantly increased, the activity of superoxide dismutase (SOD) increased, and the content of hydroxyproline (Hyp) significantly reduced in liver tissue. Liver histopathology showed that EPS could significantly reduce the formation of liver fibrosis and inflammatory cell infiltration in rats, indicating that EPS could reduce the pathological damage of compound factors to rat liver tissue.
e mechanism of EPS in the treatment of liver fibrosis was as follows: ① Protect hepatocytes, prevent the further development of liver injury, and promote the recovery of liver function. ② Inhibit the release of cytokines and promote the repair of damaged cells. ③ Scavenge oxygen free radicals and resist lipid peroxidation damage.
④ Inhibit the formation of ECM and promote the degradation of pathological deposited collagen.
Carbon tetrachloride is the most commonly used and classic model of acute liver injury in mice induced by chemical drugs. After entering hepatocytes, carbon tetrachloride dissolves the lipids of mitochondrial membrane, thus affecting the structure and function of mitochondria,  Evidence-Based Complementary and Alternative Medicine reducing the synthesis of enzyme proteins, causing the destruction of enzymes, thus affecting the metabolic function and energy generation, and denaturing and necrotizing hepatocytes. During hepatocytes degeneration and necrosis, the permeability of cell membrane increases, and a large amount of ALT and AST flows into the blood, which increases the serum ALT and AST. erefore, the determination of the content of ALT and AST in serum can reflect the degree of hepatocyte degeneration and necrosis. EPS can significantly inhibit the activities of ALT and AST in serum of mice with acute liver injury induced by carbon tetrachloride and significantly reduce the degree of liver  e liver is the main organ of alcohol metabolism in vivo. Excessive alcohol intake will lead to different degrees of damage to the liver and abnormal increase of serum ALTand AST. e high-dose EPS group can significantly reduce the activity of AST in serum of alcoholic mice and reduce the degree of pathological damage of damaged liver, indicating that it had a certain protective effect on acute liver injury caused by alcohol.
D-galactosamine is currently recognized as a relatively good experimental animal model for studying the pathogenesis of viral hepatitis and observing the curative effect of drugs. Its poisoning mechanism is that D-galactosamine enters the body and combines with uridine phosphate (UDP) to form UDP galactosamine, resulting in the  Evidence-Based Complementary and Alternative Medicine depletion of uridine phosphate, so that the nucleic acids, proteins, and the synthesis of substances such as lipopolysaccharide are inhibited, which limits the generation and supplement of organelles and enzymes. Organelles are damaged, cell membranes are damaged, and calcium ions flow into cells, resulting in hepatocyte damage. EPS can significantly reduce the activity of ALT and AST and the content of MDA in serum of D-galactosamine hydrochloride-induced liver injury model rats, increase the activity of SOD, and reduce the pathological damage of liver tissue, indicating that EPS had a protective effect on D-galactosamine-induced liver injury in rats.  In conclusion, EPS can effectively inhibit the formation of liver fibrosis and has obvious preventive and therapeutic effects on liver injury caused by various factors. In summary, our findings showed that EPS exhibits potent activities and should be considered a good option and an additional source of natural compounds for the treatment of hepatic fibrosis and hepatic injury. EPS is a promising drug candidate [26][27][28][29][30][31][32][33][34][35][36][37].
Data Availability e data are included in the manuscript.

Conflicts of Interest
e authors declare that they have no conflicts of interest.