Study on the Protective Effect and Mechanism of Tibetan Medicine Ershiwuwei Songshi Pill on Acute Alcoholic Liver Injury in Rats

Ni MA, Qi MA, Baoyu JIN, Jian GU, Puyang GONG

College of Pharmacy, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] To investigate the protective effect and mechanism of Ershiwuwei Songshi Pill on acute alcoholic liver injury in rats. [Methods] Sixty male SD rats were randomly divided into normal control group, model group, polyene phosphatidylcholine (82.08 mg/kg) positive control group and Ershiwuwei Songshi Pill high (180 mg/kg), medium (90 mg/kg) and low (45 mg/kg) dose groups. The rats in each group were given corresponding drugs by intragastric administration for 3 d and fed normally. From the 4th d, all groups except the normal control group were fed with 15 mL/kg liquor (56% vol) for 7 d after 2 h of administration. After the last modeling, the levels of AST, ALT, TC, TG, SOD, GSH in serum and IL-1β, IL-6, TNF-α in liver tissue were measured. The protein expressions of Bax, Bcl-2 and Caspase3 were determined by Western blotting. HE staining was used to observe the pathological changes of liver tissue under light microscope. [Results] Compared with the model group, the body weight and the levels of SOD and GSH in the treatment group were significantly higher than those in the model group; liver index, serum ALT, AST, TC, TG, IL-1β, IL-6 and TNF-α levels decreased significantly (P<0.01 or P<0.05); HE staining showed that there was a small amount of collagen proliferation and lymphocyte infiltration in connective tissue around the hepatic portal area in the model group, multiple inflammatory foci could be seen in the lobules, and round fat vacuoles could be seen in the cytoplasm; in each administration group, the pathological condition of the liver was mild. [Conclusions] Ershiwuwei Songshi Pill had a certain protective effect on acute alcoholic liver injury in rats, and its mechanism might be related to reducing enzyme, promoting lipid metabolism and anti-inflammation.

Key words Ershiwuwei Songshi Pill, Alcoholic liver injury, Mechanism

1 Introduction

Alcoholic liver disease (ALD) is liver damage caused by excessive drinking or alcoholic abuse, including alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and alcoholic cirrhosis. At present, alcoholic liver injury has become the second largest type of liver disease after viral hepatitis, and it is a common reason for the development of liver cirrhosis[1]. For the liver damage caused by alcohol, there is a lack of specific drugs, so it is of great significance to seek effective drugs against alcoholic liver disease[2].

Ershiwuwei Songshi Pill is a classic Tibetan medicinal variety, which is composed of 25 medicines, such as tophus, sandalwood, coral,Artemisiaannua, bezoar, clove, musk, and saffron. It has been collected inChinesePharmacopoeiaover the years and has the effects of soothing the liver and promoting gallbladder, clearing heat and detoxification, promoting blood circulation and removing blood stasis. It is a classic compound prescription for the treatment of liver diseases in Tibetan areas of China. Clinical control experiments showed that the drug could promote alanine aminotransferase and serum bilirubin to return to normal[4], and had a significant therapeutic effect on viral hepatitis and liver cirrhosis[5]. The studies of Sun Fangyunetal.[6]found that the drug could protect liver function by reducing the content of serum endotoxin. However, there are no related reports about its mechanism on acute alcoholic liver injury.

2 Materials and methods

2.1Materials

2.1.1Experimental animals. SPF male SD rats, weighing 180-200 g, purchased from Chengdu Dashuo Experimental Animal Research Center, experimental animal production license No.: SCXK (Chuan) 2015-030. The experiment began after a week of adaptive feeding.

2.1.2Drugs and reagents. Ershiwuwei Songshi Pill, lot No.181201, Tibet Jinzhu Yazhi Tibetan Medicine Co., Ltd.; positive drug Essentiale, lot No.8BJD383, Sanofi (Beijing) Pharmaceutical Co., Ltd.; liquor (56% vol), Beijing Hongxing Co., Ltd.; 0.9% NaCl injection, purchased from Sichuan Kelun Pharmaceutical Co., Ltd.; glutamic pyruvic transaminase (ALT) kit, glutamic oxaloacetic transaminase (AST) kit, superoxide dismutase (SOD) kit, total cholesterol (TC) kit, triglyceride (TG) kit and reduced glutathione (GSH) kit, all purchased from Nanjing Jiancheng Bioengineering Institute; interleukin-1β (IL-1β) kit, interleukin-6 (IL-6) kit and tumor necrosis factor (TNF-α) kit, purchased from Hangzhou MultiSciences Biotechnology Co., Ltd.; RIPA lysate, phosphorylated protease inhibitor, BCA protein quantitative detection kit, skim milk powder, developer fixing reagent and β-actin, all purchased from Wuhan Servicebio Biotechnology Co., Ltd.

2.1.3Main instruments. PL-203 electronic balance, Mettler-Toledo Instruments (Shanghai) Co., Ltd.; JY92-II ultrasonic cell crusher, Ningbo Scientz Biotechnology Co., Ltd.; Ne0fuge15R table-top high speed frozen centrifuge, Heal Force Instruments (Shanghai) Co., Ltd.; FBZ2001-UP-P pure water filter, Qingdao Flom Science and Technology Co., Ltd.; MX-F turbine mixer, Scilogex Co., Ltd. RT3100 automatic microplate washer, Rayto, USA; Epoch enzyme labeling instrument, BioTeK Instrument Co., Ltd.; TSY-B decolorizing shaker, KZ-II homogenizer, Servicebio; DYY-6C electrophoresis power supply, DYCZ-24DN double vertical electrophoresis instrument, DYCZ-40D electrophoresis instrument, Beijing Liuyi Instrument Factory; V300 scanner, EPSON; NIKON Eclipse Ci microscope, NIKON digital sight DS-FI2 imaging system.

2.2Methods

2.2.1Animals and treatment. Sixty male SD rats were randomly divided into normal control group, model group, polyene phosphatidylcholine (82.08 mg/kg) positive control group and Ershiwuwei Songshi Pill high (180 mg/kg), medium (90 mg/kg) and low (45 mg/kg) dose groups. The rats in each group were given corresponding drugs by intragastric administration for 3 d and fed normally. From the 4th d, except the normal control group which was given distilled water, all groups were fed with 15 mL/kg liquor (56% vol) for 7 d after 2 h of administration, and the model of acute alcoholic liver injury in rats was established. After the establishment of the model, the rats in each group were forbidden to eat but could drink water for 12 h. After the blood was taken from the femoral artery, the liver was quickly dissected, washed with cold normal saline and weighed. Part of it was fixed with 4% paraformaldehyde and the remaining tissue was made into liver homogenate.

2.2.2Biochemical index detection. After the blood was collected, it was left still for 30 min, and frozen and centrifuged, and the supernatant was taken. Part of the liver was treated with cold saline to make 10% of the liver homogenate. The serum indexes of AST, ALT, TC, TG, SOD and GSH, as well as the indexes of IL-1β, IL-6 and TNF-α in liver homogenate, were detected by ELISA method. It was operated according to the relevant kit instructions, and the enzyme-labeled tester was used for detection.

2.2.3Determination of protein expression. After being treated by homogenizer, the sample tube was subjected to ice bath for 30 min, and shaken every 5 min to ensure that the tissue was completely cracked. The supernatant was collected by 12 000 rpm centrifugation for 10 min at 4 ℃. According to the requirements of the determination kit, the protein was extracted and its concentration was determined, and the samples with the same protein content were taken for electrophoresis, membrane transfer, blocking, first antibody incubation, second antibody incubation, ECL luminescence, development and fixing. Finally, gel image analysis was used. The film was scanned and archived, Photoshop was used for sorting and decoloring, and the optical density of the target band was analyzed by Alpha software processing system.

2.2.4Tissue pathomorphology. The fixed liver tissue was taken for dehydration, embedding, film making, HE staining, and observed and photographed under light microscope.

2.2.5Calculation of liver index. The weight of the rats in each group was weighed before death. After dissecting and taking the liver, the liver was rinsed with cold normal saline, and wiped off with filter paper, and then the liver tissue was weighed. The liver index was calculated as follows:

Liver coefficient=[Liver weight (g)/Body weight (g)]×100.

3 Results and analysis

3.1GeneralbehaviorofratsAfter observation, it was found that the rats in each group were drunk and lethargic within 1 h, and then alleviated. In the model group, 2 rats died, the rats were dispirited, the activity was reduced, the fur was dull, and some of the rats’ droppings were thin and soft.

In the control group, the rats drank and ate normally, the body weight increased significantly, and there was no death, and the state of rats in each administration group was better than that in the model group.

3.2LiverindexandserumlevelsofALTandASTinrats

Table 1 showed that compared with the normal control group, the liver index of the model group increased, which proved that the liver damage was accompanied by swelling after alcohol administration. Compared with the model group, the body weight of rats in each dose group of Ershiwuwei Songshi Pill increased and the liver coefficient decreased, of which the high dose group was the most significant, followed by the medium dose group.

This showed that Ershiwuwei Songshi Pill could alleviate the liver swelling caused by alcohol to a certain extent, so it had a good protective effect on liver injury. Compared with the normal control group, the levels of serum ALT and AST in the model group were significantly higher than those in the normal control group (P<0.01). Except that there was no significant difference in the low dose group of Ershiwuwei Songshi Pill (P<0.05), the serum ALT and AST levels in polyene phosphatidylcholine positive group and Ershiwuwei Songshi Pill high and middle dose groups decreased to different degrees (P<0.01 orP<0.05). This suggested that the protective mechanism of Ershiwuwei Songshi Pill on acute alcoholic liver injury in rats was related to the levels of serum transaminase ALT and AST.

3.3Serumlipids(TC,TG)andliveroxidativedamageindexes(SOD,GSH)ofratsTable 2 showed that compared with the normal control group, the levels of serum TC and TG in the model group increased significantly (P<0.01), while the serum TC and TG levels in the high, middle and low dose groups of Ershiwuwei Songshi Pill decreased to varying degrees, of which it decreased significantly in the high dose group of Ershiwuwei Songshi Pill, followed by the low dose group. The levels of SOD and GSH in the model group were significantly higher than those in the model group (P<0.01 orP<0.05).

GroupDose∥mg/kgBody weight∥gLiver index∥%ALT∥IU/LAST∥IU/LNormal control-288.40±12.41∗∗3.10±0.14∗∗8.64±2.00∗∗20.39±3.32∗∗Model-262.91±8.20##3.41±0.25##72.52±15.98##105.09±12.43##Polyene phosphatidylcholine82.08281.98±6.82∗∗3.18±0.22∗19.15±4.31∗∗53.91±5.55∗∗High dose180276.60±12.27∗∗3.19±0.16∗38.98±4.86∗∗62.17±9.61∗∗Medium dose90274.65±7.95∗3.11±0.18∗∗56.96±8.55∗82.70±12.94∗Low dose45273.97±6.50∗3.25±0.1067.85±13.4095.86±11.79

Note: Compared with the normal control group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

GroupTC∥mmol/LTG∥mmol/LSOD∥U/mLGSH∥μmol/LNormal control0.82±0.09∗∗3.87±0.50∗∗16.37±0.96∗∗447.75±44.43∗∗Model2.17±0.28##7.90±0.48##8.61±0.90##102.39±18.43##Polyene phosphatidylcholine1.28±0.21∗∗5.74±0.39∗∗12.74±1.73∗∗275.46±39.31∗∗High dose1.47±0.25∗∗6.23±0.47∗∗12.49±1.24∗∗204.54±16.60∗∗Medium dose2.05±0.327.28±0.64∗11.42±1.54∗∗165.08±24.26∗Low dose1.83±0.28∗7.32±0.38∗11.03±0.87∗∗153.63±29.63∗

Note: Compared with the normal control group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

3.4Indexesofinflammationinserumandlivertissueofrats

Table 3 showed that the content of IL-1β, IL-6 and TNF-α in the model group was significantly higher than that in the normal control group. Compared with the model group, except that there was no significant difference in the level of IL-6 in the low dose group of Ershiwuwei Songshi Pill, the content of IL-1β, IL-6 and TNF-α in each group was significantly lower than that in the model group (P<0.01 orP<0.05).

GroupIL-1βIL-6TNF-αNormal control127.54±22.15∗∗47.96±6.80∗∗28.39±3.05∗∗Model403.14±37.89##287.28±32.49##49.68±9.47##Polyene phosphatidylcholine231.25±19.93∗∗256.45±52.69∗36.30±8.51∗∗High dose289.64±37.34∗∗101.68±18.81∗∗26.13±3.24∗∗Medium dose322.07±41.54∗164.89±22.59∗∗31.71±5.43∗∗Low dose387.39±54.56∗271.71±50.8529.23±7.17∗∗

Note: Compared with the normal control group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

3.5Expressionofapoptosis-relatedproteinsinratliverTable 4 showed that the expression level of Bax, Bcl-2 and Caspase3 in the model group was significantly higher than that in the normal control group. Compared with the model group, except that there was no significant difference in the levels of Bcl-2 and Caspase-3 in the low dose group of Ershiwuwei Songshi Pill, the expression level of Bax and Bcl-2 in each group was significantly higher than that in the model group (P<0.01 orP<0.05). The expression of Caspase 3 decreased significantly (P<0.01 orP<0.05).

GroupBaxBcl-2Caspase 3Normal control0.30±0.02∗∗1.10±0.15∗∗0.15±0.01∗∗Model1.08±0.05##0.34±0.06##1.14±0.07##Polyene phosphatidylcholine0.53±0.06∗∗0.90±0.16∗0.29±0.02∗∗High dose0.67±0.03∗∗0.86±0.21∗0.70±0.13∗∗Medium dose0.58±0.06∗∗0.73±0.10∗0.88±0.13∗Low dose0.64±0.12∗∗0.47±0.081.09±0.13

Note: Compared with the normal control group,#P<0.05,##P<0.01; compared with the model group,*P<0.05,**P<0.01.

3.6HistopathologyofratliverResults in Fig.1 showed that there was a small amount of connective tissue collagen proliferation around the hepatic portal area in the model group, accompanied by considerable lymphocyte infiltration. The hepatic sinusoids were slightly dilated and the Kupffer cells in the hepatic sinusoids increased. Multiple inflammatory foci could be seen in the lobules, and round fat vacuoles could be seen in the cytoplasm. In Polyene phosphatidylcholine group, the structure of hepatic lobule was clear, the nucleus of hepatocyte was clear, the cytoplasm was abundant, the hepatic cord was neatly arranged, the hepatic sinusoid was not obviously dilated or squeezed, and lymphocyte infiltration was rarely seen in the portal area. In the low dose group of Ershiwuwei Songshi Pill, the connective tissue around the portal area of the liver tissue was edematous and loose, the hepatocytes around the local central vein were edematous and the cytoplasm was loose.

Note: a. normal control group; b. model group; c. polyene phosphatidylcholine group; d. Ershiwuwei Songshi Pill high dose group; e. Ershiwuwei Songshi Pill medium dose group; f. Ershiwuwei Songshi Pill low dose group.

Fig.1 Comparison of liver histomorphology of rats in each experimental group (HE×200)

4 Discussion

The levels of serum ALT and AST are the most sensitive indicators of hepatocyte injury, and indirectly reflect the degree of hepatocyte injury[7]. When the permeability of the damaged cell membrane increases, ALT and AST will overflow from the cells and mitochondria into the bloodstream. Therefore, the higher the concentration of ALT and AST, the more serious the cell injury or necrosis. A large amount of alcohol enters the rat body, destroys the cell membrane structure of rat hepatocytes, increases the cell membrane permeability, releases intracellular ALT and AST into the blood, and increases the concentration of ALT and AST in the blood[9]. Pre-administration of Ershiwuwei Songshi Pill to rats can effectively inhibit the increase of serum ALT and AST concentration induced by alcohol. It is inferred that Ershiwuwei Songshi Pill can protect the hepatocyte membrane and maintain the normal structure and function of hepatocytes. Serum lipids are of great significance. If abnormal metabolism leads to accumulation, it is easy to form atherosclerosis, and the liver can absorb lipids from circulating plasma, resulting in fat accumulation in the liver to form fatty liver[10].

After drinking a large amount of 56 degrees of liquor, the rats could not metabolize normally, resulting in the accumulation of triglyceride and total cholesterol in the serum and could not be eliminated by normal metabolism. After administration of Ershiwuwei Songshi Pill in advance, the content of serum triglyceride and total cholesterol could be reduced. Ershiwuwei Songshi Pill regulated the metabolism of lipids, reduced the abnormal increase of TG and TC, promoted oil metabolism, and effectively reduced the disorder of lipid metabolism and oil accumulation caused by alcohol. Alcohol can cause obvious oxidative damage, such as the decrease of SOD activity and GSH level, while Ershiwuwei Songshi Pill can significantly increase the antioxidant enzyme SOD activity and GSH level, and play a protective role in antioxidant damage. Alcoholic liver injury can significantly increase the levels of IL-1β, IL-6 and TNF-α in liver tissue. These high level inflammatory factors are important factors causing liver injury. Ershiwuwei Songshi Pill can significantly reduce the levels of IL-1β, IL-6 and TNF-α, and protect the liver to some extent.

Apoptosis is an important pathological change of alcoholic liver disease. The results showed that alcohol could significantly increase the expression of Bax, Bcl-2, Caspase3 and significantly decrease the expression of Bcl-2 protein, while Ershiwuwei Songshi Pill could significantly reduce the expression of Bax, Caspase3 and increase the expression of Bcl-2 protein. Ershiwuwei Songshi Pill can effectively reduce hepatocyte apoptosis and alcoholic liver injury.

To sum up, this experiment preliminarily verified that administration of Ershiwuwei Songshi Pill before drinking could reduce the liver injury caused by alcohol and had a certain protective effect on acute alcoholic liver injury in rats, and its mechanism was related to reducing enzymes, promoting lipid metabolism and anti-inflammation.