Protective Effect and Action Mechanism of Modified Ershiwuwei Songshi Pills on Chronic Liver Injury Induced by CCL4

Xican MA, Shuai YANG, Ke BA, Zhilong SHI, Jian GU

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

Abstract [Objectives] To study the protective effect of Ershiwuwei Songshi Pills on chronic liver injury induced by carbon tetrachloride (CCL4) in rats before and after the modification conforming to the compatibility theory of Tibetan medicine, and to explore its action mechanism. [Methods] Male Wistar rats were randomly divided into the blank control group, model group, Hugan tablets group (0.490 g/kg), Ershiwuwei Songshi Pills group (0.117 g/kg), and Modified Ershiwuwei Songshi Pills group (removing cinnabaris, Aristolochia contorta, and Aconitum naviculare, 0.105 g/kg). Except the blank group, the remaining groups were injected subcutaneously with 20% carbon tetrachloride olive oil solution every 3 d, and modeled for 6 weeks. During this time, intragastrically administered corresponding drugs. Six weeks later, blood was taken from the femoral artery, and the rats were killed through dislocating the cervical spine, the liver was taken, and the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined. Then, liver fibrosis indicators tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were detected by immunohistochemical method. [Results] Compared with the model group, the pathological map of the liver section showed that liver injury was improved in each administration group. The serum ALT and AST contents in rats of each administration group were significantly reduced (P<0.05), and the protein expressions of NF-κB, TNF-α, IL-1β and IL-6 in liver tissue were also reduced by varying degrees (P<0.05). [Conclusions] Ershiwuwei Songshi Pills and its modification group have a protective effect on liver injury induced by carbon tetrachloride. The modified prescription conforms to the compatibility rules of Tibetan medicine. The mechanism may be related to reducing the damage caused by inflammatory factors through regulating the role of inflammatory signaling pathway. Thus, it can be used as a reference for future optimization proposals.

Key words Ershiwuwei Songshi Pills, Modified prescription, Liver injury induced by carbon tetrachloride (CCL4)

1 Introduction

Ershiwuwei Songsi Pills is a classic Tibetan medicine compound, consisting of 25 kinds of medicines such as Turquoise, Margarita, Cinnabari, Aristolochia contorta, and Aconitum naviculare,etc. Clinically, it is widely applied in liver poisoning, liver pain, liver cirrhosis, liver water seepage, and various acute and chronic hepatitis and cholecystitis and other hepatobiliary diseases, showing significant therapeutic effects[1-5].

Tibetan medicine has many components and complex action mechanism. In the past, studies of Tibetan medicine focused on component analysis and classic pharmacology. Compared with traditional Chinese medicine research, both the research level and research direction were relatively limited[6]. Due to the lack of "theoretical support of Tibetan medicine" and failure to reflect the compatibility rules of Tibetan medicine, the active components and action mechanism of Tibetan medicine compound are not clear[7]. The compatibility methods of Tibetan medicine are mainly the compatibility of nature and taste and compatibility of nature and effect. The taste compatibility of Ershiwuwei Songshi Pills is characterized by astringent and bitter taste medicines having functions of clearing the pathogenic heat and eliminating the fire. And the compatibility of nature and effect is based on cool nature medicines, having functions of clearing the pathogenic heat and detoxification, promoting the blood circulation, and removing blood stasis. The compatibility of nature and taste is characterized by bitter and sweet medicines, having functions of treating heat diseases. According to statistical analysis, Tibetan medicine prescription commonly used for treating liver diseases is mainly the compatibility ofCrocussativusandCalculusbovis.C.sativusis sweet in taste, andC.bovis,A.contorta,Terminaliachebula, andA.navicularehave characteristics of bitter taste and cool nature. Cinnabari, Santalum album, and Margarita are astringent medicines[8].

In the prescription, many components are bitter and astringent. Cinnabari contains heavy metal mercury,A.navicularecontains aconitine,A.contortacontains aristolochic acid. Long-term taking of these components may cause severe liver and kidney injury[9-12]. Considering this, we removed the bitter and astringent components in the prescription. Then, we explored the protective effect of Ershiwuwei Songshi Pills on chronic liver injury induced by carbon tetrachloride (CCL4) in rats before and after the modification, to find the modified prescription conforming to the compatibility theory of Tibetan medicine, and make a preliminary study of its action mechanism.

2 Materials and methods

2.1Materials

2.1.1Experimental animals and place. Forty clean wistar rats, male, body weight 180-220 g, provided by Sichuan Dossy Experimental Animal Center, animal certification No.SCXK[Chuan] 2015-030. Experimental place was in the National Medicine Basic Research and Development Laboratory of College of Pharmacy, Southwest Minzu University [No.Chuanshi 2017(011)].

2.1.2Medicines and main reagents. Ershiwuwei Songshi Pills (Tibetan Medicine Co., Ltd. of Tibetan Medical College, batch No.Z54020090); Hugan Tablets (Heilongjiang Sunflower Pharmaceutical Group Co., Ltd., batch No.201601173); Turquoise, Margarita, Cinnabari,A.contorta, andA.naviculare,Coralliumrubrum,S.album,Bostaurusdomesticus, Meconopsis,Myristicafragrans,Ewgewiacaryophyllata,Saxifragaumbellulata,Terminaliabillerica,Crocussativus,Gossampinusmalabarica,Dalbergiaodorifera,Adhatodavasica, Limestone,Bambusatertilis,Phyllanthusemblica, scrap iron (processed byTerminaliachebula), dried fruit ofT.chebula, Faeces Trogopterori, cabardine, andAucklandialappawere bought from Hehuachi traditional medicine market in Chengdu and Tibetan Medicine Co., Ltd. of Tibetan Medical College, and identified by Professor Gu Jian from College of Pharmacy of Southwest Minzu University. Among them, scrap iron and Cinnabari are processed medicines. All medicines were pulverized and crushed, screened with No.6 sieve for experiment.

CCl4was bought from Chengdu Kelong Chemical Co., Ltd. (Analytical reagents, batch No.2015042001); AST and ALT reagent kits were bought from Nanjing Jiancheng Biological Engineering Research Institute (batch No.20180105). Target gene antibodies: IL-6, IL-1β, TGF-β1, NF-κB, TNF-α were purchased from Proteintech Group, Inc. (rabbit polyclonal antibody, concentration 1∶100, batch No.21865-1-AP, 16806-1-AP, ab92486, ab16502, ab6671); Biotin secondary antibodies were purchased from Beijing Beijing Zhongshan Golden Bridge Biological Technology Co., Ltd. (goat anti-rabbit working solution, batch No.SP-9001).

2.1.3Instruments. Multi-functional microplate analyzer MB-580 (Shenzhen Huisong Technology Development Co., Ltd.); 722 visible spectrophotometer (manufactured by Shanghai Yoke Instrument Co., Ltd.); digital trinocular camera microscope (BA400 Digital, Motic China Group Co., Ltd.).

2.2Methods

2.2.1Experimental animal groups. Clean male wistar rats were randomly divided into 5 groups. According to the description of Ershiwuwei Songshi Pills in theChinesePharmacopoeia, and with reference to animal and human body surface area reduced equivalent dose ratio table, calculated daily dose of rats in the administration group. Five groups were Ershiwuwei Songshi Pills group (administration dose 0.117 g/kg), Modified Ershiwuwei Songshi Pills group (removing cinnabaris,A.contorta, andA.naviculare, 0.105 g/kg), Hugan Tablet group (administration dose 0.490 g/kg), model group and blank control group. Except for the blank group, all rats were administered with 0.5% CMCC-Na suspension.

2.2.2Liver injury model. Except the blank group, the remaining groups were injected subcutaneously with 10 mL/kg of 20% carbon tetrachloride olive oil solution every 3 d in rats. Blood was taken from the femoral artery of the rat, centrifuged at 4 000 r/min for 10 min, and the supernatant was absorbed and stored at -80 ℃ for test. The rat liver was dissected, and 0.5 cm3of the liver was fixed in neutral formaldehyde. The remaining part of liver was made into liver homogenate and stored at -80 ℃ for test.

2.2.3Indicators. The effects of biochemical indicators such as AST and ALT in the serum of each group of rats were measured according to the kit instructions. HE staining was used to detect the liver pathology of rats, and immunohistochemical methods were used to detect the expressions of TNF-α, NF-κB, IL-6 and IL-1β, and made a record of its mean optical density (MD).

2.2.4Data analysis. The Image-Pro Plus 6.0 image analysis system was used to determine the integrated optical density (IOD) and area (Area) of all collected images, and the mean optical density (MD) of each image was calculated. The mean optical density of three images were used to calculate the mean number, to obtain the average optical density of each sample. SPSS 17.0 statistical analysis software was used to perform one-way ANOVA on the data of each group, and the data were expressed in.

3 Results and analysis

3.1PathologicalobservationoflivertissueofratsFrom Fig.1, it can be seen that, in the blank group, the liver capsule was intact and the hepatic lobular boundaries were clear, the hepatic cords were arranged radially around the central vein, and the liver cells were arranged neatly, there was no obvious inflammatory cell infiltration in the hepatic sinus. In the model group, the structure of the liver lobule was damaged, and the arrangement of hepatic cords was disordered, a lot of different size of fat vacuoles appeared; some hepatocytes were degenerated, taking on long spindle-shape, squeezed to the side of the cells, and large areas of spotty necrosis appeared in the hepatocyte interstitial. Compared with the model group, hepatic cell necrosis was significantly reduced in the Hugan Tablet group, the fat vacuoles were smaller in the Ershiwuwei Songshi Pills group, while Modified Ershiwuwei Songshi Pills group had neatly arranged hepatocytes with only a few vacuoles. These indicate that Hugan Tablet, Ershiwuwei Songshi Pills, and modified Ershiwuwei Songshi Pills had a certain protective effect on chronic liver injury induced by CCl4in rats. The results of semi-quantitative analysis are listed in Table 1.

Note: A. blank group, B. model group, C. Hugan Tablet group, D. Ershiwuwei Songshi Pills group, E. modified Ershiwuwei Songshi Pills group.

Fig.1Pathologicalsectionofratlivertissue(HE × 400)

GroupDose∥mL/kg-++++++++++Control-71000Model1000152Hugan Tablet1000440Ershiwuwei Songshi Pills1000350Modified Ershiwuwei Songshi Pills1000350

Note: degeneration is divided into five levels: no lesion (-), slight (+), mild (++), moderate (+++), and severe (++++).

3.2Effectofcarbontetrachloride-inducedchemicalliverinjuryonliverfunctionofratsCompared with the blank group, the contents of AST and ALT in the model group increased significantly (P<0.01), indicating that the model was successfully reproduced. Compared with the model group, the contents of ALT and AST in each administration group significantly declined (P<0.05), as indicated in Table 2.

GroupDose∥g/kgAST∥U/LALT∥U/LControl-20.4±6.35∗∗16.7±4.33∗∗Model-102.0±23.7200.0±16.2Hugan Tablet0.49037.9±8.37∗56.2±29.4∗Ershiwuwei Songshi Pills0.11757.3±19.2∗68.9±26.1∗Modified Ershiwuwei Songshi Pills0.10554.8±11.4∗53.1±19.4∗

Note: compared with the model group,*P<0.05,**P<0.01; It is the same as below.

3.3EffectonliverfibrosisindicatorsinserumofratswithliverinjuryCompared with the blank group, the model group had significantly higher TNF-α, NF-kB, IL-6 and IL-1β (P<0.01); compared with the model group, the protein expressions of NF-kB, TNF-α, IL-1β and IL-6 in the Hugan Tablet group, Ershiwuwei Songshi Pills Group and modified Ershiwuwei Songshi Pills Group significantly declined (P<0.05). Results are listed in Table 3.

GroupAdministrationdose∥g/kgNF-κBTNF-αIL-1βIL-6Control-0.184 0±0.0114 ∗∗0.251 0±0.012 1∗∗0.181 3±0.009 8∗∗0.252 7±0.011 2∗∗Model-0.207 4±0.009 40.271 5±0.006 90.201 5±0.010 00.274 3±0.015 3Hugan Tablet0.4900.185 9±0.008 0∗∗0.257 0±0.010 4∗0.190 0±0.008 4∗0.265 1±0.013 0Ershiwuwei Songshi Pills0.1170.188 7±0.014 4∗0.259 9±0.011 7∗0.191 5±0.007 5∗0.264 2±0.012 7Modified Ershiwuwei Songshi Pills0.1050.186 3±0.01 30∗0.259 0±0.007 5∗0.191 0±0.010 7∗0.258 1±0.011 9∗∗

4 Discussions

The liver plays an important role in activities such as secretion, metabolism, and synthesis in humans and other animals[13]. Various factors such as alcohol, drugs, viruses, and endotoxins may cause different liver injury[14-18]. ALT and AST are commonly applied in clinical evaluation of liver injury. Serum ALT and AST levels can reflect liver injury[16]. In this experiment, it is found that Hugan Tablet Group, Ershiwuwei Songshi Pills Group, and modified Ershiwuwei Songshi Pills Group could significantly reduce the ALT and AST (P<0.05), proving that both the original prescription and modified prescription have the functions. The effect on ALT is as follows: modified group > Hugan Tablet group > original prescription group, and the effect on AST is as follows: Hugan Tablet group > modified group > original prescription group.

NF-κB participates in regulating the expression of various inflammatory factors and immune genes, thus inhibiting its protein expression can reduce the enlargement and persistence of inflammatory lesions[19-23]. According to the experimental results, compared with the model group, the expression of NF-κB protein in each administration group has a significant decline trend, and the protein expression of TNF-α, IL-1β and IL-6 has different degrees of decline. Hugan Tablet Group, Ershiwuwei Songshi Pills Group, and modified Ershiwuwei Songshi Pills Group had significant effect on reducing the liver injury.

According to Tibetan medicine theory, liver disease is caused by human liver, gallbladder, spleen, and stomach being affected by heat poisoning. The main treatment is to clear heat supplemented with detoxification. The components of Ershiwuwei Songshi Pills show the compatibility of heat-clearing drugs, that is, "bitter" and "astringent" components. After modification, the compatibility conforms to Tibetan medicine compatibility theory.

In summary, Ershiwuwei Songshi Pills and its modified prescription have a certain therapeutic effect on rats with chronic liver injury induced by CCL4. The action mechanism of original prescription and modified prescription may be associated with the inhibition of oxidative stress and inflammatory response and involvement in regulating the expression of inflammatory factor proteins.