Action mechanism of Yiqi Hexue Formula in the treatment of liver cirrhosis based on network pharmacology and experimental verification

ZHANG Ying-xue, SUN Feng-xia, LI Xiao-ling, XU Chun-jun, GUO Yu-fei, CHEN Zimeng

1. Beijing University of Chinese Medicine,Beijing 100029, China

2. Department of Infections ,Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University, Beijing 100010, China

Keywords:Yiqi Hexue Formula Liver cirrhosis Network pharmacology Mechanism of action

ABSTRACT

1. Introduction

Liver cirrhosis is a kind of advanced hepatic fibrosis disease caused by various factors of chronic liver damage [1]. The activation and proliferation of hepatic stellate cells (HSCs) is the central link of hepatic fibrosis, which is related to multiple signal transduction pathways [2,3].Every year, about 2 million people die from liver disease and 1 million people die from complications of liver cirrhosis. Liver cirrhosis is currently the 11th most common cause of death in the world, bringing a serious health and economic burden to the world [4].At present, the treatment of liver cirrhosis is mainly etiological treatment, anti-inflammatory and anti hepatic fibrosis,and actively prevent and treat complications. If the liver is seriously damaged, the only treatment option may be liver transplantation[5,6].Traditional Chinese medicine has played its unique advantages in the treatment of liver cirrhosis, especially in the aspect of anti liver fibrosis, which has been widely concerned and recognized, and has become one of the important means for the treatment of liver cirrhosis [7].

Professor Guan Youbo, the founder of the liver disease department of Beijing Traditional Chinese medicine hospital, believes that the occurrence, development and prognosis of the disease are inseparable from the theme of Qi and blood. Liver cirrhosis is based on Qi deficiency and blood stagnation. For chronic liver disease and chronic physical deficiency, we should first pay attention to Qi and blood, promote blood circulation and remove blood stasis.Therefore,the treatment of liver cirrhosis should be based on replenishing qi and blood, nourishing blood and softening the liver, and softening the firmness and dissipating lumps[8,9].The Department of Liver Diseases, Beijing Hospital of Traditional Chinese Medicine has long been adhering to Professor Guan Youbo’s "Qi-blood differentiation"academic thought, using the method of replenishing qi and softening the blood to dispel stagnation, formulating the basic prescription for replenishing qi and blood, and applying the addition and subtraction of Yiqi Hexue Formula to treat patients with liver cirrhosis. It has achieved remarkable curative effect and accumulated rich clinical experience. This study intends to explore the possible anti-cirrhosis mechanism of Yiqi Hexue Formula through network pharmacology and animal experiment research.

2. Materials and methods

2.1 Acquisition of effective components and targets of Yiqi Hexue Formula

Using the TCMSP database, search for Astragalus, Angelica,Radix Paeoniae Alba, and set OB≥30% and DL≥0.18. Using the BATMAN-TCM database, search the turtle shell and Chickens Gizzard-membrane, set the score cutoff≥20, and the P value after correction＜0.05, and screen the effective compounds and their targets. Then use "PERL" software to merge the obtained drug components and target points. And apply Uniprot to correct the target protein to the standard gene name.

2.2 Acquisition of genes related to cirrhosis

Taking "cirrhosis" as the keyword, the genes related to liver cirrhosis were searched from GeneCard, PharmGkb,OMIM,DrugBank, TTD databases respectively. Application R×64 4.0.2.The "Venn" installation package and script in the software take and combine the results and draw the Venn diagram.

2.3 Obtaining potential targets

The R software is used to intersect the target related to the drug active ingredient and the disease target, and draw the Venn diagram to obtain the potential target for the treatment of liver cirrhosis of the active ingredient of Yiqi Hexue Formula.

2.4 Constructing a regulatory network diagram of "active pharmaceutical ingredients-disease targets"

Firstly, the final data obtained from 1.1 and 1.2 are sorted through"PERL" software and scripts, and then Cytoscape 3.8.0 software is used to construct a network diagram of "drug components-disease targets". The greater the degree value of a node, the more it will participate in biological functions.

2.5 Construction of protein interaction network

Import the intersection genes into the STRING database, set the species condition to be human, and the confidence level is greater than 0.9, and construct a protein-protein interaction (PPI) network.Import the results into Cytoscape, and use the MCC algorithm in the cytoHubba plug-in to get the hub gene.

2.6 Enrichment analysis of KEGG pathway

Use R software to transform the id of the intersection gene, and perform KEGG pathway enrichment analysis. The P value filter condition and the corrected filter condition are both set to 0.05,and the subsequent experimental verification pathways are selected from the enrichment results of the KEGG pathway, and the pathway diagram is drawn.

2.7 Experimental verification

2.7.1 Experimental animals

Thirty-five SPF male SD rats, 6 weeks old, weighing (200±10) g,were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., license number SCXK (Beijing) 2016-0011.The animal experiment was approved by the Experimental Animal Management Committee of Beijing Institute of Traditional Chinese Medicine (Ethics No. 2021030102).

2.7.2 Main reagents and instruments

Carbon tetrachloride (CCl4), for HPLC, ≥99.0% (Shanghai Macklin Biochemical Technology Co., Ltd.); HE staining solution(Zhuhai Baso Biotechnology Co., Ltd.); Masson staining solution prepared by the laboratory; RNA extraction kit ( Thermo Fisher Scientific, PureLinkTMRNA Mini Kit); one-step fluorescent quantitative RT-PCR kit (096A) (TaKaRa); fluorescent quantitative PCR instrument (Roche Germany, model: LightCycler 480 II).

2.7.3 Preparation of Chinese Medicine Yiqi Hexue Formula consists of 30 g of raw astragalus, 12 g of angelica, 15 g of Radix Paeoniae Alba, 15 g of vinegared turtle shell,and 10 g of vinegared Chickens Gizzard-membrane. In clinical use, one pair a day. Purchased from Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University.The rat gavage dose refers to "Pharmacological Experimental Methodology"[10], converted into rat dosage according to the dosage conversion coefficient of adult and rat, and the dosage is 0.861 g/100 g.

2.7.4 Rat modeling and administration

Refer to related literature [11,12] and improve the model. A total of 35 rats were raised. After 3 days of adaptive feeding, 3 rats were randomly selected as the blank control group, and the remaining 32 model rats were treated with 50% CCl4solution (The solvent is olive oil) The dose of 2 mL/kg was injected intraperitoneally for 2 weeks, and 3 mL/kg was injected for 4 weeks, twice a week. All rats were free to drink and eat.After a total of 6 weeks of model building,3 models were randomly selected and evaluated by pathological examination. After the successful model building was evaluated by pathological examination, they were randomly divided into model group, Yiqi Hexue Formula group, 10 in each group. The traditional Chinese medicine group was given the Yiqi Hexue Formula by gavage, and the rest of the rats were gavage with the same amount of normal saline. All rats were gavage once a day, the administration volume: 1 mL/100g, for 4 weeks.

2.7.5 Collection of pathological specimens After pentobarbital sodium anesthesia, the rat was fixed, the abdominal cavity was opened, the rat liver was cut, the liver tissue at the same location was cut off, placed in 4% paraformaldehyde solution, and then several pieces of liver tissue were taken in liquid nitrogen Freeze quickly and store all samples in a refrigerator at-80 ℃ for later RT-qPCR detection.

2.7.6 Index detection

(1) Take 3 rats from each group and perform HE staining and Masson staining to observe the pathological changes of liver tissue;

(2) Take 3 mice from each group, and measure the expression levels of JAK2 and STAT3 mRNA in liver tissues by RT-qPCR.According to the kit instructions, total RNA from rat liver tissues of each group was extracted, cDNA was synthesized, and RT-qPCR analysis was performed. Primer sequence: JAK2 upstream primer 5'-ATGCTTCCTGTATTCACCTGCCTTG-3', downstream primer 5'-GCTCCTGCCCTTTCATTCCTCTG-3', STAT3 upstream primer 5'-CGGTTCAGTGAGAGCAGCAAGG-3', downstream primer 5'-AGTGAGACAAGAGGAGCAGGTGAG-3'.

2.8 Statistical processing

The data obtained were statistically analyzed and mapped with GraphPad Prism 8.0.2 software and SPSS 26.0 software. The data of experimental results are all expressed by means of Mean±SD. If the square difference is uniform, one-way analysis of variance (oneway ANOVA) is used for comparison of multiple groups. P<0.05 indicates that the difference is statistically significant.

3. Results

3.1 Screening of active ingredients and target prediction of Yiqi Hexue Formula

A total of 43 active ingredients were obtained, including 13 Radix Paeoniae Alba, 2 Angelica, 20 Astragalus, 1 turtle shell, and 7 Chickens Gizzard-membrane. A total of 1 081 predicted targets were obtained at the same time. The effective ingredients and target prediction files were merged using "PERL" software and its scripts,and then standardized annotated and sorted using Uniprot database and "PERL" software, and finally 648 targets were obtained.

3.2 Genes related to liver cirrhosis

Through GeneCard database, OMIM database, PharmGkb database,TTD database, DrugBank database, 3 158, 9, 1, 23, 86 target genes related to liver cirrhosis were obtained respectively. After sorting, a total of 3 220 target genes related to liver cirrhosis were obtained.See Figure 1 for details.

Figure 1 Map of target genes related to liver cirrhosis

3.3 The intersection of drug targets and disease targets

After sorting and analysis, a total of 648 potential drug targets and 3 220 disease targets were obtained. The intersection of the two genes was 306, as shown in Figure 2.

Figure 2 Venn diagram of common target

3.4 "active ingredient core target" network analysis

There are 336 nodes in the active ingredient core target map,including 30 from compounds and 306 from genes (see Figure 3).The active ingredients in the top 10 of degree are listed (see Table 1).

Figure 3 Network diagram of "active ingredients-core targets"

Table 1 Basic information of pharmaceutical chemical composition

3.5 PPI network construction

The intersection genes were imported into the STRING database to construct a PPI network. The PPI network was imported into Cytoscape, and the nodes were ordered according to the MCC value. The first 10 targets were JUN, RELA, IL6, IL1B, CXCL8,IL1A, CCL2, IL4, TP53, IL10 (see Figures 4 and 5) It is speculated that these targets are the hub genes of Yiqi Hexue Formula in the treatment of liver cirrhosis.

3.6 Enrichment analysis of KEGG pathway

Figure 4 PPI network of Yiqi Hexue Formula in the treatment of liver cirrhosis

After KEGG pathway enrichment analysis, a total of 180 pathway enrichment results were obtained, involving PI3K/Akt, MAPK,IL-17, JAK/STAT and other signaling pathways (Figure 6). It is suggested that Yiqi Hexue Formula may exert therapeutic effects on liver cirrhosis through the above pathways.

3.7 Experimental verification

3.7.1 Model evaluation

After the modeling, the results of HE staining and Masson staining showed that the blank control group had no obvious inflammatory cell infiltration, the hepatocyte cords were arranged in a radial state and the lobule structure was complete. In the model group,hepatocyte necrosis, inflammatory cell infiltration, severe steatosis,disordered arrangement of hepatocyte cords, diffuse fibrous hyperplasia, and false lobule formation can be seen, as shown in Figure 7 and Figure 8.

Figure 5 Hub gene (Top10) of Yiqi Hexue Formula in the treatment of liver cirrhosis

Figure 6 JAK/STAT signaling pathway (red borders represent common genes)

Figure 7 HE staining of rat liver tissue (×200)

Figure 8 Masson staining of rat liver tissue (×200)

3.7.2 Yiqi Hexue Recipe improves liver pathology in cirrhotic rats

After the treatment, the results of HE staining and Masson staining showed that the hepatocyte cords in the blank control group were arranged neatly and there was no obvious inflammatory cell infiltration. In the model group, the structure of liver lobules was damaged, and some liver cells showed fatty degeneration and inflammatory cell infiltration. Compared with the model group,the degree of liver fibrosis in the Yiqi Hexue Formula group was significantly reduced, the coloration of fibers was significantly lighter, the infiltration of inflammatory cells was reduced, and liver cells were scattered in fatty vacuoles, as shown in Figure 9 and Figure 10.

Figure 9 HE staining of rat liver tissue (×200)

Figure 10 Masson staining of rat liver tissue (×200)

3.7.3 Comparison of JAK2 and STAT3 mRNA expression in liver tissues of rats in each group

Compared with the blank control group, the expression of JAK2 and STAT3 mRNA in the model group was significantly increased,and the difference was statistically significant (P<0.05); compared with the model group, the expression of JAK2 and STAT3 mRNA in the Yiqi Hexue Formula group was significantly decreased, and the difference was statistically significant Significance (P<0.05). Figure 11 .

Figure 11 Comparison of JAK2 and STAT3 mRNA expression in liver tissues of rats in each group

4. Discussion

Our research team has accumulated a certain amount of research foundation in the treatment of cirrhosis with Yiqi Hexue Formula in the early stage. The study found that the combination of Yiqi Hexue Formula and Entecavir Tablets in the treatment of hepatitis B cirrhosis can help improve the patient's TCM syndrome score and reduce liver inflammation. Improve liver synthesis reserve function, improve spleen size, portal vein width, reduce Child score,increase recompensation rate, etc., the clinical effect is definite [13-15],But its specific mechanism is unclear. Network pharmacology can preliminarily predict the mechanism of action of traditional Chinese medicine, and has a wide range of applications in the field of traditional Chinese medicine [16]. This project intends to use network pharmacology to study the multiple active components of Yiqi Hexue Formula, explore the mechanism of Yiqi Hexue Formula in treating liver cirrhosis, and then use animal experiments to verify the signal pathway predicted by network pharmacology.

This study found that the key compounds in Yiqi Hexue Formula are mainly active ingredients such as Bilatriene, quercetin, Nicotinic Acid, kaempferol, and vitamin C. Bilatriene is one of the main chemical components of Chickens Gizzard-membrane[17]. Through the analysis of active ingredient-core target, it is found that the target of bilatriene includes PDGFB, SCNN1A, KCNJ8, etc. The specific mechanism needs to be further verified.Quercetin is a polyphenolic flavonoid. Studies have found that quercetin can resist CCl4-induced liver fibrosis [18,19]. Through network pharmacological research,it was found that the targets of quercetin include PTGS1, PTGS2,JUN, STAT1, AKT1, IL6R, etc., and related mechanism studies have found that quercetin and other flavonoids can interfere with signal pathways closely related to the occurrence of liver fibrosis[20].Internet pharmacological studies have shown that nicotinic acid is closely related to PPARA, IL13, IL6, MAPK9, TP53 and other targets, and the combination of curcumin and nicotinic acid can significantly reduce the expression of TNF-α and IL-6 in the liver [21]. Niacin can significantly improve liver cell steatosis and fibrosis by regulating the expression of PPARα, DGAT2, and SREBP1c mRNA [22].The targets of kaempferol include STAT1, JUN, MMP1, MAPK8, etc.In vitro experiments have shown that kaempferol can significantly inhibit the proliferation of HSC-T6 cells and has a strong effect [23];in vivo experiments have shown that kaempferol has anti-fibrotic effects. The mechanism of action may be through down-regulating the mRNA expression of IL-13 and COL1, inhibiting collagen synthesis, and up-regulating the mRNA expression of MMP-2 to promote collagen decomposition [24].Studies have shown that the antioxidant vitamin C may inhibit HSC proliferation by inhibiting leptin-induced oxidative stress[25]. Clinical studies have found that vitamin C combined with probiotics can help improve liver function and liver fibrosis indicators in patients with liver cirrhosis[26].

Regarding KEGG pathway enrichment, it mainly involves PI3K/Akt, MAPK, IL-17, JAK/STAT and other signaling pathways.The PI3K/Akt signaling pathway is involved in the formation of liver fibrosis, which is mainly related to the regulation of the degradation of extracellular matrix and the intervention of HSC activation [27].The MAPK signaling pathway is closely related to liver fibrosis[28],among which the ERK/MAPK signaling pathway is involved in the proliferation, differentiation and migration of HSC[29], and JNK and p38 can regulate cell differentiation during the process of fibrosis[30].IL-17 induces liver fibrosis through a variety of mechanisms, such as promoting the production of IL-6, IL-1β and TNF-α by inflammatory cells, and can increase the expression of TGF-β1.It can also directly induce HSC to produce type I collagen by activating the STAT3 signaling pathway[31].JAK/STAT signaling pathway is widely involved in cell proliferation, differentiation,apoptosis and inflammation, and it plays an important role in regulating leptin, IL-4, and IFNα in the process of liver fibrosis[32].JAK2 and STAT3 play an important role in the signal transduction of this pathway[33,34]. Relevant studies have shown that after some cytokines bind to HSC cell membrane receptors, they activate the JAK2/STAT3 pathway and expose nuclear localization signals,STAT3 Enter the HSC nucleus, combine with specific regulatory sequences on DNA and initiate its transcription, so that HSC proliferates and promotes the formation of liver fibrosis [35,36].However, there are relatively few studies on cirrhosis/liver fibrosis and the classic JAK2/STAT3 signaling pathway. Combined with the results of network pharmacology.In this study, this pathway was selected for verification, and part of the mechanism of action of Yiqi Hexue Formula in the treatment of liver cirrhosis was explored.The results of the study showed that the expression of JAK2 and STAT3 mRNA in the liver tissues of the model group was significantly upregulated, indicating that the JAK2/STAT3 signaling pathway is involved in the formation of liver cirrhosis. The expression of JAK2 and STAT3 mRNA in the Yiqi Hexue Formula treatment group was significantly down-regulated compared with the model group,indicating that Yiqi Hexue Formula inhibited the expression of JAK2 and STAT3.

In summary, this study used network pharmacology and animal experiments to study the mechanism of action of Yiqi Hexue Formula in treating liver cirrhosis. Network pharmacology found that Yiqi Hexue Formula can treat liver cirrhosis through multiple components, multiple targets, and multiple pathways. The purpose of this method is to provide a theoretical basis for further extracting effective active ingredients from Yiqi Hexue Formula to treat liver cirrhosis. Experimental verification shows that JAK2/STAT3 signaling pathway is involved in the formation of liver cirrhosis. Yiqi Hexue Formula has a clear effect on the treatment of liver cirrhosis and can improve its pathological manifestations. Its mechanism of action may be related to the inhibition of JAK2/STAT3 signaling pathway transmission.

All authors' conflicts of interest statement

The author declares that there is no conflict of interest in the publication of this paper.

Author contribution

Zhang Yingxue conceived this research, completed network pharmacology analysis, and participated in experimental design,execution and data statistics. Sun Fengxia contributed to writing review and supervision. Li Xiaoling and Xu Chunjun performed data analysis. Guo Yufei and Chen Zimeng participated in the execution of the experiment. All authors designed experiments, wrote manuscripts, and revised the papers. All authors read and approved the final version of the manuscript.