Effect of Shenlingyigan decoction on inflammatory factors related to liver injury regulated by TLR3 signaling pathway

Background

To investigate the therapeutic effect of Shenlingyigan decoction on acute liver injury. Further explored the mechanisms involved in the therapeutic properties of Shenlingyigan decoction by test several key proteins (TLR3, TRIF, TBK1, IRF3, IFNβ, IL-1 and IL-6) within the TLR3 signaling pathway.

Methods

The mouse acute liver injury model group was established by pretreatment with D-GalN and Poly (I:C) induction. The acute liver injury mouse treatment groups were gavage with different doses of Shenlingyigan decoction for 3 days. The therapeutic effects of Shenlingyigan decoction were preliminarily evaluated using organ indices, tissue images, and HE staining. Furthermore, potential associated signaling pathways and target effects were predicted through network pharmacology. Western blot experiments were conducted to examine the expression of relevant proteins (TLR3, TRIF, TBK1, IRF3, IL-1, and IL-6). In addition, immunofluorescence assays were performed to assess the localization of IRF3 and IFNβ expression in the cytoplasm and nucleus. Finally, the effects of Shenlingyigan decoction on the expression of TLR3, TRIF, TBK1 and IRF3 genes were further studied by QT-PCR.

Results

The liver organ index, the tissue photos and HE staining showed that Shenlingyigan decoction could reduce inflammation by decreasing the presence of inflammatory cells and downregulating the expression of IL-1 and IL-6. The result of network pharmacology showed 709 potential drug and disease overlapping targets. Toll-like receptor signaling pathway was related with these targets through KEGG analysis. Besides, TLR3, TBK1, IRF3, IL6, were important targets associated with viral hepatitis. Westernblot and Immunofluorescence analysis showed that Shenlingyigan decoction reduced the expression of TLR3 and TBK1 in mice with liver injury, while increasing the expression of IRF3. Shenlingyigan decoction does not significantly affect the expression of TRIF and IFNβ; however, it enhances the expression of IRF3 in the nucleus, consequently leading to increased expression of IFNβ in the nucleus. The results of QT-PCR showed that Shenlingyigan decoction could down-regulate the expression of TLR3, TRIF and TBK1 genes, and up-regulate the expression of IRF3 gene.

Conclusions

Shenlingyigan decoction participated in immune responses by effecting the expression of TLR3 signaling pathway-related factors to treat the acute liver injury.

Keywords: TLR3 signaling pathway, Shenlingyigan decoction, Acute liver injury

2.1. The preparation of SLYG

The formula of Shenlingyigan decoction (SLYG) is as follows: Panax quinquefolius L. (Araliaceae), Ophiopogon japonicas (Asparagaceae), rehmannia root (Gesneriaceae), radix paeo-niae alba (Paeoniaceae), ganoderma lucidum (Curtis), schisandra chinensis (Schisandraceae), Lithospermum erythrorhizon (Boraginaceae), Rubia cordifolia L (Rubiaceae), Lilium longiflorum Thunb. (Liliaceae), citrus chirocarpus (Rutaceae) and Glycyrrhiza glabra L. (Leguminosae). The nomenclature of all plant species mentioned in this study adheres to the latest revision based on the “World Flora Online” (www.worldfloraonline.org) and MPNS (http://mpns.kew.org) databases. All the herbs included in the prescription were procured from Beijing Tongrentang Pharmacy, and their specific sourcing information is provided in Table 1.

Upon procurement, the medicinal materials were soaked in water (with 10 times the weight of the medicinal materials) for 30 min and boiled for 20 min. This process was repeated three times, and the obtained liquid was mixed and filtered. The resultant liquid was concentrated to a concentration of 3.5 g/ml and stored in a refrigerator. The dosage of Shenlingyigan decoction in the table is adult dose. Through the conversion of body surface area formula, the dosage of mice is set as 0.4 ml (1.4 g)/20 g. In order to compare the therapeutic effect of halving the dosage, we set the traditional Chinese medicine group as SLYG (1) is 0.4 (1.4 g) ml/20 g and SLYG (2) is 0.2 (0.7 g) ml/20 g.

2.2. Animals

Institute of Cancer Research (ICR) mice which were four-week-old and weighing 15–20 g, equally male and female, were purchased from Hunan Laike Jingda Experimental Animal Co. Ltd. (SCXK (Hunan) 2019-0004). The modeling drugs are as follows: D-GalN and Poly (I:C) Sigma-Aldrich (St. Louis, MO, USA).

The mice were offered food and water freely. We housed the mice in rooms with light and dark cycle every 12 h. Experimental schemes and animal nursing have the approbation of the Institutional Animal Care and Use Committee of Xi’an Children’s Hospital. All animals are treated humanely, and every effort is made to minimize animal suffering and population. Forfty mice were randomly divided into 4 groups, each mouse in the model group and in the SLYG (1,2) groups was injected intraperitoneally with 10 mg/mouse D-GalN, followed by 1 μg/mouse Poly (I: C). Each mouse in the control group was intraperitoneally injected with the same amount of normal saline. As show in Fig. 2. (1) The control group that was treated with saline. (2) The model group (Poly (I: C) + D-GalN) that was treated with saline, and (3) The three SLYG treatment groups (Poly (I: C) + D-GalN + SLYG) that was treated with a different dose of SLYG (1,2) on the second day after modeling.

2.3. Organ index calculation

All mice received intragastric administration once a day for 3 days, and all mice were sacrificed. After killing the mice, the liver tissues were dissected and removed immediately. First, the liver in each group was photographed. The liver index was then weighed and recorded to calculate the liver index of the mice. The index was calculated according to the formula [19]: liver index = liver weight of mice (mg)/body weight of mice (g) × 100%.

2.4. Hematoxylin-eosin staining (HE)

The mice livers were washed with ice-cold saline after sacrificed, blotted with filter paper, and fixed by soaking in 10% neutral formalin. Each liver was dehydrated in different concentrations of C₂H₅OH, cleared with xylene, and embedded in paraffin. Sections were taken to 5 μm, baked at 70 °C for 5 min, dewaxed with xylene, and rehydrated with graded alcohol. 3 sections in each group were then stained with hematoxylin.

2.5. Network pharmacology analysis

The active chemical compounds of “the Shenlingyigan decoction which consist of Panax quinquefolius L., Ophiopogon japonicas, rehmannia root, radix paeoniae alba, ganoderma lucidum, schisandra chinensis, Lithospermum erythrorhizon, Rubia cordifolia L, Lilium longiflorum Thunb, citrus chirocarpus, and Glycyrrhiza glabra L.” were screened by the Traditional Chinese Medicine System Pharmacology (TCMSP) database [20]. The screening conditions were oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. We converted each compound screened into canonical SMILES via PubChem. Subsequently, these SMILES were imported into SwissTargetPrediction, which could be used to predict potential targets of the compounds. The species were selected as “Homo sapiens,” and the probability >0.11 was used as the screening condition.

With “viral hepatitis” as the keyword, the disease-related targets were retrieved through the OMIM (https://omim.org/), and GeneCards – the human gene database (www.genecards.org/). Overlapping targets between potential targets of herb compounds and disease-related targets were considered potential targets for viral hepatitis. The protein-protein interaction (PPI) network was constructed by the STRING database (https://cn.string-db.org/) and visualized by Cytoscape 3.9.1 software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted by linking targets to the Database for Annotation, Visualization, and Integrated Discovery database (DAVID) (https://david.ncifcrf.gov/). Next, the picture of KEGG pathway enrichment was created using the online tools (http://bioinformatics.com.cn/).

2.6. ELISA

The prepared liver homogenate was used for ELISA to measure the expression level of Interleukin 6 (IL-6), and interleukin 1 (IL-1) (kits from Xiamen Lunchangshuo Biological Technology Co. LTD) according to the manufacturer’s protocol.

2.7. Western blot

The liver stored at −80 °C was weighed and added with RIPA lysis buffer (Biyuntian, China), the supernatant absorbed by centrifugation was the total protein of the liver after sufficient vortex lysis on ice. The samples’ protein concentration was test by BCA protein assay kit (Biyuntian, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (20 μg) were used to separate the samples and polyvinylidene fluoride (PVDF) membrane (Merck Millipore, USA) was used to transferred the proteins. The transferred membrane is incubated and bound with a primary antibody (TLR3 (rabbit polyclonal antibody, Abcam, USA) TRIF (rabbit polyclonal antibody, Abcam, USA) TBK1(rabbit polyclonal antibody, Abcam, USA) IRF3(rabbit monoclonal antibody, Abcam, USA) IFNβ (rabbit polyclonal antibody, Abcam, USA)), then the membrane is further incubated and bound with a second antibody conjugated with the appropriate horseradish peroxidase. Finally, protein bands were displayed using ECL chromogenic solution (Biyuntian, China).

2.8. Immunofluorescence assay (IF)

The liver sections in each group were place in citrate buffer solution (pH = 6.0), heat in microwave for 8 min, then cool to room temperature. 3% H₂O₂ added for 10 min at room temperature to inactivate endogenous enzymes. 5% sheep serum placed in wet boxes, sealed for 20 min at room temperature. After that incubated with the primary anti-nuclear factor antibodies (IRF3(rabbit monoclonal antibody, Abcam, USA) IFNβ(rabbit polyclonal antibody, Abcam, USA)) at 4 °C overnight. Then wash the sections for 3 times after conjugation with primary antibodies. They were incubated using the corresponding secondary antibody labeled with Cy3 (Sulfo-Cyanine3). Cell nuclei were stained with (DAPI 4’,6-diamidino-2-phenylindole). Suitable positive controls were utilized. Finally, the neutral gum was sealed, and the image was observed and preserved under an optical microscope (Leica, Germany).

2.9. Real-time quantitative RT-PCR

For total RNA extraction from tissue, a small portion of liver tissue sample was removed, frozen in liquid nitrogen and homogenized. 500 μl Trizol was added to the cell sample. To the 1.5 ml EP tube containing Trizol, 100 μl chloroform was added, mixed by shaking, and then allowed to stand for 5 min. The tube was centrifuged at 12,000 rpm and 4 °C for 10 min. The upper colorless and transparent aqueous phase was transferred to another clean 1.5 ml EP tube. The RNA precipitate was washed with 1 ml of 75% ethanol, air-dried at room temperature for approximately 5–10 min, and then reverse transcribed into cDNA. After the reaction was detected using PerfectStart Green qPCR SuperMix (Full Gold, AQ601-04), real-time fluorescence quantitative PCR instrument (ABI, Q1) was used for RT-PCR. The expression level of mRNA was calculated using the 2^-ΔΔCt method, with GAPDH gene as the internal reference. T-test was used to evaluate the difference in expression. The primer sequences are shown in Table 2.

2.10. Statistical analysis

Data differences in the icons are represented by the mean ± standard error (SEM) for each group. Analysis of variance (one-way analysis of variance) and t-test were used to detect whether the difference between groups was significant. Unless otherwise stated, all statistical analyses were performed using Origin8.0 (Originlab, USA). P < 0.05 was considered statistically significant. P < 0.1 was considered statistical difference.

3.1. The Shenlingyigan decoction could reduce inflammatory reaction in the injured liver

During the whole process of modeling and administering acute liver injury in mice, no death was observed. The liver visceral index (n = 10) of mice was calculated and showed that the liver visceral index was significantly increased in the model group compared to the control group. The liver visceral index of mice in SYLG1 group was evidently less than that in model group (Fig. 3). As showed in Fig. 4a, the liver tissue photos of mice in the model group exhibited signs of congestion and swelling, whereas no such phenomenon was observed in the control group. However, the injured livers in the SLYG (1,2) group demonstrated an improvement in terms of congestion and swelling compared to the model group. The results of HE staining showed that lots of inflammatory cells infiltrated the liver tissue of mice in the model group. The situation was improved in the SLYG (1,2) group (Fig. 4b).

3.2. Potential signaling pathways between Shenlingyigan decoction and viral hepatitis

We confirmed that Shenlingyigan decoction could treat acuate liver injury, and further explored its mechanism. The possible signaling pathway between the Shenlingyigan decoction and viral hepatitis was predicted based on network pharmacology. We retrieved 11 active ingredients of the Panax quinquefolius L., 9 active ingredients of the Ophiopogon japonicas, 9 active ingredients of the radix paeoniae alba, 8 active ingredients of the rehmannia root, 61 active ingredients of the ganoderma lucidum, 8 active ingredients of the schisandra chinensis, 12 active ingredients of the Lithospermum erythrorhizon, 17 active ingredients of the Rubia cordifolia L, 5 active ingredients of the citrus chirocarpus, 6 active ingredients of the Lilium longiflorum Thunb., and 91 active ingredients of the Glycyrrhiza glabra L. from the TCMSP database. And then, we predicted 863 herb targets by SwissTargetPrediction. The Cytoscape software was used to analyze the effective components to obtain the target network diagram of the compound components (Fig. 5a), the shape size is positively correlated with the degree value. From Genecards and OMIM databases, we obtained 10,662 potential disease targets. We got 709 overlapping targets as showed in the Wayne diagram (Fig. 5b). According to the Molecular COmplex Detection (MCODE) score, we got the most closely connected regeions from the interactions of the 709 overlapping targets, including 57 overlapping targets (Fig. 5c). In addition, AKT1 gots the highest score in these targets. Through KEGG analysis of these targets, the first 60 relevant signal pathways were obtained and selected, as shown in the figure Fig. 5d, which included Toll-like receptor signaling pathway. We applied the criteria “Drugs & Compounds, Expression in Human Tissues, Pathways” to screen for target proteins associated with viral hepatitis. This screening process yielded 110 key targets, including TLR3, TBK1, IRF3 and IL6. The disease-associated network diagram was constructed using Cytoscape software for analysis as showed in Fig. 5e.

AKT1 is a key upstream kinase associated with NF-κB and IRF3 inflammatory signaling pathways in inflammation-related responses [21,22]. Notably, the search results in the PPI network of key target protein of the intersection targets showed that TLR3, TBK1, IRF3, IL6, were important targets associated with viral hepatitis (Fig. 5e). Toll-like receptors (TLRs) could recognize molecules unique to microbes so that distinguish self from non-self which play an important role in innate and adaptive immune responses. Toll-like receptor signaling pathway includes the Myd88-dependent pathway and the TRIF (Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β)-dependent pathway. TRIF need its associated enzymes such as TBK1(TANK-binding kinase1) to induce translocation and activation of transcription factors such as NF -κ B (nuclear factor κ B), AP -1 (activator protein 1), and IRF3(interferonregulatory factor 3), besides, the TRIF/TBK1pathway is critical for IRF-3-mediated transcription [23]. The ligands of TLR3 were Poly I: C and dsRNA which were sourced from viruses and only works via the TRIF-dependent pathway [24,25]. Based on literature research and analysis results of network pharmacology, we speculated that the Shenlingyigan decoction could improve the acute liver injury caused by virus through modulation of the TLR3 signaling pathway.

3.3. The Shenlingyigan decoction can reduce the expression of IL-1 and IL-6 in the liver

IL-1 and IL-6 expression in the liver of mice in each group was tested by ELISA. The results in Fig. 6 showed that the expression of IL-1 in the model group and SLYG (1,2) groups increased significantly than that of control group. IL-1 in the SLYG (1,2) groups significantly decreased than that of the model group. There was a significant difference between the SLYG1 group and the model group. IL1 in SLYG2 group was also lower than that in model group, but the difference was not significant. The results in Fig. 7 showed that the expression of IL-6 increased a lot in the model group and SLYG (1,2) groups than that of control group. The expression of IL-6 decreased significantly in the SLYG1 group and SLYG2 group than that of the model group. The IL-6 expression in the SLYG1 group and the SLYG2 group did not have significantly difference (Fig. 7).

3.4. The Shenlingyigan decoction regulates the expression of TLR3 signaling pathway-related proteins

To investigate the mechanism of action of Shenlingyigan decoction, we further examined several key proteins within the TLR3 signaling pathway. The results (Fig. 8) showed that compared to the control group, the expression levels of TLR3, TBK1, IRF3 and IFNβ were increased in the liver injury model group. In comparison to the model group, the expression levels of TLR3 and TBK1 were decreased in the SLYG1 and SLYG2 groups, whereas IRF3 expression was increased in both SLYG1 and SLYG2 groups, with no significant change observed in IFNβ expression. The expression level of TRIF was relatively low in all groups, with no significant differences observed. Additionally, we found that the downregulation of TLR3 and TBK1 was more pronounced in the SLYG2 group compared to the SLYG1 group, while the upregulation of IRF3 was more pronounced in the SLYG1 group compared to the SLYG2 group. These results indicate the therapeutic effects of Shenlingyigan decoction are associated with the TLR3 signaling pathway.

3.5. Shenlingyigan decoction changed the distribution of IRF3 and IFN-β between nucleus and cytoplasm

The current study aimed to investigate the impact of Shenlingyigan decoction on the distribution of IRF3 and IFN-β between the nucleus and cytoplasm, utilizing immunofluorescence analysis through confocal microscopy. In the control group, IRF3 was primarily distributed in the cytoplasm in control group and model group; (Fig. 9). However, treatment with Shenlingyigan decoction was observed to cause a significant augmentation in the amount of IRF3 present in nuclei when compared to the model group. This phenomenon was more obvious in SLYG1 group than in SLYG2 group. Similarly, in the control group, IFN-β was detected to be distributed within the cytoplasm (Fig. 10). After modeling, the expression of IFNβ in the cytoplasm was significantly increased, but there was no significant expression in the nucleus. The expression of IFNβ in the nucleus was significantly increased in the SLYG1 group and the SLYG2 group when compared to the model group. Thus, the findings of the study support the hypothesis that Shenlingyigan decoction can promote the migration of IRF3 to the nucleus, as well as cause an increase in the distribution of IFN-β in the nucleus.

3.6. Shenlingyigan decoction regulates the expression of TLR3 signaling pathway-related genes

In order to determine whether Shenlingyigan decoction intervenes in the changes of TLR3 signaling pathway-related gene levels, real-time quantitative PCR was used to detect the mRNA expression levels of TLR3, TRIF, TBK1, and IRF3 in liver tissue. The results are shown in Fig. 11. Compared to the control group, the expression level of TLR3 in the model group was significantly increased. In comparison to the model group, the expression level of TLR3 in the SLYG1 group was significantly decreased, and the SLYG2 group also showed a decrease, although there was no significant difference between the groups. Compared to the normal group, TRIF showed a significant decrease in expression in the SLYG1 group, with no significant differences between the other groups. TBK1 showed significantly higher expression in the model group compared to the normal group and SLYG group, with TBK1 in the SLYG1 group lower than that in the SLYG2 group, and the difference was significant. In comparison to the normal group, IRF3 showed a significant increase in expression in both the model group and SLYG group, with the SLYG group showing a significantly higher expression than the model group, and the SLYG2 group slightly higher than the SLYG1 group, with significant differences.

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Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.