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

  • ️Mon Jan 01 2024

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

Xiaoli Liu et al. Heliyon. 2024.

Abstract

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: Acute liver injury; Shenlingyigan decoction; TLR3 signaling pathway.

© 2024 The Authors.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yanping Shi reports financial support was provided by 10.13039/100016067Shaanxi Provincial Administration of Traditional Chinese Medicine research Project. Xiaoli Liu reports financial support was provided by Health Scientific Research Talent Project of Xi’an Municipal Health Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

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Graphical abstract
Fig. 1
Fig. 1

TLR3 signal pathway.

Fig. 2
Fig. 2

Model-making process of the animal experiment.

Fig. 3
Fig. 3

Visceral index (P < 0.1) * represents a significant difference from the model group, # represents a significant difference from the control group.

Fig. 4
Fig. 4

a. Liver tissue photos, b. Pictures of mice liver tissues (200×) in each group of HE staining.

Fig. 5
Fig. 5

(a) Active ingredient-target network diagram of Shenlingyigan decoction, xys is Panax quinquefolius L., md is Ophiopogon japonicas, bs is radix paeoniae alba, sd is rehmannia root, lz is ganoderma lucidum, wwz is schisandra chinensis, zc is Lithospermum erythrorhizon, qc is Rubia cordifolia L, fs is citrus chirocarpus, bh is Lilium longiflorum Thunb.,gc is Glycyrrhiza glabra L. (b)Venn diagram of Shenlingyigan decoction and viral hepatitis; (c) The PPI network of key target protein of the intersection targets. The circular layout is arranged counterclockwise according to the node from light to dark and small to large. (d) KEGG pathway enrichment analysis. The larger the area of dots, the more the counts; the smaller the P-value, the bluer the dot color. Red box represent Toll-like receptor signaling pathway and Hepatitis B signaling pathway. (e). Yellow nodes represent TLR3, TBK1, IRF3 and IL6 respectively.

Fig. 5
Fig. 5

(a) Active ingredient-target network diagram of Shenlingyigan decoction, xys is Panax quinquefolius L., md is Ophiopogon japonicas, bs is radix paeoniae alba, sd is rehmannia root, lz is ganoderma lucidum, wwz is schisandra chinensis, zc is Lithospermum erythrorhizon, qc is Rubia cordifolia L, fs is citrus chirocarpus, bh is Lilium longiflorum Thunb.,gc is Glycyrrhiza glabra L. (b)Venn diagram of Shenlingyigan decoction and viral hepatitis; (c) The PPI network of key target protein of the intersection targets. The circular layout is arranged counterclockwise according to the node from light to dark and small to large. (d) KEGG pathway enrichment analysis. The larger the area of dots, the more the counts; the smaller the P-value, the bluer the dot color. Red box represent Toll-like receptor signaling pathway and Hepatitis B signaling pathway. (e). Yellow nodes represent TLR3, TBK1, IRF3 and IL6 respectively.

Fig. 6
Fig. 6

The expression of IL-1(P < 0.05). * represents a significant difference from the model group (n = 6); # represents a significant difference from the control group (n = 6); △ represents a significant difference from the SLYG1 group (n = 6).

Fig. 7
Fig. 7

The expression of IL-6 (P < 0.05). * represents a significant difference from the model group (n = 6); # represents a significant difference from the control group (n = 6).

Fig. 8
Fig. 8

Effects of Shenlingyigan decoction on protein expression of vital genes related to TLR3 signaling pathway in response to acute liver injury.

Fig. 9
Fig. 9

Effects of Shenlingyigan decoction on translocation of IRF3 into the nucleus. The blue represents the nucleus, and the red represents IRF3.

Fig. 10
Fig. 10

Effects of Shenlingyigan decoction on translocation of IFN-β into the nucleus. The blue represents the nucleus, and the red represents IFN-β.

Fig. 11
Fig. 11

Shenlingyigan decoction regulates the expression of TLR3 signaling pathway-related genes mRNA levels in mice (n = 3) (P < 0.05). * represents a significant difference from the model group (n = 6); # represents a significant difference from the control group (n = 6); △ represents a significant difference from the SLYG1 group (n = 6).

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