Dihydromyricetin ameliorates lipopolysaccharide‒induced hepatic injury in chickens by activating the Nrf2/Keap1 pathway and regulating mitochondrial dynamics

Abstract

Dihydromyricetin (DHM) is a flavonoid found in vine tea that exhibits various pharmacological characteristics, including antibacterial, antiapoptotic, and antioxidant effects. Our previous study revealed that DHM can alleviate chicken hepatic injury, but the underlying mechanism has not been elucidated. To further investigate the protective mechanism of DHM, this study firstly predicted by network pharmacology that the potential regulatory pathways of DHM on hepatic injury. Subsequently, the experimental models were replicated in vivo and in vitro using Hy‒Line white broiler chickens and chicken primary hepatocytes treated with DHM and with/ without LPS. Network pharmacology results showed that the effect of DHM on hepatic injury might be related to oxidative stress and mitochondrial function. Further experiments showed that DHM significantly reduced LPS‒elicited serum ALT and AST activities, promoted antioxidant enzyme activities and scavenged ROS in chicken liver or primary hepatocytes. Molecular docking studies showed that DHM could directly bind to Nrf2 and Keap1. Furthermore, DHM treatment regulated the expression of Nrf2 and Keap1, thereby upregulating the downstream expression of antioxidant factors, including HO‒1 and NQO1, in vivo and in vitro. Moreover, DHM modulated the expression of mitochondrial dynamics related factors, including Mfn1/2, Opa1, Drp1, and Fis1, meanwhile, DHM ameliorated mitochondrial structural damage and increased the MMP. Overall, these results suggested that DHM activated the Nrf2/Keap1 pathway and regulated the balance between mitochondrial fusion and fission, ultimately alleviating chicken hepatic injury induced by LPS.

Keywords: Dihydromyricetin; Nrf2/Keap1 pathway, Mitochondrial dynamics, Oxidative stress, Hepatic injury.

Graphical abstract

Fig. 1

Effects of DHM on hepatic injury based on network pharmacology. (A) Wayne plots of DHM and hepatic injury targets. (B) Constructing PPI networks with collective targets. (C) Construction of a PPI network with core targets. (D) GO enrichment analysis of core target genes. (E) KEGG pathway enrichment analysis of DHM for the treatment of hepatic injury. (F) The Network of relationships between DHM, the target, and the top 15 KEGG pathways.

Fig. 2

DHM attenuates LPS‒induced chicken primary hepatocytes injury and oxidative stress. (A) Schematic diagram of the animal experiment process. (B) Morphology of chicken primary hepatocytes. (C) Cell viability by MTT. (D) LDH activity in cell cultures. (E‒G) Antioxidant enzyme GSH‒PX, CAT and SOD activity in hepatocyte. (H) ROS fluorescence. Magnification: 400 ×; bar: 100 μm. The significance for comparisons between the control and model groups is noted as **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^##p < 0.01.

Fig. 3

Changes in the Nrf2/Keap1 pathway in LPS‒induced chicken primary hepatocyte injury. (A) DHM and Nrf2 molecular docking results. (B) DHM and Keap1 molecular docking results, (a) band diagram, (b) 3D diagram, (c) the hydrogen‒bond interaction. (C) Nrf2, (D) Keap1, (E) HO‒1 and (F) NQO1 mRNA expression. (G) Protein bands of Nrf2, Keap1, HO‒1 and NQO1. (H) Nrf2 (nuclear), (I) Keap1, (J) HO‒1 and (K) NQO1 protein levels. The significance for comparisons between the control and model groups is noted as **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^##p < 0.01.

Fig. 4

Changes in the levels of mitochondrial fusion division related factors in LPS‒induced chicken primary hepatocyte injury. (A) Mfn1, (B) Mfn2, (C) Opa1, (D) Fis1 and (E) Drp1 mRNA expression. (F) Protein bands of Opa1, Mfn1, Mfn2, Fis1 and Drp1. (G) Mfn1, (H) Mfn2, (I) Opa1, (J) Fis1 and (K) Drp1 protein levels. (L) The MMP of chicken hepatocytes. Magnification: 100 ×; bar: 100 μm. The significance for comparisons between the control and model groups is noted as **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^##p < 0.01.

Fig. 5

Effects of DHM on LPS‒induced chicken hepatic injury and oxidative stress. (A) Schematic diagram of the animal experimentation process. (B) AST and (C) ALT activities in the serum. (D) GSH‒PX, (E) CAT, and (F) SOD activities in the liver. The significance for comparisons between the control and model groups is noted as *p < 0.05 and **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^#p < 0.05 and ^##p < 0.01.

Fig. 6

Changes in the Nrf2/Keap1 pathway in LPS‒induced chicken hepatic injury. (A) Nrf2, (B) Keap1, (C) HO‒1 and (D) NQO1 mRNA expression. (E) Protein bands of Nrf2, Keap1, HO‒1 and NQO1. (F) Nrf2 (nuclear), (G) Keap1, (H) HO‒1 and (I) NQO1 protein levels. The significance for comparisons between the control and model groups is noted as *p < 0.05 and **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^#p < 0.05 and ^##p < 0.01.

Fig. 7

Changes in the expression of mitochondrial fusion division related factors in LPS‒induced hepatic injury. (A) Mfn1, (B) Mfn2, (C) Opa1, (D) Fis1 and (E) Drp1 mRNA expression. (F) Protein bands of Opa1, Mfn1, Mfn2, Fis1 and Drp1. (G) Mfn1, (H) Mfn2, (I) Opa1, (J) Fis1 and (K) Drp1 protein levels. (L) Morphology of hepatocyte mitochondria by ultramicropathology in chickens. Magnification: 20,000 ×; scale bar: 1 μm. The significance for comparisons between the control and model groups is noted as **p < 0.01. The significance for comparisons between the model and DHM+LPS groups is noted as ^#p < 0.05 and ^##p < 0.01.

Fig. 8

Mechanism of DHM regulates LPS‒induced hepatic oxidative stress in chickens.