Immunomodulatory Effect of Artemisia annua L. Water Extract on Meat-Type Sheep via Activating TLR4/NF-κB Signaling Pathways

This experiment was designed to systematically evaluate the immunomodulatory effect of water extract of <i>Artemisia annua</i> L. (WEAA) on sheep, both in vivo and in vitro, and to determine the involvement of the TLR4/NF-κB signaling pathway in mediating these effects. In experiment 1, 32 female sheep (Dorper × Han, 3 months old, 24 ± 0.09 kg each) were designated to 4 groups, with each group receiving a basal diet supplemented with, respectively, 0 (control group), 500, 1000, and 1500 mg/kg WEAA. The serum, liver, and spleen immune indicators and related gene expressions were measured. In experiment 2, the peripheral blood lymphocytes (PBLs) were processed with WEAA (0, 25, 50, 100, 200, and 400 μg/mL), with six replicates assigned to each concentration group, then the cell viability, immune function, and related gene expressions were measured, and the optimal concentration of WEAA was determined. In experiment 3, the experimental groups consisted of PBLs subjected to treatments with or without PDTC (NF-κB inhibitor) and with or without WEAA, forming four distinct treatment groups (six replicates/group): PDTC(-)/WEAA(-) group, PDTC(-)/WEAA(+) group, PDTC(+)/WEAA(-) group and PDTC(+)/WEAA(+) group. The immune indexes and TLR4/NFκB pathway related indexes were determined. The results were as follows: WEAA dose-dependently enhanced the content of immunoglobulins (IgA, IgG, IgM) and cytokines (IL-1β, IL-2, IL-4) in the serum, liver, and spleen tissues, among which IgA, IgG, and IL-4 were the most significantly affected core indicators (<i>p</i> < 0.05). Meanwhile, WEAA dose-dependently upregulated the expression of TLR4/NF-κB pathway-related genes (<i>TLR4</i>, <i>IKKβ</i>, <i>IκBα</i>, <i>NF-κBp65</i>) and their downstream cytokine-related genes (<i>IL-1β</i>, <i>IL-4</i>) in liver and spleen tissues (<i>p</i> < 0.05). Of these genes, liver <i>IL-4</i>, <i>IκBα</i>, and spleen <i>IL-4</i> were the most prominently regulated core genes (<i>p</i> < 0.05), The optimal supplementary dose of WEAA was determined to be 1000 mg/kg. In addition, adding 100 μg/mL WEAA to the culture medium of PBLs significantly enhanced immune function and cell viability. The underlying mechanism involved the TLR4/NF-κB pathway; that is to say, WEAA enhanced sheep's immune indicators by upregulating TLR4/NF-κB pathway genes, thereby coordinately regulating humoral and innate immunity, thereby improving the immune indices of sheep. This study provided compelling experimental support for the prospective utilization of WEAA as a functional feed supplement in intensive meat-type sheep production systems.