Toll Like Receptor 4

The total RNA of liver samples was extracted using the AG RNAex Pro reagent (Accurate Biology, Hunan, China). The cDNA was acquired by the reverse transcription (RT) kit (Accurate Biology, Hunan, China). The cDNA samples were amplified by real-time quantitative polymerase chain reaction with SYBR^® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology, Dalian, China). The mRNA expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), NLRP3, sirtuin1 (Sirt1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme-oxygenase 1 (HO-1), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase 1 (GPX1), NAD(P)H quinone oxidoreductase-1 (NQO1), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) in the liver samples were assessed using a LightCycler 96 (Roch, Switzerland) with SYBR^® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology, Da Lian, China). All the primer sequences are listed in Table 2. β-actin was used as an internal reference gene, and the 2^−ΔΔCT method was used to calculate the messenger ribonucleic acid (mRNA) expression of target genes relative to β-actin (17 (link)).

Protein collection and western blotting was carried out as outlined in Kennon and Stewart [80 (link)]. Briefly, total protein was isolated with modified Hunter’s Buffer (1% Triton X-100, 75 mM NaCl, 5 mM Tris (pH 7.4), 0.5 mM orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, 0.25% NP-40 and Halt-Protease Inhibitor Cocktail (100X; 78430; ThermoFisher Scientific; Waltham, MA, USA)). Protein concentration for each sample was determined using the bicinchoninic acid (BCA) assay (23225; Pierce™ Biotechnology; ThermoFisher Scientific; Waltham, MA, USA) according to the manufacturer’s instructions. Proteins were separated on a 12% SDS-PAGE gel and transferred to methanol-activated Immobilon-P PVDF membrane (Millipore Sigma; ThermoFisher Scientific; Waltham, MA, USA). The membranes were blocked, and primary antibodies were placed on the membrane in either 5% milk or 5% BSA diluted in TBS-T (50 mM Tris-Base, 100 mM NaCl, and pH 7.4 with 0.001% Tween-20) overnight at 4 °C. The following primary antibodies were utilized: α-SMA (1:1000; A2547; Sigma-Aldrich; St. Louis, MO, USA), vimentin (1:1000; Cell Signaling, Danvers, MA, USA; 5741), osteopontin (OPN; 1:400; Abcam, ab8448), toll-like receptor 4 (TLR4; 1:400; Santa Cruz Biotechnology; Dallas, TX, USA;, sc-293072), phosphorylated ERK ½ (1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-7383), total ERK 1 (1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-271269), total ERK 2, (1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-1647), phosphorylated NF-κB p65 (1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-136548), total NF-κB p65 (1:400, Santa Cruz Biotechnology; Dallas, TX, USA; sc-8008), phosphorylated p38 MAPK (1:1000; Cell Signaling; Danvers, MA, USA; 9211), total p38 MAPK (1:1000; Cell Signaling; Danvers, MA, USA; 9212), superoxide dismutase 1 (SOD-1; 1:400; Santa Cruz Biotechnology, Dallas, TX, USA; sc-101523), superoxide dismutase 2 (SOD-2; 1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-133134), and β-tubulin (1:400; Santa Cruz Biotechnology; Dallas, TX, USA; sc-398937) as a loading control. The membrane was washed with TBS-T and then incubated at room temperature with a secondary antibody conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology; Dallas, TX, USA;). The membrane was washed and then incubated in Pierce enhanced chemiluminescent substrates (ThermoFisher Scientific; Waltham, MA, USA) for 2 min. The membrane was visualized with the iBRIGHT western blot imaging system (ThermoFisher Scientific; Waltham, MA, USA), and densitometric analysis on bands was performed with NIH Image J software.

Using the ab initio free docking approach from HDOCK server [48 (link)], the designed vaccines were docked with the human TLR4 (Human Toll-like receptor-4). This server utilizes both template- and docking-based binding models of two molecules and allows its interactive visualization. Moreover, mmGBSA analysis were also performed for each of the docking complexes using HawkDock server [49 (link)]. The server employs a hybrid docking method setting it apart from other similar servers. The HawkDock server provides experimental information on the protein–protein binding site and small-angle x-ray drip in a quick and authentic manner [49 (link)]. We docked the MD2 protein as a native ligand with TLR4. Furthermore, PDBsum server [50 (link)] was utilized to analyze the interaction patterns between the docked molecules. The vaccine design was modeled using Robetta before protein–protein docking, and the TLR4 structure was acquired from RCSB [51 (link)]. The structures were prepared before the docking by removing water molecules, heteroatom and other atoms.