Ziziphora clinopodioides flavonoids based on network pharmacology attenuates atherosclerosis in rats induced by high-fat emulsion combined with vitamin D3 by down-regulating VEGF/AKT/NF-κB signaling pathway.

Ziziphora clinopodioides flavonoids (ZCF) is a major bioactive total flavonoids compound isolated from Ziziphora clinopodioides Lam. It has been long used as an anti-atherosclerosis (AS) in clinics. However, anti-AS effects of ZCF have not been fully investigated. The objective of this study is to further investigate the anti-AS activities and mechanisms of ZCF in vivo. The main chemical components, action targets and signal pathways of Ziziphora clinopodioides Lam were predicted and analyzed by network pharmacology technology. The main bioactive components of Ziziphora clinopodioides Lam were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS). In vivo experiments, atherosclerosis in rats induced by high-fat emulsion combined with vitamin D3 and treated with simvastatin (0.45 mg/kg/d), ZCF (6.25, 12.5, 25 g/kg/d) for 7 weeks. We found that ZCF significantly reduced blood lipid levels (TG, TC, and LDL-C), and decreased lipid deposition in the aorta and atherosclerotic lesion size, inhibited mitochondrial mem- brane potential (MMP2/9/12/13) impairment. Meanwhile, ZCF may down-regulated the levels of VEGF, AKT, NF-κB, ICAM-1 and VCAM-1 proteins, indicating ZCF may play an anti-atherosclerotic role by down-regulating the VEGF/AKT/NF-κB signaling pathway. Results from this study demonstrated that ZCF have an anti-AS ability to lower lipid concentrations and protect endothelial function, antioxidant and anti-inflammatory activity, and suggested that ZCF might be a potential therapeutic drug in the prevention of AS.

A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB). The proteins Ag85B, MPB64, and ESAT-6 are the major immunogenic antigens of M. bovis; these proteins play important roles in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed various DNA vaccines with the genes encoding the three antigens mentioned above. This procedure involved the following steps: fusion of two genes (pcDNA-MPB64-Ag85B, pcMA), fusion of three genes (pcDNA-MPB64-Ag85B-ESAT-6, pcMAE), bivalent combinations (pcDNA-Ag85B+pcDNA-MPB64, pcA+M), and trivalent combinations (pcDNA-Ag85B+pcDNA-MPB64+pcDNA-ESAT-6, pcA+M+E). The immune response to the DNA vaccines was evaluated based on serum antibody titers, lymphocyte proliferation assay, and titers of the cytokines interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). The protective efficacy following challenge with a virulent M. bovis strain, C68001, was evaluated based on survival rate, bacterial loads in lung tissue, and histopathologic changes. A significant 2-fold increase in serum antibody levels was observed in mice vaccinated with fusion DNA (two or three genes). Furthermore, the lymphocyte proliferation (SI) values and the levels of IFN-gamma and IL-2 were higher in mice vaccinated with fusion DNA (two or three genes) than in those immunized with polyvalent combination DNA vaccines (P<0.05). Additionally, the fusion DNA vaccines provided protection that was superior to that provided by the polyvalent combination DNA vaccines following challenge with M. bovis strain C68001. The protective efficacy of the fusion DNA vaccines in mice immunized three times was equivalent to the protective efficacy in mice immunized once with the Bacillus Calmette-Guerin (BCG) vaccine. This suggests that fusion DNA vaccine represent a promising approach for the prevention of bTB.