ABSTRACT
Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.
Subject(s)
HMGB1 Protein , Ligusticum , Osteoarthritis , Humans , Rats , Animals , NF-kappa B/genetics , NF-kappa B/metabolism , Chondrocytes , Caspase 3/metabolism , bcl-2-Associated X Protein/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , HMGB1 Protein/genetics , HMGB1 Protein/metabolism , HMGB1 Protein/pharmacology , Dinoprostone , Myeloid Differentiation Factor 88/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolism , Signal Transduction , Inflammation/metabolism , Osteoarthritis/drug therapy , Osteoarthritis/genetics , Apoptosis , RNA, Messenger/metabolismABSTRACT
Background: Obese type 2 diabetes mellitus (obese T2DM) is one of the prime diseases that endangers human health. Clinical studies have confirmed the ability of the Huanglian Huazhuo capsule to treat obese T2DM; however, its mechanism of action is still unclear. In this study, effects and mechanisms of the Huanglian Huazhuo capsule in obese T2DM were systematically investigated using network pharmacology and molecular docking techniques. Methods: The active ingredients and targets of the Huanglian Huazhuo capsule were extracted from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Obese T2DM diabetes-related targets were retrieved from a geographic dataset combined with a gene card database. A protein-protein interaction (PPI) network was constructed to screen core targets. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using Database for Annotation Visualization and Integrated Discovery (DAVID). Interactions between potential targets and active compounds were assessed using molecular docking. Molecular docking was performed on the best core protein complexes obtained using molecular docking. Results: A total of 89 and 108 active ingredients and targets, respectively, were identified. Seven core targets were obtained using a topological analysis of the PPI network. The GO and KEGG pathway enrichment analyses showed that the effects of the Huanglian Huazhuo capsules were mediated by inflammation, lipid response, oxidative stress-related genes, and HIF-1 and IL-17 signaling pathways. Good binding ability was observed between the active compounds and screened targets using molecular docking. Conclusions: The active ingredients, potential targets, and pathways of the Huanglian Huazhuo capsule for the treatment of obese T2DM were successfully predicted, providing a new strategy for further investigation of its molecular mechanisms. In addition, the potential active ingredients provide a reliable source for drug screening in obese T2DM.
ABSTRACT
A field experiment with five treatments, control (CK, no fertilizer), conventional fertilization (U), double-effect inhibitor synergistic urea (DU), coated urea (CU) and slow/controlled release urea mixture (CDU), was conducted to investigate the effects of conventional fertilization (240 kg N·hm-2) and one-off application of different slow/controlled release fertilizers (180 kg N·hm-2) on the yield and quality of fresh maize, soil inorganic nitrogen (N), and ammonia (NH3) emissions. The results showed that the total amount of ammonia volatilization was the highest in treatment of conventional fertilization (U), with N topdressing being an important source of NH3 emission. Compared with U treatment, the NH3 volatilization in the DU, CU, and CDU treatments was reduced by 78%-81%. At harvest stage, the soil layer of 80-100 cm in the U treatment had the highest nitrate concentration (51.6 mg·kg-1), resulting in a high risk of N leaching. In contrast, the nitrate concentrations in the same soil layer in the slow/controlled release fertilizer treatments were much lower, reducing the risk of leaching. In comparison with U, three slow/controlled release fertilizer treatments with 25% lower N application did not decrease yield but increased seed Vc, soluble sugar and protein contents. The agronomic efficiency and economic benefit of DU treatment were the highest among three slow/controlled release fertilizers treatments. In conclusion, the application of new type of slow/controlled release fertilizer could improve the yield and quality of fresh maize, and significantly reduce the risk of ammonia loss and N leaching. Considering the higher cost of the polymer coated urea, the double-effect inhibitor urea has lower cost and is more convenient to make. It is therefore a better choice to fresh maize planting.