RESUMEN
Ligustilide (LIG) is the main lipophilic component of the Umbelliferae family of pharmaceutical plants, including Radix angelicae sinensis and Ligusticum chuanxiong. LIG shows various pharmacological properties associated with anti-inflammation and anti-apoptosis in several kinds of cell lines. However, the therapeutic effects of LIG on chondrocyte apoptosis remain unknown. In this study, we investigated whether LIG had an anti-apoptotic activity in sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and could delay cartilage degeneration in a surgically induced rat OA model, and elucidated the potential mechanisms. In vitro studies revealed that LIG significantly suppressed chondrocyte apoptosis and cytoskeletal remodelling, which maintained the nuclear morphology and increased the mitochondrial membrane potential. In terms of SNP, LIG treatment considerably reduced the expression levels of cleaved caspase-3, Bax and inducible nitric oxide synthase and increased the expression level of Bcl-2 in a dose-dependent manner. The LIG-treated groups presented a significantly suppressed expression of activating transcription factor 2 and phosphorylation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The inhibitory effect of LIG was enhanced by the p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125 and offset by the agonist anisomycin. In vivo studies demonstrated that LIG attenuated osteoarthritic cartilage destruction by inhibiting the cartilage chondrocyte apoptosis and suppressing the phosphorylation levels of activating transcription factor 2, JNK and p38 MAPK. This result was confirmed by histological analyses, micro-CT, TUNEL assay and immunohistochemical analyses. Collectively, our studies indicated that LIG protected chondrocytes against SNP-induced apoptosis and delayed articular cartilage degeneration by suppressing JNK and p38 MAPK pathways.
Asunto(s)
4-Butirolactona/análogos & derivados , Apoptosis/efectos de los fármacos , Cartílago Articular/patología , Condrocitos/enzimología , Condrocitos/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Óxido Nítrico/toxicidad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , 4-Butirolactona/química , 4-Butirolactona/farmacología , Animales , Forma del Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Inyecciones Intraarticulares , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Nitroprusiato/farmacología , Osteoartritis/patología , Ratas Sprague-DawleyRESUMEN
In most aptamer based stimulus response mesoporous silica nanoparticles (MSN) systems, the aptamer is modified on the MSN via electrostatic interaction, however leakage might exist after a certain time in the system and hence the stability is not good. In this study, the pores of MSN were capped by aptamer through click chemistry reaction for the first time and the system was then employed to develop a fluorescence biosensor. Specifically, the aptamer of the target (thrombin in this study) was hybridized with its complementary DNA (which was initially modified with alkyne at the terminal) to form a double strand DNA (dsDNA) firstly, and then this dsDNA was modified on N3 modified MSN via Cu(I) catalyzed alkyne-azide cycloaddition reaction. The guest molecules (fluorescein) were blocked in the pores of the MSN with high efficiency and nearly no leakage was detected. Upon the introduction of thrombin, thrombin specifically recognized its aptamer, so aptamer released from the MSN; and the single strand DNA(ssDNA) left could not cap the pores of the MSN efficiently and hence caused the releasing of fluorescein into the solution. The enhanced fluorescence intensity of the system has a good linear relationship with the thrombin concentration in the range of 50-1000ngmL-1 with a detection limit of 28.46ngmL-1. The proposed biosensor has been successfully applied to detect thrombin in serum samples with high selectivity. The same strategy can be applied to develop biosensors for different targets by changing the adopted aptamer.