Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.

Effect and mechanism of N-acetylcysteine on adjuvant arthritis rats

Objective:To investigate the anti-inflammatory effects of N-Acetylcysteine(NAC)on adjuvant arthritis(AA)rats and the changes of Toll-like receptor 2(TLR2)-inflammatory factors.Methods:50 SD rats,randomly divided into 10 control groups,40 AA model group,after 10 d,which were divided into model group,NAC groups gave low,medium and high dose NAC(lavage respectively 50,100,200 mg/kg)for 16 d,joint swelling degree and arthritis index evaluation NAC therapeutic effect on AA rats;Immunohistochemical staining and Western blot were used to observe the expression of TLR2 protein in synovial tissue of joints.Serum levels of TNF-α,IL-1βand IL-10 were detected by ELISA.Results:Compared with the control group,the joint swelling and arthritis index in the model group were significant,and the joint swelling and arthritis index in the NAC treatment groups were significantly reduced(P<0.05,P<0.01).The expression of TLR2 protein in the synovial tissue of NAC treatment groups was decreased,inflammatory factors TNF-αand IL-1βwere decreased,and IL-10 was increased(P<0.01).Conclusion:NAC can improve the symptoms of arthritis in AA rats,and its mechanism may be related to inhibition of TLR2 and regulation of downstream inflammatory factor pathways in AA rats.