Patient-related factors related to prolonged preparation-to-colonoscopy interval and insufficient purgative intake before colonoscopy: A post-hoc observational study.

OBJECTIVES: Prolonged preparation-to-colonoscopy (PC) interval and insufficient purgative intake (PI) are two important indicators for quality of bowel preparation for colonoscopy. We aimed to investigate patient-related factors associated with increased PC interval or insufficient PI. METHODS: The post-hoc regression analyses were performed using the data from two prospective studies (NCT04434625 and NCT04101097). Patients receiving reinforced instructions for bowel preparation were recruited. The co-primary outcomes included prolonged PC interval or insufficient PI. RESULTS: Altogether 1806 patients from five endoscopic centers underwent bowel preparation from September 2019 to March 2021. The cut-off values were 6 h for PC interval and 80% for PI. In all, 116 (6.4%) and 73 (4.0%) presented an extended PC interval and insufficient PI, respectively. Multivariate logistic regression analysis showed that a low education level was significantly associated with PC interval ≥6 h. Female sex, body mass index (BMI), and coronary artery disease (CAD) were found to be significantly correlated with insufficient PI in univariate analysis, while multivariate analysis demonstrated BMI <20 kg/m2 (odds ratio [OR] 4.14, 95% confidence interval [CI] 1.92-8.94, P < 0.001) and 20-25 kg/m2 (OR 2.23, 95% CI 1.33-3.73, P = 0.002) and CAD (OR 3.23, 95% CI 1.22-8.53, P = 0.018) were identified as independent risk factors for PI <80%. CONCLUSIONS: In spite of reinforced education, a number of patients did not follow the instructions for bowel preparation. The factors for a prolonged PC interval did not overlap with those for insufficient PI. Individualized interference may be considered in different subpopulations.

Smarcd1 antagonizes the apoptosis of injured MES23.5 DA cells by enhancing the effect of Six2 on GDNF expression.

Glial cell line-derived neurotrophic factor (GDNF) played critical roles in the survival and repair of dopaminergic (DA) neurons. Transcription factor Six2 could repair injured DA cells by promoting the expression of GDNF, however, the underlying molecular mechanisms remain largely unknown. In this study, we screened forty-three proteins that interacted with Six2 in MES23.5 DA cells treated with 6-OHDA by liquid chromatography - electrospray - ionization tandem mass spectrometry (LC-ESI-ITMS/MS). Among these proteins, Smarcd1 is a member of SWI/SNF chromatin-remodeling complex family. Our results confirmed that Smarcd1 formed a transcription complex with Six2, and Smarcd1 mainly binded to the 2840 bp-2933 bp region of the GDNF promoter. Furthermore, knockdown of Smarcd1 inhibited the effect of Six2 on GDNF expression, and resulted in decreased cell viability and increased the apoptosis of injured DA neurons, and the result of overexpression of Smarcd1 is opposite to knockdown. Taken together, our results indicate that smarcd1 can be recruited to the promoter region of GDNF by transcription factor Six2 to promote the effect of Six2 on GDNF expression and protect injured MES23.5 DA cells, which could be useful in identifying potential drug targets for promoting endogenous GDNF expression.

MES23.5 DA Immortalized Neuroblastoma Cells Self-protect Against Early Injury by Overexpressing Glial Cell-derived Neurotrophic Factor via Akt1/Eya1/Six2 Signaling.

Injured neurons can initiate their own neurotoxin-induced repair mechanisms by expressing protective genes and activating specific intracellular signal transduction pathways. Although glial cell-derived neurotrophic factor (GDNF) plays a key role in the repair of dopaminergic (DA) neurons, whether there is high expression of GDNF in DA neurons at an early stage of injury has not yet been reported. In this study, neurotoxin-induced GDNF overexpression was detected for the first time in MES23.5 DA immortalized neuroblastoma (MES23.5 DA) cells soon after 6-hydroxydopamine (6-OHDA) treatment. We also observed that the phosphorylation of Akt1, a member of the protein kinase B family, was increased. Further studies showed that activated Akt1 increased the phosphorylation of the protein phosphatase Eya1, which is a member of the eyes absent (Eya) family of transcriptional cofactors. Then, activated Eya1 decreased the phosphorylation of the sine oculis-related homeobox 2 (Six2) transcription factor. In addition, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) revealed that Six2 promoted GDNF transcription in MES23.5 DA cells by directly binding to the GDNF promoter. Finally, we showed that inhibiting neurotoxin-induced GDNF overexpression increased MES23.5 DA cell death, while promoting GDNF expression via Six2 overexpression decreased DA neuronal death. These results suggest that MES23.5 DA cells with early 6-OHDA-induced injury can promote the overexpression of GDNF by activating the Akt1/Eya1/Six2 signaling pathway, and this overexpression of GDNF has protective effects on injured MES23.5 DA cells. Hence, this study highlights a new target for drug development for the treatment of Parkinson's disease.

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 shRNA 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-shRNA 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-shRNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.