Research Regulation Of Wuzi Yanzong Pill For Apoptotic Pathway Of NTDs Cell Model Based On The Transcriptome Sequencing Technology

Objective:In this study,PC12 cells were used to prepare the cell model of neural tube defects.After intervention of Wuzi Yanzong Pill on PC12 Cells by atRA establishment model,we detected cell viability and cell apoptosis.RNA-seq technique was used to sequence the whole genome of PC12 cells and analyze the gene expression.The aim of this study was to investigate the regulation of Wuzi Yanzong Pill on apoptotic pathway of neural tube defect cell model and its mechanism.Methods:Wuzi Yanzong Pill group was given 125 μg/mL Wuzi Yanzong Pill and Folic Acid group was given 100 μg/mL Folic Acid 24 hours before model establishment.The viability of PC12 cells was detected by XTT assay.The apoptotic rates were detected by Annexin V-FITC/PI assay.Total RNAs were sequenced by High-throughput RNA-seq technology.To filter and obtain the differentially expressed genes.To find Interesting Target Gene Sets by Venn Diagram.GO function and KEGG pathway significance enrichment analysis were performed on the target gene set.WGCNA software was used to analyze the co-expression network of genes related to apoptotic mechanism of PC12 cells induced by atRA after Wuzi Yanzong Pill intervention.Results:1.Effect of Wuzi Yanzong Pill on the viability of PC12 cells induced by atRAWuzi Yanzong Pill can increase the activity of PC12 cells interfered by atRA,and the effect is better than folic acid.Compared with the blank group,the cell viability of the model group decreased significantly(P<0.001);compared with the model group,the cell viability of Wuzi Yanzong Pill group increased significantly(P<0.001);compared with the model group,the cell viability of Folic Acid group increased significantly(P<0.05).2.Effect of Wuzi Yanzong Pill on the apoptosis of PC12 cells induced by atRAWuzi Yanzong Pill can reduce the apoptotic rate of PC12 cells intervened by atRA,and the effect is better than Folic Acid.Compared with the blank group,the apoptotic rate of the model group was significantly higher(P<0.001);compared with the model group,the apoptotic rate of the Wuzi Yanzong Pill group was significantly lower(P<0.001);compared with the model group,the apoptotic rate of the Folic Acid group was significantly lower(P<0.01).3.Differential gene expression analysis of transcriptome sequencing in PC12 cells induced by Wuzi Yanzong Pill in atRA establishment model3.1 Identification and analysis of differentially expressed genes and target gene setsWuzi Yanzong Pill can inhibit most of up-regulation or down-regulation gene expression in PC12 cells induced by atRA.There were 625 up-regulated genes in model group compared with blank group,390 genes in Wuzi Yanzong Pill group were down-regulated,while 62 genes in Folic Acid group were down-regulated;1456 genes in model group were down-regulated compared with blank group,1073 genes in Wuzi Yanzong Pill group were up-regulated,and 903 genes in Folic Acid group were up-regulated.3.2 Genes in Apoptotic Pathway and Target Gene SetsThere are eight genes in the rno04210 Apoptosis pathway Wuzi Yanzong Pill related target gene set,including Birc5,Gadd45 g,Gadd45b,Nfkbia,Pik3r1,Casp8,Casp12 and Ern1.There are three genes in the rno04210 Apoptosis pathway in the folic acid related target gene set,namely Casp12,Ern1 and Birc5.3.3 GO analysis of Target Gene SetsThe target gene sets related to Wuzi Yanzong Pill were significantly enriched in three apoptotic related GO items GO:0043523: regulation of neuron apoptotic process,GO:0051402 neuron apoptotic process and GO:0070059 intrinsic apoptotic signaling pathway in endoplasmic reticulum stress.3.4 KEGG Pathway analysis of Target Gene SetsThe target gene sets related to Wuzi Yanzong Pill significantly enriched KEGG pathway related to the mechanism of Wuzi Yanzong Pill,such as rno04110 cell cycle,rno03030 DNA replication,rno04141 endoplasmic reticulum protein processing and so on.3.5 Analysis of Co-expression Network of Genes Related to Apoptotic Mechanisms of PC12 Cells Intervened by Wuzi Yanzong Pill in atRA establishment modelIn addition to apoptosis pathway related genes,the mechanism of Wuzi Yanzong Pill inhibiting apoptosis may be related to 26 genes such as Ttk,Sema6 b,Mcm5 and so on.Conclusion:Wuzi Yanzong Pill can inhibit apoptosis of PC12 cells induced by atRA,and the effect is better than folic acid.Its inhibitory mechanism is related to the extensive inhibition of gene overexpression in the model by Wuzi Yanzong Pill;is related to the down-regulation of Ern1 and Casp12 genes associated with endoplasmic reticulum stress;is related to the down-regulation of Caspase family members Casp8;is related to the up-regulation of Birc5,Gadd45 b and Gadd45 g genes;is related to neuron apoptotic process;the up-regulation of Pik3r1,Nfkbia gene,may further be related to PI3K/Akt signaling pathway and NF-κB signaling pathway;may also be related to Cell cycle,Axon guidance,Cholinergic synapse,cAMP signaling pathway,Protein processing in endoplasmic reticulum KEGG pathway,and 26 genes such as Ttk,Sema6 b,Mcm5,etc.with high degree of connectivity and co-expression of apoptotic genes in the target gene set.