五子衍宗丸通过调控泛素-蛋白酶体系统抑制反式维甲酸诱导的PC12细胞凋亡OA

Objective:To study the relationship between ubiquitin-proteasome system(UPS)and all-trans retinoic acid(atRA)-induced neuronal apoptosis induced by Wuzi Yanzong Pills(五子衍宗丸,WYP).Methods:Rat adrenal pheochromocytoma cells(PC12)were randomly divided into a normal control group,model control group,125 μg/mL WYP group,and MG132 proteasome inhibitor group.PC12 cells were treated with atRA to induce an apoptosis model.The cell viability was measured by the CCK-8 method,and the apoptosis rate in each group was detected by flow cytome-try.High-throughput RNA-Seq sequencing technology was used to sequence the total RNA of cells and screen the differ-entially expressed genes.GO enrichment analysis and KEGG pathway significant enrichment analysis were performed on the target trend modules to screen apoptosis-and ubiquitin-related genes.The atRA was used to induce a PC12 cell ap-optosis model.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the mRNA expression of apoptosis genes,including cell cycle checkpoint kinase2(Chek2)and aurora kinase B(Aurkb),recombinant raspase 4(Caspase4),as well as ubiquitin-related genes,such as Polo-like kinasel(Pik1),recombinant ubiquitin conjugating en-zyme E2C(Ube2C),and tribbles pseudokinase3(Trib3).Results:WYP significantly enhanced the cell viability(P<0.01)and reduced the apoptosis rate(P<0.01).In target trend module,GO enrichment analysis,and KEGG analysis,the GO items and KEGG pathways significantly enriched with MG132_down-regulation and MG132_down-regulation in Wuziyanzongwan_up-regulation and MG132_up-regulation overlapped in most of the GO items and KEGG pathways.The screened apoptosis-and ubiquitin-related genes were highly connected in PPI and co-expression network analysis.The two analysis results showed that there were apoptosis-related genes such as Chek2,Aurkb,and Caspase4,and ubiquitin-re-lated genes such as Pik1,Ube2c,and Trib3.KEGG pathways involving both apoptosis-related genes and ubiquitin-relat-ed genes,included cell cycle,fluid shear stress,atherosclerosis,and other pathways.The RT-qPCR results showed that WYP down-regulated the expression of apoptosis-related genes and Trib3 mRNA(P<0.05,P<0.01)and up-regulated the expression levels of Chek2 and Aurkb mRNA(P<0.05).MG132 up-regulated the expression of apoptosis-related genes and Trib3 mRNA(P<0.05)and down-regulated the expression levels of Chek2 and Aurkb mRNA(P<0.05).The effect of MG132+atRA+WYP on the expression of apoptosis-related genes and UPS-related genes was consistent with the trend of the MG132+atRA group(P<0.05).Conclusion:WYP can inhibit the apoptosis of PC12 cells induced by atRA and has a protective effect on neuron-like cells.The mechanism may be related to ubiquitin via regulating the mR-NA expressions of Chek2,Aurkb,Birc5,Pik1,Ube2c,Aurka,and Caspase4.