Study On The Mechanism Of Helicid Based On SiRNA Silencing PNOC Gene

Objective: The antidepressant effect of helicid was detected by a chronic unpredictable mild stress(CUMS)rat model,and the PNOC gene function was explored at the cellular level based on the results of whole transcriptome sequencing.Methods: 1.60 SD rats were randomly assigned to 2 groups,10 in the normal control group and 50 in the chronic stress model group.After 6 weeks of CUMS,the patients were randomly divided into contol,model group,fluoxetine hydrochloride group(5mg/kg)and helicid large,medium and small dose group(32 mg/kg,16 mg/kg,8 mg/kg),and were given continuous intragastric administration for 6 weeks.Except the contol group,each group received CUMS for 12 weeks.The behavioral changes of rats were evaluated by sucrose preference test,body weight measurement,open field test,forced swimming test and Morris water maze.2.Culture rat glioma C6 cells and LPS was used to induce cellular inflammation.Detected the toxicity of LPS on C6 cells and selected the optimal concentration of helicid by CCK-8 experiment,and detected the expression of PNOC by Q-PCR after 24 hours of LPS stimulating cells,and screened the optimal fragment of interfering PNOC gene.After the silencing of PNOC gene by siRNA,detect the expression of intracellular reactive oxygen species(ROS),IL-1,IL-6,TNF-? cytokines by ELISA kits,GDNF were detected by Q-PCR,and COX-2,iNOS and GFAP by Western Blot,to explore the role of inflammation model PNOC genes in cells.Results: 1.Before the beginning of the experiment,there was no significant difference in the behavior of rats in each group.After modeling with CUMS,compared with the control group,the body weight,sucrose consumption,total distance traveled,number of crossing,number of rearings and crossing platform times of rats in the CUMS group were significantly reduced(p < 0.05),and the immobility time of forced swimming and the escape latency period of morris water maze were significantly increased(p < 0.05).Compared with the rats in the CUMS group,the body weight,sucrose consumption,total distance traveled,number of crossing,number of rearings,and number of crossing platform position of the rats in the fluroxetine group with a large dose of helicid were significantly increased(p < 0.05),and the immobility time of forced swimming and the escape latency of morris water maze were significantly reduced(p < 0.05).2.In CCK-8 experiment,2?g/ml LPS showed no damage to cells(p < 0.05)and with a various concentration of helicid showed no damage to cells(p < 0.05),the optimal concentration of helicid(100 ?M)was selected.Q-PCR results showed that the expression of PNOC gene increased in the LPS-induced cell inflammation model,while helicid could down-regulate the expression of PNOC gene(p< 0.05).The best interfering fragment of siRNA-001 was selected by Q-PCR;Immunofluorescence results showed that compared with the blank group,the fluorescence intensity and expression of GFAP increased after LPS stimulation;ROS results showed that LPS induced increased ROS expression in cells,and siRNA silenced PNOC gene reduced ROS expression(p<0.05);ELISA results showed that,compared with the blank group,the expressions of IL-1,IL-6,TNF-? cytokines increased after LPS induction(p < 0.05),while the expressions of IL-1,IL-6,TNF-? cytokines decreased after siRNA silencing of PNOC gene(p < 0.05);Western Blot results showed that siRNA silencing of PNOC gene reduced the expressions of COX-2,iNOS and GFAP in the LPS-induced cell inflammation model(p < 0.05)and improved the expression of p-ERK?p-AKT(p<0.05).Conclusion: 1.CUMS can induce depression-like behaviors in rats,including decreased body mass,anhedonia,decreased voluntary action,increased degree of despair,and decreased learning and memory ability,etc.Helicid can improve depression-like behaviors caused by chronic unpredictable mild stress and play an antidepression role.2.In LPS-induced cell inflammation model,Silencing of PNOC gene by siRNA can significantly reduce the expression of ROS,GFAP,iNOS,COX-2 and cytokines,and increase the expression of GDNF,indicating that siRNA silencing PNOC may involve in the inflammatory response through oxidative stress,the expression of neurotrophic factors,cytokines,p-ERK and p-AKT;helicid may reduce the expression of PNOC gene in the cellular inflammation model.