To Explore The Antidepressant Mechanism Of Xiaoyao Powder And Paeoniflorin Based On The Inhibition Of Hippocampal Neuron Apoptosis By MiR-29b

1.Objective:This research aims to observe Xiaoyaosan and its main ingredient paeoniflorin regulate miR-29b inhibiting hippocampal neuron apoptosis program to explore the antidepressant mechanism of Xiaoyaosan.Depression and stress can induce hippocampal neuronal cell apoptosis in non-human mammals and humans.Similarly,hippocampal neuronal cell apoptosis may lead to depression.MicroRNA(microRNA)is a small non-coding RNA molecule that can regulate the stability and/or translation efficiency of target messenger RNA,and can affect about 50%or more of the activity of all protein-coding genes in mammals.miR-29b can inhibit the translation of BH3 family proteins,thereby preventing cell death under the stimulation of apoptosis.BH3 protein is a family of pro-apoptotic regulators,which is essential for inducing the release of cytochrome c from mitochondria.miR-29b mediates anti-apoptotic function by targeting and inhibiting multiple members of the BH3 pro-apoptotic gene family Paeoniflorin is a quality control component of Xiaoyaosan,a traditional Chinese medicine compound.It is a natural molecule with antidepressant potential that has been discovered in recent years.More and more studies have confirmed that paeoniflorin has anti-inflammatory.antioxidant and neuroprotective effects,and can prevent or even block lipopolysaccharide(LPS)induces apoptosis of hippocampal neurons.Using CUMS to replicate a rat model of depression,LPS induces apoptosis of HT22 cell line,combined with HPLC,monoclonal antibody technology,immunofluorescence,qPCR and other technologies in vivo and in vitro research verification Xiaoyaosan based on miR-29b inhibits hippocampal nerve cell apoptosis and exerts an antidepressant mechanism,and paeoniflorin is the key medicinal ingredient.For revealing the material basis of Xiaoyaosan's antidepressant,it is of great significance to clarify the composition of paeoniflorin corresponding to the regulatory target.2.Methods(1)In vivo experiments shared 75 SPF male healthy SD rats with a weight range of 200-240g,and were adaptively reared in a clean animal room for 7 days.The breeding environment and conditions:Put 5 rats in a cage and raise them together.The rats were given regular feed and deionized water,the room temperature was controlled at 22±2?,the humidity was maintained at 30%to 40%,the light was maintained at a normal circadian rhythm and the breeding environment was kept quiet.The CUMS method was used to make a rat model of depression.The animal model was evaluated through rat macroscopic characterization,food intake,body weight,sucrose preference experiment,and open field experiment.The hippocampus was taken on ice,and the CA3,CA1 and DG areas were microdissected.Western Blot and q PCR were used to detect the gene and protein expression levels of miR-29b,BIM,BID,BAX,Casp-9,and Casp-3.Four After the rats were perfused,whole brains were taken,and paraffin sections were subjected to TUNEL staining to observe the apoptosis of hippocampal neurons.(2)In vitro experiment,Mab-PF solution was prepared by using monoclonal antibody technology together with HPLC technology.The Xiaoyaosan liquid was filtered through the paeoniflorin antibody adsorption column,and the liquid was washed out with three times the volume of the adsorption column PBS and passed through rotary evaporation.The instrument is concentrated into medicated serum.In the LPS-induced apoptosis model of HT22 cell line,it was verified that paeoniflorin is the key pharmacodynamic component of Xiaoyaosan's neuroprotective effect against apoptosis.Intervention with 2.5%,10%Xiaoyaosan medicated serum,Mab-PF medicated serum and 100,300,500 ?mol concentration of paeoniflorin drug medium for 24 hours after intervention in HT22 cell line,4ug/ml concentration of LPS induced HT22 cell apoptosis,CCK8 method was used to screen out the optimal drug concentration,cell flow cytometry was used to detect the rate of apoptosis,RT-qPCR and Western Blot methods were used to detect the expression of miR-29b,BIM,BAX,Casp-9,and Casp-3,and immunofluorescence was used The technique detects the expression of BIM in apoptotic neuronal cells.(3)Use 2.5%,10%Xiaoyaosan medicated serum,10%Mab-PF medicated serum and 10,100,300?mol concentration of paeoniflorin drug medium to intervene HT22 cell line for 24 hours,then use 4ug/ml concentration of LPS Induce HT22 cell apoptosis,use cell flow cytometry to detect the apoptosis ratio,use Western Blot to detect the expression of BIM,use immunofluorescence to detect the expression of BIM in apoptotic neuronal cells,and observe the relationship between different drug concentrations and BIM inhibition.(4)Based on lentiviral transfection technology,after intervention of HT22 cell line with 300?mol concentration of paeoniflorin drug medium for 24 hours,4ug/ml concentration of LPS was used to induce HT22 cell apoptosis,cell flow cytometry was used to detect the apoptotic ratio and qPCR Methods The expression of BIM was detected,and the expression of BIM in apoptotic neuron cells was detected by immunofluorescence technology3.Results(1)Six-week CUMS successfully replicated an animal model of depression with syndrome of stagnation of liver qi and spleen deficiency.The apoptosis of the hippocampus of each group was detected by the TUNEL staining method,and it was found that the hippocampus of the model rats had neuronal apoptosis and CA3 The area is the most significant.qPCR was used to detect the expression levels of miR-29b in the hippocampal CA3 area of each group of rats that regulate the pro-apoptotic molecule BIM and BID-mediated apoptosis pathway related genes.Compared with the control group,the expression of miR-29b in the model group was down-regulated,and the expressions of BID,BIM,BAX,Casp-9,and Casp-3 were increased Xiaoyaosan could reverse this trend.Paeoniflorin could not down-regulate the expression of BIM.Western Blot detection of mitochondrial apoptosis pathway-related molecular protein levels:Compared with the control group,the relative expressions of BIM,BID,BAX,Cleaved-Casp-9,and Cleaved-Casp-3 in the CA3 area of the hippocampus of the model group increased significantly,Xiaoyaosan,paeoniflorin and fluoxetine can inhibit its high expression(2)In vitro experiments,through the detection of monoclonal antibody technology combined with HPLC,after double knockout,the effective knockout rate of paeoniflorin was 95%.The optimal concentration of Xiaoyaosan and Paeoniflorin was 10%and 300?mol respectively by cck8 method.The cell flow cytometry results showed that 4ug/ml LPS induced cell apoptosis Xiaoyaosan,Mab-PF,and paeoniflorin can all inhibit apoptosis.LPS induced HT22 cell apoptosis and paeoniflorin,Mab-PF,Xiaoyaosan medicated serum regulating effect qPCR results.miR-29b,mRNA in the LPS-induced apoptosis model group,the expression level decreased significantly,and each drug group had an up-regulating effect on it,and the difference was statistically significant.The up-regulated miR-29b mRNA expression level in Mab-PF and Paeoniflorin groups was lower than that of Xiaoyaosan medicated serum,and the difference was statistically significant.The mRNA expression levels of BIM,BAX,Casp-9.and Casp-3 in the apoptotic cell model group were significantly higher than those in the control group Xiaoyaosan and Mab-PF medicated serum both had a down-regulating effect.Paeoniflorin only has no regulatory effect on the expression level of BIM mRNA.(3)Xiaoyaosan has anti-LPS-induced apoptosis effects at 10%concentrations,and paeoniflorin has anti-apoptotic effects at concentrations above 100 ?mol.The inhibitory effect of Xiaoyaosan and Paeoniflorin on BIM is drug concentration-dependent.With the increase of drug concentration,the relative expression of BIM protein decreases.(4)Knock down the miR-29b mRNA expression level and increase the BIM protein expression level,which promotes spontaneous cell apoptosis.Paeoniflorin can target and regulate miR-29b,inhibit BIM protein translation,and resist neuronal apoptosis4.Conclusion:(1)In vivo experiments show that there is hippocampal neuronal apoptosis in the six-week CUMS depression rat model,and the CA3 area is the most significant.(2)There is down-regulation of miR-29b expression at the gene level in the CUMS model,and Xiaoyaosan and paeoniflorin can adjust its expression.miR-29b is the key target of Xiaoyaosan to exert neuroprotective effects.(3)In vitro experiments showed that Xiaoyaosan group was better than Mab-PF group in regulating miR-29b in inhibiting BIM,indicating that paeoniflorin plays an important role in this mechanism..(4)Paeoniflorin can target and regulate miR-29b,inhibit BIM protein translation,and resist neuronal apoptosis.