The Mechanism Of Saikosaponin A And Paeoniflorin Regulated Autophagy Function Of PC12 Cell Under Stress State

ObjectiveThe experimental study was designed to investigate the mechanism of the regulating Liver drugs for adjusting autophagy function of stress status PC12 cell. From autophagy as the microscopic point to explore the physiological and pathological mechanism of TCM LIVER visceral manifestation. In chronic psychological stress injury as the starting point, the use of Regulating the Liver method to adjust autophagy disorder state, thereby preliminary demonstration the conveyance and dispersion function of LIVER visceral manifestation and autophagy have internal relations. And prove that team proposed "cell autophagy adjustment, is the LIVER controlling conveyance and dispersion of whole body qi activityin the cellular level manifestations" theoretical hypothesis is scientific.In order to further explore hidden as the pathogenesis of liver function in the performance and mechanism of the micro level, which in turn to verify the macro theory from the microscopic view, so as to enrich and innovation of TCM viscera-state theory, and provide experimental evidence for treating some autophagy disorders by. regulating liver therapy.Methods1.Corticosterone treated PC12 cells cause stress damage state cell to establish modelExperiment on geometric proportions corticosterone 800μM,400μM,200μM, 100μM,50μM, absolute ethanol groups and control groups, and each group are detected by MTT assay method for cell activity.2. Saikosaponin A and paeoniflorin on stress damage state of PC12 cell morphological observation of autophagy2.1 With different working concentrations of paeoniflorin and Saikosaponin A effect in normal PC12 cells to observe whether the two monomers can affect cell viability.2.2 With different working concentrations of paeoniflorin and Saikosaponin A causing the state of stress injury of PC12 cells, elected an optimum cell activity among different concentrations of paeoniflorin and Saikosaponin A by MTT assay method, using of the best concentration of paeoniflorin and Saikosaponin A to conduct the following experiment.2.3 According to the above experiment, the cells were divided into control group, model group, paeoniflorin group, Saikosaponin A group, respectively, after treatment, by electron microscopy observe the autophagy structures of the groups. In addition, by autophagy double adenovirus (mRFP-GFP-LC3) transfect cells to observe the autophagy and autolysosome ratio, thereby monitoring the flow of autophagic process.3. A preliminary study of the mechanism of Saikosaponin A and paeoniflorin regulating cell autophagyUsing RT-PCR detection method to detect the expression of LC3II/I, P62, mTOR’s mRNA in each group cells. What’s more, using Western Blot testing methods to detect protein expression of LC3II/I, P62, mTOR and p-mTOR’s in each group cells. Thus further understand the situation of autophagy-autolysosome-protein degradation.ResuIts1. Corticosterone treated PC12 cells cause stress damage state cell to establish modelWith increasing of corticosterone concentration, cell activity gradually reduced. Compared with the control group, the cell activity of CORT 800μM, CORT 400μM, CORT 200μM, CORT 100μM were significantly decreased (P<0.01). The cell activity of CORT 50μM decreased (P<0.05). Absolute ethanol group compared with the control group, the cell activity have no statistical difference. When corticosterone concentration is 400μM, the cell survival rate is the most close to 40%.2. Saikosaponin A and paeoniflorin on stress damage state of PC12 cell morphological observation of autophagy2.1 With different working concentrations of paeoniflorin and Saikosaponin A effecting in normal PC12 cells, caused no significant effect on normal PC12 cells, and there is no statistical difference.2.2 With different working concentrations of paeoniflorin and SaikosaponinWith different working concentrations of paeoniflorin and Saikosaponin A causing the state of stress injury of PC12 cells, detected by MTT detection discovering that compared with the control group, the cell activity is significantly decreased in model group (P<0.01); the cell activity of DMSO group is substantially unchanged. Compared with model group,400μM,200μM, 100μM paeoniflorin will be improved cell activity (P<0.01), in which 400μM paeoniflorin has the most active cells; 50μM,25μM paeoniflorin has no difference with the model group, which is no statistically significant.10μM, 5μM,2.5μM,1.25μM Saikosaponin A also increased the cell activity significantly (P<0.01), but 0.625μM Saikosaponin A has no statistical difference with the model group. Due to the cell activity of 400μM paeoniflorin group is the best, so use 400μM paeoniflorin conduct the following experiment. And the cell activity of 10μM,5μM Saikosaponin A group is similar, so saikosaponins A 10μM group is selected for the following experiment.2.3 By the electron microscopy and autophagy double adenovirus (mRFP-GFP-LC3) transfection on PC12 cells to observe autophagy-related structures, compared with the control group, the number of autolysosome in the model group was significantly increased (P<0.01), autophagy have a more significant increased (P<0.01). However, the number of autophagy is more than autolysosome(P<0.01). Compared with model group, there is significantly reduced the number of autophagy in paeoniflorin group and saikosaponins A group (P<0.01), while the number of autolysosome has little difference. Among them, the number of autophagy and autolysosome is similar in paeoniflorin group, while the number of autophagy is more than autolysosome in Saikosaponin A group (P<0.01).3. A preliminary study of the mechanism of Saikosaponin A and paeoniflorin regulating cell autophagyAccording to the RT-PCR test results, the comparison with the control group, mTOR mRNA expression levels of the model group was increased (P<0.05). P62 mRNA expression levels and LC3II mRNA expression levels is significantly increased (P<0.01). Compared with the model group, the expression of mTOR, P62 and LC3II mRNA is significantly reduced in paeoniflorin group (P<0.01). In addition, Saikosaponin A can also reduce the expression of mTOR, P62 and the LC3II mRNA (P<0.01), but the extent of which P62 and LC3II mRNA expression is not as significantly as the paeoniflorin group. While, mTOR mRNA expression is more obvious than paeoniflorin group.According to Western Blot test results found that, compared with the control group, model group LC3II/I protein expression was significantly increased (P<0.01), P62 protein expression was significantly increased (P <0.01), mTOR protein expression did not change significantly, no statistical difference, p-mTOR expression significantly is reduced (P<0.01). Compared with model group, the expression of LC3II/I and P62 protein is significantly reduced in paeoniflorin group (P<0.01); the expression of mTOR protein is significantly reduced(P<0.05); and p-mTOR protein is no significant change. What’s more, the Saikosaponin A compared with the model group, the LC3II/I protein expression is significantly reduced (P<0.05), the expression of P62 is significantly reduced protein (P<0.05), significantly reduced the expression of mTOR protein (P<0.05); the expression of p-mTOR protein is no significant difference.Gonclusions1. By 400μM corticosterone processing Well-differentiated PC12 cells, can imitate model of neurons in the stress state, and the cell activity of model group was obviously decreased.2. The injuried PC12 cells by corticosterone found that the number of autophagy increased significantly, but the growth rate of autolysosome is not obvious, indicating that autophagy flow was significantly inhibited.3. Paeoniflorin and Saikosaponin A can effectively significantly relieve the inhibited autophagic flow in model group; and increase autophagy activity, thereby enhancing cell activity.4. This study further proved that " the conveyance and dispersion function of LIVER visceral manifestation and autophagy regulative function is related, and autophagy regulative function is the conveyance and dispersion function of LIVER in the embodiment of a cellular level " theory hypothesis, enriched liver controlling conveyance and dispersion," the essence of the theory.