Study On Protective Effects And Mechanisms Of Drug-containing Serum Of Regulating Liver Recipe JWSNS On Injury Model Induced By Corticosterone And Glutamate On PC12 Cells

Stress is a physical or psychological stimulus that can produce mental or physiological reactions that may lead to illness. Psychological stress is the body to evaluate the threat of the original stress through awareness. With the social development and the increased competitive pressures, psychological stress has become an important factor to threat human health that can not be overlooked. Therefore, the impact of psychological stress on healthy has been attached to the study. Chronic psychological stress may play a crucial role in the development of heart cerebrovascular disease, endocrine disease, neuropsychiatric diseases and autoimmune disease, which in enabling the body appeared to accelerate the normal aging process, learning and memory impairment, mental stress disorder, dementia, depression, low immunity, such as a multi-system disorder, with clinical depression is the most common mental and physical disorder. Liver has the function by regulating the Qi-blood and fluids, and emotional regulating stressed. Emotional radical changes, the liver-QI, Qi-reverse chaos, resulting in a series of psychosomatic reaction disease, so the psychological reaction to become a liver has the function of regulating the functions of emotional function of the central nervous biological mechanism of entry and the application of medical liver has the function of the theory to further explore the causes of depression and the mechanism of antidepressant drug therapy research has the very important significance. In early researchment, combined Chinese traditional medicine theory with modern psychological stress theory, we put forward the hypothesis of study. It is that the TCM theory of "Gan takes charge of dispersion and could regulate emotion" is affirmatively to have certain mechanisms of central neurobiology. So, cut-in from the point of psychological stress reaction, adopting the research thoughts of "prescription-syndrome-therapeutic effectiveness essence of Zang-Fu function", a model of chronic psychological stress reaction(CPSR) for imitating the process of comprehensive pathologic change due to Gan fails to take charge of dispersion and leads to emotional disorder was established. It is considered based on analysis of materials obtained from previous studies, that the central neurobiologic mechanism of so called dispersion, which Gan in charge of, is related to the regulation of hypothalamus-pitutary-adrenalgland axis. Concretely, the function of Gan in TCM may be, in the gross, related with the changes of multiple neuro transmitters and their synthetase produced in the process of CPSR(emotional disorder) regulation, such as neuro peptides, hormones, cyclic necleotide system and Fos protein expression, showing the characteristics of multiple links, multip lelevels and multiple targets, with the effects involve several brain regions including various clusters of nuclei in hypothalamus, hippocampus and amygdala, etc.Based on previous research and focused on the current domestic and international research, the further therapeutic effect ofregulating recipe JWSNS drug-containing serum and its molecular material foundation on cells and molecules level were investigated in this study. PC12 cells has neuronal features and functionality and was taken as a studying object in the experiment. Cultured PC12 cells in vitro with corticosterone (Cort) and glutamate (Glu) as the injury model, for simulating the high levels of glucocorticoid (GC) and the accumulation state of glutamate in depression patients in vitro. Chronic psychological stress can lead to nerve cell damage is the key issue in depression. To observe the effect of the regulating liver Recipe JWSNS (JWSNS) drug-containing serum from micro articulate and reveals mechanisms of treatment to chronic stress-induced neuronal injury with regulating liver Recipe JWSNS prescription. To provide a theoretical basis for the future research and development of new drugs of anti-depression medicine. The roles of liver to adjust the emotional changes though central nervous biological material basis are currently explored. The experimental study which focus on the liver has the regulating emotional function of cellular and molecular mechanism is a preliminary study. This study is divided into four parts: Partâ… Protective effects of regulation liver recipe JWSNS drug-containing serum on PC12 cells psychological injury model induced by Corticosterone and Glucocorticoids and mechanismsDepression patients have emotional disorders and neurological system injury, ln the long-term stress of depression patients, the HPA axis was in a continuous hyperfunctional states, so that the body has remained high level of GC consistently. After the depression of excitatory amino acid such as glutamate accumulated, leading to neuronal degeneration, caused hippocampal neurons apoptosis and death, and even damaged the function and morphology of brain. Experimental observation in this part were carried out to the protective effect of JWSNS drugt-containing serum on corticosterone and glutamate-induced injury in PC12 cells. Tiaogan recipe JWSNS (JWSNS) contains Bupleurum Rootl5g, white peony Root15g, Bitter Orange6g, Wolfberry Fruit15g, Cape jasmine 5g, dry rehmannia root15g, abalone she1130g. SPF Wistar rats were randomly divided into four groups: normal control group, JWSNS low-, middle-, high-dose group(raw drug dose is 0.846,1.69,3.384 kg/L).The serum was separated from abdominal aortic blood. Rat pheochromocytoma PC12 cells were cultured with Hams' -F12K medium and 10% dialyzed heat-inactivated horse serum and 5%bovine serum at 37℃in a humidified atmosphere of 95% air and 5%CO₂ for 2 or 3 days. Cells were passaged every 2 or 3 days. After being cultured for 24h with corticosterone, glutamate and JWSNS-containing low-, middle-, high-dose serum, PC12 cell morphological change was obseved with an inverted phase-contrast microscopy. Morphologica lobservations of cultured PC12 cells in vitro was recorded to a video tape via a color video camera and a digitized data of each pictur was stored in the computer at a regular sampling time fo later analysis. The result proved that the methed of culturing PC12 cells was correct and successful. Measurement of cell viability PC12 cell viability was measured by MTT assay which is based on the capacity of the mitochondrial enzyme succinate dehydrogenase to transform the yellow MTT tetrazolium saltin to blue MTT formazan crystals in viable ceils. The level of conversion provides an indication of mitochondrial metabolic function. After PC12 cells being incubated with 100, 200, 400μmol/LCort, 10, 50, 100μmol/h Glu, 5%, 10% JWSNS-containing serum for 24 hours, PC12 cells survival rate of different concentrations of corticosterone, Glutamate and JWSNS drug-containing serum group ware deter -minated by MTT. The results showed that the cell survival rates of 100μmol/L, 200μmol/L, 400μmol/LCort group were 72.31-26.16%. The cell survival rates of 10μmol/L, 50μmol/L, 100μmol/LGlu were 67.69-21.54%. Compared with the control group, the cell survival rates of different concentration of corticosterone and glutamate group was significant (p＜0.05-0.001).The growth of PC12 cells were inhibited and induced varying degrees of injury by different concentrations of corticosterone and glutamate. With corticosterone and glutamate concentration increased, the cell survival rates were significantly reduced also and aggravated the PC12 cell injury. The cell survival rates of 5%JWSNS-containing serum low-, middle - and high-dose group was 86.76%, 98.41% and 94.11%, the cell survival rates of 10%JWSNS-containing serum low-, high- dose group was 85.29, 97.06% and 89.71%, the cell survival rates of 5%, 10% normal serum group was 92.65% and 88.24%. compared with normal cells control group, the result has no difference (p＞0.05). This indicates that JWSNS- containing serum has non-toxi and side effects on PC12 cells. Selected 200μmol/LCort, 50μmol/LGlu asdamage model group, using MTT to observe the protective effect of JWSNS-containing serum on injury model induced by corticosterone and glutamate on PC12 cell. The results showed that the cell survival rates of 200μmol/LCort group and 50μmol/LGlu group were 51.67% and 46.77% respectively. Compared with the normal cells control group the cell survival rates of 200μmol/LCort group and 50μmol/LGlu group decreased significantly. This indicates that the injury model induced by corticosterone and glutamate on PC12 cells was established successfully. The cell survival rates of Cort+5%, Glu+5%JWSNS-containing serum low-, high- dose group were 46.77%, 52.62% 48.38%, 45.19%. 48.26% and 46.77% respectively, The survival rates of Cort+5%. Glu+5% normal serum cell group were 45.16% and 43.55% respectively. The cell survival rates of Cort+10%, Glu+10% normal serum group were 56.29% and 50.23%, Compared with the model group, the cell survival rates of Cort+5%. Glu+5%JWSNS-containing serum and of the Cort+5%, 10%, Glu+5%. 10%normal serum group had no significant (p＞0.05). The result showed that 5%JWSNS-drug-containing serum and normal serum group had no significant anti-injury effects. The cell survival rates of Cort+1O%, Glu+10%JWSNS-containing serum low-, middle-, high- dose group were 72.58%, 87.11%, 75.81%, 69.35%, 82.25% and 70.97% Compared with corticosterone, glutamate model group and normal serum group, 10%JWSNS-containing serum increased cell survival rates significantly, the difference was significant (p＜0.05-0.01), The result showed that the 10% JWSNS serum-containing serum play a important protective role in injury model induced by corticosterone and glutamate in PC12 cells, of which 10%JWSNS was strongest dose group.Partâ…¡Effects of regulation liver recipe JWSNS drug-containing serum on apoptosis and BDNF protein expression in Corticosterone and Glucocorticoids-induced PC12 cells psychological injury modelThe effects of regulation liver recipe JWSNS drug-containing serum on apoptosis and the protein expression of BDNF in Corticosterone and Glucocorticoids-induced PC12 cells injury model in vitro were observed by Annexin/PI double staining flow-cytometry method and Indirect immunofluores-cence staining method. The experimental was divided into groups: the normal PC12 cells control, 200μmol/LCort, 50μmol/LGlu injury model group, 200μmol/LCort, 50μmol/LGIu+10%JWSNS drug-containing serum low-, middle-, high-dose group and 200μmol/LCort, 50μmol/LGlu +10%normal serum group. With acridine orange(AO)and Hoechst33342(Ho) staining methods to observed PC12 cells morphological changes in apoptosis induced by corticosterone and glutamate. Fluorescence in AO can be seen under the microscope after staining of nuclei of living cells showed bright green, and the spread of nucleolar RNA in the cytoplasm was orange, the rest of the cytoplasm of pale red-brown. Death cell was dark green, the entire cytoplasm was dark green. Hoechst33342 fluorescence staining: After deleting cultured medium, cells were rinsed with PBS and stained with Hoechst 33324(fixed by formalaehyde before Hoechsat33342) for 10min, rinsed with PBS twice and observed under fluorescence microscope. Fluorescent dye Hoechst33342 can dye living cells and apoptotic cell DNA through cell membrane. The living cells had the strongest ability to absorb Hoechst33342 under the condition of iso-osmia, so Hoechst 33342 staining the living cells nuclei blue, cell with the brightest blue fluorescene were living cells and cells with the darkest blue fluorescene were apoptotic cells. Early apoptosis cells appear weaker blue fluorescence, nuclear condensation. And the chromatin condensation, formation of apoptotic bodies and fluorescence intensity as signs of apoptosis could be seen. the nuclear chromatin condensation-intensive, marginalization, formed blob was deeply stained-or crescent-rings at the nuclear membrane, budding nuclear rupture formation of apoptotic bodies and other cell morphological manifestations of apoptosis and cell death often be observed in 200μmol/L Cort and 50μmol/L Glu injury model group; It could be seen that chromatin was evenly distributed in non apoptotic cells. Apoptotic bodies and cell death were rarely seen in the normal PC12 cells control group, apoptotic bodies densely gathed in the 200μmol/LCort and 50μmol/LGlu injury model group and the apoptotic bodies were significantly reduced in the Cort+10%, Glu+10%JWSNS-containing serum low-, middle-, high- dose group.Flow cytometric analysis of apoptosis and necrosis: Twenty-four hours after treated with 200μmol/L corticosterone, 50μmol/L glutamate, Cort+10%, Glu+10%JWSNS-containing serum low-, middle-, high- dose and Cort+10%, Glu+10% normal serum, PC12 cells were harvested, washed anddouble-stained by using an AnnexinV-fluorescein isothiocyanate(AnnexinV-FITC) apoptosis detection kit. This kit is based on the phenomenon that soon after initiating apoptosis, phosphatidylserine is translocated from the cytoplasmic face of the plasma membrane to the cell surface. AnnexinV has a strong, Ca²⁺-dependent affinity to phosphatidylserine and therefore is used as a probe for detecting apoptosis. Cells that have lost membrane integrity will show red staining(by propidium iodide) throughout the nucteus and therefore will be easily distinguished from the apoptotic cells. Samples were incubated with annexinV and propidium iodide for 15 min in darkness at room temperature, and then quantitatively analyzed by a FACS can flow cytometer equipped with the Systemâ…¡analysis software. The apoptosis rate of normal conmtrol cell group was (16.24±0.78)%. The apoptosis rate of 200μmol/LCort and 50μmol/LGlu injury model group was (45.91±0.95)% and (39.58±0.7)%, compared with normal conmtrol cells group, the apoptosis rate of Cort and Glu injury model group were increased significantly(p＜0.001), indicating that corticosterone and glutamate could induce the early apoptosis in cultured PC12 cell in vitro. The apoptosis rate of Cort+10%, Glu+10%JWSNS drug-containing serum group dropped remarkbly. The apoptosis rate of middle dose group fell to (28.99±1.20)%. (23.89±0.35)% and the apoptosis rate of high dose group apoptosis rate fell to (31.82±2.04%), (29.84±0.97%), compared with the corticosterone, glutamate injury model group, the difference was significant (p＜0.05-0.01). The cell apoptosis rate of Cort+10%, Glu+10%JWSNS drug-containing serum middle, high-dose group was significantly lower than Cort+10%, Glu+10% normal serum group, compared with corticosterone, glutamate injury model group, Cort+10%, Glu+10% normal serum group, the difference was significant(p＜0.05-0.01), indicating that JWSNS drug-containing serum has the effect of anti-apoptosis induced by confrontation and Glutamate in PC12 cells, of which 10%JWSNS drug-containing serum medium dose group was the most effective. Compared with corticosterone and glutamate injury model group, the apoptosis rate of Cort+10%, Glu+10% normal serum group showed no significant difference, indicating that the normal serum was without the resistance effect to pc12 cells apoptosis induced by corticosterone and glutamate.Indirect immunofluorescence staining method was used to observe the expression of BDNF protein in PC12 cells. The colour of positive products of FITC-labeled BDNF of immunofluorescence staining was green. In the negative control samples positive BDNF immunofluorescence reaction were not observed. There was a small amount expression of BDNF in the normal PC12 cells control group. And compared to normal cells, few positive expression of BDNF waer observed in corticosterone and glutamate injury model group, mainly in small quantities and very low fluorescence intensity, and almost the same as the background color. The result show that corticosterone and glutamate inhibited the expression of BDNF in PC12 cells. The positive expression of BDNF in Cort+10%, Glu+10%JWSNS drug-containing serum group increased, espcialy the fluorescence intensity increased significantly (++-+++), higher than that of corticosterone(±), glutamate(±) and normal serum(+) remarkbly. Green fluorescent particles points, or zonal distribution around the nucleus, The positive expression of BDNF was up by Cort+10%, Glu+10%JWSNS drug-containing serum, of compared with corticosterone, glutamate and normal serum group had obvious differences, indicating that 10%JSNS drug-containing serum increased the expression of BDNF in corticosterone and glutamate-induced apoptosis PC12 cells, of which middle dose group was strongest effect.Partâ…¢Effects of regulation liver recipe JWSNS drug-containing serum on cellular [Ca²⁺]i in Corticosterone and Glucocorticoids-induced PC12 cells psychological injury modelThe intracellular [Ca²⁺]i in PC12 cells were tested in the study using Laser Scan Confocal Microscope (LSCM), effect of corticosterone. Glutamate-induced JWSNS drug-containing serum. The experimental cells were divided into groups: normal cells control group, 200μmol/LCort and 50μmol/LGlu injury model groupl, Cort+10%, Glu+10%JWSNS drug-containing serum group, Cort+10%, Glu+10%normal serum group. Using Fluo-3/AM probe. Cultured PC12 cells were incubated with Fluo 3-AM working solution (the final concentration of Fluo 3-AM is 5μmol/L) at 37℃for 45min, followed by 3 times washes with the culture medium. The cultured neurons grown on glass coverslips were placed in a cell chamber on the stage of an inverted microscope. The fluorescence signals were measured with confocal laser scanning system Fluo3-AM fluorescence in the cells was excited at 488 nm froma high-power Ar+ laser, and the emission bands at 530 nm were detected by a photomultiplier. The change in [Ca²⁺]i was represented by relative fluorescence intensity. The results showed that normal PC12 cells was generally low-intensity fluorescent, PC12 intracellular [Ca₂₊]i concentration in normal group was 53.6±7.9; [Ca²⁺]i fluorescence intensity in corticosterone, glutamate injury model group in PC12 cells increased markedly and reached 134.9±12.1, 162.3±5.3. The intracellular [Ca²⁺]i concentration was enhenced significantly (p＜0.001). Compared with corticosterone and glutamate model group, the fluorescence intensity and intracellular [Ca²⁺]i concentration of Cort+10%, Glu+10%JWSNS drug-containing serum group was significantly weakened, and lower than Cort+10%, Glu+10%normal serum group, the fluorescence intensity in the middle dose group dropped 64.7±4.8, 69.7±6.7, had a significant difference (p＜0.01-0.001). Corticosterone and glutamate can increase intracellular [Ca²⁺]i concentration in PC12 cells and result in the calcium overload in PC12 cells: Regulation liver recipe JSNS drug-containing serum had the effect of dereasing PC12 cells intracellular [Ca²⁺]i overload induced by by corticosterone and glutamate, of which the middle dose group had strongest effect.Partâ…£Effects of regulation liver recipe JWSNS drug-containing serum on transcription factor cAMP response element binding protein (CREB) and phosphorylation (P-CREB) in Corticosterone and Glucocorticoids-induced PC12 cells psychological injury modelUsing immunohistochemical methods and Western blot method to detecte the effect of JWSNS drug -containing serum on transcription factor cAMP response element binding protein (CREB) and phosphorylation (P-CREB)Immunohistochemical detection of the expression of CREB: Experimental cells were divided into four groups: normal cells control group, 200μmol/L Cort and 50μmol/L Glu injury model groupl, Cort+10%, Glu+10%JWSNS drug- containing serum group, Cort+10%, Glu+10%normal serum group. The positive expression of CREB in normal PC12 cells mainly located in the nucleus, brownish yellow color. Normal cells CREB expression was 30.27±6.2: The positive expression of CREB of corticosterone and glutamate model group were has light brown, the expression level was 17.1±8.2, 20.8±5.7, respectly, and compared to normal PC12 cells group, the expression of CREB in corticosterone and glutamate model group decreased significantly (p＜0.01); The expression of CREB in normal serum group increased to 25.7±6.5, 26.3±8.2, compared with the Cort and Glu group there were not statistically significant. 10%JWSNS drug-containing serum can be seen deepening nuclear staining, the expression of CREB significantly increased and of which middle dose group were 62.6±11.7, 79.5±7.6, compared with the corticosterone and glutamate group there was significant (p＜0.001), and compared with the normal serum group, the difference was significant (p＜0.01); The result showed that corticosterone and glutamate can reduce the expression of CREB in PC12 cells, 10%JWSNS drug-containing serum can up-regulated the expression of CREB, and the middle dose group increased significantly, normal serum has no such effect.Western blot assay phospho-CREB (Ser133) expression: Western blot Protein preparation was done according to the standard method. Protein concentration of the supernatant was determined by the bicinchoninic acid assay. The protein supernatant was run on 10%SDS-PAGE and transferred tonitrocellulose. Then nitrocellulose was incubated for 1h in TBS/T containing 5% fat-free dried milk, and then incubatedwith rabbit polyclonal antibodies p-CREB (1:500) at 4℃overnight, followed by 2h of incubation with horseradish peroxidase conjugated goat anti-rabbit(1:500)Protein bands were then detected with chemiluminescent reagent. The intensities of the obtained bands were determined by a densitometer. Each densitometric value was expressed as mean±SD.The expression of P-CREB was different in each group, compared with normal cells group, the expression of P-CREB decreased in corticosterone and glutamate model group. The expression of p-CREB protein in 10%JWSNS drug-containing serum group was significantly increased, compared with the corticosterone and glutamate model group, the difference was significant (p＜0.01), compared with normal serum group had differences (p＜0.05).This study used cellular and molecular biology methods to explore the protective effect of regulating liver recipe JWSNS drug-containing serum PC12 cells to confirm that the liver has the function of modulating the emotional function and has neurobiology molecular mechanism. The results of this study present a preliminary findings that suggests JWSNS drug-containing serum had significant protective effect on PC12 cell injured by corticosterone and glutamate. JWSNS drug-containing serum can increase the expression of BDNF in corticosterone and glutamate-induced apoptotic PC12 cells, regulating liver recipe JWSNS drug-containing serum has effect on ease intracellular calcium overload which was induced by corticosterone and glutamate in PC12 cells, of which the middle dose group has the strongest effect.JWSNS drug-containing serum could increase CREB phosphorylation level, and raising the CREB expression of PC12 cells that play akey role in protecting neurons.