Based On The No/cgmp Pathway Of Radix Paeoniae Alba Nourishing Blood, Liver Function And Molecular Mechanism Of A Preliminary Study

As Chinese traditional hematinic. Radix Paeoniae Alba has a role in nourishing blood and emolliating the liver. Based on the former studies about paeoniflorin acting on NO/cGMP signal pathway, we investigate the molecular mechanism of paeoniflorin in antistress via regulating NO/cGMP signal pathway. The model of restraint stress combined with blood deficiency induced by60Co-y irradiation is used to explore the modeling method of "Blood deficiency and liver qi stagnation syndrome". The function of "Nourishing blood and emolliating the liver"of Radix Paeoniae Alba will be studied from many aspects, such as increasing blood cells, regulating HPA axis and neurotransmitter. And in the further study, we throw light on the effects of paeoniflorin on the key material NO and cGMP on the NO/cGMP signal pathway on molecular levels, and sGC, PDE, Glu and its receptor NOS, in order to indicate pathological changes of syndrome of stagnation of liver qi and blood deficiency on the NO/cGMP signal pathway and investigate the molecular mechanism of paeoniflorin in antistress via regulating NO/cGMP signal pathway.1Pharmacodynamic tests about "nourishing blood and emolliating the liver" function of Radix Paeoniae Alba1.1Preliminary study on the effect of paeoniflorin on nourishing blood1.1.1The enriching blood effect of paeoniflorin in the mice with blood deficiency induced by compound method of bleeding.Objective:To observe the enriching blood effect of paeoniflorin in the mice with blood deficiency induced by compound method of bleeding.Methods:After given7day drugs, the mice comes to two-week modeling stage. The animal model of blood deficiency was established by a combination of starved feeding with routine food at the rate of75g/kg body weight per day, swimming20minutes and removing0.4ml blood every other day in each mouse for two weeks. The amount of WBC, RBC and PLT and the content of Hb in the peripheral blood were determined by the Automatic Blood Cell Analyzer on7d and14d.Results:compared with model group,100mg/kg paeoniflorin can increase the WBC number on7d(P<0.001);100mg/kg paeoniflorin,50mg/kg paeoniflorin,25mg/kg paeoniflorin can increase the WBC number on14d(P<0.05, P<0.001, P<0.001).The RBC number of model mice also had an increase after given paeoniflorin.Conclusion:paeoniflorin can increase the number of WBC and RBC in the peripheral blood, which indicates the effect of enriching the blood.1.1.2The effect of paeoniflorin on blood deficiency model mice induced by irradiation.Objective:The blood deficiency model was prepared by Co60gamma-ray irradiation to study the enriching blood function of paeoniflorin.Methods:Blood deficiency mice model was established by using3.5Gy Co60y-ray. We infused high, middle and low dosages of paeoniflorin into mices stomach for14days after irradiation.20μ1blood samples of each group of mice were taken from caudal vein respectively on3,7,14days and then white blood cells were counted.Results:WBC of other groups was significantly lower than the control group(P<0.001). WBC of paeoniflorin-high was significantly higher than the model group on the third day and14th day(P<0.01,P<0.05).Conclusion:High dose of paeoniflorin can increase WBC of the blood deficiency model rats at certain time points.1.2Preliminary study on relieving depression effect of paeoniflorin.Objective:To observe the antidepressant effect of paeoniflorin on the mouse TST test.Methods:Mice were divided into paeoniflorin-high, paeoniflorin-middle. paeoniflorin-low, model, fluoxetine groups. After2days administration, mouse tail-end was fixed by tape, making mouse hanged upside-down. Record the inactive time in4minutes after2minutes adaption.Results:Compared with model group, paeoniflorin-high, paeoniflorin-middle and lluoxetine groups can reduce the immobility time of tail-suspension(P<0.05).Conclusion:paeoniflorin-high and paeoniflorin-middle groups have antidepressant effects.1.3Reasearch on "enriching blood and emolliating the liver" function of paeoniflorin by the model of restraint stress combined with blood deficiency induced by60Co-y irradiation.Objective:The model of restraint stress combined with blood deficiency induced by6OC0-γ irradiation is used to explore the "’enriching blood and emolliating the liver" function of paeoniflorin. the major effective ingredient of Radix Paeoniae Alba.Methods:Rats are divided into ten groups. They are control group(group one), irradiation group(group two), irradiation plus Qian Cao group(group three), irradiation plus paeoniflorin group(group four), restraint stress group(group five), restraint stress plus fluoxetine group(group six), restraint stress plus paeoniflorin-high group(group seven), model group(group eight), model plus paeoniflorin-high group(group nine), model plus paeoniflorin-low group(group ten). Six groups were built into blood deficiency model by using3.5Gy Co60y-ray. They are group two, group three, group four, group eight, group nine and group ten. Six groups were built into restraint stress model by given restraint stress combined with single cage breeding. We observed the efleets of paeoniflorin on weight, praxiology changes, hemogram, and neurotransmitter in hippocampus on the model rats. Results:WBC of other groups significantly lower than the control group on the third day and7th day(P<0.001); paeoniflorin can increase WBC of the model group on the third day and7th day(P<0.001).The weight gain of model group rats grew slowly. Paeoniflorin didn’t play a significant role in increasing weight. Paeoniflorin-high and paeoniflorin-low can both increase the sucrose water consumption of model group. The low dosage increased the numbers of crossings and rearings in the open field test(compared with model group, P<0.001). And numbers of electric shocks in the step-down test were decreased by high and low dosage(compared with model group, P<0.001, P<0.01).Paeoniflorin-low can reduce CORT content in model rats plasma. Paeoniflorin-high can increase5-HT content in model rats hippocampus(compared with model group, P<0.05).Conclusion:Comprehensive evaluation of a number of indicators can be drawn that the model of restraint stress combined with blood deficiency induced by60Co-y irradiation has a good mix of the blood deficiency model and liver qi stagnation model, reflects "’blood deficiency and liver qi stagnation" status. Paeoniflorin has the "nourishing blood and emolliating the liver" function on this model.2The molecular mechanism of paeoniflorin in antistress via regulating NO/cGMP signal pathway.2.1The effect of paeoniflorin on Glu in hippocampus tissue.Objective:To explore the effect of paeoniflorin on Glu in hippocampus tissue by building the model of restraint stress combined with blood deficiency induced by60Co-γ irradiation.Methods:Took hippocampus of rats of group one, five, six, seven, eight, nine and ten. Selected the supernatant after the tissue was homogenized and centrifuged, then did OPA derivatization. O-phthalaldehyde (OPA) column derivatization HPLC with fluorescence was used to detect glutamate content in the hippocampus.Results:The glutamate content in the hippocampus of model rats had an increase. And80mg/kg or40mg/kg paeoniflorin can reduce glutamate content(40mg/kg is better than80mg/kg), but had no significantly differences compared with model group.Conclusion:Glu accumulated in the hippocampus of model rats. Paeoniflorin can play a reduced role in hippocampus Glu content of model rats.2.2The effect of paeoniflorin on the content of NO in hippocampus.Objective:To explore the effect of paeoniflorin on NO in hippocampus tissue by building the model of restraint stress combined with blood deficiency induced by60Co-y irradiation.Methods:Gricss method was used to detect NO content in hippocampus tissue of all groups except group three.Results:The NO content in the hippocampus of model rats had an increase. And80mg/kg or 40mg/kg paeoniflorin can reduce NO content(40mg/kg is better than80mg/kg), but had no significantly differences compared with model group.Conclusion:NO accumulated in the hippocampus of model rats. Paeoniflorin can play a reduced role in hippocampus NO content of model rats.2.3The effect of paeoniflorin on the content of cGMP in hippocampus.Objective:To explore the effect of paeoniflorin on cGMP in hippocampus tissue by building the model of restraint stress combined with blood deficiency induced by60Co-y irradiation.Methods:RIA method was used to detect cGMP content in hippocampus tissue of all groups except group three after samples were pretreatment.Results:Compared with control group, cGMP content in hippocampus tissue of model group had a significant increase. After given40mg/kg paeoniflorin, cGMP content in hippocampus tissue of model group reduced to some extent.Conclusion:40mg/kg paeoniflorin can reduce cGMP content in hippocampus tissue of model group.ConclusionBased on the Chinese medicine theory, we tested and verified the "nourishing blood and emolliating the liver" function of paeoniflorin, the major effective ingredient of Radix Paeoniae Alba, we also investigated the molecular mechanism of paeoniflorin in antistress via regulating NO/cGMP signal pathway. We used one new animal model to explore the "Blood deficiency and liver qi stagnation syndrome" and finally we successfully found this new animal model can reflect both blood deficiency syndrome and restraint stress syndrome. We found that the "nourishing blood and emolliating the liver" function of paeoniflorin was achieved by regulating HPA axis and5-IIT content in hippocampus. Further, we found the target spots of paeoniflorin on NO/cGMP pathway were NRK NR2A、 NR2B、GluR1、 mGluR5、iNOS、nNOS、PDE5A、PDE2A、PDE9A、sGCa2和sGCβ1. As a result, scientific implications of the basic theory of TCM were clarified. The experimental results can provide the basis for clinical application of Radix Paeoniae Alba, and would have a good academic value and clinical significance.