Study On The Anti-stress Mechanism Of Paeoniflorin Based On The Liver-softening And Depression-relieving Effects Of White Peony Roo

Liver-Qi stagnation syndrome is due to emotional injury.If the emotional disorders,it will lead to blood and qi disorders.In TCM,Radix Paeoniae Alba was regarded as a bitter and sour medicinal herb with "cold property",with the TCM efficiency "nourishing blood and yin,regulating liver remove pain".Antidepression is an important feature of clinical application of Radix Paeoniae Alba.Both Radix Paeoniae Alba and Radix Paeoniae Rubra are derived from Ranunculaceae,the main species are same.According the Chinese Pharmacopoeia,quality control index of Radix Paeoniae Alba and Radix Paeoniae Rubra is paeoniflorin（PF）,the content of PF in Radix Paeoniae Alba is more than 1.2%and more than 1.8%in Radix Paeoniae Rubra.Empirical evidence prescribed that PF have broad therapeutic benefits for pain,muscle spasm,inflammation,menstrual dysfunction,and neurodegenerative disorders.PF is the efficiency material basis of Radix Paeoniae Alba.Albiflorin（AF）is the characteristic active ingredient of Radix Paeoniae Alba and Radix Paeoniae Rubra.However,compared to PF,the pharmacological properties of AF are not yet well investigated.Based on our own patent,it is documented that AF possess remarkable effects for anemia,pain and depression.However,effect of AF on the anti-stress mechanism of chronic restraint stress（CRS）rats and the mechanism of neuroprotective effect by regulating NO-mediated mitochondrial pathway are not yet well inve stigated.The main cause of Liver-Qi stagnation syndrome is emotional injuries,most closely related to function of "liver".However,TCM function of "liver" is connected with the hypothalamus-pituitary-adrenal axis（HPA axis）and other body reaction axis of the physiological regulation in modern medical.HPA axis is the core of the body’s response to stress,maintaining the body’s stress balance.However,if the body is subject to sustained stress,HPA axis continued hyperactivity,not only involved in the central nervous system monoamine neurotransmitter secretion and release,but also can cause hippocampal neuronal damage.Thus,TCM efficiency disorder of "liver" and modern medical psychology on stress is basically the same.Based on this,the study of Liver-Qi stagnation syndrome can learn from the theory of stress in modern medicine,from the organization,cell,function,metabolism and other multi-level.In the preliminary study found that the anti-depression of AF in suspended stress model mice via reduce the NO/cGMP signaling pathway.Based on the theory that PF is the effective material ingredient of Radix Paeoniae Alba and our o wn patent can provide enough monomer substance.Therefore,this study,guided to TCM efficacy of Radix Paeoniae Alba,explored the anti-stress effect of AF on CRS rats and the neuroprotective effect of AF in Corticosterone（Cort）-induced injury PC 12 cell model.1 Literature Review1.1 Research on Liver Depression Syndrome and Modern Chronic StressThe relationship between TCM "Emotional Disease" and modern stress theory was studied.The mechanism of hippocampal injury induced by chronic stress was established,which provide a solid theoretical foundation to explore the anti-stress mechanism of AF.1.2 Research on the Mechanism of Nitric Oxide in the Nervous SystemThe mechanism of action of Nitric oxide（NO）in the nervous system was detected,it is clear that NO plays a physiological role with its concentration.High concentration of NO caused neurotoxicity mainly through two aspects:promoting HPA axis activation,leading to high secretion of glucocorticoids;regulating the mitochondrial-mediated apoptosis signal pathway.It is provides the modern pharmacological mechanism basis for further research.1.3 Effects of PF and AF on nervous systemData sources of PUBMED from Auguest 2010 to December 2016 were searched.Inclusion criteria clearly described the effects of PF and AF on nervous system.Developments have elucidated multiple layers（cellular and molecular）of neuroprotection actions by PF and AF.This knowledge is valuable for our review of how PF and AF by energy metabolism and molecular circuitries involved in regulating cell survival,proliferation,energy metabolism,neurodegeneration,and neuroprotection.It provides modern research ideas for the systematic study of TCM efficacy of Radix Paeoniae Alba,and also provides a modern scientific reference for further research on the anti-stress mechanism of AF.2 Experiment Study2.1 Anti-stress effect of AF on chronic restraint stress（CRS）ratsAim of study:To explore the Anti-stress effect of AF on CRS rats.Method:White peony root water extraction,Xiaoyao pill（including white peony prescription）,paeoniflorin（white peony effective ingredients）and fluoxetine as a positive control.80 SD rats were randomly divided into 8 groups:blank group,model group,White peony root water extraction group,Xiaoyao pill group,fluoxetine group,PF group,AF 30mg/kg group and AF 15mg/kg group.The changes of body mass,the intake of sugar and water were measured and recorded.The independent behavior changes of CRS rats in open field test were detected.The percentage of open arms entries and open arms entries time in elevated plus maze were checked.Results:AF increased the body weight of CRS rats（P<0.05）.In the open field test,AF increased the number of walking（P<0.01）and decreased the retention time（P<0.05,,P<0.01）.In the n elevated plus maze,AF increased the percentage of open arms entries（P<0.05）and the percentage of open arms entries time（P<0.001,P<0.01）.Discussion:AF as a characteristic effective component of white peony root,with the ability to ameliorate the anxiety and depression of CRS rats.AF could increase the body weight and changed the self-acting behavior of CRS rats and ameliorate the anxiety.2.2 Effects of AF on HPA axis,Monoamine Neurotransmitter and Hippocampal 5-HT1A,5-HT2A Receptor in Rats with Chronic DepressionAim of study:To investigate the effects of AF on HPA axis hormone levels,monoamine neurotransmitter content,5-HT1AR and 5-HT2AR receptor mRNA and protein expression in rats with chronic obstructive stress-induced liver depression syndrome.Method:Xiaoyao pill（including white peony prescription）,paeoniflorin（white peony effective ingredients）and fluoxetine as a positive control.70 SD rats were randomly divided into 7 groups:blank group,model group,Xiaoyao group,fluoxetine group,PF group,AF 30mg/kg group and AF 15mg/kg group.The contents of ACTH,CRH and CORT in serum were measured by Elisa,5-HT1AR and 5-HT2AR mRNA expression and protein expression were deteced by RT-qPCR and Western blot.Results:AF decreased the content of 5-HT（P<0.01,P<0.001）.AF could down-regulate 5-HT2AR protein expression（P<0.05、P<0.01）.Discussion:AF could regulate 5-HT1AR in hippocampus further regulate 5-HT and other neurotransmitter secretion on CRS rats.AF also could further regulate the expression of BDNF by modulating the hippocampal 5-HT2AR.2.3 Effect of AF on BDNF and NO/cGMP Signaling Pathway in Hippocampus of Rats with Liver Depression Syndrome Induced by Chronic Constraint StressAim of study:To explore the mechanism of anti-stress effect of AF on chronic restraint stress in rats via NO/cGMP signaling pathway and BDNF content and protein expression in hippocampus.Method:The content of NO,cGMP and BDNF in hippocampus were measured by Griess or Elisa,iNOS and nNOS mRNA expression was studied by RT-qPCR.Western blot was used to detect nNOS,cGMP and BDNF protein expression.Results:AF antagonized the molecular mechanism of death due to energy depletion caused by NO by decreased the content of NO（P<0.05）and down-regulated nNOS mRNA expression.AF reduced the content of cGMP（P<0.01）and BDNF（P<0.05）as well as BDNF protein expression（P<0.001）.Discussion:AF could regulate the molecular mechanism of the death of NO in the hippocampus of rats with liver depression induced by chronic restraint stress.Furthermore,the anti-stress effect mechanism of AF was regulated the expression of BDNF via NO/cGMP signal pathway.2.4 Neuroprotection of AF on Cort-induced PC12 cell apoptosisAim of study:To explore the neuroprotection of AF on Cort-induced PC 12 cell apoptosis.Method:cell viability and changes in living cell/dead cell morphology were measured by CCK8,LDH,Hoechst33342 staining and tunnel staining method to explore the neuroprotection of AF.Results:AF increased the survival cell rate（P<0.01）checked by CCK8 and decreased the number of apoptotic cells detected by Hoechst 33342 staining（P<0.05）and Tunnel staining（P<0.05）.Discussion:AF could induce cort-induced PC12 cell apoptotic rate with significant anti-apoptotic activity.2.5 Neuroprotection of AF on Cort-induced PC12 cell apoptosis via MitochondriaAim of study:To explore the mechanism of AF on mitochondria-mediated apoptosis pathway.Method:The ROS and Ca²⁺content was measured by DCFH-D and Fura-2/AM method respectively,MMP was checked by calcium chlorophyll and CoCl₂,mitochondrial membrane potential changes was detected by JC-1 method.Results:AF decreased the content of Ca²⁺（P<0.01、P<0.05）,the membrane potential of the mitochondria（ΔψM）（P<0.05）and changed MMP;AF reduced the Bax protein expression（P<0.05）.Discussion:Using a variety of methods to prove the anti-apoptotic effect of AF by increased the ROS content,regulate the MMP and ΔψM,as well as reduced the the concentration of intracellular Ca²⁺ due to stress via mitochondrial apoptotic pathway.2.6 Effect of AF on Cort-induced PC12 cell apoptosis via NO-mediated Mitochondrial Energy Metabolic PathwayAim of study:To explore the mechanism of the neuroprotection of AF on NO-mediated mitochondrial energy metabolism and the effect of mitochondrial activity via NO/cGMP signaling pathway on the model of Cort-induced PC 12 cell injury.Method:The NO content was measured by DAF-FM/DA method,3-NT protein concentration and cGMP content was detected by Elisa,iNOS and nNOS mRNA expression was studied by RT-qPCR,Western blot was used to detect Caspase3 and Caspase9 protein expression.A variety of pharmacodynamic methods were used to demonstrate the effect of AF on cortisone-induced PC 12 cell injury model,which could regulate the effect of NO on the mitochondrial cell apoptosis.Results:AF decreased the content of NO（P<0.001、P<0.01、P<0.001）and cGMP（P<0.05）;AF reduced the Bax protein expression（P<0.05）,Caspase3 protein expression（P<0.05）and Caspase9 protein expression（P<0.01、P<0.001）.Discussion:AF reduced the NO content,decreased the membrane potential of the mitochondria（ΔψM）,up-regulated the protein expression of Bax,increased the permeability of mitochondrial membrane（MMP）and decreased the Caspase-dependent apoptosis signal pathway.AF could improve the biological function of mitochondria via NO/cGMP signaling pathway to ensure the formation and supply of energy and inhibit cell apoptosis.3.ConclusionBased on the traditional Chinese medicine（TCM）efficacy of Radix Paeoniae Alba,this study explored the anti-stress effect mechanism of Albiflorin,indicating that AF was the effective material basis for the white peony "Rougan Jieyu".Enrich the basic connotation of TCM theory of white peony,providing experimental basis for the clinical application and the establishment of quality standards and industrial production of white peony.In this study,based on the close research on the progress of modern research on white peony,and the pharmacological research of paeoniflorin,and the patented extraction of albiflorin and paeoniflorin.Albiflorin,the effective component of Radix Paeoniae Alba,was explored the anti-stress effect on chronic restraint stress-induced liver depression syndrome rat model comprehensively from the different level.For the first time,the neuroprotection effect of Albiflorin was investigated via NO-mediated mitochondrial energy metabolism process and the NO/cGMP signaling pathway.It is revealed that Albiflorin is the material basis of the TCM function of white peony.