Effect of Xiaoyaosan on JNK Pathway in LPS-induced Depressive-like Behavior in Mice / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Xiaoyaosan in alleviating lipopolysaccharide （LPS）-induced depressive-like behavior in mice based on the c-Jun N-terminal kinase （JNK） pathway. MethodAfter adaptive feeding， C57BL/6J mice were randomly divided into normal group， model group， minocycline group （intrabitoneal injection， 50 mg·kg-1）， fluoxetine group （intragastric administration， 2.6 mg·kg-1）， and low-， medium-， and high-dose Xiaoyaosan groups （intragastric administration，6.012 5， 12.025， and 24.050 g·kg-1）. After 14 days of administration， the model group and each administration group were intraperitoneally injected with 2 mg·kg-1 LPS， and the normal group was intraperitoneally injected with equal volume of normal saline. Depressive-like behavior in mice was assessed using the open field test and the elevated zero maze test. High-performance liquid chromatography （HPLC） was used to measure the levels of norepinephrine （NE） and epinephrine （E） in the mouse hippocampus. Enzyme-linked immunosorbent assay （ELISA） was performed to determine serum interleukin-1β （IL-1β） levels. Immunohistochemistry was used to measure the protein expression levels of ionized calcium-binding adapter molecule-1 （Iba-1）， c-Fos， and c-Jun. Real-time polymerase chain reaction （Real-time PCR） was used to measure mRNA expression levels of IL-1β， c-Jun， c-Fos， and JNK3 in the mouse hippocampus. Protein expression levels of JNK and phosphorylated （p）-JNK in the mouse hippocampus were measured using capillary protein automated protein expression analysis system （Western）. ResultCompared with the normal group， the model group exhibited significantly reduced central area residence time， crossing times， and travel distance in the open field （P<0.01）， significantly increased serum IL-1β levels （P<0.01）， significantly decreased NE and E levels （P<0.05）， upregulated mRNA expression of IL-1β， JNK3， and c-Fos， and increased protein expression of Iba-1， c-Fos， and c-Jun （P<0.05， P<0.01）. Compared with the model group， the Xiaoyaosan groups showed increased central area residence time and open arm residence time （P<0.05）， increased NE and E levels （P<0.01）， decreased mRNA expression of IL-1β， JNK3， c-Jun， and c-Fos， and decreased protein expression of Iba-1， c-Fos， JNK， and p-JNK （P<0.05， P<0.01）. The minocycline group and the fluoxetine group showed decreased mRNA expression of JNK3， c-Jun， and c-Fos （P<0.05， P<0.01）. The minocycline group showed decreased serum IL-1β and p-JNK protein expression （P<0.01）. The fluoxetine group exhibited increased NE and E levels and decreased c-Fos protein expression （P<0.01）. ConclusionXiaoyaosan can improve depressive-like behavior induced by LPS in mice， and its mechanism may be related to the inhibition of neuroinflammatory responses and the JNK pathway.

Effect of Danzhi Xiaoyaosan on Phosphorylation of Tau Protein in Rats with Alzheimer's Disease Based on PP2A and GSK-3β / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Danzhi Xiaoyaosan on the phosphorylation of tau protein and different sites of glycogen synthase kinase-3β （GSK-3β） and phosphoseryl/suanyl phosphate protein phosphatase 2A （PP2A） in the hippocampus of rats with Alzheimer's disease （AD） and its mechanism. MethodThe rat model of AD was established by injecting okadaic acid into the bilateral hippocampus of 90 male Wistar rats in SPF grades. The rats with successful modeling were selected and randomly divided into model group， aricept group （0.5 mg·kg-1）， and Danzhi Xiaoyaosan high， medium， and low groups （17.55， 8.77， and 4.38 g·kg-1）， and then gavaged for 42 d， once a day. Morris water maze was used to detect the learning and memory ability of rats， Nissl's staining was used to observe the morphological structure of neurons in the hippocampus， and Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tau protein， GSK-3β， and PP2A. Western blot was used to determine the protein expression levels of tau protein， GSK-3β， and PP2A. ResultAs compared with the control group， the learning and memory abilities of the rats in the model group were significantly decreased （P<0.01）， and the hippocampal CA3 region cells had abnormal structure， disorderly arrangement， and decreased number. The expression levels of GSK-3β mRNA， GSK-3β， p-GSK-3β-Tyr216， p-PP2A， and p-tau were increased in the model group as compared with the control group （P<0.01）， and those of p-GSK-3β-Ser9 and PP2A decreased significantly （P<0.01）. As compared with the model group， the learning and memory ability of the Aricept group and the Danzhi Xiaoyaosan groups were improved （P<0.05， P<0.01）， and the cell morphology and the number of hippocampal CA3 regions were better. The mRNA expression levels of PP2A and tau in the Aricept group were significantly up-regulated （P<0.05）， the mRNA expression level of GSK-3β was significantly down-regulated （P<0.01）， and the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-PP2A were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A in the high-dose Danzhi Xiaoyaosan group was significantly up-regulated （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of p-PP2A， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of GSK-3β was significantly down-regulated in the medium-dose Danzhi Xiaoyaosan group （P<0.01）， the protein expression levels of GSK-3β， p-GSK-3β-Tyr216， and p-tau were down-regulated （P<0.05， P<0.01）， and the protein expression level of PP2A was significantly up-regulated （P<0.01）. As compared with the model group， the mRNA expression level of PP2A was significantly up-regulated in the low-dose Danzhi Xiaoyaosan group （P<0.01）， and that of GSK-3β was significantly down-regulated （P<0.01）， whereas the protein expression levels of GSK-3β and p-GSK-3β-Tyr216 were down-regulated （P<0.05， P<0.01）， and those of p-GSK-3β-Ser9 and PP2A were significantly up-regulated （P<0.01）. ConclusionDanzhi Xiaoyaosan can improve the learning and memory ability of rats with AD， and its mechanism may be related to the regulation of the activities of GSK-3β and PP2A protein-related sites and the phosphorylation of tau protein.

Meridian Tropism of Components in Bupleuri Radix Based on Nonalcoholic Steatohepatitis Model and Principal Component Analysis / 中国实验方剂学杂志

ObjectiveTo explore the meridian tropism of components in Bupleuri Radix （Chaihu， CH） based on the model of nonalcoholic steatohepatitis （NASH） and clarify the substance basis of the meridian tropism of CH in Xiaoyaosan （XYS） by means of principal component analysis. MethodEighty SPF male C57BL/6 mice were randomly assigned into 8 groups， with 10 mice in each group. Except that the blank group was fed with the methionine choline-sufficient （MCS） diet， the other mice were fed with methionine choline-deficient （MCD） diet for 4 weeks to establish the nonalcoholic steatohepatitis （NASH） model. After the established model was confirmed by hematoxylin-eosin （HE） staining， the mice were administrated with corresponding drugs by gavage once a day for 4 weeks. Specifically， the 8 groups were XYS group （2.874 g·kg-1）， XYS-CH group （2.445 g·kg-1）， XYS-CH+volatile oils （Vol， 0.163 mg·kg-1） group， XYS-CH+polysaccharides （Pol， 24.067 mg·kg-1） group， XYS-CH+flavones （Fla， 2.241 mg·kg-1） group， and XYS-CH+saponins （Sap， 2.746 mg·kg-1） group. The model group and the blank group were administrated with the same volume of normal saline. After the last administration， the mice were sacrificed for the collection of blood and liver tissue. The pathological changes of liver were observed by HE staining and oil red O staining. Enzyme linked immunosorbent assay （ELISA） kits were used to determine the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， triglyceride （TG）， total cholesterol （TC）， high-density lipoprotein （HDL）， and low-density lipoprotein （LDL） in serum as well as malondialdehyde （MDA）， superoxide dismutase （SOD）， catalase （CAT）， and glutathione peroxidase （GSH-Px） in liver. SPSS Statistics 23 was used for principal component analysis and comprehensive evaluation to determine the substance basis of the meridian tropism of CH in NASH mice. ResultCompared with the blank control group， the modeling led to hepatocyte swelling， increased fat vacuoles， and appearance of inflammatory cells. Further， the modeling elevated the levels of ALT， AST， TG， TC， and LDL and lowered the HDL level in serum， and it increased the MDA level and decreased the SOD， CAT， and GSH-Px levels in liver. Compared with the model group， the administration of XYS and XYS-CH in combination with the components of CH alleviated the oxidative damage in liver （P<0.05）. The comprehensive score of the pharmacological efficacy was in a descending order as follows： XYS > XYS-CH+Sap > XYS-CH+Fla > XYS-CH+Pol > XYS-CH+Vol > XYS-CH. Among the chemical components of CH， Sap had the best effect. ConclusionSap lowers the blood lipid level， regulates the abnormal lipid metabolism， and alleviates the oxidative damage of liver， which is the substance basis for CH to exert the meridian tropism in liver.

Intervention of Hei Xiaoyaosan in Alzheimer's Disease Immune Inflammation Based on Liver-kidney Homology / 中国实验方剂学杂志

Alzheimer's disease （AD） is a neurological disease highly related to age， which is the main cause of senile dementia and the most common disease leading to the loss of daily living ability of the elderly. AD brings heavy mental burden and economic pressure to patients， families， and society. Traditional Chinese medicine （TCM） ascribes AD to category of "dementia"， believing that the treatment should start from kidney because kidney deficiency is the root cause. Combined with the physiological and pathological characteristics of liver， this paper proposed that liver-kidney homology was an important idea for the prevention and treatment of AD. The main pathological manifestations of AD were amyloid β-protein （Aβ） deposition and neurofibrillary tangles （NFT）， and the pathogenesis was complex. A growing number of studies showed that immune inflammation played an important role in the pathogenesis of AD. The important target of treating AD was the regulation of neuro-immune inflammation through the nuclear factor kappa B （NF-κB）/NOD-like receptor thermal protein domain associated protein 3 （NLRP3）/Caspase-1/interleukin-1β （IL-1β） signaling pathway. Based on the idea of liver-kidney homology， this paper selected the representative formula Hei Xiaoyaosan to explore its effect on the prevention and treatment of AD and the mechanism from the perspective of regulating NF-κB/NLRP3/Caspase-1/IL-1β signaling pathway and inhibiting neuro-immune inflammation， expecting to further promote the in-depth study on the prevention and treatment of AD， and provide references for the prevention and treatment of AD by TCM.

Effect of Hei Xiaoyaosan on Neuroinflammation in AD Model Rats： Based on Wnt/β-catenin Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore whether Hei Xiaoyaosan can inhibit the inflammatory response in the hippocampi of Alzheimer's disease （AD） rats by regulating and activating the Wnt/β-catenin signaling pathway to improve the cognitive and memory dysfunction. MethodAmong the 90 male Wistar rats， 12 were randomly selected as the blank group （normal saline） and 12 as the sham operation group （normal saline）. For the remainder， amyloid β-protein42 （Aβ42） was injected in the left and right hippocampus to induce AD， and then the AD rats were randomized into model group， low-， medium-， and high-dose Hei Xiaoyaosan groups （corresponding doses of Hei Xiaoyaosan， ig）， and donepezil group （donepezil hydrochloride，ig）， with 12 in each group. The administration lasted 42 days. The pathological changes of hippocampal CA1 region was observed based on Nissl staining. The escape latency on the 1st to 5th day in Morris water maze was recorded and the spatial memory on the 6th day was tested. Enzyme-linked immunosorbent assay （ELISA） was employed to detect the expression of interleukin （IL）-6， IL-10， and tumor necrosis factor-α （TNF-α） in rat hippocampus and serum， Western blotting to examine the protein expression of glycogen synthase kinase-3β （GSK-3β）， β-catenin， and peroxisome proliferator-activated receptor gamma （PPARγ）， and real-time polymerase chain reaction （Real-time PCR） to determine the mRNA expression of rat GSK-3β， β-catenin， and PPARγ. ResultCompared with the blank group， the number of neurons in the hippocampal CA1 area of model group was significantly reduced， the arrangement was uneven， the cell body was damaged more obviously， and the Neisser body was unclear. The treatment group was significantly prolonged （P<0.01）， and the number of crossing stations was significantly reduced （P<0.01）， the levels of IL-10 in serum and hippocampus of rats in the model group were significantly decreased， while the levels of IL-6 and TNF-α were significantly increased （P<0.01）， the GSK-3β protein and mRNA in the model group were significantly increased， and the protein expressions of β-catenin and PPARγ were significantly decreased （P<0.01）， and the difference was more obvious. The number of neurons in the donepezil group was more distributed， neatly arranged， the structure was intact， and the Nissl bodies were clear and definite， the escape latency on the 3rd to 5th days in middle and high dose groups of Hei Xiaoyaosan and the donepezil group was significantly shortened （P<0.01）， the number of crossing platforms increased significantly （P<0.01）， the expression levels of IL-10 in the rat hippocampus and serum were significantly increased， while IL-6 and TNF-α were significantly decreased （P<0.01）， GSK-3β in the rat hippocampus was significantly increased. The expressions of GSK-3β protein and mRNA were significantly decreased， while the expression levels of β-catenin and PPARγ protein and mRNA were significantly increased （P<0.01）. There was no significant difference in each index between the donepezil hydrochloride group and the high-dose Hei Xiaoyaosan group. ConclusionHei Xiaoyaosan can inhibit the inflammatory response in the hippocampus of AD rats by regulating the Wnt/β-catenin signaling pathway， thereby alleviating the cognitive and memory impairment of AD rats.

Danzhi Xiaoyaosan-containing Serum Reverses Energy Metabolic Reprogramming of MDA-MB-231 Breast Cancer Cells / 中国实验方剂学杂志

ObjectiveTo observe the effect of Danzhi Xiaoyaosan-containing serum on MDA-MB-231 breast cancer cells， and to find out whether the action mechanism is related to its intervention in energy metabolism. MethodThirty six-week-old male SD rats were randomly divided into the blank group， Danzhi Xiaoyaosan （8.99 g·kg-1） group， and Xihuangwan （0.55 g·kg-1） group. The serum was isolated after drug intervention for seven days. The cell viability was detected by methyl thiazolyl-tetrazolium （MTT） assay， the cell cycle by flow cytometry， and the apoptosis by Annenxin V/propidium iodide （PI） double staining. Following the determination of intracellular glucose content using the glucose testing kit， the expression of glucose transporter 1 （GLUT1） was measured by immunofluorescence staining. Seahorse XFe cell energy metabolism analyzer was used to detect the extracellular acidification rate （ECAR） and oxygen consumption rate （OCR）. The expression levels of hexokinase 2 （HK2）， pyruvate kinase M2 （PKM2）， and lactate dehydrogenase A （LDHA） were assayed by Western blot. ResultCompared with the blank group， Danzhi Xiaoyaosan-containing serum inhibited the proliferation of MDA-MB-231 cells， with the best effect observed after intervention with 15% Danzhi Xiaoyaosan-containing serum for 48 h （P<0.01）， blocked the MDA-MB-231 cells in G0/G1 phase（P<0.01）， and down-regulated the GLUT1 expression， basal glycolysis， glycolysis capacity， glycolytic reserve， basal respiration， adenosine triphosphate （ATP） production， spare respiratory capacity（P<0.01）， as well as the protein expression of HK2， PKM2， and LDHA in MDA-MB-231 cells（P<0.05，P<0.01）. ConclusionDanzhi Xiaoyaosan-containing serum inhibits MDA-MB-231 cell proliferation， promotes apoptosis， and induces cell cycle arrest， which may be related to its reversal of energy metabolic reprogramming in MDA-MB-231 cells.

Ideas and Strategies of Hei Xiaoyaosan Regulating TMA/FMO3/TMAO Metabolic Pathway of Intestinal Flora in Prevention and Treatment of Alzheimer's Disease （AD） Based on "Simultaneous Regulation of Three Yin" of Liver， Spleen and Kidney / 中国实验方剂学杂志

Alzheimer's disease （AD） is a neurodegenerative disease. With the acceleration of aging process， the number of AD patients increases year by year. This threatens the health and even life of patients， and causes heavy economic burden and mental pressure to patients， families and society. In traditional Chinese medicine （TCM）， AD belongs to the category of dementia， and tonifying kidney is the main treatment. Based on the basic theory of TCM and combined with clinical manifestations of AD， AD is closely correlated with liver and spleen. Therefore， "simultaneous regulation of three Yin" of liver， spleen and kidney will be an important way for the prevention and treatment of AD. Hei Xiaoyaosan， a representative prescription of "simultaneous regulation of three Yin" of liver， spleen and kidney， has theoretical， experimental and clinical basis in preventing and treating AD. Modern studies have shown that neurofibrillary tangle formed by tau hyperphosphorylation is a main pathological feature of AD， and trimethylamine oxide （TMAO） is closely related to tau hyperphosphorylation. Therefore， regulating TMAO metabolism to inhibit tau hyperphosphorylation is a new target for the prevention and treatment of AD. On the basis of the above theory and previous studies， this paper put forward the hypothesis that Hei Xiaoyaosan regulates the trimethylamine（TMA）/heparin monooxygenase 3（FMO3）/TMAO metabolic pathway of intestinal flora through "simultaneous regulation of three Yin" of liver， spleen and kidney， and then inhibits tau hyperphosphorylation in brain hippocampus， thereby protecting nerve cells， improving learning and memory， and preventing AD. This paper explored the role and mechanism of Hei Xiaoyaosan in the prevention and treatment of AD from the perspective of inhibiting tau hyperphosphorylation by regulating the TMA/FMO3/TMAO metabolic pathway of intestinal flora， which provided new ideas and strategies for in-depth study of Hei Xiaoyaosan in the prevention and treatment of AD.

Antidepressant Effect of n-Butyl Alcohol Extracts from Xiaoyaosan via IGF-1Rβ/PI3K/Akt Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention effect of <italic>n</italic>-butyl alcohol extracts from Xiaoyaosan against depression-like behavior induced by chronic unpredictable mild stress （CUMS） in model mice and the role of insulin-like growth factor-1 receptor <italic>β</italic> （IGF-1R<italic>β</italic>）/phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt） signaling pathway in such intervention. Method:The effective dose of n-butyl alcohol extracts from Xiaoyaosan was preliminarily determined in model mice with behavioral despair. Then the male C57BL/6 mice were randomly divided into the blank group， model group， fluoxetine group， Xiaoyaosan group， and the low- （20 g·kg^-1） and high-dose （40 g·kg^-1） <italic>n</italic>-butyl alcohol extract groups. The mice in all groups except for the blank group were exposed to CUMS for inducing the depression-like behavior， which was judged by the sucrose preference test （SPT）. The successfully modeled mice in the medication groups were intragastrically administered with the corresponding drugs， whereas those in the blank and model groups were treated with an equal volume of solvent for five successive weeks. Following the SPT， tail suspension test （TST）， and novelty suppressed feeding test （NSFT） at the end of the fifth week， the insulin-like growth factor-1 （IGF-1） levels in mouse serum and hippocampus were measured by enzyme-linked immunosorbent assay （ELISA）. The average optical density （<italic>IA</italic>） of Nissl bodies in mouse hippocampal CA3 region was detected by toluidine blue staining. The 5-bromo-2-deoxyuridine （Brdu） and doublecortin （DCX） expression in the dentate gyrus （DG） was assayed using immunofluorescence method. The protein expression levels of IGF-1R<italic>β</italic>， PI3K， phosphorylated-PI3K （p-PI3K）， Akt， p-Akt， cysteine aspartic acid-specific protease 3 （Caspase-3）， and cleaved Caspase-3 in the hippocampus were determined by Western blot. Result:The results of forced swimming test and TST showed that n-butyl alcohol extracts from Xiaoyaosan at 9.1 and 40 g·kg^-1 both significantly shortened the immobility time of mice （<italic>P</italic><0.05， <italic>P</italic><0.01）， indicating that the effective dose ranged from 9.1-40 g·kg^-1. Compared with the model control， the n-butyl alcohol extracts from Xiaoyaosan at 20 and 40 g·kg^-1 significantly increased the sucrose preference percentage （<italic>P</italic><0.05， <italic>P</italic><0.01）， shortened the immobility time in TST （<italic>P</italic><0.01） and the feeding latency in NSFT （<italic>P</italic><0.01）， reversed the down-regulated IGF-1 content in mouse serum and hippocampus （<italic>P</italic><0.01）， increased the AOD of Nissl bodies in the hippocampal CA3 region （<italic>P</italic><0.01）， promoted the expression of Brdu and DCX in DG （<italic>P</italic><0.05， <italic>P</italic><0.01）， and down-regulated the protein expression levels of IGF-1R<italic>β</italic> （<italic>P</italic><0.05， <italic>P</italic><0.01）， p-PI3K/PI3K （<italic>P</italic><0.05， <italic>P</italic><0.01）， p-Akt/Akt （<italic>P</italic><0.05）， and cleaved Caspase-3/Caspase-3 in the hippocampus of CUMS mice. Conclusion:The n-butyl alcohol extracts from Xiaoyaosan are equivalent to Xiaoyaosan in inhibiting expression. They alleviate the depression-like behavior in CUMS mice， induce the production of Nissl bodies in hippocampal CA3 region， enhance neuronal proliferation and differentiation in DG， and facilitate neurogenesis. All these may be related to the inhibition of over-activated IGF-1R<italic>β</italic>/PI3K/Akt pathway and the reduction of neuronal apoptosis.

Xiaoyaosan Promotes Myelin Regeneration and Alleviates Depressive Symptoms in Vascular Dementia Mice with Depression via Promoting Microglia Polarization / 中国实验方剂学杂志

Objective:To investigate the effect of Xiaoyaosan on depressive behavioral phenotype in mice with vascular dementia （VaD） mice and its possible mechanism. Method:Sixty three-month-old male C57/BL6 mice were divided into the normal control group， model group， positive control group， as well as low-， medium-， and high-dose Xiaoyaosan groups. Mice in all groups except for the normal control group underwent bilateral carotid artery stenosis. Two weeks later， they were subjected to chronic restraint stress， 6 h per day， for inducing VaD complicated with depression. Mice in the low-， medium-， and high-dose Xiaoyaosan groups were treatment with intragastric administration of Xiaoyaosan decoction （5， 10， 20 g·kg^-1）， the ones in the positive control group with fluoxetine （10 mg·kg^-1）， and those in the normal control group and model group with an equal volume of normal saline for four weeks， during which the restraint stress was maintained. The depressive behavioral phenotype of mice was observed in sugar water preference test and tail suspension test. The fluorescence expression of myelin basic protein （MBP） in ventral hippocampus （vHIP） was detected by fluorescence immunoassay. The ultrastructure of myelin sheath in vHIP was observed by transmission electron microscopy. The protein expression levels of MBP， myelin oligodendrocyte glycoprotein （MOG）， myelin-associated glycoprotein （MAG）， triggering receptor expressed on myeloid cells-2 （TREM2）， inducible nitric oxide synthase （iNOS）， arginase 1 （Arg1）， interleukin-I<italic>β</italic> （IL-1<italic>β</italic>）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） were assayed by Western blot. Result:As revealed by behavioral test， compared with the normal control group， the model group exhibited prolonged immobility time and decreased percentage of sugar water preference （<italic>P</italic><0.01）. Compared with the model group， Xiaoyaosan significantly shortened the immobility time of mice （<italic>P</italic><0.05） and increased the percentage of sugar water preference （<italic>P</italic><0.01）. Western blot results showed that the protein expression levels of MBP， MOG， and MAG in vHIP of the model group were remarkably decreased as compared with those of the normal control group （<italic>P</italic><0.01）. The protein expression levels of MBP， MOG， and MAG in vHIP of the low-dose Xiaoyaosan group were increased in contrast to those in the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the protein expression of iNOS was decreased （<italic>P</italic><0.01）. The protein expression levels of MBP， MOG， MAG， TREM2， Arg1， IL-4， and IL-10 in the medium- and high-dose Xiaoyaosan groups were up-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas those of iNOS， IL-1<italic>β</italic>， and TNF-<italic>α</italic> were down-regulated （<italic>P</italic><0.01）. The immunofluorescence findings demonstrated that the mean fluorescence intensity of MBP in the model group declined in comparison with that in the normal control group （<italic>P</italic><0.01）， while the mean fluorescence intensities of MBP in the low-， medium-， and high-dose Xiaoyaosan groups were enhanced to different degrees （<italic>P</italic><0.01）. It was observed under the transmission electron microscope that the myelin structure of the model group was loosened and the dense layer was separated and irregularly arranged. Xiaoyaosan improved the structural integrity of myelin sheath and the looseness of lamellar structure. Conclusion:Xiaoyaosan ameliorates the depressive behavioral phenotype of VaD mice， which may be related to the up-regulation of TREM2， the induction of M2 polarization of microglia cells， the enhancement of their anti-inflammatory and phagocytic abilities， and the promotion of damaged myelin sheath regeneration.

Changes of Microglia in Hippocampus of Mice Induced by Maternal Separation with Restraint Stress and Regulatory Effect of Wenyang Jieyu Prescription / 中国实验方剂学杂志

Objective:To observe the activation of microglia in hippocampus of depressed and anxious mice induced by maternal separation with acute restraint stress and the expression of interleukin-1<italic>β</italic>（IL-1<italic>β</italic>），interleukin-6（IL-6），tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）， investigating the mechanism of Wenyang Jieyu prescription in treating anxiety and depression. Method:Eighty four male C57BL offspring were randomly divided into control group， acute restraint stress group and model group on postnatal day 0（PD0）. Maternal separation combined with acute restraint stress was used to prepare anxious and depressed model mice， dividing the model mice into model group， Wenyang， Jieyu， Wenyang Jieyu and fluoxetine group according to random number table method. During the period of PD21-PD90， the control， acute restraint stress and model mice were fed with normal diet， with the other groups fed with corresponding medicine mixed diet. The Wenyang， Jieyu and Wenyang Jieyu groups were given 5.85， 12.03 and 16.71 g·kg^-1·d^-1 respectively. The fluoxetine group was given 2.60 mg·kg^-1·d^-1. Open field， zero maze test and social interaction tests were used to evaluate the anxiety and depression of model mice. The expression of Iba-1 in hippocampal microglia was detected by immunohistochemistry（IHC）. The mRNA expression of IL-1<italic>β</italic>， IL-6， TNF-<italic>α</italic>， Iba-1 and glucocorticoid receptor（GR）were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with the control group， total movement distance and time spent in central zone in 5 min of the model mice significantly decreased（<italic>P</italic><0.01）， time spent in opened arm and total movement distance decreased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）， investigation time during testing and training increased significantly（<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly increased（<italic>P</italic><0.01）， the expression levels of GR mRNA significantly decreased（<italic>P</italic><0.01）. The result of IHC staining showed that microglia were over activated. Compared with the model group， total movement distance and time spent in central zone in 5 min of mice in the Wenyang Jieyu and fluoxetine group significantly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）.Time spent in opened arm significantly increased（<italic>P</italic><0.01）. Investigation time during testing and training significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of Iba-1 protein and mRNA，IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> mRNA significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA increased significantly（<italic>P</italic><0.05，<italic>P</italic><0.01）. IHC staining showed the microglia recovered. Time spent in opened arm of mice in the Wenyang group and Jieyu group significantly increased （<italic>P</italic><0.01）， time spent investigating during testing decreased significantly （<italic>P</italic><0.05）， the expression levels of Iba-1 protein and mRNA，IL-6 mRNA significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression of GR mRNA of mice in the Wenyang group significantly increased （<italic>P</italic><0.05）， the expression of TNF-<italic>α </italic>mRNA significantly decreased （<italic>P</italic><0.05）. Total movement distance of mice in the Jieyu group increased significantly （<italic>P</italic><0.01）， time spent investigating during training decreased significantly （<italic>P</italic><0.05），the expression level of IL-1<italic>β </italic>mRNA significantly decreased （<italic>P</italic><0.05）. IHC staining showed that microglia recovered partly in both groups. Conclusion:The comprehensive curative effect and pharmacological action of Wenyang Jieyu prescription were better than Wenyang prescription and Jieyu prescription. Wenyang Jieyu prescription can treat anxiety and depression in maternal separation and acute restraint stress mice， its possible mechanism may be related to the decreased activation of microglia， down-regulation of IL-1<italic>β</italic>，IL-6，TNF-<italic>α</italic> expression and up-regulation of GR expression.

Modified Xiaoyao Erxian Decoction in Treatment of Mood Disorders in Perimenopausal Syndrome / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of modified Xiaoyao Erxian decoction in the treatment of mood disorders among perimenopausal syndrome due to kidney deficiency and liver depression and its effects on monoamine neurotransmitters and brain-derived neurotrophic factor （BDNF）. Method:According to the random number table， 108 patients were divided into a control group （54 cases） and an observation group （54 cases）. Control group were treated with Shugan Jieyu capsule orally， two capsules per time， two times per day， while those in the observation group received the modified Xiaoyao Erxian decoction， one bag per day， for 12 consecutive weeks. Before and after treatment， the Hamilton Anxiety Scale （HAMA）， 17-item Hamilton Depression Rating Scale （HAMD-17）， modified Kupperman Index （KI）， Pittsburgh Sleep Quality Index （PSQI）， Menopause-specific Quality of Life （MENQOL） and kidney deficiency and liver depression syndrome scores were calculated. The levels of estradiol （E₂）， follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， BDNF， 5-hydroxytryptamine （5-HT）， norepinephrine （NE）， and dopamine （DA） were detected， followed by safety evaluation. Result:The HAMA， HAMD-17， KI， kidney deficiency and liver depression syndrome， PSQI， and MENQOL scores of the observation group were lower than those of the control group （<italic>P</italic><0.01）， whereas the 5-HT， E₂， DA， NE， and BDNF levels in the observation group were higher （<italic>P</italic><0.01）. The clinical efficacy of the observation group was superior to that in the control group （<italic>Z</italic>=2.184， <italic>P</italic><0.05）. No adverse reactions occurred after the oral administration of Chinese medicinal preparations. Conclusion:The modified Xiaoyao Erxian decoction effectively alleviates the mood disorders and other related symptoms of perimenopausal syndrome due to kidney deficiency and liver depression by regulating the monoamine neurotransmitters， BDNF， and E₂， without inducing obvious side effects.

Metabolic module identification and biological mechanism of Xiaoyaosan for treatment of depression / 药学学报

The antidepressant effect of Xiaoyaosan has been demonstrated. It is of value to explore the biological mechanism of Xiaoyaosan in the treatment of depression from the perspective of functional modules by using the method of functional module division of the metabolic network. The differential metabolites and related enzymes and proteins regulated by Xiaoyaosan were identified in the database. Pathway enrichment analysis and crosstalk pathway analysis of Xiaoyaosan regulated metabolites was carried out. A network of differentially regulated metabolites and their enzymes and proteins was constructed by using the STRING tool. The CNM decomposition algorithm was used to extract the functional modules of the network and enrichment analysis of functional modules was carried out. The results show that Xiaoyaosan regulates 97 differential metabolites, 234 related enzymes and 258 depression-related proteins. The pathways crosstalk analysis was divided into two sub-networks, one of which is related to the neural system and cell signal transduction, the other is related to the endocrine system and metabolic pathways. KEGG pathway enrichment analysis of the network and 9 functional modules extracted by the CNM algorithm shows that module 1 and module 3 belong to the pathways that can be enriched into more pathways with fewer proteins. The corresponding functions of these pathways include the endocrine system, amino acid metabolism, the nervous system and signal transduction. In this study, pathway crosstalk analysis and metabolic network module division strategies were used to explain the biological mechanism of Xiaoyaosan in the treatment of depression, providing ideas and methods for in-depth study of the pharmacological mechanism of this traditional Chinese medicine from the perspective of metabolic regulation.

Clinical Efficacy of Liuwei Dihuangwan Combined with Danzi Xiaoyaosan on H-type Hypertension with Syndrome of Yin Deficiency and Yang Hyperactivity / 中国实验方剂学杂志

Objective::To discuss the clinical efficacy of Liuwei Dihuangwan combined with Danzi Xiaoyaosan on H-type hypertension with syndrome of Yin deficiency and Yang hyperactivity and its effect on vascular endothelial function and inflammatory factors. Method::One hundred and fifty patients were randomly divided into control group (75 cases) and observation group (75 cases) by random number table.Patients in control group got enalapril maleate and folic acid tablets, 1 tablet/time, 1 time/day, and patients with uncontrollable blood pressure were also given nifedipine sustained-release tablets, 20-30 mg/time, 2 times/days.In addition of the therapy of control group, patients in observation group were also given modified Liuwei Dihuangwan combined with Danzi Xiaoyaosan, 1 dose/day.The course of treatment was 12 weeks.Before and after treatment, systolic blood pressure (SBP), diastolic blood pressure (DBP) and up-to-standard blood pressure were detected.And ambulatory blood pressure, standard deviation of 24-hours systolic blood pressure (24 h SSD), standard deviation of 24-hour diastolic blood pressure 24 h DSD), 24 h mean systolic blood pressure (24 h SBP), 24 h mean diastolic blood pressure (24 h DBP) were recorded.And ankle brachial index (ABI) and brachial artery blood flow mediated diamete (FMD) were discussed, syndrome of Yin deficiency and Yang hyperactivity was scored, and levels of iterleukin-6 (IL-6), IL-10, high-sensitivity C-reactive protein (hsCRP) and homocysteine (Hcy) were detected. Result::The levels of SBP, DBP, 24 h SSD, 24 h DSD, 24 h SBP and 24 h DBP were all lower than those in control group (P<0.01). The achievement rate of accidental blood pressure in observation group was 81.19%, which was higher than 66.98% in control group (χ2=29.81, P<0.01). And levels of ABI, FMD and IL-10 were higher than those in control group (P<0.01), while the score of syndrome of Yin Deficiency and Yang Hyperactivity and the levels of Hcy, leptin, IL-6 and hs-CRP were lower than those in control group (P<0.01). Conclusion::In addition to the therapy of antihypertensive and folic acid, Liuwei Dihuangwan combined with Danzi Xiaoyaosan can be given to control the level of blood pressure and Hcy, relieve the variability of blood pressure, alleviate clinical symptoms, raise the rate of achievement rate of blood pressure, improve the function of vascular endothelium, and regulate inflammatory factors.

Mechanism of Xiaoyaosan in Improving Abnormal Lipid Metabolism and Steatohepatitis of Ovariectomized Rats / 中国实验方剂学杂志

Objective::To investigate the effect of Xiaoyaosan (XYS) on hepatic lipid metabolism and steatohepatitis in ovariectomized (OVX) female SD rats and its mechanism. Method::Forty female SD rats were randomly divided into sham surgery group, OVX group, low-dose XYS group (3 g·kg-1), and high-dose XYS group (9 g·kg-1). Bilateral ovaries of rats were excised to replicate the obesity model of ovariectomized rat. After 6 weeks of intragastric administration, the change rate of body mass in each group, the levels of blood lipids and liver function of rats were detected. Hematoxylin-eosin (HE) staining and oil red staining were used to observe the hepatocyte histomorphology and the intrahepatic fatty deposits. The expressions of hepatic proinflammatory cytokines and estrogen receptor beta (ERβ) were determined by quantitative real time polymerase chain reaction (Real-time PCR). Result::Compared with sham surgery group, the change rate of body mass of OVX group was significantly increased (2-6 weeks) with the changes in the course of drug administration and the levels of serum total cholesterol (TC), alanine aminotransferase (ALT) (P<0.05), aspartate amino transferase (AST) (P<0.01), low-density lipoprotein (LDL) (P<0.05) were markedly increased too (P<0.05, P<0.01). By histological method, in OVX group, the structure of hepatic cord became disordered, and there were new lipid droplets in hepatocyte cytoplasm, transcription levels of hepatic interleukin-1β (IL-1β) and interleukin-6 (IL-6) in OVX group were significantly increased (P<0.05). Compared with OVX group, the growth rate of body weight in low-dose and high-dose XYS group showed significant decreases with the increase of the cycle of drug administration (3-6 weeks). XYS significantly reduced levels of serum TC, ALT, AST, and LDL levels of OVX rats (P<0.05) in a dose-dependent manner, while serum triglyceride (TG), alkaline phosphatase (AKP) and high-density lipoprotein (HDL) levels in the four groups showed no statistical significance, XYS can improve hepatocyte structure and steatosis of OVX rats, XYS could reduce the transcription hepatic levels of IL-6 and IL-1β of OVX rats in a dose-dependent manner (P<0.05, P<0.01), but there was no significant difference in the transcription level of tumor necrosis factor-α (TNF-α) among groups, both low and high-dose XYS can increase the transcription hepatic level of ERβ in OVX group (P<0.05, P<0.01). Conclusion::XYS can improve the growth rate of body mass, the hepatic lipid metabolism abnormalities and steatohepatitis of OVX rats. The mechanism may be related to the elevated expression of hepatic ERβ by XYS, so as to inhibit the hepatic pro-inflammatory factors expressions.

Research Progress on Antidepressant Pathway and Active Ingredients of Xiaoyaosan and Its Analogous Prescriptions / 中国实验方剂学杂志

Depression is a psychiatric disease that seriously affects human life in the context of rapid social development and increased living pressure, and brings serious healthproblems and economic burdens to human society.Traditional Chinese medicine has a long history in the treatment of depression, and Xiaoyaosan has been repeatedly confirmed as a classic prescription for the intervention in depression, but its antidepressant mechanism and its active ingredients are still unclear.Through the search of China Knowledge Network(CNKI) and PubMed database, the literature and data on the clinical and mechanism research of Xiaoyaosan and its analogous prescriptions on depression in the past 10 years are systematically sorted out.It is found that Xiaoyao San and its analogous prescriptions are mainly focusing on the following aspects: regulating hypothalamic-pituitary-gonad, hypothalamic-pituitary-thyroid, and hypothalamic-pituitary-adrenal axis, affecting 5-hydroxytryptamine(5-HT), norepinephrine(NE) neurotransmitters in hippocampus, interfering with inflammatory reaction process, modulating the BDNF/CREB signaling pathway, regulating intestinal flora and gastrointestinal function, regulating endothelial window foramen of hepatic sinus, repairing chronic stress-induced blood-brain barrier permeability, affecting autophagy to reduce neuronal apoptosis, and regulating the leukoplakia-nuclear adrenal gland (LC-NE) system to play an antidepressant role.The main active ingredient including, saikosaponin A which could down-regulate interleukin expression level, saikosaponin D which alleviates HPA axis dysfunction, quercetin which inhibits corticotropin-releasing-factor(CRF) mRNA expression, paeoniflorin which regulates neurotransmitters, and paeoni lactone which inhibits hyperfunction of HPA axis Glycosides, reduce inflammatory cytokines, atractylenolide I, atractylenolide Il, atractylenolide Ill, which could reduce the expression of pro-inflammatory cytokine mRNA and inhibit NF-κB activated, curcumin which could play the neuroprotective and anti-inflammatory and antioxidant roles, L-menthol which could suppress the HPA axis overactivation, liquiritin isoliquiritin which could regulate the monoamine neurotransmitters, and isoliquiritigenin which could inhibit the monoamine oxidase.

Effect of Hei Xiaoyaosan on Expression of CaMKⅡα Protein and Its Phosphorylation in Hippocampus and Cerebral Cortex of APP/PS1 Double Transgenic Mice / 中国实验方剂学杂志

Objective::To study the effect of Hei Xiaoyaosan on the expression of calcium calmodulin-dependent protein kinase Ⅱ alpha(CaMKⅡα) and its phosphorylation in hippocampus and cortex of mice with Alzheimer's disease. Method::After weighing, 30 APP/PSI transgenic male mice were divided into model group, donepezil hydrochloride group and Hei Xiaoyaosan group according to random principle and 10 in each group.At the same age, wild-type C57BL/6 10 mice of the same species were treated as blank group. Donepezil hydrochloride group (6 g·kg-1) and Hei Xiaoyaosan group (3.25 mg·kg-1) were administered for 90 days, then the behavior of all the mice were detected by Morris water maze, the expression of CaMKⅡα, p-CaMKⅡα proteins in hippocampus and cortex by immunohistochemical technique and Western blot. Result::After intervention 3 months, compared with blank group, the average escaping latency periods prolonged significantly and the number of cross-platform and effective areas were decreased distinctly in model group mice(P<0.01), CaMKⅡα protein relative expression decreased significantly(P<0.01), p-CaMKⅡα protein relative expression increased significantly(P<0.01). Compared with the model group, the escape latency of donepezil hydrochloride and Hei Xiaoyaosan group were significantly shortened, and the number of crossing platforms and effective areas was significantly increased (P<0.05, P<0.01), the expression of CaMKⅡα protein in the hippocampus and cortex of drug groups was significantly increased (P<0.01), p-CaMKⅡα protein in the hippocampus and cortex of drug groups was significantly decreased (P<0.05, P<0.01). Conclusion::Hei Xiaoyaosan can improve the learning and memory ability of AD mice by regulating the expression of CaMKⅡα and its phosphorylation, which are key proteins involved in the mechanism of cell memory formation in different brain regions of AD mice.

Effect of Xiaoyaosan on Changes of JAK/STAT Pathway in Hypothalamic Arcuate Nuclei of Rats With Chronic Mild Unpredictable Stress / 中国实验方剂学杂志

Objective:To observe the changes of leptin receptor-tyrosine kinase Janus2 （JAK2）/signal transducer and activator of transcription 3 （STAT3） pathway and the regulatory effect of Xiaoyaosan on the hypothalamic arcuate nuclei of rats with chronic mild unpredictable stress model （CUMS）. Method:Sixty male sprague-dawley rats were randomly divided into normal group， model group， Xiaoyaosan group， and fluoxetine group. After one-week adaptive feeding， the rats in model group， Xiaoyaosan group and fluoxetine group were uesd to replicate the chronic psychological stress rat model through mild unpredictable stimulation. Meanwhile ， they were simultaneously administered the corresponding drugs， Xiaoyaosan 19.27 g·kg-1·d-1， Fluoxetine 2 mg·kg-1·d-1 （based on the average adult body weight of 60 kg）， the rats in the normal group and the model group were given the same volume of normal saline for 6 weeks. The body weight， food intake， sucrose consumption ratios， and the experimental behavior in the open field test （OFT） of the groups were observed. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the expressions of ob-R， JAK2， and STAT3 in the arcuate nucleus of rat hypothalamus. Result:Compared with the normal group， the body weight and food intake of the model group were significantly decreased （P<0.05， P<0.01）， the sucrose consumption ratios ， the total behavioral distance of the experimental field and the total distance of the central area were significantly reduced， the protein and mRNA expressions of ob-R， JAK2， STAT3 in the arcuate nucleus of hypothalamus in rats increased significantly （P<0.05， P<0.01）. Compared with the model group， the body weight of Xiaoyaosan group increased significantly on the 7th， 14th， 21st and 28th days （P<0.05， P<0.01）， the food intake of rats increased significantly on the 21st and 35th days of the experiment （P<0.05）， and the sucrose consumption ratios， the total distance of the experimental behavior in the open field test （OFT） and the total distance of the central area were significantly improved. Xiaoyaosan had a corresponding regulatory effect on the protein and mRNA expressions of ob-R， JAK2， STAT3 in the arcuate nucleus of hypothalamus in model rats （P<0.05， P<0.01）. Conclusion:Xiaoyaosan regulates the body weight， appetite， and energy metabolism of chronically mild and unpredictable stress rats， which may be related to the ob-R-JAK2/STAT3 pathway in the hypothalamic arcuate nucleus.

Effect of Addition and Subtraction Therapy of Xiaoyaosan Combined with Simotang to Gut-brain Axis of Patients with Irritable Bowel Syndrome with Predominant Constipation and Syndrome of Stagnation of Liver Qi / 中国实验方剂学杂志

Objective:To observe clinical effect of addition and subtraction therapy of Xiaoyaosan combined with Simotang to irritable bowel syndrome with predominant constipation （IBS-C） and syndrome of stagnation of liver Qi and to study influence to gut-brain axis. Method:One hundred and forty-four patients were randomly divided into control group （72 cases） and observation group （72 cases） by random number table. The 66 patients in control group completed the therapy （2 patients were falling off or missing visit， 5 patients were eliminate）， 65 patients in observation group completed the therapy （4 patients were falling off or missing visit， 2 patients were eliminate）. Patients in control group got Simotang， 20 mL/time， 3 times/day. In control group， patients in observation group added addition and subtraction therapy of Xiaoyaosan combined with Simotang， 1 dose/day. And courses of treatment in two groups were 4 weeks. Before and after treatment， degree of abdominal pain， irritable bowel syndrome （IBS） symptom severity scale （IBS-SSS）， quality of life in irritable bowel syndrome （IBS-QOL）， syndrome of stagnation of liver Qi， scores of Hamilton anxiety scale-14（HAMA-14）， Hamilton depression scale-17（HAMD-17）， complete spontaneous bowel movement （CSBM）， response rate of abdominal pain and defecation， remission rate of IBS-SSS were all recorded. And levels of vasoactive intestinal peptide （VIP）， substance P （SP）， neuropeptide Y （NPY）， 5-hydroxytryptamine （5-HT）， somatostatin （SS） and calcitonin related gene peptide were detected， and safety was evaluated. Result:Degree of abdominal pain， scores of IBS-SSS， syndrome of stagnation of liver Qi ， HAMA-14， HAMD-17 and levels of VIP， NPY， 5-HT， SS and CGRP in observation group were all lower than those in control group （P<0.01）. And times of CSBM， score of IBS-QOL and level of SP were all higher than those in control group （P<0.01）. Besides， response rate of abdominal pain and defecation and remission rate of IBS-SSS in observation group 95.38%（62/65）， 93.85%（61/65） and 90.77%（59/65）were higher than 83.33%（55/66），78.79%（52/66）and 75.76%（50/66） in control group （P<0.05）. And curative effect of traditional Chinese medicine （TCM） syndromes in was better than that in control group （Z=2.1034， P<0.05）. No serious adverse events happened and no adverse reaction caused by TCM. Conclusion:Addition and subtraction therapy of Xiaoyaosan combined with Simotang can significantly ameliorate IBS-C symptoms， reduce bad mood， improve patients' quality of life， regulate a variety of brain gut peptide factors， and improve brain gut axis disorder. It has good clinical efficacy and safety.

Feasibility of Hei Xiaoyaosan in Preventing and Treating Alzheimer's Disease Based on Theory of 'Correlation of Liver and Kidney' Based on Ca2+-CaM/CaMKⅡ-CREB Signal Pathway / 中国实验方剂学杂志

Alzheimer's disease（AD） is an incipient aging neurodegenerative disease， which increases rapidly along with the development trend of social aging and seriously threatens the health of the people. In the absence of effective preventive measures， it will have an enormous impact on the socio-economic and healthcare system. The study found that abnormal cell signal transduction is a key link in many diseases. Cell signal transduction theory has been widely used to clarify the essence of traditional Chinese medicine visceral image and the mechanism of traditional Chinese medicine. 'Correlation of Liver and Kidney' is one of the core plates of the theory of 'Correlation of Five Organs'， which is suitable for explaining the pathogenesis of complex diseases and the correlation of multiple syndromes， and guiding the prescription of clinical syndrome. Hei Xiaoyaosan， as the first choice compound for the prevention and treatment of AD based on the theory of "Correlation of Liver and Kidney' in our team， can play the effects of prevention and treatment by soothing liver and nourishing blood， strengthening spleen and tonifying kidney， and promoting brain collaterals and dredging viscerab spirit. Based on the theory of 'Correlation of Liver and Kidney'， this paper expounds the pathogenesis of AD from the perspective of traditional Chinese medicine， and puts forward the methods and ideas of the preventing and treating of AD from Ca2+-calcium/calmodulin dependent protein （CaM）/calcium/calmodulin dependent protein kinaseⅡ（CaMKⅡ）-cyclic adenosine phosphate reactive element binding protein （CREB） cell signal transduction pathway by consulting literatures and previous studies.

Mechanism of Xiaoyaosan in Preventing and Treating Hepatic Injury Caused by Tripterygium Glycosides / 中国实验方剂学杂志

Objective:To observe the preventive and therapeutic effects of 5-week administration with Xiaoyaosan on rat liver injury caused by tripterygium Glycosides. Method:Thirty-one SD rats were randomly divided into 4 groups， namely normal group， tripterygium glycosides group， tripterygium glycosides+Xiaoyaosan group （treatment group）， and tripterygium glycosides+Xiaoyaosan for 1 week in advance group （prevention group）. Tripterygium glycosides （37.5 mg·kg-1） was administered intragastrically， and Xiaoyaosan （water decoction， 19.270 g·kg-1） was administered intrastrically. First， the rats of prevention group were intragastrically administrated with Xiaoyaosan at 8：00-9：00 am， and the rats of other groups were given an equal volume of normal saline. After 1 week， the rats of tripterygium glycosides group were administered intragastrically with tripterygium glycosides suspension at 8：00-9：00 am. The rats of the treatment group and the prevention group were intragastrically administrated with Xiaoyaosan at 8：00-9：00 am， and then tripterygium glycosides suspension 2 hours later. All the drugs were given once a day for 5 weeks. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of liver， enzyme-linked immunosorbent assay （ELISA） was used to detect serum interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）， chemical method was used to detect the content changes of aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， glutathione peroxidase （GSH-Px）， superoxide dismu-tase （SOD）， trace malondiamine aldehyde （MDA）. Immunohist-ochemical staining was used to observe the expressions of IL-6， IL-1β and TNF-α in liver tissue. Result:Compared with the normal group， the tripterygium glycosides group showed inflammatory cell infiltration， hepatocyte edema， hepatic sinuses squeezing and narrowing， liver plate widening， and liver cell necrosis， the contents of serum IL-1β， IL-6， TNF-α， ALT， AST and MDA were significantly increased（P<0.01）， but the contents of GSH-Px and SOD were decreased significantly （P<0.05， P<0.01）， while the positive expressions of IL-6， TNF-α， IL-1β in liver tissue were significantly increased （P<0.01）. Compared with tripterygium glycosides group， rats in the treatment group and the prevention group had less inflammatory cells infiltration and reduced edema in the liver tissue， and disorders in some cell， the contents of serum IL-1β， IL-6， TNF-α， ALT， AST， MDA were significantly decreased （P<0.01）， the contents of GSH-Px and SOD were significantly increased （P<0.05）， and the positive expressions of IL-6， TNF-α， IL-1β in liver tissues were significantly decreased （P<0.01）， with a better efficacy in the prevention group. Conclusion:Xiaoyaosan can obviously alleviate the long-term liver toxicity caused by tripterygium polyglycoside to a certain extent， with a better prophylactic effect.