Effects of Erxian Decoction （二仙汤） on Choroid Plexus Folate Transport-Hippocampal Neurogenesis in Rat Brain Tissue of Late-Onset Depression Model / 中医杂志

ObjectiveThis study aims to investigate the effectiveness and potential mechanism of Erxian Decoction （二仙汤） for late-onset depression （LOD）. MethodsForty Wistar male rats of 7-8 weeks were divided into 20 each of normal group and youth depression group. Sixty Wistar male rats of 20 months were divided into 20 each of elder group， LOD group and Erxian Decoction group. The rats in youth depression group were modelled with chronic unpredictable mild stress （CUMS） for 6 weeks to build a depression model， and the elder rats in LOD group and Erxian Decoction group were modelled with CUMS for 6 weeks to build a LOD model， with no interventions in the normal group and the elder group. Gastric administration was carried out at the same time of modelling， rats in Erxian Decoction group were given 8 g/（kg·d） of Erxian Decoction by gavage， and rats in the normal group， youth depression group， elder group， and LOD group were given 4 ml/（kg·d） of pure water by gavage， for 6 weeks in each group. Sugar water preference experiment and forced swimming experiment were used to evaluate the depression-like behaviour of rats， absent field experiment was used to evaluate the spontaneous activity ability of rats， and T-maze experiment was used to evaluate the cognitive function of rats； Western blot assay was used to detect neuronal nuclei （NeuN）， nestin， doublecortin （DCX） in hippocampal tissue， and zonula occludens-l （ZO-1）， folate receptor α （FRα）， reduced folate carrier （RFC）， and protoncoupledfolate transporter （PCFT） protein expression in choroid plexus； immunofluorescence was used to detect the number of double-positive cells for Ki-67/nestin， the number of double-positive cells for 5-bromodeoxyuracil nucleoside （BrdU）/DCX in hippocampal dentate gyrus， and the protein expression of ZO-1， FRα， RFC， and PCFT in choroid plexus tissues； ELISA technique was used to detect plasma， cerebrospinal fluid， 5-methyltetrahydrofolate （5-MTHF） in hippocampal tissues； Pearson correlation analysis was used to correlate the 5-MTHF content in cerebrospinal fluid and hippocampal tissues and the expression of nestin， DCX， and NeuN proteins in hippocampal tissues of rats in youth depression group and LOD group. ResultsCompared with normal group， rats in youth depression group and LOD group showed reduced sugar water preference， reduced total distance travelled in the absent field， reduced number of times through the grid， prolonged forced swimming immobilisation， reduced hippocampal NeuN， nestin and DCX protein expression， reduced number of Ki-67/nestin double-positive cells in hippocampal dentate gyrus， reduced number of BrdU/DCX double-positive cells in hippocampal dentate gyrus， and reduced expression of ZO-1 and FRα proteins and fluorescence intensity （P＜0.05 or P＜0.01）； the correct rate of T-maze on days 3 and 4 in the rats of youth depression group and on days 2 to 4 in the rats of LOD group significantly decreased， and the content of 5-MTHF in the cerebrospinal fluid and hippocampal tissues of the rats of LOD group significantly decreased as well （P＜0.05 or P＜0.01）. Compared with youth depression group， rats in LOD group showed a decrease in total distance travelled in absenteeism， number of times through the grid， a significant decrease in the correct rate of the T-maze on day 1-4， a decrease in NeuN， nestin， DCX protein expression of hippocampal tissue and the number of Ki-67/nestin double-positive cells， BrdU/DCX double-positive cells of hippocampal dentate gyrus （P＜0.05 or P＜0.01）. Compared with LOD group， rats in Erxian Decoction group had elevated sugar-water preference and total distance travelled in absenteeism and number of times through the grid， shorter forced swimming immobility time， significantly higher correct rate of the T-maze on day 3-4， elevated expression of NeuN， nestin， and DCX proteins in hippocampal tissues， increased number of Ki-67/nestin double-positive cells and BrdU/DCX double-positive cells in hippocampal dentate gyrus， and increased 5-MTHF content in cerebrospinal fluid and hippocampal， and ZO-1， FRα， RFC， PCFT protein expression and fluorescence intensity increased in choroid plexus （P＜0.05 or P＜0.01）. Correlation analysis showed that there was a positive correlation between 5-MTHF content in cerebrospinal fluid （r = 0.466）， hippocampal tissue （r = 0.522） and nestin expression in hippocampal tissue （P＜0.05）. ConclusionErxian Decoction could improve depressive-like behaviour and cognitive impairment in LOD model rats， and its mechanism may promote hippocampal neurogenesis by alleviating structural damage to the choroid plexus of the brain tissue， and improving impaired choroid plexus folate transport.

Effect of Erxian Decoction-containing serum on H_2O_2-induced proliferation and osteogenic differentiation of MC3T3-E1 cells via BK channels / 中国中药杂志

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.

Molecular Mechanism of Erxian Decoction in Treatment of Anxiety Disorder Based on Network Pharmacology and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo predict the potential molecular mechanism of Erxian decoction in the treatment of anxiety disorder based on network pharmacology， and to verify the efficacy and mechanism using the animal model of maternal separation combined with restraint stress. MethodActive components and related targets of Erxian decoction were obtained by traditional Chinese medicine system pharmacology database and analysis platform （TCMSP） and SwissTargetPrediction. The targets related to anxiety disorder were screened out through GeneCards， therapeutic target database （TTD）， online mendelian inheritance in man database （OMIM）， and DrugBank， and the drug-disease intersection targets were obtained by taking intersections with the drug targets. The protein-protein interaction （PPI） network was constructed by the STRING database， and the core targets were screened out based on topological parameter analysis. Gene Ontology （GO） enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were carried out for the intersection targets through the Metascape platform. Maternal separation combined with restraint stress was used to induce the mouse model of anxiety disorder. From the end of lactation on the 21st postnatal day （PD21） to the completion of restraint stress on the 97th postnatal day （PD97）， the mice were fed with Erxian decoction mixed with diet. The anxiety state of mice was evaluated by open field test and elevated O-maze test. The content of plasma corticosterone （CORT） in mice was detected by enzyme-linked immunosorbent assay （ELISA）. The expression levels of protein kinase B （Akt1）， mammalian target of rapamycin （mTOR）， brain-derived neurotrophic factor （BDNF）， postsynaptic density-95 （PSD95）， and synaptophysin in the hippocampus of mice were detected by Western blot and real-time quantitative polymerase chain reaction （Real-time PCR）. ResultNinty-seven active components and 227 action targets of Erxian decoction were obtained. There were 3 863 targets related to anxiety disorder， with 161 drug-disease intersection targets. Among these intersection targets， core targets such as Akt1， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor （TNF）， and mTOR were presumedly closely related to anxiety disorder. The results of KEGG pathway analysis showed that Erxian decoction mainly treated anxiety disorder through phosphatidylinositol 3-kinase （PI3K）/Akt， mitogen-activated protein kinase （MAPK）， and neuroactive ligand-receptor interaction signaling pathways. The results of animal experiments showed that compared with the model group， the Erxian decoction group significantly increased the time of mice spent in the central zone and central crossing times and time spent in the opened arm and opened arm crossing times， with significantly increased expression levels of p-Akt1， p-mTOR， BDNF， PSD95， and synaptophysin （Syp）. ConclusionErxian decoction has the multi-target and multi-pathway characteristics in the treatment of anxiety disorder， and its mechanism may be related to the improvement of synaptic plasticity and neuroinflammation by affecting Akt1， IL-1β， IL-6， TNF， mTOR， and other core targets and modulating PI3K/Akt， MAPK， as well as neuroactive ligand-receptor interaction signal pathways.

Molecular Mechanism of Erxian Decoction in Treatment of Anxiety Disorder Based on Network Pharmacology and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo predict the potential molecular mechanism of Erxian decoction in the treatment of anxiety disorder based on network pharmacology， and to verify the efficacy and mechanism using the animal model of maternal separation combined with restraint stress. MethodActive components and related targets of Erxian decoction were obtained by traditional Chinese medicine system pharmacology database and analysis platform （TCMSP） and SwissTargetPrediction. The targets related to anxiety disorder were screened out through GeneCards， therapeutic target database （TTD）， online mendelian inheritance in man database （OMIM）， and DrugBank， and the drug-disease intersection targets were obtained by taking intersections with the drug targets. The protein-protein interaction （PPI） network was constructed by the STRING database， and the core targets were screened out based on topological parameter analysis. Gene Ontology （GO） enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were carried out for the intersection targets through the Metascape platform. Maternal separation combined with restraint stress was used to induce the mouse model of anxiety disorder. From the end of lactation on the 21st postnatal day （PD21） to the completion of restraint stress on the 97th postnatal day （PD97）， the mice were fed with Erxian decoction mixed with diet. The anxiety state of mice was evaluated by open field test and elevated O-maze test. The content of plasma corticosterone （CORT） in mice was detected by enzyme-linked immunosorbent assay （ELISA）. The expression levels of protein kinase B （Akt1）， mammalian target of rapamycin （mTOR）， brain-derived neurotrophic factor （BDNF）， postsynaptic density-95 （PSD95）， and synaptophysin in the hippocampus of mice were detected by Western blot and real-time quantitative polymerase chain reaction （Real-time PCR）. ResultNinty-seven active components and 227 action targets of Erxian decoction were obtained. There were 3 863 targets related to anxiety disorder， with 161 drug-disease intersection targets. Among these intersection targets， core targets such as Akt1， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor （TNF）， and mTOR were presumedly closely related to anxiety disorder. The results of KEGG pathway analysis showed that Erxian decoction mainly treated anxiety disorder through phosphatidylinositol 3-kinase （PI3K）/Akt， mitogen-activated protein kinase （MAPK）， and neuroactive ligand-receptor interaction signaling pathways. The results of animal experiments showed that compared with the model group， the Erxian decoction group significantly increased the time of mice spent in the central zone and central crossing times and time spent in the opened arm and opened arm crossing times， with significantly increased expression levels of p-Akt1， p-mTOR， BDNF， PSD95， and synaptophysin （Syp）. ConclusionErxian decoction has the multi-target and multi-pathway characteristics in the treatment of anxiety disorder， and its mechanism may be related to the improvement of synaptic plasticity and neuroinflammation by affecting Akt1， IL-1β， IL-6， TNF， mTOR， and other core targets and modulating PI3K/Akt， MAPK， as well as neuroactive ligand-receptor interaction signal pathways.

Changes in Intestinal Microflora and Lipid Metabolism in Ovariectomized Rats and Regulatory Effect of Erxian Decoction / 中国实验方剂学杂志

ObjectiveTo explore the intervention effect of Erxian decoction on intestinal microflora after ovariectomy in rats by 16S rRNA gene sequencing. MethodThirty-two female healthy SD rats were randomly divided into a Sham operation （Sham） group， a model （OVX） group， an estrogen （E） group， and an Erxian decoction （EXD） group， with 8 rats in each group. The rats in the E group and the EXD group received 1.8×10-4 g·kg-1 estradiol valerate solution and 9 g·kg-1 Erxian decoction， respectively， and those in the Sham group and the OVX group received an equal volume of distilled water once a day for 16 weeks. After 16 weeks， the levels of serum estrogen and blood lipid were detected. The fecal DNA was extracted， followed by 16S rRNA gene sequencing and analysis. ResultCompared with the Sham group， the OVX group showed reduced serum estrogen level （P<0.01） and increased serum levels of total cholesterol （TC） and low-density lipoprotein cholesterol （LDL-C） （P<0.05）. Compared with the OVX group， the E group and the EXD group showed increased serum estrogen level （P<0.01） and reduced TC and LDL-C （P<0.05）. Alpha diversity showed that there was no significant change in intestinal microflora diversity after ovariectomy. Beta diversity showed that there were significant differences in the structure of intestinal microflora in the four groups. The intervention of Erxian decoction could improve the changes in intestinal microflora after ovariectomy. LEfSe was used to analyze the differential flora in the four groups. The results showed that the Sham group and the OVX group had 3 differential bacterial phyla and 18 differential bacterial genera， the OVX group and the E group had 1 differential bacterial phylum and 12 differential bacterial genera， and the OVX group and the EXD group had 3 differential bacterial phyla and 5 differential bacterial genera. Estrogen intervention could reverse the change trend of Ruminococcus 1， Anaerovibrio， and Turicibacter in the OVX group. Erxian decoction intervention could reverse the change trend of Bacteroidetes， Firmicutes， Prevotella 9， Ruminococcaceae UCG-014， Ruminococcus 1， and Fusicatenibacter in the OVX group. ConclusionThe structure and function of intestinal microflora in ovariectomized rats changed obviously， and Erxian decoction could ameliorate the change.

Feasibility of Erxian Decoction and Wenshen Prescription in Treatment of Depression Based on Network Pharmacology and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo predict the molecular mechanism of Erxian decoction and Wenshen prescription （modified Erxian decoction） in the treatment of depression based on network pharmacology and explore the feasibility of Wenshen prescription in the treatment of depression by comparing the efficacy and mechanism of the two decoctions based on a depression model induced by maternal separation combined with chronic restraint stress. MethodActive components and targets of Erxian decoction and Wenshen prescription were collected through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine （BATMAN-TCM）. Targets related to depression were screened out from databases such as GeneCards， Online Mendelian Inheritance in Man database （OMIM）， and DrugBank. Common targets of drugs and disease were obtained and imported to Cytoscape 3.8.2 to plot the drug-active component-target-disease network. STRING platform was used to construct a protein-protein interaction （PPI） network and core targets and related core components were screened out. Gene Ontology （GO） enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） functional enrichment analysis were performed on common targets through Metascape platform. The depression model was induced in mice by maternal separation combined with chronic restraint stress. From the 21st day of maternal separation （PD21） to the 111th day of restraint stress completion （PD111）， mice were fed with the diet mixed with Erxian decoction or Wenshen prescription for intervention. The depressive state of mice was evaluated according to the sucrose preference test， tail suspension test， open field test， and elevated O-maze test. The expression of ionized calcium-binding adapter molecule 1 （Iba1） in the microglia was observed by immunohistochemistry （IHC）. Western blot and Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） were used to detect the expression levels of protein kinase B1（Akt1）， brain-derived neurotrophic factor （BDNF）， postsynaptic density-95 （PSD95）， and synaptophysin （Syn）. ResultA total of 126 and 118 targets of Erxian decoction and Wenshen prescription in the treatment of depression were screened out， with only eight more targets of Erxian decoction than Wenshen prescription. The two decoctions shared the same core targets， mainly including Akt1， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α）. KEGG pathway enrichment analysis predicted that Erxian decoction and Wenshen prescription mainly treated depression through the phosphatidylinositol-3 kinase （PI3K）/Akt signaling pathway， mitogen-activated protein kinase （MAPK） signaling pathway， and neuroactive ligand-receptor interaction pathway. Animal experiments showed that compared with the results in the model group， Erxian decoction and Wenshen prescription could up-regulate the sucrose preference index， prolong the time spent in the central zone， increase the number of crossings， prolong the time spent in opened arm， increase the number of crossings in the opened arm， elevate the expression levels of p-Akt1， BDNF， PSD95， and Syn （P<0.05， P<0.01）， shorten the immobility time of tail suspension， and reduce the expression level of Iba-1 in the hippocampal microglia （P<0.05， P<0.01）. No significant difference between the two decoctions was found. ConclusionUnder the pathogenesis and syndrome law of depression dominated by kidney yang deficiency， Wenshen prescription modified from Erxian decoction is feasible in the treatment of depression. The mechanism may be attributed to the fact that both decoctions can improve neuroinflammation and synaptic plasticity in the hippocampus by affecting Akt1， IL-1β， IL-6， TNF-α， and other core targets and regulating the PI3K/Akt， MAPK， and neuroactive ligand-receptor interaction signaling pathways.

Clinical Observation of Modified Buzhong Yiqitang Combined with Erxian Decoction in Treatment of Stress Urinary Incontinence in Perimenopausal Women / 中国实验方剂学杂志

Objective:To observe the clinical efficacy of the modified Buzhong Yiqitang combined with Erxian decoction in treating stress urinary incontinence （SUI） of perimenopausal women due to spleen and kidney Qi deficiency. Method:One hundred and six patients were randomly divided into a control group （52 cases） and an observation group（54 cases）. Patients in both groups received lifestyle intervention and pelvic floor muscle training （PFMT）. On this basis， patients in the observation group were further treated with the modified Buzhong Yiqitang combined with Erxian decoction， 1 bag/day， while those in the control group were provided with Suoquan pills， 6 g/time， 2 times/day， for eight weeks. Following the international consultation on incontinence questionnaire-short form （ICIQ-SF） scoring before and after treatment， the urodynamic parameters such as maximum urinary flow rate （Q_max）， maximum urethral closure pressure （MUCP）， residual urine volume （RUV）， abdominal pressure leakage point pressure （ALPP）， and bladder capacity （BC） were measured. The number of incontinence episodes per 24 h， the degree of urinary incontinence， the amount of 1 h urine leakage， and the spleen and kidney Qi deficiency syndrome score were recorded before and after treatment. The levels of estradiol （E₂）， follicle stimulating hormone （FSH）， pituitary adenylate cyclase activating peptide （PACAP）， and vasoactive intestinal peptide （VIP） were measured before and after treatment. Result:The ICIQ-SF sub-scores of the urinary incontinence frequency， severity， and impact on quality of life as well as the total score in the observation group were all lower than those in the control group （<italic>P</italic><0.01）. Q_max， MUCP， ALPP and BC in the observation group were elevated in contrast to those in control group （<italic>P</italic><0.01）， while the RUV declined （<italic>P</italic><0.01）. Compared with the control group， the observation group exhibited a decreased number of incontinence episodes per 24 h， milder degree of urinary incontinence， reduced amount of 1 h urine leakage， and lower spleen and kidney Qi deficiency syndrome score （<italic>P</italic><0.01）. The E₂， PACAP， and VIP in the observation group were up-regulated as compared with those in the control group （<italic>P</italic><0.01）， whereas the FSH was down-regulated （<italic>P</italic><0.01）. The cure and effective rates of the observation group were （29/50） 58.00% and （47/50）94.00%， respectively， significantly better than （18/48）37.50% and （38/48）79.17% of the control group （<italic>χ</italic>²=4.124， <italic>χ</italic>²=4.683， <italic>P</italic><0.05）. Conclusion:On the basis of the lifestyle intervention and PFMT， the modified Buzhong Yiqitang combined with Erxian decoction obviously alleviates urinary incontinence， adjusts sex hormones， PACAP and VIP， ameliorates urodynamic parameters， and enhances the quality of life of patients with SUI due to spleen and kidney Qi deficiency. The resulting cure and effective rates are superior to those of the positive control.

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.

Effects of Shenling-Baizhu Powder combined with modified Guilu-Erxian Decoction on bone metabolism and inflammation response in osteoporosis patients / 国际中医中药杂志

Objective:To explore the effects of Shenling-Baizhu Powder combined with modified Guilu-Erxian Decoction on bone metabolism and inflammation response in osteoporosis patients. Methods:A total of 82 patients with osteoporosis of spleen-kidney deficiency, meeting the inclusion criteria in the hospital, were enrolled between June 2018 and October 2019. They were divided into observation group and control group by random number table method, 41 in each group. The control group was treated with oral calcium carbonate D3 tablets and alendronate sodium, while the observation group was treated with Shenling-Baizhu Powder combined with modified Guilu-Erxian Decoction on basis of control group. Both groups were treated for 6 months. Before and after treatment, scores of TCM symptoms were conducted. The bone mineral density (BMD) values of lumbar vertebra L 2-4, femoral neck and distal radius1/3 site were detected by dual-energy X-ray BMD analyzer. The levels of serum tartrate-resistant acid phosphatase 5b (TRACP 5b) and type I C-terminal cross linked peptide (CTX-I) were detected by electrochemiluminescence analyzer. The level of bone gla protein (BGP) was detected by radioimmunoassay. The levels of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α (TNF-α) and C-reactive protein (CRP) were detected by full-automatic biochemical analyzer. The adverse events during treatment were observed. And clinical curative effect was evaluated. Results:The differences in response rate between observation group and control group was statistically significant [95.1% (39/41) vs. 78.0% (32/41); χ2=5.145, P=0.023]. After treatment, scores of clinical symptoms (pain in back and loin, soreness and weakness of waist and knees, limb fatigue, debilitation, dizziness and tinnitus, frequent nocturia, loose stools, poor complexion) in observation group were significantly lower than those in the control group ( t=14.268, 10.732, 20.720, 7.564, 9.055, 15.975, 10.826, 6.552, all Ps<0.001). After treatment, BMD values of lumbar vertebra L 2-4 (0.89 ± 0.06 g/cm 3vs. 0.81 ± 0.04 g/cm 3, t=7.104), femoral neck (0.80 ± 0.08 g/cm 3vs. 0.72 ± 0.06 g/cm 3, t=5.122) and distal radius 1/3 site (0.65 ± 0.12 g/cm 3vs. 0.56 ± 0.14 g/cm 3, t=3.125) in observation group were significantly greater than those in the control group ( P<0.01). After treatment, levels of serum BGP and IL-10 in observation group were significantly higher than those in the control group ( t=3.875, 3.714, P<0.01), while levels of TRACP-5b, CTX-I, IL-6, TNF-α and CRP were significantly lower than those in the control group ( t=3.169, 5.849, 9.412, 4.606, 5.430, all Ps<0.001). Conclusion:Shenling-Baizhu Powder combined with modified Guilu-Erxian Decoction can improve clinical symptoms in patients with primary osteoporosis of spleen-kidney deficiency, increase BMD, regulate bone metabolism, alleviate inflammatory response and improve clinical curative effect.

Preliminary study on cerebrospinal fluid proteomics of Erxian Decoction against neurogenesis impairment in late-onset depression / 中国中药杂志

This study aims to elucidate the underlying mechanism of Erxian Decoction(EXD) against neurogenesis impairment in late-onset depression(LOD) rats based on cerebrospinal fluid(CSF) proteomics. A total of 66 20-21-month-old male Wistar rats were randomized into naturally aged(AGED) group, LOD group, and EXD group. All rats received chronic unpredictable mild stress(CUMS) for 6 weeks for LOD modeling except for the AGED group. During the modeling, EXD group was given EXD(ig, twice a day at 4 g·kg~(-1)) and other groups received equivalent amount of normal saline(ig). After modeling, a series of behavioral tests, such as sucrose preference test(SPT), open-field test(OFT), forced swimming test(FST), and Morris water maze test(MWMT) were performed. Immunofluorescence method was used to detect the number of Ki-67/Nesti-positive cells and BrdU/DCX-positive cells in the hippocampal DG area of each group. High-concentration corticosterone(CORT) was combined with D-galactose(D-gal) to simulate the changes of LOD-related stress and aging and the proliferation and differentiation of primary neural stem cells of hippocampus in each group were observed. Data independent acquisition(DIA)-mass spectrometry(MS) was used to analyze the differential proteins in CSF among groups and bioinformatics analysis was performed to explore the biological functions of the proteins. Behavioral tests showed that sucrose consumption in SPT, total traveling distance in OFT, and times of crossing the platform in MWMT were all reduced(P<0.01) and the immobility time in FST was prolonged(P<0.01) in the LOD group compared with those in the AGED group, suggesting that LOD rats had developed depression symptoms such as anhedonia, decreased locomotor activity ability, and cognitive dysfunction. Behavioral abnormalities were alleviated(P<0.01, P<0.05) in the EXD group as compared with those in the LOD group. Immunofluorescence results demonstrated that Ki-67/Nesti-positive cells and BrdU/DCX-positive cells in the hippocampal DG area were fewer(P<0.05) in LOD group than in the AGED group, and the positive cells in the EXD group were more(P<0.05) than those in the LOD group. In vitro experiment showed that the proliferation and differentiation of primary hippocampal neural stem cells under the CORT+D-gal treatment were reduced(P<0.01). The proliferation rate of neural stem cells decreased(P<0.05) in CORT+D-gal+LOD-CSF group but increased(P<0.01) in CORT+D-gal+EXD-CSF group compared with that in the CORT+D-gal group. A total of 2 620 proteins were identified from rat CSF, with 135 differential proteins between the LOD group and AGED group and 176 between EXD group and LOD group. GDF11, NrCAM, NTRK2, and GhR were related to neurogenesis and 39 differential proteins were regulated by both LOD and EXD. EXD demonstrated obvious anti-LOD effect, as it improved the locomotor activity ability and cognitive function of LOD rats and protected the proliferation and differentiation of hippocampal neural stem cells. EXD exerts anti-LOD effect by regulating the proteins related to neurogenesis in CSF, such as GDF11, NrCAM, NTRK2, and GhR and maintaining hippocampal neurogenesis.

Effect of Erxian Decoction on proteomics and PI3K signaling pathway of osteoblasts stimulated by H2O2 / 中国中药杂志

The present study aimed to explore the effect of Erxian Decoction on proteomics of osteoblasts stimulated by hydrogen peroxide(H_2O_2) and its protective mechanism with the H_2O_2-induced cell model of oxidative stress. The primary osteoblasts were cultured from the skulls of newborn rats(within 24 hours) and divided into a control group, a model group, a Fosamax group, and an Erxian Decoction group. Blank serum was added in the control group and model group, and the drug-containing serum was added correspondingly to the remaining two groups. After 45 hours, H_2O_(2 )stimulation was conducted for three hours except for the control group, followed by protein extraction. Nano-LC-LTQ-Orbitrap system was used for protein detection, Protein Discovery for protein identification, and SIEVE for quantitative and qualitative analysis. Furthermore, following the blocking of PI3 K signaling pathway by LY294002(10 μmol·L~(-1)), a control group, a model group, an LY294002 group, an Erxian Decoction group, and an Erxian Decoction + LY294002 group were set up to observe the effect of Erxian Decoction on cell proliferation, alkaline phosphatase(ALP) activity, and the relative expression of BMP-2, OPG, p-Akt, p-FoxO1 of osteoblasts stimulated by H_2O_2 under LY294002 intervention. The results revealed that 78 differential proteins were discovered between the Erxian Decoction group and model group, which were involved in the regulation of PI3 K/Akt, glucagon, estrogen, insulin, and other signaling pathways. LY294002 blunted the promoting effect of Erxian Decoction on osteoblast proliferation and significantly down-regulated the expression of OPG and p-FoxO1, whereas its down-regulation on the expression of BMP-2 and p-Akt was not significant. Both LY294002 and Erxian Decoction increased the ALP activity of osteoblasts, which may be related to the cell state and the cell differentiation. The above results suggest that Erxian Decoction can protect osteoblasts stimulated by H_2O_2, with the PI3 K/Akt signaling pathway as one of the internal mechanisms.

Effect of Erxian Decoction on Myocardial Microvessels and Hemorheology in Ovariectomized Rats / 中国实验方剂学杂志

Objective:To observe the changes of myocardial microvessel density， microvascular endothelial cell morphology and hemorheology in ovariectomized rats and explore the interventional effects of Erxian decoction. Method:Thirty-two healthy 10 week-old female SPF SD rats were randomly divided into sham operation group， model group， estrogen group （estradiol valerate， 0.18 mg·kg-1） and Erxian decoction group （9 g·kg-1）. The rats were intragastrically administered 2 weeks after ovariectomy， once a day for 16 weeks. Sham operation groups and model groups were given equal volumes of purified water. After 16 weeks of administration， the cardiac function was measured by noninvasive ultrasound cardiogram （UCG）， CD34 in the myocardial tissue was tested by immunofluorescence staining to measure the microvessel density， the morphological structure of microvessels of myocardial tissue were detected by transmission electron microscope， the levels of estrogen （E2） in rat plasma were detected by radioimmunoassay， the levels of endothelin-1 （ET-1）， prostacyclin I2 （PGI2）， thromboxane A2 （TXA2）， endothelial growth factor （VEGF）， and von Willebrand Factor （vWF） in rat plasma were detected by enzyme-linked immuno sorbent assay （ELISA）， four items of coagulation was detected by blood coagulation analyzer， whole blood viscosity and plasma viscosity were detected by hemorheology. Result:Compared with sham operation group， the ejection fraction （EF） decreased （P<0.01）， the left ventricular short axis shortening rate （FS） decreased （P<0.01）， and the left ventricular end systolic volume （LVVols） increased （P<0.01）， myocardial microvessel density significantly reduced （P<0.01）， the endothelial cells were swollen and the cytoplasm was cavitation， E2 in rat plasma decreased （P<0.01）， ET-1， VEGF， vWF increased （P<0.01）， prostacyclin I2 /thromboxane A2 （PGI2/TXA2） decreased （P<0.01）， plasma activated partial prothrombin time （APTT） decreased （P<0.01）， fibrinogen （FIB） increased （P<0.01）， whole blood viscosity， plasma viscosity， and cassone viscosity increased （P<0.01）， whole blood high-cut， low-cut index， and red blood cell （RBC） aggregation index increased （P<0.05） in model group. Compared with model group， EF and FS increased （P<0.05）， LVVols decreased （P<0.05）， myocardial microvessel density significantly increased （P<0.01）， the endothelial cell edema was improved， and transport vesicles were clearly visible， E2 in rat plasma increased （P<0.01）， ET-1， VEGF， decreased （P<0.01）， PGI2/TXA2 increased （P<0.01）， APTT increased （P<0.01）， whole blood viscosity， whole blood high shear relative index， RBC aggregation index decreased （P<0.05）， Kasson viscosity and plasma viscosity decreased （P<0.01） in Erxian decoction group. Conclusion:Erxian decoction increases myocardial microvessel density， protects the structural integrity of microvascular endothelial cells， improves its endothelial secretion function and hemorheology in ovariectomized rats， and protects heart function.

Clinical Efficacy and Mechanism of Modified Erxian Decoction in Treating Osteoporosis with Yang Deficiency / 中国实验方剂学杂志

Objective::To observe the effect of modified Erxian decoction on the physical condition, pain in the lower back and joints, limb activity, bone density, bone metabolism and biochemical indexes in patients with osteoporosis caused by Yang deficiency, in order to explore the possible mechanism. Method::Totally 100 cases of osteoporosis with Yang deficiency were randomly divided into treatment group and control group, with 50 cases in each group. The control group was treated with basic anti-osteoporosis therapy, and the treatment group was give modified Erxian decoction combined with basic therapy for 6 weeks. Short physical performance battery (SPPB) scores of low back and joint pain and Yang deficiency symptom score before and after treatment were recorded and analyzed. before and after treatment, the changes of blood serum calcium (Ca), phosphorus (P), L1-4, femoral neck bone mineral density(BMD), osteocalcin (BGP) and type I collagen peptide amino end level (P1NP) were measured. Result::The scores of lumbar back and joint pain, Yang deficiency symptom score, limb function and activity, lumbar spine 1-4 (L1-4), femoral neck BMD, Ca, P, BGP and P1NP between two groups before treatment had no statistically significant difference. After 6 weeks of treatment, limb activity score, L1-4, femoral neck BMD, serum Ca and P levels in treatment group were significantly higher than those in control group (P<0.05). The scores of the pain in the lower back and joints, Yang deficiency symptom score, BGP and P1NP in treatment group were significantly lower than those in control group (P<0.05). Conclusion::Modified Erxian decoction can significantly alleviate the pain in the lower back and joints of patients with osteoporosis caused by Yang deficiency, enhance the limb function and activity status, improve the physical condition and bone density of patients, reduce bone conversion, with a good effect in treating and alleviating symptoms of osteoporosis caused by Yang deficiency.

Effect of Erxian Decoction on Cardiac Electrophysiology in Peri-menopausal Rats / 中国实验方剂学杂志

Objective: To explore the effect and mechanism of Erxian decoction on peri-menopausal cardiac electrophysiology in rats. Method: Female sprague-dawley rats were randomly divided into six groups:sham operation group, model group, estradiol valerate group, and low, medium and high-dose Erxian decoction groups. Except the sham operation group, the rats in the other groups were completely removed from the ovarian replication peri-menopausal rat model. At the same time, estradiol valerate group (8×10-4 g·kg -1·d-1), low-dose Erxian decoction group (4 g·kg-1·d-1), middle-dose Erxian decoction group (8 g·kg-1·d-1) and high-dose Erxian decoction group (12 g·kg-1·d-1), sham operation group and model group were given the same amount of normal saline. The administration was given once a day for 80 consecutive days. The electrocardiogram of rats was recorded by biosignal detector connected to lead Ⅱ electrode. The content of estradiol (E2) in rat serum was detected by enzyme-linked immuno sorbent assay (ELISA). The pathological changes of rat uterus were observed by hematoxylin-eosin. The expression of the estrogen alpha receptor (ERα) protein was detected by Western blot method in myocardial tissue of rat. Result: Compared with the sham operation group, the amplitudes of P wave, R wave and T wave in the electrocardiogram of the model group were significantly decreased (P2 level was significantly lower (Pα receptor protein in myocardial tissue was significantly decreased (P2 level (Pα receptor protein (PConclusion: Erxian decoction can improve cardiac electrophysiological changes in peri-menopausal rats, and its mechanism may be related to the increase of estrogen activity and estrogen receptor expression in rats.

Effect of Erxian Decoction on cyclophosphamide-induced oligospermia in mice / 中华男科学杂志

<p><b>Objective</b>To study the effect of Erxian Decoction (EXD) on oligospermia (OS) induced by cyclophosphamide in mice.</p><p><b>METHODS</b>Eighty 6-week-old male Kunming mice were randomly divided into five groups of equal number, normal control, OS model control, and low-, medium- and high-dose EXD, the former two groups treated intragastrically with normal saline and the latter three with EXD at 3, 6 and 12 g per kg of the body weight qd for 30 days. From the 21st day of administration, the mice of the normal control group were injected intraperitoneally with saline and those of the other four groups with cyclophosphamide at 80 mg per kg of the body weight qd for 5 consecutive days. At 24 hours after the last gavage, the bilateral epididymides of the mice were collected and sperm suspension prepared for determination of the sperm count and motility, and the bilateral testes were harvested for histomorphological observation and measurement of the concentrations of superoxide dismutase (SOD), malondialdehyde (MAD) and glutathione (GSH) in the testis tissue.</p><p><b>RESULTS</b>Compared with the normal controls, the mice of the OS model control group showed significant decreases in epididymal sperm concentration (［9.31 ± 1.32］ vs ［3.32 ± 1.13］×107/ml, P <0.01) and motility (［44.75 ± 8.12］% vs ［25.95 ± 11.41］， P<0.01) and the concentrations of SOD (［37.27 ± 0.99］ vs ［14.23 ± 1.99］ U/mg prot, P <0.01) and GSH (［101.55 ± 8.74］ vs ［58.77 ± 8.93］ μmol/L, P <0.01) but an obvious increase in the MDA level (［2.21 ± 0.65］ vs ［2.61 ± 0.15］ nmol/mg prot, P <0.05) in the testis tissue. In comparison with the OS model controls, the mice treated with low-, medium- and high-dose EXD exhibited significantly increased epididymal sperm concentration (［8.34 ± 2.59］, ［8.59 ± 1.10］ and ［8.41 ± 1.47］×107/ml) (P <0.01) and motility (［36.04 ± 12.33］%, ［38.87 ± 13.13］% and ［41.90 ± 8.09］%) (P <0.01) and concentrations of SOD (［22.99 ± 1.11］, ［20.82 ± 1.81］ and ［21.33 ± 1.66］ U/mg prot) (P <0.01) and GSH (［104.74 ± 2.47］, ［98.61 ± 12.98］ and ［108.89 ± 5.85］ μmol/L) (P <0.01) but decreased level of MDA (P <0.05).</p><p><b>CONCLUSIONS</b>Erxian Decoction can improve cyclophosphamide-induced reduction of sperm concentration and motility, which might be associated with its abilities of resisting oxidation and reducing oxidative stress injury.</p>

Clinical Study of Acupoint Catgut-embedding Therapy for Prevention and Treatment of Bone Marrow Depression in Breast Cancer Patients Induced by FEC Chemotherapy / 广州中医药大学学报

Objective To compare the clinical efficacy of acupoint catgut-embedding therapy plus fundamental treatment versus fundamental treatment alone for the prevention and treatment of bone marrow depression in breast cancer patients induced by FEC(fluorouracil, epirubicin and cyclophosphamide) chemotherapy. Methods A total of 46 post-operation breast cancer patients accepting the first cycle of FEC chemotherapy were randomly divided into treatment group and control group, 23 cases in each group. The control group was given fundamental treatment including oral use of Chinese medicine of modified Xiangsha Liujunzi Decoction for regulating stomach and arresting vomiting, modified Guipi Decoction and Gui Lu Erxian Decoction for nourishing blood and generating blood, and western medicine of Batilol Tablets and Vitamin B4 Tablets. The treatment group was given acupoint catgut-embedding therapy on bilateral Zusanli(ST36) and Shenshu(BL23) plus fundamental treatment. The effect on bone marrow depression in the two groups was observed after treatment. Results(1) On post-chemotherapy day 7 and 8, the count of white blood cells (WBC) and neutrophils (NE) in the treatment group was higher than that in the control group, the difference being significant(P < 0.05 or P < 0.01).(2) The utilization of granulocyte colony-stimulating factor(G-CSF) in the treatment group was less than that in the control group, the difference being significant (P < 0.05). (3) On post-chemotherapy day 10, the effect on improving bone marrow depression in the treatment group was superior to that in the control group, the difference being significant (P<0.01) . Conclusion The acupoint catgut-embedding therapy plus fundamental treatment is more effective and safe for the prevention and treatment of bone marrow depression in breast cancer patients induced by FEC chemotherapy than fundamental treatment alone.

Clinical Study onErxian Decoction Combined with Elcatonin in the Treatment of Postmenopausal Osteoporosis / 中国中医药信息杂志

Objective To observe the clinical efficacy ofErxian Decoction combined with elcatonin for the treatment of postmenopausal osteoporosis.Methods Eighty cases of female patients with postmenopausal osteoporosis were randomly divided into treatment group and control group, 40 cases in each group. The control group received intramuscular injections elcatonin treatment, and the treatment group received oralErxian Decoction on the basis of the treatment of the control group, for 12 weeks. The main clinical symptom score, VAS score and bone mineral density (BMD) of L2-L4 and in the tibia of the two groups were observed before and after treatment to assess the therapeutic effect.Results After treatment, VAS scores of the two groups were significantly lower (P<0.05). The total effective rate of the treatment group was 92.5% (37/40); the control group was 75.0 (30/40), with statistical significance (P<0.05). The treatment group was significantly better than the control group in main symptom scores, especially in improving body symptoms (P<0.05). BMD of the treatment group was significantly higher than the control group.ConclusionErxian Decoction combined with elcatonin therapy for postmenopausal women with osteoporosis can significantly improve clinical symptoms and increase BMD.