[Percentages and functions of natural killer cell subsets in peripheral blood of patients with severe aplastic anemia].

OBJECTIVE: To analyze the percentage and functional changes of natural killer (NK) cell subsets in peripheral blood of severe aplastic anemia (SAA) patients before and after immunosuppressive therapy (IST) so as to evaluate the relationships between these changes and hematopoietic functions and explore the role of NK cells in the pathogenesis of SAA. METHODS: By flow cytometry, the percentages of NK cells (CD3(-)CD56(+)CD16(+)) and its subsets [CD3(-)CD56(bright)CD16(-)(CD56(bright)), CD3(-)CD56(dim) CD16(+)(CD56(dim)), CD3(-)CD56(-)CD16(+)] in peripheral blood lymphocytes were detected in 12 untreated patients, 30 recovered patients and 13 normal controls respectively from April 2010 to December 2010 in our hospital. NK cells activating receptors (NKG2D and NKp46), perforin and granzyme-ß of patients and normal controls were also detected. The correlation between these changes and hematopoietic functions, including the percentages of neutrophil granulocyte (ANC%), lymphocyte and reticulocyte absolute value in peripheral blood, and hyperplasia degree, percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes absolute value in bone marrow were evaluated. RESULTS: (1) The percentages of NK cells (10.30% ± 6.08%) and CD56 bright cells (0.11%) in untreated patients were significantly lower than those of recovered patients (16.47% ± 8.29%, 0.68%, both P < 0.05) or normal controls (19.45% ± 6.88%, 0.53%, both P < 0.05). The percentage of CD56(dim) cells in untreated patients was significantly lower than that of normal controls (9.62% ± 6.04% vs 18.21% ± 7.16%, P < 0.05). The percentage of CD3(-)CD56(-)CD16(+) cells was significantly higher in recovered patients than that of untreated patients or normal controls (0.79% vs 0.37%, 0.41%, both P < 0.05). (2) The expression of NKp46 and perforin of NK cells in untreated (88.23%, 64.97% ± 21.61%) and recovered patients (82.97%, 66.14% ± 20.73%) were significantly higher than those of healthy controls (40.99%, 42.11% ± 27.25%, all P < 0.05). (3) The percentage of NK CD56(bright) and CD3(-)CD56(-)CD16(+) cells of patients was positively correlated with ANC% (r = 0.423, 0.609, 0.468 respectively, all P < 0.05) and the percentage of granulocytes in bone marrow (r = 0.357, 0.517, 0.434 respectively, all P < 0.05). The percentages of NK, CD56(bright), CD56(dim) and CD3(-)CD56(-)CD16(+) cells were positively correlated with the hyperplasic degree of bone marrow (r = 0.455, 0.412, 0.404, 0.451 respectively, all P < 0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow (r = -0.522, -0.435, -0.411, -0.547 respectively, all P < 0.05). The expression of NKG2D, NKp46, perforin and granzyme-ß of NK cells had no correlation with hematopoiesis (all P > 0.05). CONCLUSION: The lowered percentage of NK CD56(bright), CD56(dim) cells and a higher expression of perforin may cause the over-function of T lymphocytes and thus lead to hematopoietic failure in SAA.

[Expression Level and Clinical Significance of OTC4 Gene in Patients with Myelodysplastic Syndromes].

OBJECTIVE: To investigate the expression characteristics and clinical value of OTC4 gene in patients with myelodysplastic syndrome (MDS). METHODS: Sixty-five patients with MDS were selected from June 2017 to April 2018, and 39 healthy subjects were selected as control group. Mononuclear cells were isolated from bone marrow collected by aseptic puncture. The OTC4 gene level of MDS patients was detected by RT-PCR, and the OTC4 protein of MDS patients was detected by Western blot. The survival curve of MDS patients was drawn by Kaplan-Meier. Cox multivariate analysis was used to analyze the independent prognostic factors. RESULTS: The relative expression level of OTC4 gene in MDS patients was significantly higher than that in the control group (P＜0.05). Western blot showed that the expression level of OTC4 protein in MDS patients was higher than that in the control group (P＜0.05). OTC4 gene expression level closely related with the leukocyte count, and the level of hemoglobin, and lactate dehydrogenase and platelet count in MDS patients (P＜0.05). CR rate of MDS patients with low OTC4 gene expression was 54.8%, which was higher than that of high OTC4 gene expression group (P＜0.05), while HI, SD and PD rates of MDS patients with low OTC4 gene expression were 9.7%, 12.9% and 6.5% respectively, which were lower than those of high OTC4 gene expression group (P＜0.05). Kaplan-Meier survival analysis showed that OS and DFS in patients with low OTC4 gene expression were superior to those with high OTC4 gene expression (P＜0.05). Multivariate Cox regression analysis showed that leukocyte count and OTC4 gene were independent influencing factors for OS (P＜0.05), platelet level and OTC4 gene expression were independent influencing factors for DFS (P＜0.05). CONCLUSION: OTC4 gene closely relates with the severity of MDS. The patients with lower expression of OTC4 gene have better prognosis, the detection of OTC4 gene has higher clinical value for evaluating the prognosis of MDS patients.

[Expression of SHP-1 mRNA in Patients with Myelogenous Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the expression and clinical significance of SHP-1 mRNA in patients with myelogenous leukemia. METHODS: The SYBR Green-based qRT-PCR was used to assess SHP-1 mRNA levels in 54 patients with chronic myelogenous leukemia (CML), 30 cases of de novo acute myelogenous leukemia (AML) and 10 persons without malignancy as controls. RESULTS: The relative expression levels of SHP-1 mRNA in control group (CG), chronic phase CML (CP-CML) group, advanced phase of CML (including accelerated phase CML and blastic phase CML) group and AML group were 1.15±0.62, 4.96±1.76, 2.60±0.90 and 0.45±0.20, respectively. The expression of SHP-1 mRNA in patients with CML significantly increased in comparison with that in CG(P<0.05). Meanwhile, the expression of SHP-1 mRNA in CP-CML group very significantly increased as compared with that in advanced stage of CML group(P<0.0001). The expression of SHP-1 mRNA in AML group significantly decreased as compared with that in CG group(P=0.0442). In CP-CML group, statistical analysis showed that SHP-1 mRNA expression at baseline in optimal responders (5.712±0.4476) was significantly higher than that in the suboptimal or failed responders (4.044±0.3701)(P=0.0090). Meanwhile, the SHP-1 mRNA expression in AML patients was higher than that in CR group (0.4984±0.05164) and non-CR group (0.3537±0.02388)(P=0.0017). CONCLUSION: The SHP-1 mRNA levels in CML patients are higher than that in AML patients, and probably correlats with disease progression of CML. The mRNA expression level of SHP-1 may be a molecular marker to predict early response to inatinib treatment in CP-CML and AML.

Action Mechanisms of Zhongjing's Antidepressant Prescriptions： A Review / 中国实验方剂学杂志

Depression is a threat to human health due to high incidence， recurrence， and disability rates. At present， the complex etiology and pathogenesis of depression are still unclear， and such hypotheses as monoamine neurotransmitters and their receptor abnormality， hyperactivity of hypothalamic-pituitary-adrenal （HPA） axis， inflammation， and neuron damage and remodeling have been put forward. Anti-depressants developed based on the pathogenesis have a certain effect， but there also exist some problems like low cure rate， poor compliance， relapse after discontinuation， and obvious side effects. According to Zhongjing's theory， depression falls into the categories of depression syndrome， visceral agitation， insomnia， and lily disease， and it is mainly located in the liver， involving the spleen， heart， and kidney. The pathogenesis mainly lies in qi stagnation and zang-fu organ dysfunction. Attention should be focused on regulating qi and relieving depression. Zhongjing's antidepressant prescriptions have exhibited good clinical efficacy in the treatment of depression， reflecting the multi-pathway action advantages of Chinese herbs. Based on the pathogenesis of depression and domestic and foreign literature on the intervention of depression with Zhongjing's prescriptions available in the past 20 years， this paper summarized the mechanisms of Zhongjing's prescriptions against depression from the experimental and clinical research aspects， in order to provide reference for clinical treatment of depression with Zhongjing's prescriptions.

Effect of Sinisan on BDNF/TrkB， 5-HT/5-HT1AR， and HPA axis in Depression Model Rats / 中国实验方剂学杂志

Objective:To observe the effect of Sinisan on the brain-derived neurotrophic factor （BDNF）/tyrosine kinase receptor B （TrKB）， 5-hydroxytryptamine （5-HT）/5-HT1A receptor （5-HT1AR）， and hypothalamus-pituitary-adrenal （HPA） axis in depressed rats， and explore the antidepressant mechanism of Sinisan based on BDNF/TrKB， 5-HT/5-HT1AR， and HPA axis. Method:A total of 120 male Wistar rats were randomly divided into a normal group， a model group， a fluoxetine （0.01 g·kg^-1） group， and low- （1.25 g·kg^-1）， medium- （2.5 g·kg^-1）， and high-dose （5 g·kg^-1） Sinisan groups， with 20 rats in each group. The depression model was induced by isolation combined with chronic unpredictable mild stimulation（CUMS） in rats except for those in the normal group for 21 days. Rats were then treated correspondingly once a day for 21 days by gavage. Those in the normal group and the model group received an equal volume of normal saline. During the intervention， the model rats were stimulated continuously. The depressive state of CUMS model rats was evaluated by sucrose preference test and open field test. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of corticotropin-releasing hormone （CRH）， adrenocorticotropic hormone （ACTH）， and corticosterone （CORT） in the plasma and BDNF and 5-HT levels in the hippocampal homogenate. The mRNA expression of hippocampal TrKB， 5-HT1AR， glucocorticoid receptor （GR）， and mineralocorticoid receptor （MR） was detected by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. The protein expression of hippocampal TrKB， 5-HT1AR， GR， and MR was detected by Western blot. The histomorphological changes of the hippocampus were observed by hematoxylin-eosin （HE） staining. Result:Compared with the normal group， the model group showed decreased sucrose preference rate （<italic>P</italic><0.01）， reduced horizontal and vertical scores in the open field test （<italic>P</italic><0.01）， increased plasma content of CRH， ACTH， and CORT （<italic>P</italic><0.01）， declining content of BDNF and 5-HT in the hippocampus （<italic>P</italic><0.01）， dwindled mRNA and protein expression levels of TrKB， 5-HT1AR， and GR （<italic>P</italic><0.01）， elevated mRNA and protein expression of MR （<italic>P</italic><0.01）， and damaged hippocampal neurons revealed by HE staining. Compared with the model group， the groups with drug intervention showed increased sucrose preference rate （<italic>P</italic><0.01） and horizontal and vertical scores in the open field test （<italic>P</italic><0.05， <italic>P</italic><0.01）， decreased content of plasma CRH， ACTH， and CORT （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated content of hippocampal BDNF and 5-HT （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated mRNA and protein expression levels of hippocampal TrKB， 5-HT1AR， and GR （<italic>P</italic><0.05， <italic>P</italic><0.01）， reduced mRNA and protein expression levels of hippocampal MR （<italic>P</italic><0.05， <italic>P</italic><0.01）， and recovered hippocampal neurons as revealed by HE staining. Conclusion:Sinisan can exert a significant antidepressant effect by increasing hippocampal BDNF and 5-HT content， up-regulating TrKB， 5-HT1AR， and GR mRNA and protein expression， down-regulating MR mRNA and protein expression， inhibiting HPA axis hypertrophy， and enhancing the regeneration and repair of hippocampal neurons.

Clinical Observation of Suanzaoren Tang Combined with Fluoxetine in Treatment of Depression of Liver Stagnation and Blood Deficiency Accompanied by Insomnia / 中国实验方剂学杂志

Objective:To observe the clinical therapeutic effect of Suanzaoren Tang combined with fluoxetine in the treatment of patients with depression of liver stagnation and blood deficiency accompanied by insomnia. Method:The patients with depression of liver stagnation and blood deficiency accompanied by insomnia （120 cases） were randomly divided into an observation group and a control group， with 60 cases in each group. The patients in the observation group received Suanzaoren Tang combined with fluoxetine， and those in the control group received fluoxetine. The course of treatment was eight weeks. The clinical efficacy was evaluated with Hamilton Depression Rating Scale （HAMD）， Pittsburgh Sleep Quality Index（PSQI）， and Activities of Daily Living （ADL） score. Enzyme-linked immunosorbent assay （ELISA） was used to determine the plasma levels of 5-hydroxytryptamine （5-HT）， norepinephrine （NE），brain-derived neurotrophic factor （BDNF）， glial cell-derived neurotrophic factor （GDNF）， neuron-specific enolase （NSE）， and S100<italic>β</italic>. Result:After eight weeks of treatment， the scores of HAMD and PSQI were reduced（<italic>P</italic><0.01）， while the scores of ADL were elevated（<italic>P</italic><0.01），and the levels of 5-HT， NE， GDNF and BDNF were up-regulated （<italic>P</italic><0.01） in the plasma of patients in the observation group as compared with those before treatment. After treatment， compared with the control group， the observation group showed increased total effective rate（<italic>P</italic><0.01）， decreased scores of HAMD and PSQI （<italic>P</italic><0.01）， elevated score of ADL（<italic>P</italic><0.01）， up-regulated levels of 5-HT， NE， GDNF and BDNF in plasma， and declining NSE and S100<italic>β</italic>（<italic>P</italic><0.01）. Conclusion:Suanzaoren Tang combined with fluoxetine is superior to fluoxetine alone in treating the depression of liver stagnation and blood deficiency accompanied by insomnia. Its therapeutic effect is achieved by increasing the release of monoamine neurotransmitters and promoting the secretion of BDNF and GDNF in the brain.

Effect of Zuoguiwan on Wnt/β-catenin Signaling Pathway in Ovariectomized Osteoporosis Model Rats / 中国实验方剂学杂志

Objective:To observe the effect of Zuoguiwan on bone metabolism and Wnt/<italic>β</italic>-catenin signaling pathway in ovariectomized osteoporotic rats model， and to explore the molecular biological mechanism of Zuoguiwan in the prevention and treatment of osteoporosis. Method:The rat model of postmenopausal osteoporosis was established by bilateral ovariectomy， 60 female SD rats were randomly divided into sham operation group， model group， positive group （estradiol valerate tablet 0.05 mg·kg^-1·d^-1） and low， middle and high dose groups of Zuoguiwan （5.5，11，22 g·kg^-1·d^-1）.After successful establishment of the model in the 13^th week， intragastric administration （<italic>ig</italic>） was given once a day for a total of 12 weeks. After administration， the histomorphological changes of femur in rats were observed by hematoxylin-eosin （HE） staining， the bone mineral density （BMD） and bone mineral content（BMC） of femur were measured by dual energy X-ray apparatus， and the biomechanical properties of bone were measured by MTS Acumen3 biomechanical testing system. The contents of bone alkaline phosphatase （BALP）， bone glaprotein（BGP），estradiol （E₂） ，and tartrate-resistant acid phosphatase （TRAP）， type Ⅰ procollagen N-terminal propeptide （PINP） in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the protein level of Wnt2，<italic>β</italic>-catenin，low density lipoprotein related receptor protein 5 （LRP5） and the phosphorylation level of glycogen synthase kinase-3<italic>β</italic>（GSK-3<italic>β</italic>） in rat tibia. Result:Compared with sham operation group， the maximum load and stiffness of BMD，BMC， in the model group decreased significantly（<italic>P</italic><0.01）， the contents of E₂ and PINP in serum decreased significantly（<italic>P</italic><0.01）， the content of BALP，BGP，TRAP increased significantly（<italic>P</italic><0.01）， the expression levels of Wnt2，p-GSK-3<italic>β </italic>Ser9，LRP5 and <italic>β</italic>-catenin protein in bone tissue decreased significantly（<italic>P</italic><0.01）， the trabecula of femur became thinner and thinner， the number of bone trabeculae decreased. Compared with model group， the maximum load and stiffness of BMD，BMC， in estradiol group and Zuoguiwan group were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the contents of serum E₂ and PINP were significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the content of BALP，BGP，TRAP was significantly decreased （<italic>P</italic><0.01）， and the expression level of Wnt2，p-GSK-3<italic>β</italic> Ser9，LRP5， <italic>β</italic>-catenin protein in bone tissue was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01） ， the trabeculae of femur became thicker， the number increased， the structure was basically clear. Conclusion:Zuoguiwan has a certain preventive and therapeutic effect on osteoporosis in ovariectomized rats， and its mechanism may be related to increasing the level of estrogen， activating Wnt/<italic>β</italic>-catenin signaling pathway， up-regulating the expression of Wnt2 and LRP5 protein， inhibiting the activity of GSK-3<italic>β</italic>， reducing the degradation of <italic>β</italic>-catenin， coordinating the dynamic coupling balance between bone formation and bone resorption， correcting the disorder of bone metabolism and improving bone morphology.

Immunoregulatory Effect of Chaihu Jia Longgu Mulitang Modified with Bupleuri Radix on Hippocampal NLRP3 Pathway in Depressed Rats / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of Chaihu Jia Longgu Mulitang （CJLM） on hippocampal NOD-like receptor protein 3 （NLRP3）inflammasome pathway in rats with depression. Method:Sixty male SD rats were randomly divided into a normal group，a model group， a MCC950 （1 mg·kg^-1） group， and high- （13 g·kg^-1）， medium- （6.5 g·kg^-1）， and low-dose （3.25 g·kg^-1） Chaihu Jia Longgu Mulitang groups， with 10 rats in each group．The depression model was induced by isolation combined with chronic unpredictable mild stimulation（CUMS） in rats except for those in the normal group. Rats were treated correspondingly for 21 days by intraperitoneal injection in the MCC950 group and gavage in other groups. The normal group and the model group received an equal volume of normal saline. The depression-like behaviors of rats were observed by sucrose preference test （SPT） and novelty-suppressed feeding test. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of interleukin-1<italic>β </italic>（IL-1<italic>β</italic>） and IL-18 in the hippocampus of depressed rats. Western blot was used to detect the protein levels of NLRP3， apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain （ASC）， and Caspase-1. Result:Compared with the normal group， the model group showed decreased sucrose preference rate （<italic>P</italic><0.01）， prolonged novelty-suppressed feeding time （<italic>P</italic><0.01）， enhanced protein expression of NLRP3，ASC， and caspase-1<italic> </italic>（<italic>P</italic><0.05， <italic>P</italic><0.01）， and elevated expression of IL-1<italic>β</italic> and IL-18 （<italic>P</italic><0.01）. Conclusion:CJLM can alleviate depression-like behaviors in CUMS-induced model rats， and the underlying mechanism is related to the inhibition of the NLRP3 inflammasome pathway．

[Effects of Scutellaria Barbata on VEGF Expression in K562 Cells].

OBJECTIVE: To investigate the anti-angiogenesis effect of Scutellaria barbata extract(SBE) on chronic myeloid leukemia(CML) K562 cell line in vitro. METHODS: The proliferating activity after treating K562 cells with 0.5,1.0,2.0 and 4.0 g/ml SB for 24, 36, 48 hours were assessed by MTT assay. The level of vascular endothelial growth factor(VEGF) in the culture supematant of K562 cells was determined by ELISA; and the expression of VEGF mRNA was detected by RT-PCR. RESULTS: MTT assay showed that SBE could inhibit the proliferation of K562 cells in a dose-dependent manner (r=0.56); ELISA displayed that the concentration of VEGF in K562 cells in blank-control group was most high; after intervention of K562 cells by SBE (0.5,1.0,2.0 and 4.0 g/ml) for 48 h, the concentration of VEGF decreased, the comparison between different groups showed significant differences (P<0.05); after treatment with SBE for 48 h, the expression of VEGF mRNA in K562 cells decreased, the gray scale ratio of target gene/ß-actin declined, and the difference between various groups was statistically significant (P<0.05). Conclution: SBE can inhibit K562 cell proliferation, its action mechanism may related with the VEGF level concentration in K562 cells and down-regulation of VEGF mRNA expression.

[Overexpression of SHP-1 Enhances the Sensitivity of K562 Cells to Imatinib].

OBJECTIVE: To explore the effect of overexpression of SH2-containing tyrosine phosphatase 1 (SHP-1) on sensitivity of chronic myelogenous 1eukemia (CML) K562 cell line to imatinib and its related mechamism. METHODS: K562 cells were infected with the lentiviral plasmids containing the specified retroviral vector (pEX-SHP-1-puro-Lv105) or the mock vector (pEX-EGFP-puro-Lv105). The expression of SHP-1 in K562 cells treated with 0.2 µmol/L imatinib (IM) for 72 h was determined by Western blot. After transfection the CCK-8 assay was used to determine the proliferation of the tramfected K562 cells (K562(SHP-1) and K562(EGFP) cells) at 72 h after exposure to different doses of IM, the half inhibitary concentration (IC50) was calculated. The mechanisms of the overexpression effects of SHP-1 and IM on the proliferation in K562 cells was investigated, the BCR-ABL1 activity and the level of tyrosine phosphorylation of CrkL (pCrkL) was measured by flow cytometry; the Western blot was used to detect the expression and activity of these molecules controlling cell growth, including MAPK, AKT, STAT5 and JAK2. RESULTS: After exposure of K562 cells to 0.08 µmol/L IM for 72 h, there was no significant change of SHP-1 expression in K562 cells. After exposure to 0.2 µmol/L of IM for 72 h, the inhibitory rate of K562(SHP-1) group was higher than that of K562(EGFP) group (P < 0.05), indicating that overexpression of SHP-1 in K562 cells could enhance the proliferation inhtibition effect of IM on K562 cells. The IC50 of IM in K562(SHP-1) cells was the lower as compared with that of K562(EGFP) cells (P < 0.05) after exposure to different concentrations of IM for 72 h. The slope of K562(SHP-1) cells was the largest ranging 0.02 - 0.16 µmol/L of IM. Overexpression of SHP-1 and IM could inhibit the activity BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in the K562 cell line and displayed a synergistic effect. CONCLUSION: SHP-1 inhibits BCR-ABL1, MAPK, AKT, STAT5 and JAK2 signaling pathways in K562 cells, the overexpression of SHP-1 can enhance the sensitivity of K562 cells to IM.

[Clinicopathological and immunohistochemical study of extra-gastrointestinal stromal tumors arising from the omentum and mesentery].

OBJECTIVE: To explore the clinicopathological and immunohistochemical features of extra-gastrointestinal stromal tumors (EGIST) arising from the omentum and mesentery and to investigate the cellular origin of these tumors, prognostic factors, and the relationships with gastrointestinal stromal tumors. METHODS: Nineteen cases of mesenchymal neoplasms arising from the omentum and mesentery (previously diagnosed as smooth-muscle tumors or schwannomas) were studied morphological with a panel of immunohistochemistry including CD117 and CD34. RESULTS: Among the 19 cases, 14 tumors were confirmed to be EGIST, of which 6 tumors arose from the omentum and 8 cases located at the mesentery. The size of tumors ranged from 3.5cm to 29.0 cm (mean 12.4cm) in diameter. Histologically, there were 9 cases of mainly spindle cell type, 2 cases of mainly epithelioid cell type and 3 cases of mixed cell type. all EGIST expressed CD117 (14/14) and a percentage of them expressed also CD34 (8/14) and/or SMA (6/14), anyhow, all EGIST were negative for desmin and S-100 protein. Six patients with tumors arising from the omentum were all alive without evidence of disease (tumor-free). Among 7 cases with tumors of the mesentery, three patients died of the disease, 1 alive with the disease and 3 patients alive without evidence of the disease. CONCLUSIONS: EGIST were identical by their histological and immunohistochemical features with gastrointestinal stromal tumors (GIST). This tumor may arise from the multipotential mesenchymal stem cells. EGIST have various clinical behavior, and the parameters used for predicting the prognosis of GIST may not be completely suitable for EGIST evaluation.

Research Progress of ZHANG Zhong-jing's Classical Formulae in Treatment of Depression / 中国实验方剂学杂志

Depression is a kind of emotional and mental disorder， which is related to many pathogenic factors， such as abnormal metabolism of monoamine neurotransmitters， immune inflammatory reaction， neuroendocrine disorder and so on. it has the characteristics of high disability rate and high recurrence rate， which is seriously harmful to human health. It brings huge economic burden to patients and their families. At present， the commonly used antidepressants in clinic are monoamine oxidase inhibitors， tricyclic antidepressants and selective 5-hydroxytryptamine reuptake inhibitors. Long-term use causes adverse reactions such as cardiovascular， urinary and digestive dysfunction. The multi-target， multi-pathway and multi-mechanism of traditional Chinese medicine has played a great role in the treatment and rehabilitation of depression. At the same time， traditional Chinese medicine has gradually become a hot spot in the research and development of antidepressants because of its small side effects and good drug compliance. Especially the ZHANG Zhong-jing's classical formulae with precise medication and rigorous prescription has unique advantages in the treatment of depression. For thousands of years， it has been used clinically by countless doctors in the treatment of various types of depression， with definite curative effects and few side effects. In recent years， clinical research， experimental research， and pharmacological research around ZHANG Zhong-jing's classical formulae for depression have been continuously expanded and deepened. This article reveals the rules of medication of the treatment of depression with ZHANG Zhong-jing's classical formulae from three aspects of Bupleurum class prescription， Gardenia class prescription， and other prescriptions， which is reflected in the study on the composition of prescription， the etiology， and pathogenesis and the symptoms of the treatment. Integrating and classifying ZHANG Zhong-jing's classical formulae's experience in the treatment of depression， mainly expounded from two aspects， clinical research and pharmacological research， summarizing the research perspective and progress of Zhongjing prescription medicine in the treatment of depression in recent years， to excavate， inherit and develop ZHANG Zhong-jing's classical formulae， and provide experience and reference for the treatment of depression from multiple angles and ideas.

Research Progress of Banxia Houpu Tang in Treatment of Depression / 中国实验方剂学杂志

Depression is a common emotional disease，causing a heavy burden of disease on patients' families and society.Its pathogenesis is not yet fully understood，andmay be related to inflammation，neurotransmitters，neurotrophicfactors，hippocampal neuronal synaptic plasticity，oxidative stress，etc.Clinical application of Western medicine to treat depression has serious side effects，poor patient compliance，the effective rate is not high，rebound after withdrawal，etc.Traditional Chinese medicine treatment of depression has the characteristics of low side effects and high patient compliance. Depression belongs to the category of depression syndromein traditional Chinese medicine.Traditional Chinese medicine believes that the main symptoms of depression include depressed liver Qi，stagnated Qi transforming into fire，deficiency in heart and spleen，deficiency in heart and lung，stagnation of phlegm and Qi，deficiency of liver and kidney，etc. Banxia Houpu Tang is a classic recipe contained in"Synopsis of the Golden Chamber" by Zhang Zhongjing in the Han Dynasty， it has the effect of treating depression syndrome of stagnation of phlegm and Qi.By searching the literatures of Banxia Houpu Tang in the treatment of depression in Chinese knowledge network database （CNKI），Chinese biomedical literature database （CBM）， Wanfang Data， Pubmed， Embase， Web of Science and other databases，we found that both clinical application and experimental research suggest that Banxia Houpu Tang has a significant antidepressant effect. Clinical studies have shown that Banxia Houpu Tang can improve the scores of anxiety scales and depression scales of patients with depression. The antidepressant effect is significant， and the advantages are prominent. Experimental research shows that the antidepressant effect of Banxia Houpu Tang and its effective components may be related to the factors such as intervention of inflammatory response， influence of neurotransmitters， regulation of neurotrophic factors， improvement of synaptic plasticity of hippocampal neurons and reduce oxidative stress，etc. Therefore，this paper reviews several types of depression in the clinical treatment of Banxia Houpu Tang and the related experimental studies of Banxia Houpu Tang.

Effect of Chaihu Jia Longgu Mulitang on Signaling Pathway of PI3K/Akt/GSK3β/β-catenin in Hippocampus Tissue of Rats with Depression / 中国实验方剂学杂志

Objective:To study the effect of Chaihu Jia Longgu Mulitang on phosphatidylinositol-3-kinases （PI3K）/protein kinase B （Akt）/glycogen synthase kinase 3β （GSK3β）/β-catenin signaling pathway of hippocampus in rats with depression. Method:A total of 120 SD rats were randomly divided into normal group，model group， and low， middle and high-dose Chaihu Jia Longgu Mulitang groups（3.25，6.5，13 g·kg-1）， and fluoxetine group， with 20 rats in each group. Except normal group， the depression model was prepared through chronic unpredictable mild stimulation（CUMS）. The normal group and the model group were given normal saline with 6.5 g·kg-1 by gavage. Chaihu Jia Longgu Mulitang groups were intragastrically given corresponding herbal drugs 3.75，6.5，13 g·kg-1， while fluoxetine group was intragastrically given fluoxetine 10 mg·kg-1 for 21 days， once a day. Then the depressive behaviors of rats were observed by sucrose preference test （SPT） and forced swimming test （FST）. Western blot was used to detect the protein expressions of PI3K，Akt，GSK3β and phosphorylation level. Result:Compared with normal group，the sucrose preference index was decreased significantly，while the immobility time in FST was increased significantly（P<0.01）， the protein expressions of PI3K， p-PI3K p110， p-PI3K p85 were decreased significantly （P<0.01）， and expressions of Akt， p-Akt Thr308，p-Akt Ser473， p-GSK3β Ser9 and β-catenin were decreased significantly（P<0.01）， while the level of GSK3β， p-GSK3β Tyr216 were increased significantly in model group（P<0.05，P<0.01）. Compared with model group，Chaihu Jia Longgu Mulitang could increase sucrose preference index and decrease the immobility time in FST（P<0.01）， the protein expressions of PI3K p110 and PI3K p85 was increased significantly （P<0.01）， levels of Akt Thr308，Akt Ser473， p-GSK3β Ser9， β-catenin were increased significantly （P<0.01）， whereas levels of GSK3β， and GSK3β Tyr216 were decreased significantly. Conclusion:Chaihu Jia Longgu Mulitang could increase protein expression and activity of PI3K in rat hippocampus， activate Akt， inhibit GSK3β kinase activity and prevent β-catenin from degradation， so as to increase PI3K/Akt pathway activity in rat hippocampus， and protect hippocampal neurons.

Effect of Modified Suanzaoren Tang on Expression of Glutamate Receptor in Hippocampus of Chronic Restraibt Stress Depression Model Rat / 中国实验方剂学杂志

Objective:To study the effect of modified Suanzaoren Tang on the expression of excitatory amino acids receptor（EAARs） in hippocampus of rats with chronic depression， and to explore the anti-depressant mechanism of modified Suanzaoren Tang based on excitatory amino acids receptor. Method:Sixty SD rats were randomly divided into normal group，model group，and low，middle and high-dose modified Suanzaoren Tang groups，and ketamine group，with 10 rats in each group．Except normal group，the depression model of rats was prepared by using chronic restraint stress（CRS）.The normal group and model group were intragastrically（ig） given normal saline．the modified Suanzaoren Tang groups were intragastrically given corresponding herbal drugs 6，12，24 g·kg-1， ketamine group group were given ketamine 0.015 g·kg-1 through intraoeritoneal injection，for 21 days，once a day．Then the depressive behaviors of rats were observed by Morris water maze and novelty feeding experiment.Western blot was used to detect the levels of DAR1，NMDAR2A，NMDAR2B，GluR1，mGluR1，CaMKⅡα and CaMKⅡβ protein expression in rat hippocampus tissue. Result:Compared with normal group，the time of novel ingestion and escape latencywere prolonged significantly（P<0.01）， and the time of space exploration was shortened significantly（P<0.01）.The levels of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ expression were increased significantly（P<0.01），while the levels of GluR1 and CaMKⅡα expression were decreased significantly（P<0.01）in model group. Compared with model group，the time of novel ingestion and escape latency were shortened significantly （P<0.01）， and the time of space exploration was prolonged significantly（P<0.01）.The levels of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ protein expression were decreased significantly（P<0.01），but the levels of GluR1 and CaMKⅡα expression were increased decreased significantly（P<0.01）in middle and high-dose modified Suanzaoren Tang groups. Conclusion:Modified Suanzaoren Tang can improve the behavior of chronic depression rats effectly. Its mechanism may be related with reduction the expression of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ protein ，increase the expression of GluR1and CaMKⅡα protein.

Regulatory Effect of Modified Erchentang on Levels of Lymphokines Relating to Th1,Th2 and Th9 Cells and Expressions of IL-4R1/IL-13RA1 in Bronchioles of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective:To study the effect of modified Erchentang on levels of interleukin-12 (IL-12), interferon-γ (IFN-γ), interleukin-9 (IL-9), interleukin-4 (IL-4) and interleukin-13 (IL-13) in plasma and bronchoalveolar lavage fluid (BALF) of all rats, as well as expressions of interleukin-4 (IL-4) receptor (IL-4R1) and interleukin-13 (IL-13) receptor (IL-13RA1) in bronchioles tissue of rats with chronic obstructive pulmonary disease (COPD). Method:Fifty SD rats were randomly divided into 5 groups, namely normal group, model group, and low, middle and high-dose modified Erchentang groups (5, 10, 20 g·kg-1), with 10 rats in each group. COPD in rat was prepared by using cigarette smoke combined with dripping lipopolysaccharide (LPS) in trachea. After the modeling, normal and model groups were given normal saline solution through intragastric (ig) administration, while other groups were given corresponding herbal drugs (5, 10, 20 g·kg-1) intragastrically (ig) for 14 days. The levels of IL-12, IFN-γ, IL-9, IL-4 and IL-13 in plasma and BALF were detected by Enzyme-linked immunosorbent assay (ELISA) method， and immunohistochemistry (IHC) method was used to detect the expressions of IL-4R1 and IL-13RA1 in bronchioles tissue of all of the groups. Result:Compared with the normal group, the levels of IL-12 and IFN-γ were decreased significantly (P<0.01), but the levels of IL-9, IL-4 and IL-13 in plasma and BALF were significantly increased (P<0.01), and the expressions of IL-4R1 and IL-13RA1 in bronchioles tissue were increased significantly (P<0.01) in model group. Compared with the model group, the levels of IL-12 and IFN-γ were increased significantly, while the levels of IL-9, IL-4 and IL-13 in plasma and BALF were decreased significantly (P<0.01), and the expressions of IL-4R1 and IL-13RA1 in bronchioles tissue were decreased significantly (P<0.01) in modified Erchentang groups (10, 20 g·kg-1). Conclusion:Modified Erchentang has effects in resisting inflammatory and protecting tissue structure of bronchioles. Its mechanism may be correlated with increasing the levels of IL-12, IFN-γ and reducing the levels of IL-9, IL-4 and IL-13 in plasma and BALF, and inhibiting the expressions of IL-4R1 and IL-13RA1 in bronchioles tissue.

Mechanism of Modified Erchentang on Expression of CXCL8-CXCR1/2 Axle Genes in Lung Tissue of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective:To observe the effect of modified Erchentang on the expression of CXC chemokine ligand (CXCL) 8-CXC chemotaxis factor receptor (CXCR) 1/2 genes in the lung tissue of rats with chronic obstructive pulmonary disease (COPD), in order to explore the anti-inflammatory molecular mechanism of Erchentang on COPD. Method:Forty SD rats were randomly divided into normal group, model group, Jizhi syrup group and modified Erchentang group. COPD models in rats were prepared by cigarette smoke and dripping lipopolysaccharide (LPS) in the trachea. After modeling, normal and model groups were intragastrically given normal saline solution, Jizhi syrup group was given Jizhi syrup（10 g·kg-1），and modified Erchentang group was given intragastrically corresponding herbal drugs (10 g·kg-1) for 14 days. The levels of chemokines CXCL1, CXCL8 were detected by enzyme-linked immunosorbent assay in rat bronchoalveolar lavage fluid (BALF). The mRNA expressions of CXCL8, CXCR1 and CXCR2 were detected by quantitative real time PCR (Real-time PCR). Western blot was used to detect the levels of CXCL8, CXCR1 and CXCR2 protein, the pathological changes of lung tissues were observed by hematoxylin-eosin(HE) staining,and immunohistochemistry (IHC) method was used to detect the expressions of CXCL8, CXCR1 and CXCR2 protein in the lung tissue of all the groups. Result:The levels of chemokines CXCL1, CXCL8 in rats BALF were increased significantly (P<0.01), the expressions of CXCL8，CXCR1 and CXCR2 mRNA and protein were increased significantly (P<0.05, P<0.01) in model group compared with normal group. Compared with model group, the expressions of CXCL8, CXCR1 and CXCR2 mRNA and protein were decreased significantly (P<0.05), and the levels of chemokines CXCL1, CXCL8 in rats BALF were decreased significantly (P<0.01) in modified Erchentang. Conclusion:Modified Erchentang has an anti-inflammatory effect on COPD. The mechanism may be related to inhibiting the expressions of CXCL8, CXCR1, CXCR2 mRNA and protein, and reducing the release of chemokines CXCL1, CXCL8.

Effect of Modified Xiao Chaihutang on Expression of EAATs and VGLUTs in Hippocampus of Chronic Restraibt Stress Depression Model Rat / 中国实验方剂学杂志

Objective:To study the effect of modified Xiao Chaihutang on the expressions of excitatory amino acid transporters（EAATs） and vesicle glutamate transporters（VGLUTs）in hippocampus of rats with chronic depression， in order to explore the anti-depressant mechanism of modified Xiao Chaihutang based on glutamate transport. Method:A total of 120 SD rats were randomly divided into normal group， model group， and low， middle and high-dose modified Xiaochaihutang groups （6.5， 13， 26 g·kg-1） and riluzole group， with 20 rats in each group．Except normal group， the depression model of rats was prepared through Chronic restraint stress（CRS）. The normal group and the model group were intragastrically （ig） given normal saline. The modified Xiao Chaihutang groups were intragastrically given corresponding herbal drugs （6.5， 13， 26 g·kg-1）， and the Riluzole group was given Riluzole 20 mg·kg-1 through intraoeritoneal injection for 21 days， once a day. Then the depressive behaviors of rats were observed by forced swimming test （FST） and tail suspension test （TST）. The level of glutamic acid （Glu） in rats hippocampus was determined by high performance liquid chromatography （HPLC）. The mRNA expressions of EAAT1， EAAT2 and EAAT3 in hippocampus were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）method. Western blot was used to detect the protein expressions of EAAT1， EAAT2， EAAT3， VGLUT1 and VGLUT2 in rat hippocampus tissue. Nissl staining was used to observe the morphology of hippocampal neurons in rats. Immunohistochemical（IHC）S-P method were used to detect the location expressions of EAAT1， EAAT2 and NeuN proteins in rat hippocampal CA1 region tissue. Result:The immobility times in FST and TST were increased significantly（P<0.01）， the mRNA and protein expressions of EAAT1，EAAT2，EAAT3 were decreased significantly （P<0.01）， and as well as the expressions of VGLUT1 and NeuN were decreased significantly（P<0.01）， while the level of Glutamate and the expression of VGLUT2 were increased significantly（P<0.01） in model group， compared with normal group. Compared with model group，the immobility times in FST and TST were decreased significantly（P<0.05， P<0.01）， mRNA and protein expressions of EAAT1，EAAT2，EAAT3 were increased significantly（P<0.01）， and expressions of VGLUT1 and NeuN were increased significantly（P<0.01）. However， the level of Glutamate and the expression of VGLUT2 were decreased significantly（P<0.01）， and the damage of hippocampal neurons in rats was mild in middle and high-dose modified Xiao Chaihutang groups. Conclusion:Modified Xiao Chaihutang has an anti-depressive effect. Its mechanism may be related to its up-regulation of expressions of EAAT1， EAAT2， EAAT3 genes and VGLUT1 protein in the hippocampus of depression model rats.

[Expression and Clinical Significance of DNMT in Patients with Chronic Myeloid Leukemia].

OBJECTIVE: To investigate the expression and clinical significance of DNA methyltransferases (DNMT) mRNA in patients with chronic myeloid leukemia (CML). METHODS: The expression levels of DNMT mRNA in mononucllear cells (MNC) of bone marrow or in peripheral blood of 93 CML patients in 3 different phases and 10 normal controls (NC) were detected by SYBR Green flurescent quatitative PCR. RESULTS: The relative expression levels of DNMT1 mRNA in NC, chronic phase CML (CML-CP), accelerated phase (CML-AP) and blastic phase (CML-BP) were 1.45 ± 0.22, 1.83 ± 0.63, 2.95 ± 0.87 and 3.24 ± 1.39 resectively. The expression of DNMT1 mRNA showed no statistically significant difference between CML-CP and NC (P = 0.28). The expression of DNMT1 mRNA in advanced stages (including CML-AP and CML-BP) of CML obviously increased in comparison with CML-CP and NC (P < 0.05). The expression of DNMT1 mRNA in CML-AP was not significantly different from that in CML-BP (P = 0.336). The relative expression levels of DNMT3a mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.29 ± 0.34, 1.34 ± 0.46, 2.33 ± 1.05 and 3.18 ± 1.23 resectively. And the expression levels of DNMT3a mRNA were not statistically significantly different between CML-CP and NC (P = 0.844). The results showed that the expression of DNMT3a mRNA in the advanced phase of CML significantly increased in comparison with that in CML-CP and NC (P < 0.05). Meanwhile, the expression of DNMT3a mRNA in CML-AP was not different from that in CML-BP (P = 0.304). The relative expression levels of DNMT3b mRNA in NC, CML-CP, CML-AP and CML-BP groups were 1.37 ± 0.31, 16.41 ± 22.50, 9.36 ± 5.50 and 12.17 ± 13.44 resectively. It was also found that the level of DNMT3b mRNA in CML significantly increased in comparison with NC (P < 0.05), and that the between the 3 different phase of CML was not statistically significantly different (P >0.05). CONCLUSION: The expression of DNMT mRNA increases in advanced CML as compared with normal controls and CML-CP, and the increased levels of DNMT mRNA probably correlate with disease progression in CML.

Effect of Modified Erchentang on PPARγ Expression in Lung Tissue of Rats with Chronic Obstructive Pulmonary Disease / 中国实验方剂学杂志

Objective: To explore the effect of modified Erchentang on peroxisome proliferator-activated receptor gamma (PPARγ) protein and gene expressions in lung tissue of chronic obstructive pulmonary disease (COPD) rat model, and the expressions of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α) in serum, lung homogenate and bronchoalveolar lavage fluid. Method: Seventy SD rats were randomly divided into seven groups:normal group, model group, high, medium and low-dose modified Erchentang groups (40, 20, 10 g · kg-1 · d-1), Xiaokechuan group (5 g · kg-1 · d-1), and Erchentang group (5 g · kg-1 · d-1). The rat COPD model was established through smoking and intratracheal instillation of lipopolysaccharide (LPS). After successful modeling, the treatment group was given drug by gavage, while the normal group and the model group were given the same amount of saline. The concentrations of IL-6, IL-10 and TNF-α in serum, lung homogenate and bronchoalveolar lavage fluid(BALF) of rats were measured by enzyme-linked immunosorbent assay (ELISA). Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expression of peroxisome proliferator-activated receptor gamma (PPARγ), and immunohistochemistry (IHC) and Western blot were used to detect the expression of PPARγ in lung tissue. Result: Compared with the normal group, the levels of IL-6 and TNF-α in serum, lung homogenate and BALF of the model group rats increased significantly (PPγ mRNA in lung tissue of rats in model group were significantly decreased (Pγ protein was significantly inhibited(Pα in serum, lung homogenate and BALF of each treatment group decreased to varying degrees(Pα in the middle-dose Erchentang group were particularly significant. The PPARγ mRNA and protein expressions in lung tissue of rats in each treatment group were increased to varying degrees (PConclusion: Modified Erchentang may improve pulmonary inflammation and pulmonary function in COPD rats by increasing the expression of PPARγ and the content of IL-10 and decreasing the contents of IL-6 and TNF-α.