Objective To investigate the effects of Xiao Yao San on intracellular Ca2 + concentration in cultured rat hippocampal neurons in the state of chronic stress and study the mechanism of chronic stress injuring and XiaoYao San protecting. Methods MK-801 acts as tool,cultured rat hippocampal neurons were divided into seven groups, those were group 1 (control), group 2 (normal serum), group 3 (normal serum + Glu), group 4 (model serum + Glu), group 5 (model serum + Glu + MK-801), group 6 (Xiaoyaosan + Glu), group 7 (Xiaoyaosan + Glu + MK-801). to detect intracellular Ca2+ concentration in cultured hippocampal neurons in the simulated micro - environment of chronic stress and after intervention with the serum treated with Xiao Yao San by confocal laser microscope at the same period of time. Results Compared with group 1 (779.97 ± 36.81), concentration of Ca2+ intracellular of group 2 (1092.38 ± 36.41), group 3 (1472.49 ± 76. 19), group 4 (1509.52 ±104.69) and group 5 (1186.97 ±41.92) all increased significantly (P＜0.01) ,group 6 (908.74 ±40.24) increased too (P ＜ 0.05), compared with group 2, concentration of Ca2 + intracellular of group 3,4 and 5 all increased significantly (P ＜ 0.01), but group 7 (721.99 ± 60.33) decreased significantly (P ＜ 0.01). Compared with group 4, concentration of Ca2+ intracellular of group 6 and 7 decreased significantly (P＜ 0.01), group 5 decreased too (P ＜ 0.05), compared with group 6, concentration of Ca2 + intracellular of group 5 increased significantly (P ＜ 0.01),when group 7 decreased significantly (P＜0.01). Conclusion Serum of chronic stress treated with Xiao Yao San has the effect of inhibiting Ca2+ overload in hippocampal neurons,it may work through a variety of signaling pathways including Glu-NR-Ca2+ to maintain the steady-state of Ca2+ concentration in hippocampal neurons, and then to protect neurons from the neurotoxic effects of excitatory.

AIM: To investigate the effect of venous marker chicken ovalbumin upstream promoter-transcription factor Ⅱ (COUP-TFⅡ) expression on vascular endothelial cell senescence and its molecular mechanism.METHODS: The mRNA expression of COUP-TFⅡ in the human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC) was detected by RT-qPCR.After transfection with a COUP-TFⅡ siRNA (siCOUP-TFⅡ) to inhibit COUP-TFⅡ expression in the HUVEC, the senescence and proliferation of endothelial cells were evaluated by β-galactosidase staining, Western blot, CCK-8 assay and cell counting after treatment with 10-5 mol/L angiotensin Ⅱ (AngⅡ).The protein levels of Akt and p-Akt were determined by Western blot.RESULTS: Compared with the HCAEC, COUP-TFⅡ was significantly highly expressed in the HUVEC.Knockdown of COUP-TFⅡ via siCOUP-TFⅡ significantly induced endothelial cell senescence and inhibited endothelial cell proliferation and p-Akt level after treatment with AngⅡ at 10-5 mol/L.Furthermore, an Akt activator SC79 at 4 mg/L partly reversed the effect of siCOUP-TFⅡ on AngⅡ-induced endothelial cell senescence and proliferation.CONCLUSION: Knockdown of COUP-TFⅡ promotes endothelial cell senescence and inhibits endothelial cell proliferation, which might be partly regulated by Akt signaling.

Objective To observe the mechanisms that Xiaoyao powder influences the hypothalamus-pituitary-adrenal (HPA) axis of chronic-stress rats. Methods Chronic stress rats were as researching object,and RU-38486 acted as tool drugs. The serum-GC density of rats were tested with ELISA,and the glucocortcoid(GR) in hippocampus neuron were tested with immunofluorescence,the CRH mRNA in hypothalamus were tested by in situ hybridization (ISH). Results Compared with normal group ( 1.09 ± 0.11 ;0.57 ± 0.10), the expression of GR in hippocampus of model group decreased(0.65 ± 0. 10; P ＜ 0. 01 ), and the expression of CRH mRNA in hypothalamus of model group increased ( 1.12 ±0. 11; P＜0. 0l ) ,the GR in hippocampus of RU-38486 group increased ( 1.59 ± 0. 11; P ＜ 0. 01 ), and the expression of CRH mRNA in hypothalamus of RU-38486 group reduced (0.48±0.10; P＜0.05) ,but both the expression of GR in hippocampus and the CRH mRNA in hypothalamus of Xiaoyao powder group were no change (0.62 ±0.08;0.97 ±0.13; P＞0.05). Compared with model group,both the expression of GR in hippocampus of RU-38486 and Xiaoyao powder group increased (P＜0. 01) ,and both the expression of CRH mRNA in hypothalamus of RU-38486 and Xiaoyao powder group reduced (P＜0.01). Conclusion Multi-stress can result in the expression of GR in hippocampus of rats decreasing and the expression of CRH mRNA in hypothalamus increasing, but those changes can be restrained by Xiaoyao powder, and it is the maybe mechanism of Xiaoyao Powder resisting chronic stress in HPA axis.

To identify the amino alcohols related substances in atenolol. The related substances in atenolol and its stressed samples were pre-column derivatized with 9-fluorenylmethyl chloroformate. The separation was carried out on an Inertsil ODS-SP column (250 mm×4.6 mm, 5 μm) with linear gradient elution by methanol-ammonium acetate solution as the mobile phases. Electrospray positive ionization high-resolution Q-TOF/MS was used for the determination of the accurate masses and elemental compositions of the parent and fragment ions of these related substance derivatives. The structures of all the detected substances were identified by spectral analysis and synthetic analysis. Under the established conditions, atenolol and its amino alcohols related substances were well separated, and a total of 14 impurity peaks were detected and identified, of which 12 were related substances and 2 were derivatization reaction by-products. The established LC-MS method provides a reference for the examination and quality control of atenolol related substances.

Objective:To investigate the role of transforming growth factor β1 (TGF-β1) and matrix metalloproteinase (MMP)-2 in pancreatic tissue repair and reconstruction in rats with acute pancreatitis and its potential mechanism.Methods:114 male SD rats were randomly divided into normal control group (CON group) and acute edematous pancreatitis model group (AEP group), acute necrotic pancreatitis model group (ANP group), ANP control group and ANP intervention group. The rat AEP model was constructed by subcutaneous injection of caerulein, and the rat ANP model was prepared by intraperitoneal injection of L-arginine. The ANP intervention group and ANP control group were prepared by intraperitoneal injection of TGF-β1 inhibitor SB431542 or DMSO 30 min before, 24 h and 48 h after pancreatitis induction, respectively. Hydroxyproline content in pancreatic tissue was determined by hydroxyproline kit. The expression of TGF-β1, phosphorylated Smad3 (p-Smad3), type Ⅲ collagen and MMP-2 in pancreatic tissue was detected by immunohistochemical method. The activity of MMP-2 was determined by gelatin enzyme spectrometry. The expression levels of MMP-2 and p-Smad3 proteins in pancreatic tissue were detected by Western blot.Results:The hydroxyproline content in CON group was (61.71±8.56)μg/mg protein. The hydroxyproline content in AEP group reached the peak (116.72±8.53)μg/mg on the 3rd day. The peak value of hydroxyproline content in ANP group was (174.93±11.75)μg/mg on day 5. The peak value in ANP group was significantly higher than that in AEP group, and the peak value of hydroxyproline content in AEP group was significantly higher than that in CON group. The hydroxyproline content at day 3, 5 and 7 in the ANP intervention group was (108.07±10.48)μg/mg, (137.14±8.66)μg/mg and (112.35±13.16)μg/mg, respectively, and that at day 3, 5 and 7 in the ANP control group was (132.35±14.2)μg/mg, (175.43±13.75)μg/mg and (137.92±12.65)μg/mg, respectively. TGF-β1 immunohistochemical peak score in control group, AEP group and ANP group was (0.12±0.03), (1.96±0.21) and (3.00±0.28), respectively. p-Smad3 immunohistochemical peak score was (0.15±0.05), (2.05±0.20), and (3.05±0.24), while type Ⅲ collagen immunohistochemical peak score was (0.11±0.04), (1.56±0.15), and (3.10±0.17). MMP-2 immunohistochemical peak score was (0.05±0.03), (1.45±0.20), and (2.45±0.15), respectively. The immunohistochemical peak scores of TGF-β1, p-Smad3, type Ⅲ collagen and MMP-2 in ANP group were significantly higher than those in AEP group. The immunohistochemical peak scores of TGF-β1, p-Smad3, type Ⅲ collagen and MMP-2 in pancreatic tissue of ANP intervention group and ANP control group were (2.36±0.21), (2.25±0.22), (2.47±0.19), (2.00±0.10) and (3.02±0.21), (3.01±0.19), (3.05±0.24), (2.43±0.11), respectively, which in ANP intervention group was significantly lower than those in the ANP control group. The peak value of MMP-2 activity in pancreatic tissue of CON group, AEP group and ANP group was (10.85±1.73), (85.78±7.16) and (115.43±8.7), respectively, which in ANP group was significantly higher than that in AEP group, and in AEP group was significantly higher than that in CON group. In ANP intervention group and ANP control group 3 and 5 days after molding, the expression levels of MMP-2 protein in pancreas were 0.20±0.01, 1.19±0.02, 0.52±0.01, 1.54±0.05, respectively; p-Smad3 protein expression levels were 0.30±0.04, 0.66±0.11, 1.95±0.05, 1.30±0.01, respectively; and MMP-2 and p-Smad3 in ANP intervention group was significantly lower than those the ANP control group. All the differences among the groups above were statistically significant (all P value <0.001). Conclusions:TGF-β1 and MMP-2 play an important role in tissue remodeling and extracellular matrix deposition after acute pancreatitis inflammation.