Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway / 中国中药杂志

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.

Study of a-Asarone protecting BV2 cells damaged by OGD/R by regulating of NLRP3 pathway / 中国药理学通报

Aim To evaluate the protective effect of α-asarone on microglials with cerebral ischemia/reperfusion injury by measuring the expression of polar transformation and related inflammatory proteins in BV2 cells in vitro and its mechanisms.Methods The cerebral ischemia/reperfusion injury BV2 cells were pretreated by α-asarone in vitro and simulated by OGD/R model.The effect of α-asarone on the viability of damaged cells in OGD/R model was determined by CCK-8; the morphological changes of cells were observed to analyze the general morphology of cells; the levels of proinflammatory factor IL-1β, IL-18 and anti-inflammatory factor IL-10, IL-4, and ROS activity secreted by BV2 cells were detected by ELISA; the protein expressions of TGF-β, TNF-α and inflammatory related protein NLRP3, caspase 1, p-NF-κB were detected by Western blot.Results The results of in vitro experiments were as follows: the activity of damaged cells in OGD/R model was significantly increased by α-asarone, with the increase of administration dose, the cells in the low, medium and high dose groups of α-asarone decreased, and the "amoeba-like" cells and the cell body were gradually became stereoscopic and full.From the results of cell morphology, it could be seen that α-asarone had a certain proliferative effect on normal cells; the release was significantly reduced of proinflammatory factor IL-1β, IL-18 and TNF-α in OGD/R injured BV2 cells pretreated with α-asarone, also increased the release of IL-10, IL-4 and TGF-β, with a dose-effect relationship, and the high dose(16 μmol·L-1)was the best; the expressions of inflammatory related protein NLRP3, caspase 1, NF-κB and ROS activity in injured cells of OGD/R model were significantly reduced after pretreatment with α-asarone.Conclusions α-asarone has a significant protective effect on cerebral ischemia/reperfusion injury, mainly by regulating ROS activity and inhibiting phosphorylation of NF-κB, in order to reduce the excessive activation of NLRP3 inflammatory corpuscles reducing the secretion of proinflammatory factor IL-1β and IL-18, promoting the secretion of anti-inflammatory factor IL-10 and IL-4, so as to protect cerebral ischemia/reperfusion injury by anti-inflammatory reaction.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Effects of casein kinase 2 interacting protein-1 on the osteogenic differentiation ability of human periodontal ligament stem cells / 口腔疾病防治

Objective @#To investigate the effects of casein kinase 2 interacting protein-1 (CKIP-1) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs).@*Methods @#The hPDLSCs were obtained by primary culture with periodontal ligament tissues that were collected from normal humans. Then, a lentiviral vector containing a CKIP-1-specific siRNA sequence was constructed, and the transcriptional level of CKIP-1 in hPDLSCs was downregulated after vector infection. The P4 cells were divided into four groups: the control group, negative control group (infected with a control vector), CKIP-siRNA group (infected by a CKIP-1 siRNA lentivirus) and CKIP-1 group (infected by a CKIP-1 overexpression virus). All of the cells were cultured under osteogenic induction for 21 days. Then, alizarin red staining and quantitative determination were performed to detect the osteogenic differentiation ability of the hPDLSCs. In addition, qPCR was used to detect the transcriptional level of osteogenesis-related regulatory factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL), and the osteogenesis-related regulatory factors of the bone morphogenetic protein (BMP) signaling pathway.@*Results@#There were no differences in the indexes between the negative control group and the control group (P > 0.05). Compared with the negative control group, the CKIP-siRNA group demonstrated more mineralized nodules (P < 0.05), significantly increased calcium salt deposition (P < 0.05), and increased mRNA levels of osteogenesis-related regulatory factors, such as Runx2 , ALP, OCN, and RANKL, and the osteogenesis-related regulatory factors of BMP signaling pathway (P < 0.05). @*Conclusion@#Downregulation of CKIP-1 could promote the osteogenic differentiation of hPDLSCs, which is related to the transcription level of osteogenic-related regulatory factors.

Memory-improving Substances Basis and Mechanism of Polygalae Radix, Acori Tatarinowii Rhizoma and Its Couplet Medicines / 中国实验方剂学杂志

Polygalae Radix and Acori Tatarinowii Rhizoma were first recorded in Shennong's Herbal Classic. Both of them can "improve people's memory". Long-term administration can make body light and macrobian. They have often been used as couplet medicines and the core combination of nootropic and memory improvement prescriptions. At present, traditional Chinese medicine clinicians believes that the principle of Polygalae Radix and Acori Tatarinowii Rhizoma in improving memory or intelligence is to supplement the deficiency, remove phlegm and unblock nine orifices, with sufficient evidences for the traditional theory. However, its material basis and mechanism for improving memory have not been fully elucidated. In this paper, we searched the literatures about pharmacological and pharmacodynamics mechanism of Polygalae Radix,Acori Tatarinowii Rhizoma and their chemical components on nervous system in recent ten years from Pubmed database and CNKI. The main material basis for improving memory of Polygalae Radix-saponins, oligosaccharides and alone, the main material basis for improving memory of Acori Tatarinowii Rhizoma-α-asarone,β-asarone and eugenol, the changes of the quality and quantity of the active substances after combination, and the mechanism of improving memory of the single drugs and their couplet medicines, such as scavenging free radicals, regulating cholinergic system, clearing β-amyloid protein(Aβ), decreasing the level of phosphorylation of Tau protein, improving the rate of apoptosis and regulating synaptic plasticity, were systematically collected, analyzed and summarized. In view of the current research situation, this paper points out the possible shortcomings, with the aim to further explore the mechanism of Polygalae Radix combined with Acori Tatarinowii Rhizoma with the mechanism of "1+1>2".

Simultaneous determination of four constituents in Liujing Toutong Tablets by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of four constituents in Liujing Toutong Tablets (Angelicae dahuricae Radix,Magnoliae Flos,Ligustici Rhizoma et Radix,etc.).METHODS The analysis of 30％ ethanol extract of this drug was performed on a 35 ℃ thermostatic Waters C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of methanol-4％ acetic acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 320 nm.RESULTS Puerarin,ferulic acid,imperatorin and isoimperatorin showed good linear relationships within the ranges of 60.6-303 μg/mL (r=0.999 9),1.59-7.95 μg/mL (r =0.999 9),1.57-7.85 μg/mL (r =0.999 9) and 0.752 5-3.762 5 μg/mL (r =0.999 7),whose average recoveries (RSDs) were 97.75％ (1.7％),97.68％ (2.3％),97.94％ (1.0％) and 98.29％ (1.6％),respectively.CONCLUSION This stable and reliable method can be used for the quality control of Liujing Toutong Tablets.

Mechanism of PC12 cell differentiation induced by total salvianolic acid / 中国药理学与毒理学杂志

OBJECTIVE To study the differentiation of PC12 cells induced by total salvianolic acid (Tsa) and the mechanism. METHODS MTT assay was used to detect the effect of Tsa 0.01, 0.1 and 1.0 μg·L-1on proliferation of PC12 cells and on the cells damaged by oxygen and glucose deprivation (OGD).The number of projections of PC12 cells was statistically analyzed.Western blotting was applied to detect the levels of microtubule-associated protein2 (MAP-2), extracellular signal-regulated kinase1/2 (ERK1/2), phosphorylated ERK1/2(p-ERK1/2), mitogen-activated protein kinase kinase1/2(MEK1/2) and p-MEK1/2 proteins.MEK inhibitor U0126 was examined for its effect on expressions of p-ERK1/2 and ERK1/2 protein in PC12 cells induced by Tsa 1.0 μg·L-1.RESULTS Compared with normal control group, Tsa 1.0 μg·L-1could promote PC12 cell proliferation, and the survival rate was increased by 90%, but the survival rate of PC12 cells was not affected by Tsa 0.01 or 0.1 μg·L-1. Compared with OGD injured group,PC12 cells injured by OGD could be repaired by Tsa 0.1 or 1.0 μg·L-1,and the survival rate was increased to (47.7±1.8)% and (63.2±13.0)%, respectively (P<0.05, P<0.01). Compared with normal control group,Tsa 0.01,0.1 and 1.0 μg·L-1could promote the growth of PC12 cell projections (P<0.01). Western blotting results showed that Tsa could promote the expressions of MAP-2, p-ERK1/2 and p-MEK1/2 proteins, and this effect could be blocked by U0126 inhibitor (P<0.01). CONCLUSION Tsa can induce the proliferation and differentiation of PC12 cells, the mechanism of which is possibly the activation of p-MEK and p-ERK1/2.

Effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism.@*METHODS@#Thirty-six male ICR mice were randomly divided into three groups (=12):sham group, TCP wear particles (TCP) group and N-acetyl-L-cysteine (NAC) group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2nd day post-operation, NAC (1.0 mg/kg) was locally injected to the calvarium under the periosteum every other day for 2 weeks. Then, all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase (TRAP) staining; serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6); total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) activity were examined by ELISA and chemical colorimetry, respectively; protein levels of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α) and phospho-eIF2α (p-eIF2α) in periprosthetic bone tissue were detected by Western blot.@*RESULTS@#Compared with sham group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were increased obviously in TCP group (<0.05), and serum level of T-AOC and SOD activity were decreased significantly in TCP group (<0.05), GRP78 expression, the ratio of p-PERK and PERK, p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were decreased markedly in NAC group (<0.05), serum level of T-AOC and SOD activity were increased obviously in NAC group (<0.05), and GRP78 expression, the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated.@*CONCLUSIONS@#Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles, which may be mediated by inactivation of PERK/eIF2α signaling pathway.

Annual point-prevalence of healthcare-associated infection surveys in a university hospital in China, 2007-2011

Successive point-prevalence surveys were conducted annually from 2007 to 2011 to monitor the prevalence of healthcare-associated infections [HAIs] in a university hospital in Hubei Province in China. The surveys used the case definition criteria established by the Ministry of Health of the People's Republic of China. In the 5 surveys, the overall frequency of HAIs was 3.16% [301/9533]. No significant differences were identified in the point prevalence measurements of HAIs in any of the years from 2007 to 2011. Of all the cases, proportionally, the most frequent infection site was the respiratory tract [2.34%], followed by surgical sites [0.43%] and urinary tract sites [0.28%]. Gram-negative aerobic bacilli were the most common organisms mentioned; the most frequently isolated organism was Pseudomonas aeruginosa, followed by Escherichia coliand Acinetobacter baumannii. Approximately one-half of the patients were receiving antibiotics at the time of the surveys. Cephalosporin, penicillin, and quinolone were most commonly used for treatment or prevention. The differences found in HAI prevalence data across the 5 surveys given in the hospital were not statistically significant. In conclusion, this successive point-prevalence survey provides information about the trend of HAI prevalence, epidemical character, and the use of antibiotics among the university hospital's in-patients. This information allows us to initiate targeted programs for infection prevention and control