[Identification of chemical components and network pharmacology of Huanglian Decoction based on UPLC-Q-TOF-MS/MS technology].

The chemical components of Huanglian Decoction were identified by ultra-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) technology. The gradient elution was conducted in Agilent ZORBAX Extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the MS data were collected under the scanning range of m/z 100-1 500. Through high-resolution MS data analysis, combined with literature comparison and confirmation of reference substances, this paper identified 134 chemical components in Huanglian Decoction, including 12 alkaloids, 23 flavonoids, 22 terpenes and saponins, 12 phenols, 7 coumarins, 12 amino acids, 23 organic acids, and 23 other compounds, and the medicinal sources of the compounds were ascribed. Based on the previous studies, 7 components were selected as the index components. Combined with the network pharmacology research and analysis me-thods, the protein and protein interaction(PPI) network information of the intersection targets was obtained through the STRING 11.0 database, and 20 core targets of efficacy were screened out. In this study, UPLC-Q-TOF-MS/MS technology was successfully used to comprehensively analyze and identify the chemical components of Huanglian Decoction, and the core targets of its efficacy were discussed in combination with network pharmacology, which laid the foundation for clarifying the material basis and quality control of Huanglian Decoction.

[Identification of chemical components based on UPLC-Q-TOF-MS/MS and network pharmacology of Zhuru Decoction].

This study analyzed the main chemical components of Zhuru Decoction via ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS), and then predicted the mechanism of Zhuru Decoction in clearing heat, resolving phlegm, detoxifying, and treating vomiting and alcohol-related vomiting caused by heat in stomach based on network pharmacology. The gradient elution was conducted in Agilent ZORBAX extend-C_(18) column(2.1 mm×100 mm, 1.8 µm) with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B) at a flow rate of 0.3 mL·min~(-1) and the column temperature of 35 ℃. The MS adopted the positive and negative ion mode of electrospray ionization(ESI), and the data were collected in the scanning range of m/z 100-1 500. A total of 98 compounds in Zhuru Decoction were identified via BATMAN, SYMMAP, TCMSP, and relevant literature, including 36 flavonoids, 7 triterpenoids, 8 gingerols, 20 organic acids, 5 amino acids, and 22 other compounds. On the basis of the available studies, 9 components were selected as index components, and the protein-protein interaction(PPI) network of the common targets was established with STRING 11.0. Finally, 10 core targets associated with the pharmacodynamic effect were screened out. This study established the UPLC-Q-TOF-MS/MS method for identifying the chemical components in the classic prescription Zhuru Decoction, and employed network pharmacology to explore the core targets of its efficacy, which provided a refe-rence for the quality control and the research of the pharmacodynamic substances of Zhuru Decoction.

[Study on characteristic chromatogram and content determination of Wuzhuyu Decoction reference sample].

In this study, the critical quality attributes of Wuzhuyu Decoction reference sample were explored by using characteristic chromatogram, index component content and dry extract rate as indexes.The dissemination relationship of quantity value between medicinal materials-decoction pieces-reference sample was investigated to preliminarily formulate the quality standard of the reference sample.The characteristic chromatogram of 15 batches of Wuzhuyu Decoction was established by high performance liquid chromatography(HPLC) and the similarity analysis was conducted.Common peaks were demarcated and assigned to medicinal materials.Moreover, quantitative determination of limonin, evodiamine, rutaecarpine and ginsenoside Rb_1 of Wuzhuyu Decoction were performed.The dissemination of quantity value was explored combined with dry extract rate, similarity of characteristic chromatogram and transfer rate of index component content.A total of 18 common peaks were identified in the corresponding materials of Wuzhuyu Decoction reference sample, with the similarity of characteristic chromatogram greater than 0.9, and Fructus Evodiae, Radix Ginseng, Rhizoma Zingiberis Recens and Fructus Jujubae contributed 9, 5, 8 and 2 chromatographic peaks, respectively.The index component content of corresponding materials and the transfer rates of medicinal materials-decoction pieces and decoction pieces-reference sample of different batches of Wuzhuyu Decoction reference sample were as follows: the content of limonin was 0.16%-0.51%, and the transfer rates were 83.66%-115.60% and 38.54%-54.58%, respectively; the content of evodiamine was 0.01%-0.11%, the transfer rated were 80.80%-116.15% and 3.23%-12.93%, respectively; the content of rutaecarpine was 0.01%-0.05%, the transfer rates were 84.33%-134.53% and 5.72%-21.24%, respectively; the content of ginsenoside Rb_1 was 0.06%-0.11%, and the transfer rates were 90.00%-96.92% and 32.45%-67.24%, respectively.The dry extract rate of the whole prescription was 22.58%-29.89%.In this experiment, the dissemination of quantity value of Wuzhuyu Decoction reference sample was analyzed by the combination of characteristic chromatogram, index component content and dry extract rate.A scientific and stable quality evaluation method of the reference sample was preliminarily established, which provided basis for the subsequent development of Wuzhuyu Decoction and the quality control of related preparations.

[Quality value transmitting of substance benchmarks of Zhuru Decoction].

A total of 18 batches of Zhuru Decoction samples were prepared. Chromatographic fingerprints were established for Zhuru Decoction and single decoction pieces, the content of which was then determined. The extraction rate ranges, content, and transfer rate ranges of puerarin, liquiritin, and glycyrrhizic acid, together with the common peaks and the similarity range of the fingerprints, were determined to clarify key quality attributes of Zhuru Decoction. The 18 batches of Zhuru Decoction samples had 25 common peaks and the fingerprint similarity higher than 0.95. Puerariae Lobatae Radix, Glycyrrhizae Radix et Rhizoma, and Zingiberis Rhizoma Recens had 21, 3, and 1 characteristic peaks, respectively. The 18 batches of samples showed the extraction rates within the range of 18.45%-25.29%. Puerarin had the content of 2.20%-3.07% and the transfer rate of 38.5%-45.9%; liquiritin had the content of 0.24%-0.85% and the transfer rate of 15.9%-37.5%; glycyrrhizic acid had the content of 0.39%-1.87% and the transfer rate of 16.2%-32.8%. In this paper, the quality value transmitting of substance benchmarks of Zhuru Decoction was analyzed based on chromatographic fingerprints, extraction rate, and the content of index components. A scientific and stable method was preliminarily established, which provided a scientific basis for the quality control and formulation development of Zhuru Decoction.

[Quality value transfer of substance benchmarks in Huanglian Decoction].

Following the preparation of substance benchmarks in Huanglian Decoction from 18 batches, the method for detecting their characteristic spectra was established to identify the similarity range and peak attribution. The content and transfer rate ranges of the index components coptisine, palmatine, berberine, liquiritin, glycyrrhizic acid, 6-gingerol, and cinnamaldehyde and the extraction amount were combined for analyzing the quality value transfer from the Chinese medicinal pieces to substance benchmarks and clarifying the key quality attributes of substance benchmarks in Huanglian Decoction. The results showed that the substance benchmarks in Huang-lian Decoction of 18 batches exhibited good similarity in characteristic spectra(all greater than 0.98). There were 17 characteristic peaks identified in the substance benchmarks of Huanglian Decoction, including 10 from Coptidis Rhizoma, 3 from Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle(processed with water), 1 from Zingiberis Rhizoma, and 3 from Cinnamomi Ramulus. The contents and average transfer rates of the index components were listed as follows: coptisine 2.20-6.46 mg·g~(-1) and 18.50%±2.93%; palmatine 3.03-8.13 mg·g~(-1) and 26.56%±4.69%; berberine 7.71-22.29 mg·g~(-1) and 17.34%±3.00%; liquiritin 0.88-2.18 mg·g~(-1) and 9.88%±4.88%; glycyrrhizic acid 1.83-4.44 mg·g~(-1) and 8.50%±3.72%; 6-gingerol 0.56-1.43 mg·g~(-1) and 11.36%±2.37%; cinnamaldehyde 1.55-3.48 mg·g~(-1) and 19.02%±4.36%. The extraction amount of the substance benchmarks from the 18 batches was controlled at 10.65%-13.88%. In this paper, the quality value transfer of substance benchmarks in Huanglian Decoction was analyzed based on the characteristic spectra, the index component contents and the extraction amount, which has provided a basis for the subsequent development of Huanglian Decoction and the quality control of its related preparations.

[Mechanism of "herb soaking with exact amount of water" during moistening process of ginseng based on needle pressure sensor].

In this study, the texture analyzer acupuncture pressure sensor was used to objectively characterize the "herb soaking with exact amount of water" for moistening process of ginseng. The single factor rotation experiment was used to investigate the effects of puncture speed, puncture depth and puncture site on puncture force and work. According to ginseng processing method in Chinese Pharmacopoeia, ginseng medicinal materials with diameters of about 1 cm and 2 cm were selected, and puncture experiments were carried out at the set measurement time to determine the hardness, work and water absorption of the ginseng moistening process. The endpoint threshold for the ginseng softening process was determined and verified. To reflect the actual internal conditions of the ginseng softening process, the puncture depth was preferably 70%, and the puncture speed was 30 mm·min~(-1). In the ginseng moistening process, the softening hardness and the puncture work were in accordance with the first-order kinetic equation y=a×exp(-k×x). The 0 h initial hardness a of 1 cm and 2 cm ginseng herbs were 289.8 N and 1 227 N, and the rate constants K were 0.149 4 N·h~(-1) and 0.100 7 N·h~(-1), respectively. After the ginseng was completely softened, the force required for puncture was 10 N, which can be used as the standard for "drug penetration". At this time, the water absorption rate of ginseng was 70%-100%. The softening time of ginseng with a diameter of 1 cm was about 20-22 h, and the softening time of ginseng with a diameter of 2 cm was about 40-46 h. A needle-type pressure sensor was used to accurately determine the end point of the softening process of ginseng and reduce the loss of active ingredients. The study results provide reference for the softening process kinetics and the process intelligent monitoring of other dried roots and rhizomes.

YiQi Tongluo Granule against Cerebral Ischemia/Reperfusion Injury in Rats by Freezing GluN2B and CaMK II through NMDAR/ERK1/2 Signaling.

Yiqi Tongluo Granule (YQTL) is a kind of proprietary Chinese medicine, manufactured by China Shineway Pharmaceutical Group Ltd., under the authority of China Food and Drug Administration (CFDA) treating cardiovascular and cerebrovascular diseases such as ischemic stroke in China, however the underlying mechanism of YQTL on treating ischemic stroke has not been revealed. This study is aimed to evaluate the protective effect of YQTL on cerebral ischemia/reperfusion (I/R) injury and inquire into its underlying mechanisms. Cerebral I/R injury was induced by occluding the middle cerebral artery for 2 h followed by 24 h reperfusion. And regional cerebral flow was monitored by Laser Doppler flow during ischemia phase. The infarct volume was evaluated by Triphenyte-trazolium chloride staining. The protective effects of YQTL were assessed by a number of parameters, including neurological scores, regional cerebral blood flow, pathological changes of neuron in hippocampuses and hippocampus calcium level. The proteins of extracellular signal-regulated kinase (ERK), N-methyl D-aspartate receptor subtype 2B (GluN2B) and p-calcium-dependent protein kinaseII (CaMKII) response were assayed by Western blotting. I/R caused significant change in neurological deficit scores, regional cerebral flow and infarct volume. However results in YQTL groups and Nimodipine Tablets (NMDP) group were reversed. Subsequently YQTL reduced I/R-induced calcium influx. Results of hematoxylin-eosin staining manifested that YQTL significantly improved neuronal injury after I/R in rats. Meanwhile, microdialysis data demonstrated that extracellular glutamate was increased in the striatum during ischemia reperfusion, which was reduced by YQTL. YQTL and mitogen-activated protein extracellular kinase (MEK) inhibitor suppressed the I/R-mediated over-expression of GluN2B, p-ERK, ERK and p-CaMKII proteins expression. Putting these together, our results suggest that YQTL played a neuroprotective role in cerebral I/R injury, which might be exerted by inhibiting the excitotoxicity and expression of GluN2B, p-CaMKII and MEK/ERK signal pathway.

[Calcium mobilizing effect of hawthorn leaf procyanidins in vascular endothelial cells].

The hawthorn leaves have the effect of activating blood, removing blood stasis, regulating qi through the veins, dissolving turbidity and lowering lipid. Procyanidinis is one of its main active components and plays an important role in regulating vasoactivity. Previous studies showed that the regulating effect of procyanidins was related to its regulation on nitric oxide secretion from vascular endothelial cells, and this effect was dependent on the extracellular calcium concentration, suggesting that the changes in intracellular calcium ion concentration in endothelial cells may play a key role in this process. However, the research on this issue is still insufficient so far. This study is aimed to observe the effect of hawthorn leaf oligomeric procyanidins (HLP) on calcium mobilization of vascular endothelial cells, and investigate the underlying mechanism. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and labeled with Fura-2. HUVEC were treated with HLP at concentrations of 6.25, 12.5, 25 and 50 mg·L⁻¹, and the intracellular calcium concentrations were measured with a living cell microscope for 30 min. HLP increased the intracellular calcium concentration of HUVEC in a concentration dependent manner; and the intracellular calcium concentrations in 25 and 50 mg·L⁻¹ HLP groups were significantly higher than that in the normal group. With the use of calcium-free incubation buffer, addition of calcium chelating agent EGTA in incubation buffer, or use of inhibitors for sodium calcium exchanger, the effect of HLP was significantly inhibited. On the other hand, the effect of HLP could also be weakened by inhibiting the calcium release from the intracellular storage. In conclusion, these results suggest that HLP can elicit calcium mobilization in vascular endothelial cells, which may be one of the mechanisms for its vascular modulatory activity; and this calcium mobilizing effect may be achieved through promoting both extracellular calcium influx and intracellular calcium release, additionally the former may be related to activating the reverse transport of Na⁺-Ca²âº exchangers on the cell membrane.

[A network pharmacology approach to explore mechanisms of Buyang Huanwu decoction for treatment of cerebral infarction].

The point of this study is to explore and investigate mechanisms of Buyang Huanwu decoction for treatment of cerebral infarction (CI) using a network pharmacology approach. First, TCMSP database, DrugBank database and PharmMapper server were used and combined with oral bioavailability and drug analysis to screen the components of Buyang Hanwu decoction and predict the potential targets. Then, Cytoscape 3.5.1 software was used to construct compounds-targets network and the protein-protein interaction (PPI) networks for targets of compounds and CI-related targets and merge the two PPI networks to acquire active targets. Finally, gene ontology (GO) and KEGG pathway analysis of active targets were carried out by DAVID online analysis tool and KOBAS 3.0 software. In total of 150 screened compounds and 232 potential targets were obtained. And in total of 208 active targets were finally determined by merging networks. Results indicated that Buyang Huanwu decoction might have a role in treating CI by regulating some biological processes including response to drug, aging, response to hypoxia, and blood coagulation, and some molecular function, such as protein binding, enzyme binding and serine-type endopeptidase activity. The mechanisms might be concerned with PI3K-Akt signaling pathway, TNF signaling pathway, HIF-1 signaling pathway and cAMP signaling pathway. Among them, the regulation of PI3K-Akt signaling pathway might be one of the most crucial mechanisms.

[Effect of Guanmaitong Tablet on ERK and p38 Protein of TLR2 Pathway Expression in Cerebral Ischemia/Reperfusion Rats: an Experimental Study].

OBJECTIVE: To explore the inflammatory cascade mechanism through Toll like receptor 2 (TLR2) pathway after cerebral ischemia/reperfusion, and to study molecular mechanisms of Guanmaitong (GMT) Tablet for protecting brain damage. METHODS: We used bolt-line method to block/release the middle cerebral artery, causing cerebral ischemia/reperfusion (I/R) injury model. GMT Tablet was given by gastrogavage. Rats were then divided into the high dose GMT group (1200 mg/kg), the middle dose GMT group (600 mg/kg), the low dose GMT group (300 mg/kg), the positive control group (Tanakan, 20 mg/kg). Their right brain tissues were fixed in 10% neutral formalin. TLR2 expressions were detected by immunofluorescence staining. The total protein was extracted from right brain tissues by ultrasonica- tion. Expression levels of extracellular regulated protein kinases (ERK), phospho-extracellular regulated protein kinases (p-ERK), p38-mitogen activated protein kinases (p-ERK), phospho-p38-mitogen activated protein kinases [p-p38-MAPKs(p-p38)] were assessed by Western blot. Abdominal aortic blood was withdrawn. IL-6 and IL-1ß levels were detected by ELISA in brain tissues and serum. RESULTS: Compared with the sham-oepration group, expression levels of TLR2, ERK, p-ERK, p38, p-p38 protein were up-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1ß in brain tissues and serum were increased in the model group (P < 0.01). Expression levels of TLR2, ERK, p-ERK, p38, p-p38 were down-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1ß were reduced in brain tissues and serum in middle and high dose GMT groups (P < 0.05, P < 0.01). CONCLUSIONS: TLR2 pathway was involved in cerebral I/R injury. GMT protected neurons by down-regulating protein expressions of TLR2, ERK, p-ERK, p38, p-p38 and contents of IL-1ß and IL-6.

[Advance in prevention and treatment of ischemia cardio-cerebrovascular disease through increased therapeutic angiogenesis induced by traditional Chinese medicine].

Remaining organic and functional damage of ischemia cardio-cerebrovascular disease is always a main trouble puzzling the clinicians. After the discovery of endothelial progenitor cells (EPCs), researchers realize that postnatal angiogenesis is an important biological process, which play a key role to repair the ischemia tissue and improve the function. So a new concept which names therapeutic angiogenesis supply a new treament way for the ischemia cardio-cerebrovascular disease. Traditional Chinese medicine (TCM) has accumulated rich experience on treating the ischemia disease, studies found that many Chinese medicine prescriptions and effective ingredients can increase the therapeutic angiogenesis, howerer the mechanisms were not the same, they mainly manifest in regular the secretion of angiogenic factors, increase the proliferation and differentiation etc. In this paper, we review recent studies, summary the Chinese medicine prescriptions and effective ingredients which can increase the therapeutic angiogenesis, and analyze the differernt pathway. We view to provide reference for the later researchers.

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway.

AIM: To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI). METHODS: Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg(-1)·d(-1), ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg(-1)·d(-1), ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting. RESULTS: Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg(-1)·d(-1)) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 µmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002. CONCLUSION: Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.

[Intervention effect of quercetin on inflammatory secretion of cardiac fibroblasts].

To establish neonatal rat cardiac fibroblast inflammatory secretion model by using LPS 100 microg x L(-1) combined with ATP 5 mmol x L(-1), in order to study the inhibitory effect of quercetin on the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts, further investigate the effect of quercetin on the protein expression of p-NF-kappaB p65 (S276) and p-Akt (S473) by western blot, and discuss the inhibitory effect of quercetin on the inflammatory secretion of cardiac fibroblasts. According to the findings, quercetin with the concentrations between 51.74 micromol x L(-1) and 827.81 micromol x L(-1) had no significant effect on the activity of cardiac fibroblasts. Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of TNF-alpha and IL-1beta induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.01 or P < 0.05). Quercetin with the concentrations of 82.78, 41.39 micromol x L(-1) could notably inhibit the increase of IL-6 induced LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 36 h (P < 0.05), without any notable effect of quercetin with the concentration of 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20. 70 micromol x L(-1) could notably inhibit the NF-kappaB p65 (S276) activation induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 15 min, with the most significant effect in 20.70 micromol x L(-1). Quercetin with the concentrations of 82.78, 41.39, 20.70 micromol x L(-1) could notably inhibit the increase of p-Akt(473) expression induced by LPS 100 microg x L(-1) for 3 h and then ATP 5 mmol x L(-1) for 240 min (P < 0.05). Therefore, this study believes that quercetin could attenuate the secretion of inflammatory factors TNF-alpha, IL-1beta and IL-6 of cardiac fibroblasts by inhibiting the activation of NF-kappaB p65 (S276) and Akt (473).

[Protective effect of formula of removing both phlegm and blood stasis on myocardial tissues of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome].

OBJECTIVE: To study the protective effect of formula of removing both phlegm and blood stasis (TYTZ) on myocardial tissues of Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. METHOD: Totally 36 Chinese mini-swine were randomly divided to six groups: the normal control group, the model group, the Danlou tablet group, and TYTZ groups with doses of 2.0, 1.0, 0.5 g x kg(-1), with six in each group. Except for the normal control group, all of other groups were fed with high-fat diet for 2 weeks. Interventional balloons are adopted to injure their left anterior descending artery endothelium. After the operation, they were fed with high-fat diet for 8 weeks to prepare the coronary heart disease model of phlegm-stasis cementation syndrome in Chinese mini-swine. After the operation, they were administered with drugs for 8 weeks. The SOD activity and MDA content of each group were observed at the 0th week (before the experiment), the 2nd week after the high-fat diet (before the operation or drug administration) , the 6th week after the high-fat diet (4 weeks after the drug administration) and the 10th week after the high-fat diet (8 weeks after the drug administration). Meanwhile, the myocardial enzymogram test and the HE staining pathological observation were performed at the end of the experiment. The changes in the myocardial cell ultra-structure were observed under transmission electron microscope. RESULT: Compared with the normal control group, the model group showed significant decrease in serum SOD activity and notable increase in MDA content from the 2nd week to the end of experiment (P < 0.05 and P < 0.01). In the 10th week, the CK, LDH and CK-MB levels in serum also significantly increased in the model group (P < 0.05 and P < 0.01), with obvious structural abnormality in myocardial tissue pathologic morphology and ultra-structure. Compared with the model group, TYTZ groups showed specific increase in serum SOD activity and oblivious decrease in the MDA level (P < 0.05 or P < 0.01). Meanwhile, TYTZ could significantly decrease serum CK and LDH levels in the model group (P < 0.05 or P < 0.01), attenuate the ischemia injury of myocardial tissue, and improve the ultra-structure of cardiomyocytes. CONCLUSION: TYTZ shows an obvious protective effect on the myocardial injury in Chinese mini-swine with coronary heart disease of phlegm-stasis cementation syndrome. Its mechanism is related to the resistance against free radical oxidation injury and the inhibition of the lipid per-oxidation.

[Sacrococcygeal gap injection for the treatment of failed back surgery syndrome].

OBJECTIVE: To explore the clinical effect of the sacrococcygeal space injection for the treatment of failed back surgery syndrome. METHODS: From July 1998 to October 2012,47 patients with failed back surgery syndrome were treated and included 39 males and 8 females with an average age of 61.5 years old ranging from 35 to 89 years old. Among them,41 patients experienced one time of operation, 6 patients with twice of operation. Forty-one patients underwent single,bilateral fenestration or central laminectomy decompression, discectomy. Six patients underwent total laminectomy discectomy and inter body fusion and pedicle screw fixation. All patients were examined by X-ray plain film, CT or MRI before treatment. The anticoagulation was discontinuation before treatment. The needle was put into the sacrococcygeal gap at prone position in the sense of frustration,suction without cerebrospinal fluid and blood,with injection of Mailuoning (Chinese characters: see text) 15 ml. The pain was assessed by VAS before and after treatment. The Oswestry low back pain disability index and survival quality interference degree were evaluated. RESULTS: At 1 month after treatment,the pain VAS decreased from 59.24 +/- 17.35 before treatment to 19.19 +/- 11.19 after treatment (P < 0.05); The Oswestry low back pain disability index decreased from (41.35 +/- 9.87)% before treatment to (23.17 +/- 17.56)% after treatment (P < 0.05); The survival quality interference degree decreased from 6.5 +/- 2.2 before treatment to 2.6 +/- 1.4 after treatment (P < 0.05). CONCLUSION: The sacrococcygeal gap injection for treatment of failed back surgery syndrome has advantages of simple, safe, fewer complications, and low treatment cost.

[Compatibility of geniposide and ginsenoside rgl: their regulating roles in secretion of anoxia induction injured microglia inflammatory cytokines].

OBJECTIVE: To clarify the protective roles of compatibility of geniposide and ginsenoside (Rg1) in regulating ischemia injured microglia homeostasis by comparing the difference in regulatory roles of geniposide, Rg1, or ginsenoside + Rg1 in balancing secretion of oxygen glucose deprivation induced microglia inflammatory cytokines. METHODS: The mimic ischemia injured microglia model was induced by oxygen-glucose deprivation (OGD). Then geniposide, Rg1, or ginsenoside + Rg1 (Tongluo Jiunao Injection, TJI) was respectively added. The NO content was determined by Griess Reagent. The cyto activity was detected using cell count kit. Contents of TNF-alpha and TGF-beta and their expression levels were detected by ELISA and Western blot. RESULTS: Geniposide + Rg1 could significantly inhibit the release of NO, elevate the TGF-beta level, and decrease the content of TNF-alpha without influencing the cell survival. The two active ingredients played different therapeutic roles. The compatible use was obviously superior to use any one of the two active ingredients alone. CONCLUSIONS: Geniposide, Rg1, or Ginsenoside + Rg1 had regulating roles in balancing ischemia injured microglia homeostasis. Its mechanisms might be related to up-regulating the TGF-beta expression and down-regulating TNF-alpha expression.

Jolkinolide B induces apoptosis in MCF-7 cells through inhibition of the PI3K/Akt/mTOR signaling pathway.

The aim of this study was to explore the molecular mechanisms of jolkinolide B (JB), which is extracted from the root of Euphorbia fischeriana Steud. In this study, we found that JB, a diterpenoid from the traditional Chinese medicinal herb, strongly inhibited the PI3K/Akt/mTOR signaling pathway. Furthermore, we evaluated the effects of JB on the proliferation and apoptosis of MCF-7 human breast cancer cells. Our results showed significant induction of apoptosis in MCF-7 cells incubated with JB. The viability of the MCF-7 cells was assessed by MTT assay. Flow cytometry was used to detect apoptosis and cell cycle analysis. Transmission electron microscopy (TEM) analysis was used to observe cell morphology. MCF-7 cells were subcutaneously inoculated into nude mice to study the in vivo antitumor effects of JB. The growth of MCF-7 cells was inhibited and arrested in the S phase by JB. The data showed significantly decreased tumor volume and weight in nude mice inoculated with MCF-7 cells. In addition, treatment with JB was able to induce downregulation of cyclinD1, cyclinE, mTOR, p-PI3K and p-Akt, and upregulation of PTEN and p-eIF4E. Collectively, JB-induced apoptosis of MCF-7 cells occurs through the PI3K/Akt/mTOR signaling pathway. Furthermore, the PI3K/Akt signaling cascade plays a role in the induction of apoptosis in JB-treated cells. These observations suggest that JB may have therapeutic applications in the treatment of cancer.

[Protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury].

To establish cardiomyocyte hypoxia/reoxygenation injury model by culturing primary cardiomyocytes from suckling SD rats, in order to study the effect of succinic acid on LDH leakage rate cardiomyocyte ischemia/reperfusion injury. Furthermore, flow cytometry and western blot were conducted to detect the effect of succinic acid on cardiomyocyte apoptosis, cleaved caspase-3 and p-Akt, and discuss the protective effect of succinic acid on primary cardiomyocyte hypoxia/reoxygenation injury of primary cardiomyocytes from neonatal SD rats. According to the findings of the study, succinic acid at the concentrations ranging between 31.25 mg x L(-1) and 500 mg x L(-1) had no significant effect on primary cardiomyocyte activity, and succinic acid at the concentrations of 400, 200, 100, 50 mg x L(-1) could notably reduce cardiomyocyte ischemia/reperfusion LDH leakage rate (P < 0.01 or P < 0.05, respectively). Succinic acid at the concentrations of 400 mg x L(-1) and 200 mg x L(-1) could significantly reduce the percentage of cardiomyocyte apoptosis (P < 0.05), and inhibit the protein expression of cleaved caspase-3 caused by cardiomyocyte ischemia/reperfusion (P < 0.05). Succinic acid at the concentration of 400 mg x L(-1) could remarkably increase the protein expression of cardiomyocyte Akt (P < 0.05), while succinic acid at the concentration of 200 mg x L(-1) had no obvious effect on the protein expression of cardiomyocyte Akt. Therefore, this study demonstrated that succinic acid could inhibit necrosis and apoptosis caused by cardiomyocyte hypoxia/reoxygenation by activating Akt phosphorylation.

Herbal Extracts Combination (WNK) Prevents Decline in Spatial Learning and Memory in APP/PS1 Mice through Improvement of Hippocampal Aß Plaque Formation, Histopathology, and Ultrastructure.

To investigate the cognitive enhancement effect of WNK, an extracts combination of P. ginseng, G. biloba, and C. sativus L. and possible mechanisms, 5-month-old APP/PS1 transgenic mice were used in this study. After 3 months of administration, all mice received Morris water maze (MWM) training and a probe test. Mouse brain sections were detected by immunohistochemistry, HE staining, and transmission electron microscopy. MWM results showed significant difference between transgenic mice and nontransgenic littermates (P < 0.05, P < 0.01). WNK-treated mice exhibited enhanced maze performance over the training progression, especially better spatial memory retention in probe test compared to transgenic mice (P < 0.05, P < 0.01) and better spatial learning and memory at the fourth day of MWM test compared to EGB761- (G. biloba extract-) treated ones (P < 0.05). Hippocampal Aß plaque burden significantly differed between APP/PS1 and littermate mice (P < 0.001), while decreased Aß plaque appeared in WNK- or EGB761-treated transgenic brains (P < 0.05). Neurodegenerative changes were evident from light microscopic and ultrastructural observations in transgenic brains, which were improved by WNK or EGB761 treatment. These data indicate WNK can reduce the decline in spatial cognition, which might be due to its effects on reducing Aß plaque formation and ameliorating histopathology and ultrastructure in hippocampus of APP/PS1 mouse brain.

Neuroprotective effects of tongluojiunao in neurons exposed to oxygen and glucose deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongluojiunao (TLJN) is an herb extract that mainly contains ginsenoside Rg1 and geniposide, which are clinically used for treating ischemic damages in the brain. AIM OF THE STUDY: In the stroke, cerebral ischemia followed by oxygen reperfusion induced apoptosis in hippocampal neurons, while extension of axons and dendrites in neurons may compensate for and repair damages of neuronal network in the hypoxia brain. In this study, we investigated whether TLJN can protect neurons against damages by ischemia in brain vasculature. MATERIALS AND METHODS: We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons before and after the neurons were deprived of oxygen and glucose (OGD). In addition, the effects were evaluated with cell viability and neurite outgrowth before or after OGD. RESULTS: We found that TLJN could play a neuroprotective role to cultured primary rat hippocampal neurons under both normal and oxygen/glucose-deprivation (OGD) conditions. TLJN could protect both cultured primary rat hippocampal neurons and brain microvascular endothelial cells (BMECs) from cell death under both normal and oxygen/glucose-deprivation (OGD) conditions. Moreover, under the same conditions, BMECs-conditioned media pretreated by TNJN could also promote neuron viability and neurite outgrowth, indicating that TLJN stimulated BMECs to secret some neuroprotective/neurotrophic factors. CONCLUSION: These findings suggest that TLJN has a marked neuroprotective and neurotrophic roles by either direct or indirect operation, and provide insight into the mechanism of clinical efficacy of this drug against stroke.