Neuroprotective effect of tetramethylpyrazine on mice after spinal cord injury / 中国中药杂志

This study aims to investigate the neuroprotective effect of tetramethylpyrazine on mice after spinal cord injury and its mechanism. Seventy-five female C57BL/6 mice were randomly divided into 5 groups, namely, a sham operation group, a model group, a tetramethylpyrazine low-dose group(25 mg·kg~(-1)), a tetramethylpyrazine medium-dose group(50 mg·kg~(-1)), and a tetramethylpyrazine high-dose group(100 mg·kg~(-1)), with 15 mice in each group. Modified Rivlin method was used to establish the mouse model of acute spinal cord injury. After 14 d of tetramethylpyrazine intervention, the motor function of hind limbs of mice was evaluated by basso mouse scale(BMS) and inclined plate test. The levels of inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) in the spinal cord homogenate were determined by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe the histology of the spinal cord, and Nissl's staining was used to observe the changes in the number of neurons. Western blot and immunofluorescence method were used to detect the expression of glial fibrillary acidic protein(GFAP) and C3 protein. Tetramethylpyrazine significantly improved the motor function of the hind limbs of mice after spinal cord injury, and the BMS score and inclined plate test score of the tetramethylpyrazine high-dose group were significantly higher than those of the model group(P<0.01). The levels of TNF-α, IL-6, and IL-1β in spinal cord homogenate of the tetramethylpyrazine high-dose group were significantly decreased(P<0.01). After tetramethylpyrazine treatment, the spinal cord morphology recovered, the number of Nissl bodies increased obviously with regular shape, and the loss of neurons decreased. As compared with the model group, the expression of GFAP and C3 protein was significantly decreased(P<0.05,P<0.01) in tetramethylpyrazine high-dose group. In conclusion, tetramethylpyrazine can promote the improvement of motor function and play a neuroprotective role in mice after spinal cord injury, and its mechanism may be related to inhibiting inflammatory response and improving the hyperplasia of glial scar.

Protective mechanism of tetramethylpyrazine on cardiovascular system / 中国中药杂志

Tetramethylpyrazine is the main component of Ligusticum chuanxiong. Studies have found that tetramethylpyrazine has a good protective effect against cardiovascular diseases. In the heart, tetramethylpyrazine can reduce myocardial ischemia/reperfusion injury by inhibiting oxidative stress, regulating autophagy, and inhibiting cardiomyocyte apoptosis. Tetramethylpyrazine can also reduce the damage of cardiomyocytes caused by inflammation, relieve the fibrosis and hypertrophy of cardiomyocytes in infarcted myocardium, and inhibit the expansion of the cardiac cavity after myocardial infarction. In addition, tetramethylpyrazine also has a protective effect on the improvement of familial dilated cardiomyopathy. Besides, the mechanisms of tetramethylpyrazine on blood vessels are more abundant. It can inhibit endothelial cell apoptosis by reducing oxidative stress, maintain vascular endothelial function and homeostasis by inhibiting inflammation and glycocalyx degradation, and protect vascular endothelial cells by reducing iron overload. Tetramethylpyrazine also has a certain inhibitory effect on thrombosis. It can play an anti-thrombotic effect by reducing inflammatory factors and adhesion molecules, inhibiting platelet aggregation, and suppressing the expression of fibrinogen and von Willebrand factor. In addition, tetramethylpyrazine can also reduce the level of blood lipid in apolipoprotein E-deficient mice, inhibit the subcutaneous deposition of lipids, inhibit the transformation of macrophages into foam cells, and inhibit the proliferation and migration of vascular smooth muscle cells, thereby reducing the formation of atherosclerotic plaque. In combination with network pharmacology, the protective mechanism of tetramethylpyrazine on the cardiovascular system may be mainly achieved through the regulation of phosphatidylinositol 3 kinase/protein kinase B(PI3K/Akt), hypoxia-inducible factor 1(HIF-1), and mitogen-activated protein kinase(MAPK) pathways. Tetramethylpyrazine hydrochloride and sodium chloride injection has been approved for clinical application, but some adverse reactions have been found in clinical application, which need to be paid attention to.

Study on protective effects of twin drugs of tetramethylpyrazine-scutellarein on cerebral ischemia-reperfusion injury model rats and its mechanism / 中国药房

OBJECTIVE To study the protective effects of twin drugs of tetramethylpyrazine-scutellarein （TMSC4） on cerebral ischemia-reperfusion injury （CIRI） model rats and its mechanism. METHODS One hundred and five SD rats were randomly divided into sham operation group， model group， scutellarein group （0.7 mmol/kg）， tetramethylpyrazine group （0.7 mmol/kg）， and TMSC4 low-dose， medium-dose and high-dose groups （0.35， 0.7， 1.4 mmol/kg）， with 15 rats in each group. Sham operation group and model group were given constant volume of normal saline intragastrically， and other groups were given relevant drug intragastrically， once a day， for consecutive 14 d. Except for sham operation group， all other groups were treated to establish the CIRI model using the thread occlusion method. After 2 hours of ischemia and 22 hours of reperfusion， the brain index and brain water content of the rats were measured. Serum levels of interleukin 1β （IL-1β）， IL-6 and tumor necrosis factor α （TNF-α）， the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px） and catalase （CAT） in brain tissues， the situation of neuronal cell apoptosis， and the protein expressions of B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved-caspase-3 were evaluated. RESULTS Compared with sham operation group， the brain index， brain water content， the serum levels of IL-1β， IL-6 and TNF-α， the levels of MDA in brain tissues， the brain cell apoptosis and the protein expressions of Bax and cleaved-caspase-3 in model group were significantly increased （P＜0.05）； the levels of SOD， GSH- Px and CAT and the protein expression of Bcl-2 in brain tissues were significantly decreased （P＜0.05）. Compared with model group， the above indexes of rats were reversed significantly in administration groups （P＜0.05）， while the reverse effects of TMSC4 medium-dose and high-dose groups were significantly better than those of scutellarein group and tetramethylpyrazine group （P＜0.05）. CONCLUSIONS TMSC4 has a certain protective effect in CIRI model rats， the mechanism of which may be related to relieving inflammatory reaction and oxidative stress， inhibiting cell apoptosis.

TBN improves motor function and prolongs survival in SOD1G93A and TDP-43M337V mouse model of ALS / 中国药理学与毒理学杂志

OBJECTIVE Amyotrophic lateral sclerosis(ALS)is a fetal neurodegenerative disease characterized by the progressive loss of upper and lower motor neu-rons,leading to skeletal muscle atrophy,weakness,and paralysis.Oxidative stress plays a crucial role in ALS pathogenesis,including the familial forms of the disease arising from mutations in the gene coding for superox-ide dismutase(SOD1).Additionally,the abnormal accu-mulation of TAR DNA-binding protein of 43 ku(TDP-43)is a pathological feature present in almost all patients,even though the pathogenesis of ALS is unclear.Current-ly,there is no drug that can cure ALS/FTLD.Tetramethyl-pyrazine nitrone(TBN)is a derivative of tetramethylapyr-azine,derived from traditional Chinese medicine Ligusti-cum chuanxiong,which has been extensively proven to have therapeutic effects on various models of neurode-generative diseases.METHODS We investigated the therapeutic effect of TBN in the SOD1G93A and TDP-43M337V ALS mouse models.In the SOD1G93A trans-genic mouse model,TBN was administered to mice via intraperitoneal or intragastric injection after the onset of motor deficits.We injected the TDP-43M337V virus into the striatum of mice unilaterally and bilaterally,and then administered TBN 30 mg·kg-1 intragastrically to observe changes in behavior and survival rate of mice.RESULTS TBN slowed the progression of motor neuron disease,as evidenced by improved motor performance,reduced spi-nal motor neuron loss and associated glial response,and decreased skeletal muscle fiber denervation and fibrosis.TBN treatment activated mitochondrial antioxidant activity through the PGC-1α/Nrf2/HO-1 pathway and decreased the expression of human SOD1.In the mice with unilateral injection of TDP-43M337V into the striatum,TBN improved motor deficits and cognitive impairment in the early stages of disease progression.In mice with bilateral injection of TDP-43M337V into the striatum,TBN not only improved motor function but also prolonged survival.Moreover,we demonstrate that its therapeutic effect may be through activation of the Akt/mTOR/GSK-3β and AMPK/PGC-1α/Nrf2 signaling pathways.CONCLUSION TBN shows promise as an agent for the treatment of ALS/FTLD.TBN is currently undergoing clinical investigation for several indications,including a Phase Ⅱ trial for ALS.

Protective mechanism of tetramethylpyrazine on pancreatic tissue in rats with acute necrotizing pancreatitis based on network pharmacology / 中华胰腺病杂志

Objective:To explore the core targets and potential molecular mechanisms of tetramethylpyrazine in the treatment of rats with acute necrotizing pancreatitis (ANP) based on network pharmacology.Methods:The related co-targets of tetramethylpyrazine and ANP were screened out by traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) and human disease information-related databases (CTD, DisGeNET, GeneCards, OMIM); Uniprot data were used to co-link and put into the STRING database to build protein-protein interaction (PPI) networks; the Cytoscape software was used for further analysis and the key targets were obtained by using the cytoHubba plug-in. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on these key targets, and finally the molecular docking models were constructed by using PyMol and AutoDockTools software. 30 male SD rats were randomly divided into the control group, ANP group, and tetramethylpyrazine treatment group (tetramethylpyrazine group). ANP rats were induced by retrograde infusion of 4% sodium taurocholate into the biliary-pancreatic duct, and the tetramethylpyrazine group rats were injected with 10 ml/kg tetramethylpyrazine through the abdominal cavity after ANP was induced. After 12 h, pancreatic tissue was taken, a pathological examination was performed routinely, and immunohistochemical staining was performed to observe the protein expression of key targets in pancreatic tissue. Blood was taken from orbits, and then the serum IL-6 and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay (ELISA).Results:The drug platform screened 137 tetramethylpyrazine action targets, and the disease database screened out 513 ANP-related targets; then 25 targets were obtained through intersection, finally resulting in a total of 5 key targets: albumin (ALB), epidermal growth factor receptor (EGFR), caspase 3 (CASP3), mitogen-activated protein kinase 1 (MAPK1) and B-cell lymphoma-2-like protein 1 (BCL2L1). GO functional enrichment analysis of biological processes mainly involved reproductive structure or system development, response to antibiotics, chemical stress and reactive oxygen species, and the cellular components were mainly vesicle lumen, membrane raft, membrane microdomain, and secretory granule lumen; molecular functions mainly included SH2 domain, phosphotyrosine residue, protease binding, protein tyrosine kinase and nuclear receptor activity; KEGG pathway enrichment analysis were mainly enriched in Ras signaling pathway, PI3K-Akt signaling pathway, platinum drug resistance, phospholipase D signaling pathway, and EGFR tyrosine kinase inhibitor resistance. The average binding energy of the 5 key targets molecule docking was -4.20 kcal/mol. After hematoxylin-eosin staining, it could be seen that the gland structure of rats in the ANP group was disordered, the interlobular space was significantly increased, and neutrophil infiltration was observed in the acinar, perivascular and gland space. The pancreatic lobule space of tetramethylpyrazine group rats was slightly increased, with mild neutrophil infiltration. The protein expressions of EGFR, CASP3 and MAPK1 in the ANP group were significantly higher compared with those in the control group, and EGFR, CASP3 and MAPK1 expression in tetramethylpyrazine group was significantly lower than those in ANP group ( P<0.01); the protein expression of BCL2L1 in the ANP group were significantly higher than that in control group, and the protein expression of BCL2L1 in tetramethylpyrazine group were significantly higher than that in ANP group (all P value <0.05). The serum levels of IL-6 and TNF-α in the ANP group were significantly higher than those in the control group, and IL-6 and TNF-α in tetramethylpyrazine group were significantly lower than those in the ANP group (all P value <0.01). Conclusions:Tetramethylpyrazine could reduce the inflammatory response and oxidative stress injury after ANP by activating a variety of signaling pathways, enhancing the expression of anti-apoptotic genes, and blocking the enzymatic cascade reaction of apoptotic caspase, thus playing a protective role in pancreatic tissues of rats with ANP.

Effects of 2,3,5,6-Tetramethylpyrazine on alcohol-induced injury in liver cells and on the early life stages of zebrafish (Danio rerio Hamilton, 1822)

Background@#Alcoholic liver disease (ALD) is a major health problem referring to the collection of liver damage caused by excessive alcohol intake. The search for effective and safe alternatives for compounds from plants to protect the liver from extensive damages and delay the progress to a disease is still a big effort done in the scientific community. 2,3,5,6-Tetramethylpyrazine (TMP) is a compound found in a Chinese herbal medicinal plant, Ligusticum chuanxiong Hort and in some other plants. @*Objective@#This study was done to assess the hepatoprotective effects of TMP against ALD using histopathological analysis of zebrafish livers subjected to different exposure groups. TMP has been mainly used for the treatment of cardio- and cerebrovascular diseases due to its antioxidant, anti-inflammatory, and antiapoptotic properties. @*Methodology@#Adult male zebrafish were exposed to three TMP concentrations (40, 60, and 80 mg/L TMP) and to 1% v/v of ethanol. The dissected livers of the zebrafish were processed for fixing on glass slides using the H&E stains and were observed under the light compound microscopes for scoring. The safety of the TMP to the early life stages of the zebrafish was tested using the Zebrafish Embryotoxicity Test (ZFET). @*Results@#Results showed that TMP was able to dose-dependently decrease mean scores for the four parameters diagnostic of ALD, i.e., steatosis, inflammation, cell death, and ballooning degeneration. These scores were comparable to those of the untreated group (no ethanol + no treatment) and positive control (ethanol + Hepasil DTXTM), with all groups' scores being statistically different from those of the negative control group (ethanol + no treatment) (p<0.05). Results for the ZFET showed that incidence of embryo mortality as well as teratogenic malformations of embryos exposed to TMP were significantly lower compared to the positive control group. @*Conclusion@#The hepatoprotective role of TMP was implied because anomalies such as cholestasis, vessel congestion, and hemorrhage were only observed in the ethanol-treated group and not in the other groups. In the analysis of the early development of the embryos using the Zebrafish Embryotoxicity Test (ZFET), TMP was found to be non-toxic and non-teratogenic at concentrations used for liver treatment. These initial findings on TMP provided justification for its plausibility as a hepatoprotective compound against alcoholic liver diseases (ALD).

Role of p38 MAPK/CREB signaling pathway in tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with sepsis-associated encephalopathy / 中华麻醉学杂志

Objective:To evaluate the role of p38 mitogen-activated protein kinase (MAPK)/cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway in tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with sepsis-associated encephalopathy (SAE).Methods:Sixty healthy male C57BL6 mice, weighing 24-27 g, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (group Sham), sepsis group (group Sep), tetramethylpyrazine group (group TMP) and p38 MAPK inhibitor SB203580 group (group SB). The model of SAE was established by cecal ligation and puncture in anesthetized mice.Tetramethylpyrazine 10 mg/kg was injected intraperitoneally once a day at 3 days before the establishment of the model in TMP group, and SB203580 2.0 mg/kg was intraperitoneally injected at 30 min after the establishment of the model in SB group.The equal volume of normal saline was given intraperitoneally in Sham and Sep groups.At 1 day after operation, cognitive function was assessed by Morris water maze, and the escape latency and ratio of time spent in the target quadrant were recorded.The animals were sacrificed after the test, and hippocampal tissues were taken for determination of the contents of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6 (by enzyme-linked immunosorbent assay) and for detection of the expression of phosphorylation of p38 MAPK, GSK3 and CREB and expression of brain-derived neurotrophic factor (BDNF) (by Western blot). Results:Compared with group Sham, the escape latency was significantly prolonged, the ratios of time spent in the target quadrant were decreased, the contents of IL-1β, TNF-α and IL-6 were increased, the phosphorylation of hippocampus p38 MAPK was increased, the phosphorylation of GSK3 and CREB were decreased, and the expression of BDNF was down-regulated in Sep, TMP and SB groups ( P<0.05). Compared with group Sep, the escape latency was significantly shortened, the ratios of time spent in the target quadrant were increased, the contents of IL-1β, TNF-α and IL-6 were decreased, the phosphorylation of hippocampus p38 MAPK was decreased, the phosphorylation of GSK3 and CREB were increased, and the expression of BDNF was up-regulated in TMP and SB groups ( P<0.05). Compared with group TMP, no significant change was found in the parameters mentioned above in group SB ( P>0.05). Conclusion:p38 MAPK/CREB signaling pathway is involved in the process of tetramethylpyrazine-induced reduction of hippocampal inflammatory responses in mice with SAE.

Protective Effect and Mechanism of Tetramethylpyrazine Against t-BHP-induced PC12 Cell Injury / 中国实验方剂学杂志

Objective:To study the protective effect of tetramethylpyrazine (TMP) on PC12 cells induced by tert-butyl hydroperoxide (t-BHP) and the regulatory mechanism on signaling pathway of phosphatidylinositol-3-kinases (PI3K)/kinase B (Akt)/mammalian target of rapamycin(mTOR). Method:PC12 cells cultured in vitro were treated with t-BHP (200 μmol·L-1) for 6 h to establish a model of oxidative damage in PC12 cells. The experiment was divided into blank group, model group (200 μmol·L-1t-BHP), TMP group. PC12 cells were pretreated with TMP(25, 50, 100 μmol·L-1) for 12 h, and then treated with t-BHP for 6 h. The cell viability was detected by cell counting kit-8(CCK-8) method, and lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, reactive oxygen species (ROS) and glutathione peroxidase (GSH-Px) activity were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was observed by annexin V-FITC/PI double staining. B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), total protein kinase B (Akt), and phosphorylated protein kinase B (p-Akt), mTOR and p-mTOR expressions were detected by Western blot. Result:The cell viability of PC12 cells treated with 200 μmol·L-1 t-BHP decreased to about 50%after 6 h. This condition was suitable for the establishment of oxidative damage model. Compared with the model group, TMP (25, 50, 100 μmol·L-1) pretreatment for 12 h significantly increased the survival rate of PC12 cells (PPPPPPP-1) pretreatment group increased significantly (PConclusion:Ligustrazine protects PC12 cell injury induced by t-BHP by activating PI3K/Akt/mTOR signaling pathway.

The clinical effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders / 中国医师杂志

Objective To investigate the clinical therapeutic effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders.Methods 80 elderly patients with senile hypertensive cerebral infarctiontreated in our hospital were selected and randomly divided into the single treatment group and the combination treatment group,40 cases in each group.Both groups received the routine treatment.The single treatment group additionally received Shenxiong glucose injection (100 ml/d),while patients in the combination treatment group additionally received Shenqiong glucose injection combined with citicoline sodium intravenous infusion therapy (0.5 g/d),both groups were treated for 2 weeks.The levels of serum inflammatory factors,the neurological deficit score,the cognitive function score were compared and analyzed before and after treatment between two groups.Results After systemic treatment,the blood pressure and blood lipid levels of two groups were significantly improved,but there was no significant difference between the two groups (P ＞ 0.05);The serum levels of interleukin (IL)-6,IL-8 and tumor necrosis factor-α (TNF-α) of the combination treatment group improved more significantly (P ≤0.05).After treatment,the oxidative stress indexes were significantly improved in the two groups (P ≤ 0.05).The content of malondialdehyde (MDA) was decreased,while the superoxide dismutase (SOD) activity and nitric oxide (NO) content were increased significantly (P ≤ 0.05);and the improvement degree in the combination treatment group was better than in the single treatment group (P ≤0.05).The degree of improvement in the Modified Edinburgh-Scandinavia Stroke Scale (MESSS) and Hasegawa Dementia Scale (HDS) scores of the combination treatment group was more significant than those in the single treatment group (P ≤ 0.05).The total effective rate of the combination treatment group was 92.5％,which was significantly higher than that of the single group (75.0％),with statistically significant difference (P ≤ 0.05).No obvious adverse reactions happened in two groups during treatment.Conclusions Combination of citicoline sodium and shenxiang glucose on the basis of routine treatment can significantly reduce oxidative stress and inflammation levels,promote the recovery of neurological and cognitive functions,and improve the clinical efficacy and safety.It is worth popularizing and applying in the clinical treatment of senile hypertensive cerebral infarction.

Tetramethylpyrazine promotes bone marrow repair in a C57 mouse model of X-rayinduced immune-mediated bone marrow failure / 南方医科大学学报

OBJECTIVE@#To observe the therapeutic effect of tetramethylpyrazine on immune-mediated bone marrow failure (BMF) induced by different doses of X-ray exposure in C57 mice.@*METHODS@#C57BL6 mice were randomized into 4 groups, including a blank control group and 3 X-ray exposure groups with X-ray exposure at low (5.0 Gy), moderate (5.75 Gy), and high (6.5 Gy) doses. After total body irradiation with 0.98 Gy/min X-ray. The mice as recipient received injections of 4×10 lymphocytes from DBA/2 mice via the tail vein within 4 h. The survival rate of the recipient mice, peripheral blood cell counts, bone marrow nucleated cell count, and bone marrow pathology were examined at 14 days after the exposure. In the subsequent experiment, C57 mice were exposed to 5.0 Gy X-ray and treated with intraperitoneal injection of tetramethylpyrazine at the low (5 mg/mL), moderate (10 mg/mL), or high (20 mg/mL) doses (12 mice in each group) for 14 consecutive days, and the changes in BMF were observed.@*RESULTS@#X-ray exposure, especially at the high dose, resulted in significantly lowered survival rate in the mouse models of BMF at 14 days. As the X-ray dose increased, the mice showed significantly reduced peripheral blood counts of red blood cells, white blood cells, platelets and lowered bone marrow nucleated cell counts with obvious bone marrow congestion and reduction of nucleated cells ( < 0.05 or 0.001). In the mice exposed to 5.0 Gy X-ray, tetramethylpyrazine at the high dose most obviously increased bone marrow nucleated cells ( < 0.01) and red blood cells ( < 0.001), and even at the low dose, tetramethylpyrazine significantly increased the counts of white blood cells ( < 0.05) and platelets ( < 0.01) following the exposure. Tetramethylpyrazine dose-dependently alleviated bone marrow hyperemia, increased bone marrow nucleated cell counts, and lowered Fas protein expression in the bone marrow.@*CONCLUSIONS@#X-ray irradiation at 5.0 Gy is suitable for establish mouse models of immune-mediated BMF. Tetramethylpyrazine promotes bone marrow repair by regulating Fas cell apoptosis signals, which further expands the traditional Chinese medicine theory of "removing blood stasis to create new."

Tetramethylpyrazine Improves Postoperative Tissue Adhesion: A Drug Repurposing / 中国结合医学杂志

Plants are known to possess plenty of pharmacological activities as a result of various phytoconstituents. Tetramethylpyrazine (TMP), one of the most widely used medicinal compound isolated from traditional Chinese herb, is usually employed for anti-oxidation, anti-inflammation, anti-platelet aggregation, anti-lipid, anti-fibrosis, as well as activating blood, removing stasis, dilating small arteries, improving microcirculation and antagonizing calcium. In the present paper, the anti-adhesion effect of TMP were reviewed. TMP was found to play a multi-target and muti-link role in anti-adhesion by inhibiting hyperplasia of collagen and overexpression of adhesion-related factors and reducing the concentration of white blood cells and fibrin in plasma. Because previous studies mostly focused on in vitro experiments and animal experiments, there is an urgent need for clinical research with abundant indicators to further prove its anti-adhesion potency. Future basic research should concentrate on the development of TMP as a biological material.

Intervention effect of tetramethylpyrazine on inflammatory response of endothelial cells induced by activated complement alternative pathway / 中国药理学通报

Aim To study the intervention effect of tet- ramethylpyrazine(TMP) on human microvascular endothelial cells (HMECs) inflammatory response induced by activated complement alternative pathway and the possible molecular mechanisms. Methods HMECs were pretreated with different concentrations of TMP, and then exposed to the activated products of the complement alternative pathway which was prepared by cobra venom factor( CVF). The supernatant was removed and assayed for expression of the adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and the inflammatory mediator(IL-6 and TNF-ot) by using ELISA reagent kits. The nucleus transcriptional activity of NF- kB was measured by the dual luciferase reporter assay ? system. Results The adhesion molecules, inflammato ry mediator and nucleus transcriptional activity of NF- kB increased after HMECs were exposed to the products of the activated complement alternative pathway. The up-regulation of ICAM-1, VCAM-1, E-selectin, IL-6, TNF-a and the nucleus transcriptional activity of NF-kB were inhibited by various concentrations of TMP in a dose-dependent manner. Conclusions TMP can effectively reduce inflammatory response of HMECs in-duced by the activated complement alternative pathway , and the mechanism may be highly related to inhibition of nucleus transcriptional activity of NF-kB.

Effect of tetramethylpyrazine on renal oxidative stress in immune-mediated bone marrow failure mice / 中国药理学通报

; A i m To explore the effects of tetramethylpyrazine (T M P) on renal oxidative stress in immune-mediated bone marrow failure (B M F) C57 mice. Methods C57 mice were randomly divided into normal control group, total body irradiation group (T B I), model group, TMP low dose group (5 g • L " 1), T M P medium dose group (10 g • L " 1), and T M P high dose group (2 0 g • L " 1) . The B M F model was established by total body irradiation with 5. 0 Gy X-ray and l y m - phocyte infusion. The peripheral blood triline cells, bone marrow nucleated cells, bone marrow pathology, kidney pathology and oxidative stress indexes were observed 14 days later. Results Bone marrow pathology of the model group showed that the bone marrow nucleated cells decreased and the peripheral blood triline cells decreased significantly, indicating that the i m - mune-mediated B M F model was successfully established. Renal pathology of model group showed tubular edema, and the oxidative stress index M DA increased, and the antioxidant stress indicators GSH, SOD, GSHPx decreased, indicating that there was renal oxidative stress damage in BMF mice. After treatment with TMP, there was no obvious abnormality in renal pathology, the oxidative stress index MDA of the kidney decreased and the antioxidant stress index GSH, SOD increased. Immunohistochemistry showed that the expression of HIF-1 a decreased and VEGF existence was observed. Conclusions TMP can alleviate renal oxidative stress injury in immune-mediated BMF mice, which may be related to the regulation of HIF-1 oc/ VEGF expression.

Tetramethylpyrazine reverses anxiety-like behaviors in a rat model of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a trauma-induced psychiatric disorder characterized by impaired fear extermination, hyperarousal, and anxiety that may involve the release of monoamines in the fear circuit. The reported pharmacological properties of tetramethylpyrazine (TMP) include anti-cancer, anti-diabetic, anti-atherosclerotic, and neuropsychiatric activities. However, the anxiolytic-like effects of TMP and its mechanism of action in PTSD are unclear. This study measured several anxiety-related behavioral responses to examine the effects of TMP on symptoms of anxiety in rats after single prolonged stress (SPS) exposure by reversing the serotonin (5-HT) and hypothalamic-pituitary-adrenal (HPA) axis dysfunction. Rats were given TMP (10, 20, or 40 mg/kg, i.p.) for 14 days after SPS exposure. Administration of TMP significantly reduced grooming behavior, increased the time spent and number of visits to the open arm in the elevated plus maze test, and significantly increased the number of central zone crossings in the open field test. TMP administration significantly reduced the freezing response to contextual fear conditioning and significantly restored the neurochemical abnormalities and the SPS-induced decrease in 5-HT tissue levels in the prefrontal cortex and hippocampus. The increased 5-HT concentration during TMP treatment might be partially attribute to the tryptophan and 5-hydroxyindoleacetic acid mRNA level expression in the hippocampus of rats with PTSD. These findings support a role for reducing the altered serotonergic transmission in rats with PTSD. TMP simultaneously attenuated the HPA axis dysfunction. Therefore, TMP may be useful for developing an agent for treating psychiatric disorders, such those observed in patients with PTSD.

Effects of tetramethylypyrazine nitrone on proliferation and differentiation of neural stem cells / 中国应用生理学杂志

OBJECTIVES@#To investigate the role of tetramethylpyrazine(TMP) nitrone in proliferation and differentiation of neural stem cells (NSCs).@*METHODS@#We separated and cultivated the original generation of NSCs from cerebral cortex of 14 days rat embryo, and the phenotype characteristics of the third-generation NSCs was tested by immunofluorescence. The experiment was divided into control group, β-mercaptoethanol positive control group, tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + ethylene glycol tetraacetic acid(EGTA) group (=4). The third-generation cultivation of NSCs was used in the experiment. The effect of tetramethylpyrazine nitrone on the number of NSCs proliferation was determined by BrdU and MTT, and the differentiation of NSCs was determined by Western blot.@*RESULTS@#The primary NSCs was isolated successfully, neurospheres with typical NSCs morphology and expressing nestin was formed at 3-5 days. As BrdU and MTT assay results shown, compared with the control group andβ-mercaptoethanol positive control group, the NSCs proliferation numbers of tetramethylpyrazine nitrone group increased significantly(<0.05). The results of Western blot showed that the neuronal differentiation rate of NSCs was increased significantly in both the tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + EGTA group, and the differentiation rate of NSCs in tetramethylpyrazine nitrone + EGTA group increased more significantly(<0.05).@*CONCLUSIONS@#Tetramethylpyrazine nitrone can significantly enhance the proliferation and neuronal differentiation rate of NSCs. Decrease in extracellular Ca can promote the differentiation of NSCs into neurons induced by tetramethylpyrazine nitrone. Ca signaling plays an important role in the differentiation of NSCs into neurons.

Tetramethylpyrazine inhibits PM2.5-induced vascular smooth muscle cell proliferation by down-regulating JNK phosphorylation / 中国药理学与毒理学杂志

OBJECTIVE To investigate the inhibitory effect and the possible mechanism of tetra-methylpyrazine (TMP) in preventing vascular smooth muscle cells (VSMCs) proliferation induced by fine particulate matter (PM2.5). METHODS PM2.520, 200 and 400 mg · L-1 was added to VSMCs for 24 h, the survival of VSMCs was measured by MTT assay, the protein levels of p-c-Jun N-terminal kinase (JNK) and fibroblast growth factor receptor-1 (FGFR-1) in the VSMCs were detected by Western blotting, while the levels of vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1) and nitric oxide (NO) in the VSMCs were analyzed by ELISA, radioimmunoassay and nitrate reductase method, respec-tively. TMP 20, 200 and 2000 mg·L-1 or a specific inhibitor of JNK SP60012510μmol·L-1 was added into the VSMCs to observe the effect of TMP. RESULTS Compared with the normal control group, PM2.5200 and 400 mg·L-1 significantly increased the A570 nm vaule, the protein levels of p-JNK and FGFR-1,the levels of VCAM-1 and ET-1, but decreased the level of NO (P<0.01), while there were no significant changes in PM2.520 mg·L-1 group. Compared with the PM2.5200 mg·L-1 group, TMP 200 and 2000 mg·L-1 pre-treatment markedly decreased the A570 nm vaule, the protein levels of p-JNK and FGFR-1, the levels of VCAM-1 and ET-1, but increased the level of NO (P<0.01), while there were no significant changes in TMP 20 mg · L-1 pre-treated group. Moreover, the effects of TMP were significantly enhanced by the co-incubation of TMP 2000 mg · L-1 with SP60012510 μmol · L-1, compared to the TMP 2000 mg · L-1 pre-treated group (P<0.05, P<0.01). CONCLUSION TMP displays a significant inhibitory effect against VSMC proliferation induced by PM2.5. The mechanism may be related to the inhibition of JNK phosphor-ylation, and the regulation of FGFR-1 protein expression and VCAM-1, ET-1 and NO levels.

Effect of tetramethylpyrazine on autophagy in hippocampal neurons of rats with sepsis-associated en-cephalopathy / 中华麻醉学杂志

Objective To evaluate the effect of tetramethylpyrazine on autophagy in the hippocam-pal neurons of rats with sepsis-associated encephalopathy. Methods Sixty SPF healthy male Sprague-Daw-ley rats, aged 11-14 weeks, weighing 200-240 g, were divided into 3 groups(n=20 each)using a ran-dom number table: sham operation group(group Sham), sepsis group(group Sep)and tetrameth-ylpyrazine group(group TMP). Sepsis was induced by cecal ligation and puncture(CLP), and group Sham only underwent simple laparotomy. Tetramethylpyrazine 10 mg∕kg was injected intraperitoneally at 1 h before CLP in group TMP. Morris water maze test was performed in 10 rats randomly selected at 12 and 36 h after CLP. Then the rats were sacrificed, and hippocampi were isolated for determination of the expres-sion of microtubule-associated protein 1 light chain 3Ⅰ(LC3Ⅰ), LC3Ⅱ, Beclin-1 and p62 in hipp-ocampal tissues by Western blot, and the LC3Ⅱ∕LC3Ⅰratio was calculated. Results Compared with group Sham, the escape latency was significantly prolonged, the rate of time spent in the target quadrant was decreased, the LC3Ⅱ∕LC3Ⅰratio was increased, the expression of Beclin-1 was up-regulated, and the expression of p62 was down-regulated at 12 and 36 h after CLP in group Sep and group TMP(P<005). Compared with group Sep, the escape latency was significantly shortened, the rate of time spent in the target quadrant was increased, the LC3Ⅱ∕LC3Ⅰratio was decreased, the expression of Beclin-1 was down-regulated, and the expression of p62 was up-regulated at 12 and 36 h after CLP in group TMP(P<005). Conclusion The mechanism by which tetramethylpyrazine reduces sepsis-associated encephalopa-thy is related to inhibiting autophagy in the hippocampal neurons of rats.

Tetramethylpyrazine inhibits LPS-inducedinflammation of human type Ⅱ alveolar epithelial cells / 基础医学与临床

Objective To study the protective effect of TMP(Tetramethylpyrazine) on LPS(Lipopolysaccharides)-induced inflammatory response of human type Ⅱ alveolar epithelial cells (HAECⅡ) and its corresponding mechanism.Methods HAECⅡ (A549 cells derived from human lung adenocarcinoma cells) were cultured in vitro.Inflammation model was established using A549 cells after LPS stimulation.TMP and FK866 (a specific inhibitor for pre-B cell colony-enhancing factor), were added to intervene respectively.Expression level of mRNA, inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1β(IL-1β), interleukin-8 (IL-8) and PBEF(pre-B cell colony-enhancing factor) were detected by q-PCR and Western blot, respectively.The activation of NF-κB(Nuclear factor κB) was examined by Western blot to find the changes in phosphorylated P65 protein level in both nucleus and cytoplasm.Results Both the mRNA and protein level of TNF-α, IL-1β, IL-8 and PBEF in A549 cells were significantly higher after LPS stimulation than those in the control group(P<0.001).Meanwhile, the phosphorylation of P65 protein in the nucleus and cytoplas was higher(P<0.001).The expression of the aforementioned inflammatory factors and the phosphorylation of P65 protein were significantly lower after TMP inter-vention than those of LPS group(P<0.05).In comparison, after FK866 was added, the expression of TNF-α, IL-1β and IL-8 and the phosphorylation of P65 protein were also decreased(P<0.01).Conclusions TMP may be involved in the reduction of PBEF expression, which therefore inhibits NF-κB activation, antagonizes alveolar epithelial cell inflammatory response.

The protective effect of tetramethylpyrazine on rat myocytes with transient oxygen-glucose deprivation followed by reoxygenation injury and mechanism study / 中国生化药物杂志

Objective This study explored the protective effect of tetramethylpyrazine on myocardial ischemia/reperfusion injury via inhibiting oxidative stress. Methods Primary cultured neonatal myocytes were applied to explore the anti-ischemia/reperfusion injury property in vitro. The survival viability of myocytes was determined by MTT; enzyme activities such as lactate dehydrogenase, creatine kinase, superoxide dismutase, malondialdehyde, and nitric oxide were analyzed with assay kits; inducible nitricoxide synthase and endothelial nitric oxide synthase expressions were determined by Westernblot. Results Tetramethylpyrazine significantly improved the beating frequencies of myocytes after oxygen-glucose deprivation/reoxygenation procedure, decreased lactate dehydrogenase, creatine kinase, and malondialdehyde levels and enhanced superoxide dismutase activity.Tetramethylpyrazine also inhibited excessive production of nitric oxide through downregulating inducible nitric oxide synthase as well as upregulating endothelial nitric oxide synthase during ischemia/reperfusion injury. Conclusion Tetramethylpyrazine could significantly improve the oxidative-stress tolerance of myocytes to keep cell membrane integrity and protect the myocardial tissue of normal physiological function via an antioxidant effect and by restoring the balance between inducible nitric oxide synthase and endothelial nitric oxide synthase, while inhibiting the generation of cytotoxic concentrations of NO.

Dissolution Method Study of Ligustrazine Phosphate Pills / 中国药学杂志

OBJECTIVE: To establish a new dissolution method of Ligustrazine Phosphate Pills, which provides reference for revising the quality standard. METHODS: The dissolution media were hydrochloric acid solution(pH 1.2), acetate buffer solution(pH 4.5), phosphate buffer solution(pH 6.8) and water. The dissolution curves of six batches of Ligustrazine Phosphate Pills in the four kinds of dissolution media were compared to establish the dissolution method. Apparatus 2 was used for the new method of dissolution test, using 500 mL water as the dissolution medium, at the rate of 75 r·min-1. The dissolution solution was taken at 20 min and analyzed by HPLC. The dissolution limit was set at 80%. RESULTS: The dissolution curves of the six batches of samples were similar in the four kinds of dissolution media. The pills were dissolved completely within 15 min. CONCLUSION: The determination method is highly reproducible, accurate and reliable, which can objectively reflect the dissolution of ligustrazine phosphate pills, and provides a basis for the reasonable unification and revision of the dissolution test of the current quality standard.