PMID: 

Korean J Physiol Pharmacol. 2018 Sep ;22(5):525-538. Epub 2018 Aug 27. PMID: 30181699

Abstract Title: 

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

Abstract: 

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,) 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.

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PMID: 

Arch Pharm Res. 2019 Sep ;42(9):824-831. Epub 2018 Nov 17. PMID: 30448958

Abstract Title: 

Ligustrazine attenuates inflammation and oxidative stress in a rat model of arthritis via the Sirt1/NF-κB and Nrf-2/HO-1 pathways.

Abstract: 

Inflammation responses and oxidative stress are closely involved in the pathogenesis of arthritis. Ligustrazine (Lig), a natural four methyl which is isolated from Chinese herb ligusticum chuanxiong hort, has been proved significantly anti-inflammation and anti-oxidative stress effects. The present study aimed to evaluate the effect of Lig on inflammation and oxidative stress in Freund's complete adjuvant (FCA)-induced arthritis in rats. The treatment of Lig significantly decreased the hind-paw volume change and alleviated the histopathological changes in sections of rat paws induced by arthritis. Lig also reduced the serum levels of pro-inflammatory cytokines (interleukin [IL]-6, IL-1 beta, and tumor necrosis factor-alpha), increased the activity of superoxide dismutase (SOD) and reduced the concentration of malondialdehyde (MDA). Besides that, the protein expressions of the sirtuin 1 (Sirt1)/nuclear factor kappa B (NF-κB) and nuclear factor (erythroid-derived 2)-like-2 factor (Nrf-2)/heme oxygenase (HO)-1 pathways determined by western bolt further confirmed that Lig effectively inhibited the Sirt1/NF-κB pathway and activated the Nrf-2/HO-1 pathway. Taken together, our results suggest Lig has therapeutic potential for arthritis, which might be via the regulation of Sirt1/NF-κB and Nrf-2/HO-1 pathways.

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PMID: 

Mol Med Rep. 2019 Apr ;19(4):2561-2568. Epub 2019 Feb 1. PMID: 30720104

Abstract Title: 

Tetramethylpyrazine reduces inflammation in the livers of mice fed a high fat diet.

Abstract: 

The present study aimed to assess the protective effects of tetramethylpyrazine (TMP) on the livers of mice fed a high fat diet. The mice were divided into five groups: Regular diet; high fat diet; simvastatin‑treated; and low and high dose TMP‑treated groups. The results demonstrated that, compared with the control group, serum glucose, total cholesterol (TC) and low‑density lipoprotein cholesterol levels were increased in the model group. Additionally, compared with the model group, simvastatin lowered the TC level, whereas TMP did not. Compared with the control group, the level of malondialdehyde (MDA) in the liver tissue was increased and the level of glutathione peroxidase (GSH‑pX) in the liver tissue was decreased in the model group. Furthermore, compared with the model group, TMP decreased the level of MDA and increased the level of GSH‑Px; however, simvastatin did not have these effects. Immunohistochemistry and western blotting were performed; the results showed that, compared with the control group, the levels of inflammatory factors (tumor necrosis factor‑α and interleukin‑6) in the liver tissue were increased, and the ratio of phosphorylated (p)‑nuclear factor κB (NF‑κB)/NF‑κB was also increased in the model group. Theaddition of TMP and simvastatin demonstrated that, compared with the model group, the inflammatory factor levels and the ratio of p‑NF‑κB/NF‑κB were decreased. In addition, liver lipid deposition was examined in the model group using hematoxylin and eosin staining and Oil Red O staining,and the results showed that TMP and simvastatin reduced liver lipid deposition. Furthermore, compared with the control group, the reactive oxygen species (ROS) level in the liver tissue was increased. Compared with that in the model group, TMP and simvastatin decreased the ROS level. In conclusion,TMP, similar to simvastatin, exerted a notable hepatoprotective effect on mice fed a high fat diet with non‑alcoholic fatty liver disease, by inhibiting inflammatory factors and the p‑NF‑κB/ROS signaling pathway.

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PMID: 

J Cell Physiol. 2019 Feb 15. Epub 2019 Feb 15. PMID: 30770559

Abstract Title: 

Tetramethylpyrazine partially relieves hypoxia-caused damage of cardiomyocytes H9c2 by downregulation of miR-449a.

Abstract: 

Inadequate oxygen supply is probably one of the most important pathophysiological mechanisms of cardiomyocyte damage in ischemic heart disease. Tetramethylpyrazine (TMP, also known as ligustrazine) is the main active ingredient isolated from the rhizome of Ligusticum chuanxiong Hort. A previous study reported that the TMP could exert cardioprotective activity. This study aimed to explore the molecular mechanism of the protective effects of TMP on cardiomyocyte damage caused by hypoxia. The viability and apoptosis of cardiomyocytes H9c2 were detected using cell counting kit-8 assay and annexin V-FITC/PI staining, respectively. Quantitative reverse transcription polymerase chain reaction was conducted to measure the expression level of microRNA-449a (miR-449a). Cell transfection was performed to upregulate the expression level of miR-449a or downregulate the expression level of sirtuin 1 (Sirt1). The protein expression levels of Sirt1 and key factors involved in cell apoptosis and phosphatidylinositol 3-kinase/protein kinase 3 (PI3K/AKT) pathway were evaluated using western blot analysis. We found that the hypoxia incubation inhibited H9c2 viability, induced cell apoptosis, and inactivated the PI3K/AKT pathway. TMP treatment partially relieved the hypoxia-caused H9c2 cell viability loss and apoptosis, as well as reversed the hypoxia-caused inactivation of the PI3K/AKT pathway. Moreover, TMP partially alleviated the upregulation of miR-449a in H9c2 cells caused by hypoxia. Overexpression of miR-449a weakened the effects of TMP on hypoxia-treated H9c2cells. Furthermore, Sirt1 was a target gene of miR-449a. Knockdown of Sirt1 also weakened the effects of TMP on hypoxia-treated H9c2 cells. In conclusion, TMP partially relieved hypoxia-caused cardiomyocytes H9c2 viability loss and apoptosis at least through downregulating miR-499a, upregulating Sirt1, and then activating the PI3K/AKT pathway.

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PMID: 

Psychopharmacology (Berl). 2019 Jul ;236(7):2173-2185. Epub 2019 Mar 7. PMID: 30847567

Abstract Title: 

Tetramethylpyrazine ameliorates depression by inhibiting TLR4-NLRP3 inflammasome signal pathway in mice.

Abstract: 

Depression is a common but serious mental illness; meanwhile, it is also an inflammatory disorder. Toll-like receptor 4 (TLR4), as the pattern recognition receptor, has been shown to play a vital role in neuroinflammation. The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome acts as an important signaling molecule downstream of TLR4 and can promote the maturation of inflammatory cytokines, such as interleukin-1β (IL-1β). Tetramethylpyrazine (TMP) is a natural compound with neuroprotective effects but with unknown mechanisms on its antidepressant-like effect. In this study, we hypothesized that TMP ameliorates depression may be through the inhibition of the TLR4-NF-κB-NLRP3 signal pathway. Our results have shown that chronic unpredictable mild stress (CUMS) that induced the decreased sucrose preference and increased immobile time was prominently reversed by TMP and fluoxetine. Additionally, we also found that CUMS induced the upregulation of proinflammatory cytokines; TLR4 and NLRP3-associated proteins were significantly suppressed by TMP in the prefrontal cortex and hippocampus. TMP also exhibited potent antioxidant effects and increased the monoamine levels in the serum and brain, such as increasing the activity of SOD and GSH-Px, and reducing the activity of MDA in the serum, and elevating the 5-HT and NE concentration in the serum and brain. Moreover, treatment with Cli-095 (TLR4 inhibitor) also markedly inhibited CUMS-induced depression-like behaviors. Taken together, our findings suggested that TMP exerted a potential antidepressant-like effect in CUMS mice, and the molecular mechanisms may relate to inhibit the TLR4-NF-κB-NLRP3 signaling pathway in the brain.

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PMID: 

Cell Mol Biol Lett. 2019 ;24:17. Epub 2019 Feb 26. PMID: 30858867

Abstract Title: 

Tetramethylpyrazine exerts a protective effect against injury from acute myocardial ischemia by regulating the PI3K/Akt/GSK-3β signaling pathway.

Abstract: 

Objective: We investigated the protective effect of tetramethylpyrazine (TMP) on injury related to acute myocardial ischemia (AMI) induced by isoproterenol (ISO).Materials and methods: Rats were randomly assigned to five groups: control, ISO, ISO + propranolol (10 mg/kg), ISO + TMP (10 mg/kg) and ISO + TMP (20 mg/kg). The rats in the three ISO + groups were pretreated with propranolol or TMP, while the rats in the control and ISO groups were pretreated with an equal volume of saline. Afterwards, the rats in the four administration groups were subcutaneously injected with ISO for two consecutive days. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the serum were measured usingELISA. The expressions of B-cell lymphoma-associated X-2 (Bax-2), B-cell lymphoma-2 (Bcl-2), phosphoinositide-3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase 3β (GSK-3β), MDA5 and SOD1 were determined using western blotting assay. The phosphorylation of PI3K, Akt and GSK-3β werealso determined using western blotting assay. The left ventricles of the rats were extracted and stained using hematoxylin and eosin (H&E). The ST segment was recorded using electrocardiograms (ECGs).Results: Administration of TMP (10, 20 mg/kg) reduced the levels of MDA and CK and the activities of SOD and LDH in the serum. Pretreatment with TMP significantly reduced the levels of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α. Treatment with TMP also improved the histopathological alteration and decreased the ST elevation. Furthermore, TMP ameliorated the expressions of Cu, SOD1, MDA5, Bax-2, Bcl-2, p-PI3K, p-Akt and p-GSK-3β in ISO-induced rats.Conclusions: Tetramethylpyrazine protected against injury due to AMI by regulating the PI3K/Akt /GSK-3β signaling pathway.

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PMID: 

Neuromolecular Med. 2019 Sep ;21(3):262-274. Epub 2019 May 27. PMID: 31134485

Abstract Title: 

Tetramethylpyrazine Nitrone Reduces Oxidative Stress to Alleviate Cerebral Vasospasm in Experimental Subarachnoid Hemorrhage Models.

Abstract: 

Cerebral vasospasm is one of the deleterious complications after subarachnoid hemorrhage (SAH), leading to delayed cerebral ischemia and permanent neurological deficits or even death. Free radicals and oxidative stress are considered as crucial causes contributing to cerebral vasospasm and brain damage after SAH. Tetramethylpyrazine nitrone (TBN), a derivative of the clinically used anti-stroke drug tetramethylpyrazine armed with a powerful free radical scavenging nitrone moiety, has been reported to prevent brain damage from ischemic stroke. The present study aimed to investigate the effects of TBN on vasospasm and brain damage after SAH. Two experimental SAH models were used, a rat model by endovascular perforation and a rabbit model by intracisternal injection of autologous blood. The effects of TBN on SAH were evaluated assessing basilar artery spasm, neuronal apoptosis, and neurological deficits. TBN treatment significantly attenuated vasospasm, improved neurological behavior functions and reduced the number of apoptotic neurons in both the SAH rats and rabbits. Mechanistically, TBN suppressed the increase in 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine immuno-positive cells in the cortex of SAH rat brain. Western blot analyses indicated that TBN effectively reversed the altered expression of Bcl-2, Bax and cytochrome C, and up-regulated nuclear factor erythroid-derived 2-like 2 (Nrf2) and hemeoxygenase-1 (HO-1) protein expressions. In the in vitro studies, TBN inhibited HO-induced bEnd.3 cell apoptosis and reduced ROS generation. Additionally, TBN alleviated the contraction of rat basilar artery rings induced by HOex vivo. In conclusion, TBN ameliorated SAH-induced cerebral vasospasm and neuronal damage. These effects of TBN may be attributed to its anti-oxidative stress effect and up-regulation of Nrf2/HO-1.

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PMID: 

Oxid Med Cell Longev. 2019 ;2019:5820415. Epub 2019 Dec 3. PMID: 31885804

Abstract Title: 

Tetramethylpyrazine Attenuates the Endotheliotoxicity and the Mitochondrial Dysfunction by Doxorubicin14-3-3/Bcl-2.

Abstract: 

Doxorubicin (Dox) with cardiotoxicity and endotheliotoxicity limits its clinical application for cancer. The toxicitic mechanism involves excess ROS generation. 14-3-3s have the protective effects on various injured tissues and cells. Tetramethylpyrazine (TMP) is an alkaloid extracted from the rhizome of Ligusticum wallichii and has multiple bioactivities. We hypothesize that TMP has the protective effects on vascular endothelium by upregulating 14-3-3. To test the hypothesis, Dox-induced endotheliotoxicity was used to establish vascular endothelium injury models in mice and human umbilical vein endothelial cells. The effects of TMP were assessed by determining thoracic aortic strips' endothelium-dependent dilation (EDD), as well as LDH, CK, caspase-3, SOD, CAT, GSH-Px activities and MDA level in serum, apoptotic rate, and histopathological changes of vascular tissue (). Also, cell viability, LDH and caspase-3 activities, ROS generation, levels of NAD/NADH and GSH/GSSG, MMP, mPTP opening, and apoptotic rate were evaluated (). The expression of 14-3-3and Bcl-2, as well as phosphorylation of Bad (S112), were determined by Western blot. Our results showed that Dox-induced injury to vascular endothelium was decreased by TMPupregulating 14-3-3expression in total protein and Bcl-2 expression in mitochondria, activating Bad (S112) phosphorylation, maintaining EDD, reducing LDH, CK, and caspase-3 activities, thereby causing a reduction in apoptotic rate, and histopathological changes of vascular endothelium (). Furthermore, TMP increased cell viability and MMP levels, maintained NAD/NADH, GSH/GSSG balance, decreased LDH and caspase-3 activities, ROS generation, mPTP opening, and apoptotic rate (). However, the protective effects to vascular endothelium of TMP were significantly canceled by pAD/14-3-3-shRNA, an adenovirus that caused knockdown 14-3-3expression, or ABT-737, a specific Bcl-2 inhibitor. In conclusion, this study is the first to demonstrate that TMP protects the vascular endothelium against Dox-induced injury via upregulating 14-3-3expression, promoting translocation of Bcl-2 to the mitochondria, closing mPTP, maintaining MMP, inhibiting RIRR mechanism, suppressing oxidative stress, improving mitochondrial function, and alleviating Dox-induced endotheliotoxicity.

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PMID: 

Br J Pharmacol. 2020 Jan 24. Epub 2020 Jan 24. PMID: 31976548

Abstract Title: 

Tetramethylpyrazine, a Promising Drug for Treatment of Pulmonary Hypertension.

Abstract: 

RATIONALE: Tetramethylpyrazine (TMP) is a compound isolated from the traditional Chinese herb ligusticum and the fermented Japanese food natto. TMP can also be chemosynthesized in pharmacy and has long history of proven effects in the treatment of many cardiovascular diseases.OBJECTIVES: To evaluate the potential therapeutic role of TMP on pulmonary hypertension (PH) both in experimental animal models and in clinical patients with pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH).RESULTS: TMP (100 mg•kg•d) not only prevented rats from developing experimental PH, but also ameliorated PH in three models of established PH: chronic hypoxia- (chronic-HPH), sugen/hypoxia- (SuHx-PH) or monocrotaline-(MCT-PH) induced PH. The therapeutic effect of TMP was likely due to its inhibition of intracellular calcium homeostasis in pulmonary arterial smooth muscle cells. In a small cohort of patients with PAH or CTEPH, oral administration of TMP (100 mg, t.i.d. for 16 weeks) significantly increased the 6-minute walk distance (6MWD, from 385±83 to 446±80 meters; p=0.04 vs baseline, p=0.001 vs control). The 1-minute heart rate recovery (HRR1) was also improved from 13±6 to 19±8 bpm (p=0.03 vs baseline, p=0.001 vs control).CONCLUSION: Our results suggest that TMP is potentially a novel, potential and inexpensive medication for treatment of PH. Clinical trial is registered with http://www.chictr.org.cn (ChiCTR-IPR-14005379).

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PMID: 

Nutr Metab (Lond). 2020 ;17:12. Epub 2020 Jan 31. PMID: 32021640

Abstract Title: 

Tetramethylpyrazine reverses high-glucose induced hypoxic effects by negatively regulating HIF-1α induced BNIP3 expression to ameliorate H9c2 cardiomyoblast apoptosis.

Abstract: 

Background: Diabetic patients are highly vulnerable to hypoxic injury, which is associated with hypoxia induced BNIP3 expression that subsequently activate apoptosis. Our previous research show that Tetramethylpyrazine (TMP), a food flavoring agent, represses the hypoxia induced BNIP3 expression attenuate myocardial apoptosis. In this study, we evaluate the effect of TMP to provide protection against hypoxia aggravated high-glucose associated cellular apoptosis.Methods: The cytoprotective effect of TMP against high glucose induced cellular damages was determined on embryo derived H9c2 cardiomyoblast cells that were subjected to 5% hypoxia for 24 h and subjected to different duration of 33 mM high glucose challenge. Further, the involvement of HIF-1α and BNIP3 in cellular damage and the mechanism of protection of TMP were determined by overexpression and silencing HIF-1α and BNIP3 protein expression.Results: The results show that hypoxic effects on cell viability aggravates with high glucose challenge and this augmentative effect is mediated through BNIP3 in H9c2 cardiomyoblast cells. However, TMP administration effectively reversed the augmented HIF-1α levels and BNIP3 elevation. TMP improved the survival of H9c2 cells and effectively suppressed apoptosis in H9c2 cells. Further comparison on the effects of TMP on H9c2 cells challenged with high glucose and those challenged with hypoxia show that TMP precisely regulated the hypoxic intensified apoptotic effects in high-glucose condition.Conclusion: The results clearly show that flavoring agent-TMP attenuates cytotoxicity amplified by hypoxia challenge in high glucose condition by destabilizing HIF-1α.

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