CircACTR2 attenuated the effects of tetramethylpyrazine on human kidney cell injury.

Tetramethylpyrazine (TMP) is one of the active ingredients of Chuan Xiong that has been reported to have effects on numerous diseases, including diabetic nephropathy (DN). Whereas, related molecular mechanisms are not fully elucidated. We aimed to explore circACTR2's role in TMP-mediated protective effects on DN. In vitro DN condition was established in human kidney cells (HK-2) by treating high glucose (HG). CCK-8 assay and flow cytometry assay were used to observe cell viability and survival. Oxidative stress was determined by the associated markers using kits. The release of inflammatory factors was detected using ELISA kits. Quantitative real-time PCR (qPCR) and western blot were utilized for expression analysis of cricACTR2, miR-140-5p, and GLI pathogenesis-related 2 (GLIPR2). The binding between miR-140-5p and circACTR2 or GLIPR2 was confirmed by dual-luciferase, RIP, and pull-down studies. HG largely induced HK-2 cell apoptosis, oxidative stress, and inflammation, which were alleviated by TMP. CircACTR2's expression was enhanced in HG-treated HK-2 cells but attenuated in HG + TMP-treated HK-2 cells. CircACTR2 overexpression attenuated the functional effects of TMP and thus restored HG-induced cell apoptosis, oxidative stress, and inflammation. CircACTR2 bound to miR-140-5p to enhance the expression of GLIPR2. MiR-140-5p restoration or GLIPR2 inhibition reversed the role of circACTR2 overexpression. CircACTR2 attenuated the protective effects of TMP on HG-induced HK-2 cell damages by regulating the miR-140-5p/GLIPR2 network, indicating that circACTR2 was involved in the functional network of TMP in DN.

Attenuation of biofilm and virulence factors of Pseudomonas aeruginosa by tetramethylpyrazine-gold nanoparticles.

Pseudomonas aeruginosa is often identified as the causative agent in nosocomial infections. Their adapted resistance makes them strong towards antimicrobial treatments. They protect and empower their survival behind strong biofilm architecture that works as their armor toward antimicrobial therapy. Additionally, P. aeruginosa generates virulence factors, contributing to chronic infection and recalcitrant phenotypic characteristics. The current study utilizes the benevolence of nanotechnology to develop an alternate technique to control the spreading of P. aeruginosa by limiting its biofilm and virulence development. This study used a natural compound, tetramethylpyrazine, to generate gold nanoparticles. Tetramethylpyrazine-gold nanoparticles (Tet-AuNPs) were presented in spherical shapes, with an average size of 168 ± 52.49 nm and a zeta potential of -12.22 ± 2.06 mV. The minimum inhibition concentration (MIC) of Tet-AuNPs that proved more than 90 % effective in inhibiting P. aeruginosa was 256 µg/mL. Additionally, it also shows antibacterial activities against Staphylococcus aureus (MIC, 256 µg/mL), Streptococcus mutans (MIC, 128 µg/mL), Klebsiella pneumoniae (MIC, 128 µg/mL), Listeria monocytogenes (MIC, 256 µg/mL), and Escherichia coli (MIC, 256 µg/mL). The sub-MIC values of Tet-AuNPs significantly inhibited the early-stage biofilm formation of P. aeruginosa. Moreover, this concentration strongly affected hemolysis, protease activity, and different forms of motilities in P. aeruginosa. Additionally, Tet-AuNPs destroyed the well-established mature biofilm of P. aeruginosa. The expression of genes linked with the biofilm formation and virulence in P. aeruginosa treated with sub-MIC doses of Tet-AuNPs was shown to be significantly suppressed. Gene expression studies support biofilm- and virulence-suppressing effects of Tet-AuNPs at the phenotypic level.

Tetramethylpyrazine inhibits the inflammatory response by downregulating the TNFR1/IκB-α/NF-κB p65 pathway after spinal cord injury.

Previous studies have demonstrated that tetramethylpyrazine (TMP) can enhance the recovery of motor function in spinal cord injury (SCI) rats. However, the underlying mechanism involved in this therapeutic effect remains to be elucidated. We conducted RNA sequencing with a network pharmacology strategy to predict the targets and mechanism of TMP for SCI. The modified Allen's weight-drop method was used to construct an SCI rat model. The results indicated that the nuclear transfer factor-κB (NF-κB) pathway was identified through the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and an inflammatory response was identified through the Gene Ontology (GO) enrichment analysis. Tumor necrosis factor (TNF) was identified as a crucial target. Western blotting revealed that TMP decreased the protein expression of TNF superfamily receptor 1 (TNFR1), inhibitor κB-α (IκB-α), and NF-κB p65 in spinal cord tissues. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) demonstrated that TMP inhibited TNF-α, interleukin-1ß (IL-1ß), reactive oxygen species (ROS), and malondialdehyde (MDA) expression and enhanced superoxide dismutase (SOD) expression. Histopathological observation and behavior assessments showed that TMP improved morphology and motor function. In conclusion, TMP inhibits inflammatory response and oxidative stress, thereby exerting a neuroprotective effect that may be related to the regulation of the TNFR1/IκB-α/NF-κB p65 signaling pathway.

Tetramethylpyrazine attenuates renal tubular epithelial cell ferroptosis in contrast-induced nephropathy by inhibiting transferrin receptor and intracellular reactive oxygen species.

Contrast-induced nephropathy (CIN) is a leading cause of hospital-acquired acute kidney injury (AKI). Recently, ferroptosis was reported to be crucial for AKI pathogenesis. Our previous studies indicated antioxidant tetramethylpyrazine (TMP) prevent CIN in vivo. However, whether ferroptosis is involved in TMP nephroprotective mechanism against CIN is unclear. In the present study, we investigated the role of renal tubular epithelial cell ferroptosis in TMP reno-protective effect against CIN and the molecular mechanisms by which TMP regulates ferroptosis. Classical contrast-medium, Iohexol, was used to construct CIN models in rats and HK-2 cells. Results showed that tubular cell injury was accompanied by ferroptosis both in vivo and in vitro, including the typical features of ferroptosis, Fe2+ accumulation, lipid peroxidation and decreased glutathione peroxidase 4 (GPX4). Ferroptosis inhibition by classic inhibitors Fer-1 and DFO promoted cell viability and reduced intracellular ROS production. Additionally, TMP significantly inhibited renal dysfunction, reduced AKI biomarkers, prevented ROS production, inhibited renal Fe2+ accumulation and increased GPX4 expression. Expressions of various proteins associated with iron ion metabolism, including transferrin receptor (TFRC), divalent metal transporter 1, iron-responsive element binding protein 2, ferritin heavy chain 1, ferroportin 1, and heat shock factor binding protein 1, were examined using mechanistic analyses. Among these, TFRC changes were the most significant after TMP pretreatment. Results of siRNA knockdown and plasmid overexpression of TFRC indicated that TFRC is essential for TMP to alleviate ferroptosis and reduce LDH release, Fe2+ accumulation and intracellular ROS. Our findings provide crucial insights about the potential of TMP in treating AKI associated with ferroptosis.

Neuroprotection by tetramethylpyrazine and its synthesized analogues for central nervous system diseases: a review.

BACKGROUND: Due to the global increase in aging populations and changes in modern lifestyles, the prevalence of neurodegenerative diseases, cerebrovascular disorders, neuropsychiatrcic conditions, and related ailments is rising, placing an increasing burden on the global public health system. MATERIALS AND METHODS: All studies on tetramethylpyrazine (TMP) and its derivatives were obtained from reputable sources such as PubMed, Elsevier, Library Genesis, and Google Scholar. Comprehensive data on TMP and its derivatives was meticulously compiled. RESULTS: This comprehensive analysis explains the neuroprotective effects demonstrated by TMP and its derivatives in diseases of the central nervous system. These compounds exert their influence on various targets and signaling pathways, playing crucial roles in the development of various central nervous system diseases. Their multifaceted mechanisms include inhibiting oxidative damage, inflammation, cell apoptosis, calcium overload, glutamate excitotoxicity, and acetylcholinesterase activity. CONCLUSION: This review provides a brief summary of the most recent advancements in research on TMP and its derivatives in the context of central nervous system diseases. It involves synthesizing analogs of TMP and evaluating their effectiveness in models of central nervous system diseases. The ultimate goal is to facilitate the practical application of TMP and its derivatives in the future treatment of central nervous system diseases.

Whole genome sequencing and analysis of Bacillus sp. TTMP2, a tetramethylpyrazine-producing bacterium.

BACKGROUND: Tetramethylpyrazine has been extensively studied as an anticancer substance and a flavor substance in the fields of medicine and food industry. A strain with high tetramethylpyrazine production was screened from the fermented grains of Danquan winery. Genome sequencing can reveal the potential roles of bacteria by thoroughly examining the connection between genes and phenotypes from a genomic perspective. METHODS AND RESULTS: In this study, whole genome of this strain was sequenced and analyzed. This paper summarized the genomic characteristics of strain TTMP2 and analyzed genes related to the synthesis of tetramethylpyrazine. Bacillus sp. TTMP2 has a complete metabolic pathway for acetoin and tetramethylpyrazine metabolism. Gene function was analyzed by COG annotation, GO annotation, KEGG annotation and functional annotations for lipoproteins, carbohydrate-active enzymes, and pathogen-host interactions. Phylogenetic analysis indicated that Bacillus velezensis had the high homology with Bacillus sp. TTMP2. Genomes of 16 Bacillus species cover all genes of Bacillus, suggesting that genus Bacillus has an open pan-genome and can survive in diverse environments. CONCLUSION: The analysis of genome sequencing data from Bacillus sp. TTMP2 showed that its metabolic characteristics could be deeply understood, indicating that this bacterium had a particular role in tetramethylpyrazine synthesis.

Tetramethylpyrazine Antagonizes the Subchronic Cadmium Exposure-Induced Oxidative Damage in Mouse Livers via the Nrf2/HO-1 Pathway.

Hepatic oxidative stress is an important mechanism of Cd-induced hepatotoxicity, and it is ameliorated by TMP. However, this underlying mechanism remains to be elucidated. To investigate the mechanism of the protective effect of TMP on liver injuries in mice induced by subchronic cadmium exposure, 60 healthy male ICR mice were randomly divided into five groups of 12 mice each, namely, control (CON), Cd (2 mg/kg of CdCl2), Cd + 100 mg/kg of TMP, Cd + 150 mg/kg of TMP, and Cd + 200 mg/kg of TMP, and were acclimatized and fed for 7 d. The five groups of mice were gavaged for 28 consecutive days with a maximum dose of 0.2 mL/10 g/day. Except for the control group, all groups were given fluoride (35 mg/kg) by an intraperitoneal injection on the last day of the experiment. The results of this study show that compared with the Cd group, TMP attenuated CdCl2-induced pathological changes in the liver and improved the ultrastructure of liver cells, and TMP significantly decreased the MDA level (p < 0.05) and increased the levels of T-AOC, T-SOD, and GSH (p < 0.05). The results of mRNA detection show that TMP significantly increased the levels of Nrf2 in the liver compared with the Cd group as well as the HO-1 and mRNA expression levels in the liver (p < 0.05). In conclusion, TMP could inhibit oxidative stress and attenuate Cd group-induced liver injuries by activating the Nrf2 pathway.

Progress on the Synthesis Pathways and Pharmacological Effects of Naturally Occurring Pyrazines.

As one of the most essential types of heterocyclic compounds, pyrazines have a characteristic smell and taste and have a wide range of commercial applications, especially in the food industry. With the development of the food industry, the demand for pyrazines has increased. Therefore, understanding the properties, functions, and synthetic pathways of pyrazines is one of the fundamental methods to produce, control, and apply pyrazines in food or medical systems. In this review, we provide an overview of the synthesis pathways and physiological or pharmacological functions of naturally occurring pyrazines. In particular, we focus on the biosynthesis and pharmacological effects of 2,3,5,6-Tetramethylpyrazine (TTMP), 2,5-Dimethylpyrazine (2,5-DMP), and 2,3,5-trimethylpyrazine (TMP). Furthermore, areas where further research on pyrazines is needed are discussed in this work.

[Mechanism of tetramethylpyrazine combined with neural stem cells transplantation on cerebral ischemia-reperfusion rats].

This study aimed to investigate the intervention effect of tetramethylpyrazine(TMP) combined with transplantation of neural stem cells(NSCs) on middle cerebral artery occlusion(MCAO) rat model and to explore the mechanism of TMP combined with NSCs transplantation on ischemic stroke based on the regulation of stem cell biological behavior. MCAO rats were randomly divided into a model group, a TMP group, an NSCs transplantation group, and a TMP combined with NSCs transplantation group according to neurological function scores. A sham group was set up at the same time. The neurological function score was used to evaluate the improvement of neurological function in MCAO rats after TMP combined with NSCs transplantation. The proliferation, migration, and differentiation of NSCs were evaluated by BrdU, BrdU/DCX, BrdU/NeuN, and BrdU/GFAP immunofluorescence labeling. The protein expression of stromal cell-derived factor 1(SDF-1), C-X-C motif chemokine receptor 4(CXCR4), as well as oxidative stress pathway proteins nuclear factor erythroid 2-related factor 2(Nrf2), Kelch-like ECH-associated protein 1(KEAP1), heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) was detected by Western blot to study the migration mechanism of TMP combined with NSCs. The results showed that TMP combined with NSCs transplantation significantly improved the neurological function score in MCAO rats. Immunofluorescence staining showed a significant increase in the number of BrdU~+, BrdU~+/DCX~+, BrdU~+/NeuN~+, and BrdU~+/GFAP~+ cells in the TMP, NSCs transplantation, and combined treatment groups, with the combined treatment group showing the most significant increase. Further Western blot analysis revealed significantly elevated expression of CXCR4 protein in the TMP, NSCs transplantation, and combined treatment groups, along with up-regulated protein expression of Nrf2, HO-1, and NQO1, and decreased KEAP1 protein expression. This study showed that both TMP and NSCs transplantation can promote the recovery of neurological function by promoting the proliferation, migration, and differentiation of NSCs, and the effect of TMP combined with NSCs transplantation is superior. The mechanism of action may be related to the activation of the Nrf2/HO-1/CXCR4 pathway.

[Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro].

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

[Mechanism of tetramethylpyrazine intervention with ischemia-reperfusion rats based on Nrf2/HO-1/CXCR4 pathway through regulating neural stem cell migration].

This study aims to decipher the mechanism of tetramethylpyrazine(TMP) in regulating the migration of neural stem cells(NSCs) in the rat model of middle cerebral artery occlusion(MCAO) via the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase 1(HO-1)/C-X-C motif chemokine receptor 4(CXCR4) pathway. SD rats were randomized into sham, MCAO(model), and tetramethylpyrazine(TMP, 20 mg·kg~(-1) and 40 mg·kg~(-1)) groups. The neurological impairment was assessed by the modified neurological severity score(mNSS). The immunofluorescence assay was employed to detect the cells stained with both 5-bromodeoxyuridine(BrdU) and doublecortin(DCX) in the brain tissue. The effect of TMP on the migration of C17.2 cells was observed. Western blot was employed to determine the protein levels of Nrf2, HO-1, p62, NAD(P)H quinone oxidoreductase 1(NQO1), stromal cell-derived factor 1(SDF-1), and CXCR4 in the brain tissue and C17.2 cells. The results showed that after 7 days and 21 days of mode-ling, the mNSS and BrdU~+/DCX~+ cells were increased, and the expression of Nrf2 and CXCR4 in the brain tissue was up-regulated. Compared with the model group, TMP(40 mg·kg~(-1)) reduced the mNSS, increased the number of BrdU~+/DCX~+ cells, and up-regulated the expression of Nrf2, CXCR4, and SDF-1. In addition, TMP promoted the migration of C17.2 cells and up-regulated the expression of p62, Nrf2, HO-1, and NQO1 in a time-and dose-dependent manner. The expression was the highest at the time point of 12 h in the TMP(50 µg·mL~(-1)) group(P<0.01). In conclusion, TMP activates the Nrf2/HO-1/CXCR4 pathway to promote the migration of NSCs to the ischemic area, thus exerting the therapeutic effect on the ischemia-reperfusion injury. This study provides experimental support for the application of TMP in ischemic stroke.

[Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury].

This study aims to explore the effects of tetramethylpyrazine(TMP) on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI), and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate. Male SD rats were randomized into Sham, SNI, and SNI+TMP groups. Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats. Another two groups, Sham+TMP and SNI+TMP, were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC), respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg~(-1), automatic blood collection and intracerebral microdialysis(perfusion rate of 1 µL·min~(-1)) systems were used to collect the blood and brain dialysate for 24 h. HSS T3 C_(18) reversed-phase chromatographic column(2.1 mm×50 mm, 2.5 µm) was used for liquid chromatographic separation. Gradient elution was carried out with the mobile phase of methanol-water(containing 0.005% formic acid) at a flow rate of 0.25 mL·min~(-1). Electrospray ion source was used for mass spectrometry, and the scanning mode was multi-reaction monitoring under the positive ion mode. The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137, respectively. DAS 2.11 was used to calculate the pharmacokinetic parameters. The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment. The TMP in the plasma and brain dialysate of SNI rats showed the T_(max) of 15 min and 30 min, the C_(max) of(2 866.43±135.39) and(1 462.14±197.38) µg·L~(-1), the AUC_(0-t) of(241 463.30±28 070.31) and(213 115.62±32 570.07) µg·min·L~(-1), the MRT_(0-t) of(353.13±47.73) and(172.16±12.72) min, and the CL_Z of 0.73 and 0.36 L·min·kg~(-1), respectively. The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC, which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate. The method is accurate, reliable, and sensitive and can realize the important value of the application of correlation analysis theory of "automatic blood collection-microdialysis/PK-PD" in the research on neuropathic pain.

[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

[Mechanism of tetramethylpyrazine attenuates inflammatory injury in endothelial cells by activating the SIRT1 signaling pathway]. / 川芎嗪通过激活SIRT1ä¿¡å·é€šè·¯å‡è½»å† çš®ç»†èƒžç‚Žç—‡æŸä¼¤çš„æœºåˆ¶ç ”ç©¶.

OBJECTIVES: To study the effects and mechanisms of tetramethylpyrazine (TMP) on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human coronary artery endothelial cells (HCAEC). METHODS: HCAEC were randomly divided into four groups: the control group (no treatment), the model group (treated with TNF-α, 50 ng/mL for 24 hours), the TMP group (pre-treated with TMP, 80 µg/mL for 12 hours followed by TNF-α treatment for 24 hours), and the SIRT1 inhibitor group (pre-treated with TMP and the specific SIRT1 inhibitor EX527 for 12 hours followed by TNF-α treatment for 24 hours). Cell viability was assessed using the CCK-8 method, lactate dehydrogenase (LDH) activity was measured using an LDH assay kit, reactive oxygen species (ROS) levels were observed using DCFH-DA staining, expression of pyroptosis-related proteins was detected by Western blot, and SIRT1 expression was analyzed using immunofluorescence staining. RESULTS: Compared to the control group, the model group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). Compared to the model group, the TMP group exhibited increased cell viability, decreased LDH activity, ROS level and expression of pyroptosis-related proteins, and increased SIRT1 expression (P<0.05). In comparison to the TMP group, the SIRT1 inhibitor group showed decreased cell viability, increased LDH activity, ROS level and expression of pyroptosis-related proteins, and decreased SIRT1 expression (P<0.05). CONCLUSIONS: TMP may attenuate TNF-α-induced inflammatory injury in HCAEC, which is associated with the inhibition of pyroptosis and activation of the SIRT1 signaling pathway.

HMGB1-induced activation of ER stress contributes to pulmonary artery hypertension in vitro and in vivo.

BACKGROUND: HMGB1 and ER stress have been considered to participate in the progression of pulmonary artery hypertension (PAH). However, the molecular mechanism underlying HMGB1 and ER stress in PAH remains unclear. This study aims to explore whether HMGB1 induces pulmonary artery smooth muscle cells (PASMCs) functions and pulmonary artery remodeling through ER stress activation. METHODS: Primary cultured PASMCs and monocrotaline (MCT)-induced PAH rats were applied in this study. Cell proliferation and migration were determined by CCK-8, EdU and transwell assay. Western blotting was conducted to detect the protein levels of protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor-4 (ATF4), seven in absentia homolog 2 (SIAH2) and homeodomain interacting protein kinase 2 (HIPK2). Hemodynamic measurements, immunohistochemistry staining, hematoxylin and eosin staining were used to evaluate the development of PAH. The ultrastructure of ER was observed by transmission electron microscopy. RESULTS: In primary cultured PASMCs, HMGB1 reduced HIPK2 expression through upregulation of ER stress-related proteins (PERK and ATF4) and subsequently increased SIAH2 expression, which ultimately led to PASMC proliferation and migration. In MCT-induced PAH rats, interfering with HMGB1 by glycyrrhizin, suppression of ER stress by 4-phenylbutyric acid or targeting SIAH2 by vitamin K3 attenuated the development of PAH. Additionally, tetramethylpyrazine (TMP), as a component of traditional Chinese herbal medicine, reversed hemodynamic deterioration and vascular remodeling by targeting PERK/ATF4/SIAH2/HIPK2 axis. CONCLUSIONS: The present study provides a novel insight to understand the pathogenesis of PAH and suggests that targeting HMGB1/PERK/ATF4/SIAH2/HIPK2 cascade might have potential therapeutic value for the prevention and treatment of PAH.

Tetramethylpyrazine Attenuates Radiation-Induced Ototoxicity in a Rat Model.

INTRODUCTION: Tetramethylpyrazine (TMP) is a chemical compound, which has been shown to possess numerous biological features such as anticoagulation, inhibition of platelet aggregation, anti-inflammation, capillary dilatation, improvement in microcirculation, and protection against reactive oxygen radicals. The aim of the present study was to investigate the protective effect of TMP against radiation-induced ototoxicity. MATERIALS AND METHODS: 40 rats were divided into four groups. The first group was irradiated for 5 days. The second group received a single dose of 140 mg/kg/day intraperitoneal TMP given to the rats 30 min before radiotherapy (RT) for 5 days. The third group received a single dose of 140 mg/kg/day i.p. TMP for 5 days, whereas the fourth group was administered saline. All rats underwent distortion product otoacoustic emission (DPOAE) and auditory brainstem response measurements before and after the application. The temporal bulla of animals was removed for immunohistopathological examination. RESULTS: Signal-noise ratio values were significantly decreased in the RT group for the frequencies of 2-32 kHz after RT (p < 0.05), whereas the difference was not significant in terms of pre- and posttreatment values for the other groups. Also in the RT group, the ABR thresholds were significantly increased after treatment. In H&E staining, the mean scores for outer hair cells (OHCs), stria vascularis (SV), and spiral ganglion (SG) injuries were significantly higher in RT and RT + TMP groups than in the other groups. The mean OHCs and SV injury scores were also significantly higher in the RT group than in the RT + TMP group (p < 0.05). The number of cochleas that showed cytoplasmic caspase-3 immunoreactivity in the OHC, SV, and SG was significantly higher in RT and RT + TMP groups than in the other groups. CONCLUSION: The findings of the present study suggest that TMP may have a therapeutic potential for preventing sensorineural hearing loss (SNHL) related to RT.

Tetramethylpyrazine ameliorates endotoxin-induced acute lung injury by relieving Golgi stress via the Nrf2/HO-1 signaling pathway.

PURPOSE: Endotoxin-induced acute lung injury (ALI) is a severe disease caused by an imbalanced host response to infection. It is necessary to explore novel mechanisms for the treatment of endotoxin-induced ALI. In endotoxin-induced ALI, tetramethylpyrazine (TMP) provides protection through anti-inflammatory, anti-apoptosis, and anti-pyroptosis effects. However, the mechanism of action of TMP in endotoxin-induced ALI remains unclear. Here, we aimed to determine whether TMP can protect the lungs by inhibiting Golgi stress via the Nrf2/HO-1 pathway. METHODS AND RESULTS: Using lipopolysaccharide (LPS)-stimulated C57BL/6J mice and MLE12 alveolar epithelial cells, we observed that TMP pretreatment attenuated endotoxin-induced ALI. LPS + TMP group showed lesser lung pathological damage and a lower rate of apoptotic lung cells than LPS group. Moreover, LPS + TMP group also showed decreased levels of inflammatory factors and oxidative stress damage than LPS group (P < 0.05). Additionally, LPS + TMP group presented reduced Golgi stress by increasing the Golgi matrix protein 130 (GM130), Golgi apparatus Ca2+/Mn2+ ATPases (ATP2C1), and Golgin97 expression while decreasing the Golgi phosphoprotein 3 (GOLPH3) expression than LPS group (P < 0.05). Furthermore, TMP pretreatment promoted Nrf2 and HO-1 expression (P < 0.05). Nrf2-knockout mice or Nrf2 siRNA-transfected MLE12 cells were pretreated with TMP to explore how the Nrf2/HO-1 pathway affected TMP-mediated Golgi stress in endotoxin-induced ALI models. We observed that Nrf2 gene silencing partially reversed the alleviating effect of Golgi stress and the pulmonary protective effect of TMP. CONCLUSION: Our findings showed that TMP therapy reduced endotoxin-induced ALI by suppressing Golgi stress via the Nrf2/HO-1 signaling pathway in vivo and in vitro.

Bone marrow mesenchymal stem cells-derived exosomes mediated delivery of tetramethylpyrazine attenuate cerebral ischemic injury.

OBJECTIVES: Tetramethylpyrazine (TEP) can protect the brain from ischemic damage, but it has defects such as short half-life, fast absorption, wide distribution, and rapid elimination, which limits its application. Exosomes (Exos) have the property of loading drugs and transporting signal substances. Here, we elucidated the effect of TEP-loaded bone marrow mesenchymal stem cell (BMSC)-derived Exos (Exo-TEP) on cerebral ischemic injury. MATERIALS AND METHODS: The Exos were extracted by ultracentrifugation and TEP was loaded into the Exos by electroporation. Oxygen-glucose deprivation (OGD) induced-primary cortical neurons and middle cerebral artery occlusion (MCAO)-induced mouse models were used to determine the effect of Exo-TEP on cerebral ischemic injury in vitro and in vivo. RESULTS: Exo-TEP exhibited a stable and sustained release pattern compared to free TEP. Exo-TEP treatment was more significant in improving OGD-mediated decrease in cell activity, as well as a elevation in apoptosis and ROS production in cortical neurons. In comparison with Exo and free TEP treatment, Exo-TEP treatment significantly improved pathological changes, shrunk cerebral infarction volume, as well reduced neurological deficit scores and neuronal apoptosis, and oxidative stress. CONCLUSIONS: Exo-TEP was superior to free TEP in improving cerebral ischemic injury by reducing neuronal apoptosis and oxidative stress.

The Pathophysiological Changes and Clinical Effects of Tetramethylpyrazine in ICR Mice with Fluoride-Induced Hepatopathy.

The excessive intake of fluoride, one of the trace elements required to maintain health, leads to liver injury. Tetramethylpyrazine (TMP) is a kind of traditional Chinese medicine monomer with a good antioxidant and hepatoprotective function. The aim of this study was to investigate the effect of TMP on liver injury induced by acute fluorosis. A total of 60 1-month-old male ICR mice were selected. All mice were randomly divided into five groups: a control (K) group, a model (F) group, a low-dose (LT) group, a medium-dose (MT) group, and a high-dose (HT) group. The control and model groups were given distilled water, while 40 mg/kg (LT), 80 mg/kg (MT), or 160 mg/kg (HT) of TMP was fed by gavage for two weeks, with a maximum gavage volume for the mice of 0.2 mL/10 g/d. Except for the control group, all groups were given fluoride (35 mg/kg) by an intraperitoneal injection on the last day of the experiment. The results of this study showed that, compared with the model group, TMP alleviated the pathological changes in the liver induced by the fluoride and improved the ultrastructure of liver cells; TMP significantly decreased the levels of ALT, AST, and MDA (p < 0.05) and increased the levels of T-AOC, T-SOD, and GSH (p < 0.05). The results of mRNA detection showed that TMP significantly increased the mRNA expression levels of Nrf2, HO-1, CAT, GSH-Px, and SOD in the liver compared with the model group (p < 0.05). In conclusion, TMP can inhibit oxidative stress by activating the Nrf2 pathway and alleviate the liver injury induced by fluoride.

Production of Tetramethylpyrazine from Cane Molasses by Bacillus sp. TTMP20.

2,3,5,6-Tetramethylpyrazine (TTMP) is an active ingredient of Ligusticum wallichii Franch. It can be used in medicine and food fields. In this study, Bacillus sp. TTMP20 was applied to produce TTMP using cane molasses as a carbon source. After pretreatment with phosphoric acid, 170 mL/L treated molasses, combined with 10 g/L yeast powder, 30 g/L tryptone and 30 g/L (NH4)2HPO4 were used for fermentation. After 36 h, TTMP output reached the highest value of 208.8 mg/L. The yield of TTMP using phosphoric acid-treated molasses as carbon source was 145.59% higher than control. Under the sulfuric acid treatment process of molasses (150 g), the maximum yield of TTMP was 895.13 mg/L, which was 183.18% higher than that of untreated molasses (316.1 mg/L). This study demonstrated that molasses is a high-quality and inexpensive carbon source for the manufacture of TTMP, laying the groundwork for the future industrial production of TTMP.