Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Gut microbiota controls the development of chronic pancreatitis: A critical role of short-chain fatty acids-producing Gram-positive bacteria.

Chronic pancreatitis (CP) is a progressive and irreversible fibroinflammatory disorder, accompanied by pancreatic exocrine insufficiency and dysregulated gut microbiota. Recently, accumulating evidence has supported a correlation between gut dysbiosis and CP development. However, whether gut microbiota dysbiosis contributes to CP pathogenesis remains unclear. Herein, an experimental CP was induced by repeated high-dose caerulein injections. The broad-spectrum antibiotics (ABX) and ABX targeting Gram-positive (G+) or Gram-negative bacteria (G-) were applied to explore the specific roles of these bacteria. Gut dysbiosis was observed in both mice and in CP patients, which was accompanied by a sharply reduced abundance for short-chain fatty acids (SCFAs)-producers, especially G+ bacteria. Broad-spectrum ABX exacerbated the severity of CP, as evidenced by aggravated pancreatic fibrosis and gut dysbiosis, especially the depletion of SCFAs-producing G+ bacteria. Additionally, depletion of SCFAs-producing G+ bacteria rather than G- bacteria intensified CP progression independent of TLR4, which was attenuated by supplementation with exogenous SCFAs. Finally, SCFAs modulated pancreatic fibrosis through inhibition of macrophage infiltration and M2 phenotype switching. The study supports a critical role for SCFAs-producing G+ bacteria in CP. Therefore, modulation of dietary-derived SCFAs or G+ SCFAs-producing bacteria may be considered a novel interventive approach for the management of CP.

Further terpenoids from the Chloranthaceae plant Chloranthus multistachys and their anti-neuroinflammatory activities.

Three labdane-type [multisins A-C (1-3)], two guaiane-type [multisins D (4) and E (5)], and one eudesmane-type [multisin F (6)] previously undescribed terpenoids, together with 14 mono- (7-20) and seven dimeric- (21-27) known terpenoids, were isolated from the 90% MeOH extract of the whole plant of Chloranthus multistachys. Their structures and absolute configurations were determined by extensive spectroscopic methods and electronic circular dichroism (ECD) calculations. Compounds 4 and 5 are rare trinor-sesquiterpenes with a de-isopropyl guaiane skeleton, whereas compound 6 is a rearranged dinor-eudesmene featuring an uncommon octahydro-1H-indene ring system. Among the isolates, the dimeric lindenane sesquiterpenoid shizukaol C (25) exhibited the most potent (IC50 = 8.04 µM) anti-neuroinflammatory activity by inhibiting the nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells.

Modulation of Gut Microbiota by Low Methoxyl Pectin Attenuates Type 1 Diabetes in Non-obese Diabetic Mice.

Intestinal homeostasis underpins the development of type 1 diabetes (T1D), and dietary manipulations to enhance intestinal homeostasis have been proposed to prevent T1D. The current study aimed to investigate the efficacy of supplementing a novel specific low-methoxyl pectin (LMP) dietary fiber in preventing T1D development. Female NOD mice were weaned onto control or 5% (wt/wt) LMP supplemented diets for up to 40 weeks of age, overt diabetes incidence and blood glucose were monitored. Then broad-spectrum antibiotics (ABX) treatment per os for 7 days followed by gut microbiota transfer was performed to demonstrate gut microbiota-dependent effects. Next-generation sequencing was used for analyzing the composition of microbiota in caecum. Concentration of short chain fatty acids were determined by GC-MS. The barrier reinforcing tight junction proteins zonula occludens-2 (ZO-2), claudin-1 and NOD like receptor protein 3 (NLRP3) inflammasome activation were determined by Western blot. The proportion of CD25+Foxp3+CD4+ regulatory T cell (Foxp3+ Treg) in the pancreas, pancreatic and mesenteric lymph nodes was analyzed by flow cytometry. We found that LMP supplementation ameliorated T1D development in non-obese diabetic (NOD) mice, as evidenced by decreasing diabetes incidence and fasting glucose levels in LMP fed NOD mice. Further microbiota analysis revealed that LMP supplementation prevented T1D-associated caecal dysbiosis and selectively enriched caecal bacterial species to produce more SCFAs. The LMP-mediated microbial balance further enhanced caecal barrier function and shaped gut-pancreatic immune environment, as characterized by higher expression of tight junction proteins claudin-1, ZO-2 in caecum, increased Foxp3+ Treg population and decreased NLRP3 inflammasome activation in both caecum and pancreas. The microbiota-dependent beneficial effect of LMP on T1D was further proven by the fact that aberration of caecal microbiota by ABX treatment worsened T1D autoimmunity and could be restored with transfer of feces of LMP-fed NOD mice. These data demonstrate that this novel LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment. This finding opens a realistic option for gut microbiota manipulation and prevention of T1D in humans.

Low Methoxyl Pectin Protects against Autoimmune Diabetes and Associated Caecal Dysfunction.

SCOPE: This study aims to examine the protective effects of specific low-methoxyl pectin (LMP) on the development of type 1 diabetes (T1D). METHODS AND RESULTS: Female non-obese diabetic (NOD) mice are weaned onto either control or 5% LMP supplemented diets for up to 22 weeks of age. T1D incidence, gut barrier function, and pancreatic-gut immune responses are analyzed. LMP supplementation significantly dampened the onset of T1D in NOD mice. LMP supplementation induces caecal homeostasis, as indicated by the increasing SCFAs production, higher expression of tight junction proteins claudin 1, zonula occludens-2 in caecum. Furthermore, LMP-mediated caecal homeostasis impacts gut-pancreatic immunity, as evidenced by increased regulatory T cell population, modulated inflammatory cytokine expression, and suppressed NOD like receptor protein 3 (NLRP3) inflammasome activation in both caecum and pancreas. CONCLUSION: The data demonstrate that LMP limits T1D development by inducing caecal homeostasis to shape pancreatic immune environment, providing a scientific basis for using LMP as a novel functional supplementation to intervene T1D.

Corrigendum: Recent Advances on Nutrition in Treatment of Acute Pancreatitis.

[This corrects the article on p. 762 in vol. 8, PMID: 28713382.].

Recent Advances on Nutrition in Treatment of Acute Pancreatitis.

Acute pancreatitis (AP) is a common abdominal acute inflammatory disorder and the leading cause of hospital admission for gastrointestinal disorders in many countries. Clinical manifestations of AP vary from self-limiting local inflammation to devastating systemic pathological conditions causing significant morbidity and mortality. To date, despite extensive efforts in translating promising experimental therapeutic targets in clinical trials, disease-specific effective remedy remains obscure, and supportive care has still been the primary treatment for this disease. Emerging evidence, in light of the current state of pathophysiology of AP, has highlighted that strategic initiation of nutrition with appropriate nutrient supplementation are key to limit local inflammation and to prevent or manage AP-associated complications. The current review focuses on recent advances on nutritional interventions including enteral versus parenteral nutrition strategies, and nutritional supplements such as probiotics, glutamine, omega-3 fatty acids, and vitamins in clinical AP, hoping to advance current knowledge and practice related to nutrition and nutritional supplements in clinical management of AP.

Boehmenan, a Lignan From the Chinese Medicinal Plant Clematis armandii, Inhibits A431 Cell Growth via Blocking p70S6/S6 Kinase Pathway.

Previously, we have shown that boehmenan, a natural product isolated from the dried stem of Caulis clematidis armandii, exhibits various biological activities. The current study investigated the effects of boehmenan on the growth of human epidermoid carcinoma A431 cells. Cell viability and 50% inhibiting concentration (IC50) were assessed by CellTiter-Glo luminescent cell viability assay. Cell cycle arrest was measured by flow cytometry. Intracellular reactive oxygen species production and mitochondrial membrane potential (ΔΨm) collapse were analyzed by a fluorescence spectrophotometer. The activation of epidermal growth factor receptor signaling pathway was evaluated by Western blot. The results showed that boehmenan significantly inhibited the growth of A431 cells (IC50 = 1.6 µM) in a concentration- and time-dependent manner. This compound also blocked cell cycle progression at G2/M phase and modulated mitochondrial apoptosis-related proteins, as evidenced by upregulating p21, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase protein levels and by downregulating Bcl-2, pro-caspase-9 levels. In addition, boehmenan also markedly induced intracellular reactive oxygen species production and ΔΨm depolarization in a concentration-dependent manner. Furthermore, boehmenan-attenuated epidermal growth factor mediated the phosphorylation of signal transducer and activator of transcription 3 (STAT3), p70 ribosomal protein S6 kinase (p70S6)/S6 in a concentration-dependent manner. Taken together, our results suggest that boehmenan-mediated antiproliferative property in A431 cells was mediated partially by modulation of mitochondrial function and inhibition of STAT3 and p70S6 signal pathways.

S-propargyl-cysteine attenuates inflammatory response in rheumatoid arthritis by modulating the Nrf2-ARE signaling pathway.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder. Hydrogen sulfide (H2S), the third physiological gasotransmitter, is well recognized as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the protective effects of S-propargyl-cysteine (SPRC, also known as ZYZ-802), an endogenous H2S modulator, on RA and determined the underlying mechanisms. In the present study, SPRC concentration-dependently attenuated inflammatory mediator expression, reactive oxidase species generation, and the expression and activity of matrix metalloproteinases (MMP)-9 in interleukin (IL)-1ß-induced human rheumatoid fibroblast-like synoviocytes MH7A. In addition, SPRC blocked IL-1ß-mediated migration and invasion of MH7A cells. As expected, the protective effects of SPRC were partially abrogated by DL-propargylglycine (PAG, a H2S biosynthesis inhibitor). In vivo study also demonstrated that SPRC treatment markedly ameliorated the severity of RA in adjuvant-induced arthritis rats, and this effect was associated with the inhibition of inflammatory response. We further identified that SPRC remarkably induced heme oxygenase-1 expression associated with the degradation of Kelch-like ECH-associated protein 1 (Keap1) and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2); this effect was attributed to the sulfhydrylation of the cysteine residue of Keap1. Our data demonstrated for the first time that SPRC, an endogenous H2S modulator, exerted anti-inflammatory properties in RA by upregulating the Nrf2-antioxidant response element (ARE) signaling pathway.

Biginkgosides A-I, Unexpected Minor Dimeric Flavonol Diglycosidic Truxinate and Truxillate Esters from Ginkgo biloba Leaves and Their Antineuroinflammatory and Neuroprotective Activities.

Nine unexpected new flavonol glycoside cyclodimers in the truxinate (1-7, biginkgosides A-G, respectively) or truxillate [biginkgosides H (8) and I (9)] forms were isolated as minor components from the extract of Ginkgo biloba leaves. The new dimers possess an unusual cyclobutane ring formed by a [2+2]-cycloaddition between two symmetric (for compounds 1-5 and 7-9) or nonsymmetric (for 6) flavonol coumaroyl glucorhamnosides. A plausible biosynthetic pathway for these new compounds based on the frontier molecular orbital theory of cycloaddition reactions is briefly discussed. An antineuroinflammatory screening revealed that biginkgosides E (5) and H (8) inhibited nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells, with IC50 values of 2.91 and 17.23 µM, respectively. Additionally, biginkgoside F (6) showed a significant neuroprotective effect (34.3% increase in cell viability at 1 µM) against Aß25-35-induced cell viability decrease in SH-SY5Y neuroblastoma cells.

Boehmenan, a lignan from the Chinese medicinal plant Clematis armandii, induces apoptosis in lung cancer cells through modulation of EGF-dependent pathways.

BACKGROUND: Epidermal growth factor receptor (EGFR) is an effective molecular target for cancer treatment. Boehmenan, a lignan from the dried stems of Clematis armandii, exhibited the potent cytotoxic effects against many cancer cell lines in previous studies. However, the effects and underlying mechanism of boehmenan on non-small cell lung cancer (NSCLC) remains unclear. PURPOSE: The present study was designed to determine the in vitro anti-cancer properties and underlying molecular mechanisms of boehmenan on A549 NSCLC cells. STUDY DESIGN/METHODS: Cellular viability and chemoattractive properties of macrophages were investigated by using MTT and transwell migration assay, respectively. Mitochondrial membrane potential (ΔΨm), apoptotic ratio, and cell cycle were measured by flow cytometry. Protein expression was visualized by Western blot using specific antibodies. RESULTS: Boehmenan concentration-dependently suppressed proliferation and induced G1 phase arrest in A549 NSCLC cells, which were accompanied by reduction of migration, colony formation and increase of apoptosis in A549 cells. In addition, boehmenan treatment markedly modulated apoptosis-related protein (p53, p21, cleaved caspase 3, and cleaved PARP) and cyclin D1 expression and induced ΔΨm collapse in a concentration dependent manner. Furthermore, boehmenan concentration-dependently inhibited EGF-induced activation of EGFR and its downstream signaling molecules, including MEK, Akt, ERK1/2, and STAT3. CONCLUSION: Taken together, our results suggested that boehmenan-mediated anti-tumor property was mediated by modulation of mitochondria and EGFR signaling pathway in A549 NSCLC cells.

(7R,8S)-9-acetyl-dehydrodiconiferyl alcohol inhibits inflammation and migration in lipopolysaccharide-stimulated macrophages.

BACKGROUND: (7R, 8S)-9-Acetyl-dehydrodiconiferyl alcohol (ADDA), a novel lignan compound isolated from Clematis armandii Franch (Ranunculaceae) stems, has been found to exert potential anti-inflammatory activities in vitro. PURPOSE: To investigate the pharmacological effects and molecular mechanisms of ADDA on lipopolysaccharide (LPS)-induced activation and migration of macrophages. STUDY DESIGN/METHODS: Macrophages were stimulated with LPS in the presence or absence of ADDA. Expression of inflammatory mediators, including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) were measured by Western blot and commercial NO detection kit. Cellular viability and chemotactic properties of macrophages were investigated using MTT and transwell migration assays. The activation and expression of mitogen activated protein kinases, nuclear factor-κB (NF-κB), protein kinase B (Akt), Src, and focal adhesion kinase (FAK) were analyzed by Western blot. RESULTS: Non-toxic concentrations (12.5-50 µM) of ADDA concentration-dependently inhibited expression/release of inflammatory mediators (COX-2, iNOS, and NO), suppressed Akt and c-jun N-terminal kinase 1/2 (JNK) phosphorylation, and NF-κB activation in LPS-stimulated macrophages. In addition, ADDA blocked LPS-mediated macrophage migration and this was associated with inhibition of LPS-induced Src and FAK phosphorylation as well as Src expression in a concentration dependent manner. Notably, the inhibitory effects of ADDA on iNOS, NO, and Src could be mimicked by a Src inhibitor PP2 or an iNOS inhibitor l-NMMA. CONCLUSION: Our results suggested that ADDA attenuated LPS-induced inflammatory responses in macrophages and cell migration, at least in part, through inhibition of NF-κB activation and modulation of iNOS/Src/FAK axis.

Sesquiterpenoids and further diterpenoids from the rare Chloranthaceae plant Chloranthus sessilifolius.

Two new dimeric lindenane-type sesquiterpenoids (1 and 2, named chlorasessilifols A and B, resp.), one new ent-podocarpane-type C17 norditerpenoid (3), and one new ent-torarane-type diterpenoid (4), along with seven known terpenoids, were isolated from the whole plant of Chloranthus sessilifolius. The new structures were established by means of spectroscopic methods and/or observed cotton effects in the circular dichroism spectra. Among the isolates, 3α,7ß-dihydroxy-ent-abieta-8,11,13-triene (11) exhibited significant anti-neuroinflammatory activity by inhibiting the nitric oxide production in lipopolysaccharide-stimulated murine BV-2 microglial cells, with an IC50 value of 4.3 µM.

ent-Abietane-Type and Related Seco-/Nor-diterpenoids from the Rare Chloranthaceae Plant Chloranthus sessilifolius and Their Antineuroinflammatory Activities.

Fourteen new ent-abietane-type diterpenoids, sessilifols A-N (1-14), and three related new norditerpenoids (15-17) were isolated from Chloranthus sessilifolius. The absolute configurations were determined by single-crystal X-ray diffraction analysis, the modified Mosher's method, and/or the observed Cotton effects in their electronic circular dichroism spectra. Sessilifols A (1) and B (2) possess an uncommon five-membered C-ring rearranged by oxidative cleavage of the C-13/C-14 bond in abieta-7,13-diene followed by the formation of a new C-C bond between C-12 and C-14. Sessilifol C (3) is a rare 7,8-seco-9-spiro-fused ent-abietane, whereas sessilifol O (15) represents the first example of a naturally occurring 14-norabietane-type diterpenoid. Compounds 6 and 9 were found to have moderate antineuroinflammatory activities by inhibiting the nitric oxide production in lipopolysaccharide-stimulated murine BV-2 microglial cells, with IC50 values of 8.3 and 7.4 µM, respectively.

Asymmetric synthesis and evaluation of danshensu-cysteine conjugates as novel potential anti-apoptotic drug candidates.

We have previously reported that the danshensu-cysteine conjugate N-((R)-3-benzylthio-1-methoxy-1-oxo-2-propanyl)-2-acetoxy-3-(3,4-diacetoxyphenyl) propanamide (DSC) is a potent anti-oxidative and anti-apoptotic agent. Herein, we further design and asymmetrically synthesize two diastereoisomers of DSC and explore their potential bioactivities. Our results show that DSC and its two diastereoisomers exert similar protective effects in hydrogen peroxide (H2O2)-induced cellular injury in SH-SY5Y cells, as evidenced by the increase of cell viability, superoxide dismutase (SOD), and reduced glutathione (GSH) activity, and glutathione peroxidase (GPx) expression, and the decrease of cellular morphological changes and nuclear condensation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) production. In H2O2-stimulated human umbilical vein endothelial cells (HUVEC), DSC concentration-dependently attenuates H2O2-induced cell death, LDH release, mitochondrial membrane potential collapse, and modulates the expression of apoptosis-related proteins (Bcl-2, Bax, caspase-3, and caspase-9). Our results provide strong evidence that DSC and its two diastereoisomers have similar anti-oxidative activity and that DSC exerts significant vascular-protective effects, at least in part, through inhibition of apoptosis and modulation of endogenous antioxidant enzymes.

A novel compound derived from danshensu inhibits apoptosis via upregulation of heme oxygenase-1 expression in SH-SY5Y cells.

BACKGROUND: Heme oxygenase-1 (HO-1) has potential anti-apoptotic properties. A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2- ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)] was synthesized by joining danshensu and cysteine through an appropriate linker. This study investigated if the cytoprotective properties of DSC involved the induction of HO-1. METHODS: We evaluated the cytoprotective effects of DSC on H2O2-induced cell damage, apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) loss, and apoptosis-related proteins expression and its underlying mechanisms. RESULTS: DSC concentration-dependently attenuated cell death, lactate dehydrogenase release, intracellular and mitochondrial ROS production, and ΔΨm collapse, modulated apoptosis-related proteins (Bcl-2, Bax, caspase-3, p53, and cleaved PARP) expression, and inhibited phosphorylation of extracellular signal-regulated kinase 1/2 in SH-SY5Y cells induced by H2O2. In addition, DSC concentration-dependently induced HO-1 expression associated with nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf-2), while the effect of DSC was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, the protective effect of DSC on H2O2-induced cell death was abolished by HO-1 inhibitor ZnPP, but was mimicked by carbon monoxide-releasing moiety CORM-3 or HO-1 by-product bilirubin. Finally, DSC inhibited H2O2-induced changes of Bcl-2, Bax, and caspase-3 expression, and all of these effects were reversed by HO-1 silencing. CONCLUSIONS: Induction of HO-1 may be, at least in part, responsible for the anti-apoptotic property of DSC, an effect that involved the activation of PI3K/Akt/Nrf-2 axis. GENERAL SIGNIFICANCE: DSC might have the potential for beneficial therapeutic interventions for neurodegenerative diseases.

Leonurine attenuates lipopolysaccharide-induced inflammatory responses in human endothelial cells: involvement of reactive oxygen species and NF-κB pathways.

Leonurine, an active alkaloid of Traditional Chinese Medicine Herba leonuri, displayed cardioprotective effects by anti-oxidative and anti-apoptotic activities in vitro and in vivo. Herein, we explored the effects and possible mechanisms of leonurine on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVEC). We found that leonurine pretreatment concentration-dependently attenuated LPS-induced mRNA expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and monocyte chemoattractant protein-1. Meanwhile, LPS-mediated expression/release of ICAM-1, VCAM-1, and cyclooxygenase-2, and tumor necrosis factor-α was also reduced by leonurine. In addition, we confirmed that leonurine suppressed degradation of IκBα and phosphorylation of nuclear factor-κB (NF-κB) p65 as well as production of intracellular reactive oxygen species in a concentration dependent manner. Furthermore, the cytoprotective enzyme heme oxygenase-1 could be upregulated in leonurine-treated HUVEC. Our present results indicated leonurine exerted beneficial effects in inflammatory conditions partly through inhibition of reactive oxygen species and NF-κB signaling pathways.

Antiapoptotic effect of novel compound from Herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells.

Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H2O2)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H2O2 significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H2O2-stimulated cell. Moreover, H9c2 cells stimulated by H2O2 was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (ΔΨ(m)) and activation of caspase 3. Furthermore, H2O2 also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H2O2-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2.

4-Guanidino-n-butyl syringate (Leonurine, SCM 198) protects H9c2 rat ventricular cells from hypoxia-induced apoptosis.

In the present study, we examined the ability of a chemically synthesized compound based on the structure of leonurine, a phytochemical component of Herba leonuri, to protect H9c2 rat ventricular cells from apoptosis induced by hypoxia and serum deprivation, as a model of ischemia. The results revealed a concentration-dependent increase in cell viability associated with leonurine treatment, accompanied by a consistent decline in lactate dehydrogenase leakage into the culture medium. The fraction of annexin V-fluorescein isothiocyanate-positive cells was increased by hypoxia but reduced by leonurine. These changes were associated with increased expression of the antiapoptotic gene, Bcl-2, and reduced expression of the proapoptotic gene, Bax. Leonurine also reduced the cytosolic Ca overload induced by hypoxia. These results suggest that leonurine elicits potent cardioprotective effects in H9c2 cells, and these effects may be mediated by inhibition of intracellular Ca overload and apoptosis during hypoxia.