scholarly journals Increased Production of Proinflammatory Cytokines following Infection with Porcine Reproductive and Respiratory Syndrome Virus and Mycoplasma hyopneumoniae

2004 ◽  
Vol 11 (5) ◽  
pp. 901-908 ◽  
Author(s):  
Roongroje Thanawongnuwech ◽  
Brad Thacker ◽  
Patrick Halbur ◽  
Eileen L. Thacker
Keyword(s):  
Proinflammatory Cytokines ◽  
Mycoplasma Hyopneumoniae ◽  
Tracheal Ring ◽  
In Vivo Model ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Tnf Α

ABSTRACT Induction of the proinflammatory cytokines interleukin-1 (IL-1) (α and β), IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha (TNF-α) in pulmonary alveolar macrophages (PAMs) was assessed following experimental infection with porcine reproductive and respiratory syndrome virus (PRRSV) and/or Mycoplasma hyopneumoniae by using in vivo and in vitro models. The in vivo model consisted of pigs infected with PRRSV and/or M. hyopneumoniae and necropsied at 10, 28, or 42 days postinfection. Pigs infected with both pathogens had a greater percentage of macroscopic lung lesions, increased clinical disease, and slower viral clearance than pigs infected with either pathogen alone. The pigs infected with both PRRSV and M. hyopneumoniae had significantly increased levels of mRNA for many proinflammatory cytokines in PAMs collected by bronchoalveolar lavage (BAL) at all necropsy dates compared to those in uninfected control pigs. Increased levels of IL-1β, IL-8, IL-10, and TNF-α proteins in BAL fluid, as measured by enzyme-linked immunosorbent assay, confirmed the increased cytokine induction induced by the pathogens. An in vitro model consisted of M. hyopneumoniae-inoculated tracheal ring explants cultured with PRRSV-infected PAMs. PAMs were harvested at 6 or 15 h postinfection with either or both pathogens. The in vitro study detected increased IL-10 and IL-12 mRNA levels in PAMs infected with PRRSV at all time periods. In addition, IL-10 protein levels were significantly elevated in the culture supernatants in the presence of M. hyopneumoniae-inoculated tracheal ring explants. The increased production of proinflammatory cytokines in vivo and in vitro associated with concurrent M. hyopneumoniae and PRRSV infection may play a role in the increased rates of pneumonia associated with PRRSV infection. The increased levels of IL-10 may be a possible mechanism that PRRSV and M. hyopneumoniae use to exacerbate the severity and duration of pneumonia induced by PRRSV and modulate the respiratory immune response.

scholarly journals TheBDKRB2 +9/-9 Polymorphisms Influence Pro-Inflammatory Cytokine Levels in Knee Osteoarthritis by Altering TLR-2 Expression: Clinical and in Vitro Studies

2016 ◽  
Vol 38 (3) ◽  
pp. 1245-1256 ◽  
Author(s):  
Shuo Chen ◽  
Lei Zhang ◽  
Ruonan Xu ◽  
Yunfan Ti ◽  
Yunlong Zhao ◽  
...  
Keyword(s):  
Knee Osteoarthritis ◽  
Inflammatory Cytokine ◽  
Molecular Mechanisms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Knee Oa ◽  
Tnf Α ◽  
Cytokine Levels ◽  
Underlying Mechanisms

Background/Aims: The bradykinin B2 receptor (BDKRB2) +9/-9 gene polymorphisms have been shown to be associated with the susceptibility and severity of osteoarthritis (OA); however, the underlying mechanisms are unclear. In this study, we investigated the correlation between the BDKRB2 +9/-9 polymorphisms and pro-inflammatory cytokine levels in OA and the molecular mechanisms involved. Methods: A total of 156 patients with primary knee OA and 121 healthy controls were enrolled. The BDKRB2 +9/-9 polymorphisms were genotyped. The tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 levels were determined using Enzyme-linked immunosorbent assay (ELISA). The toll-like receptor (TLR)-2 and TLR-4 mRNA levels were determined by quantitative real-time PCR. The basal and bradykinin-stimulated pro-inflammatory cytokine secretion in human OA synoviocytes and the involvement of TLR-2 and mitogen-activated protein kinases (MAPKs) were investigated. Results: The presence of -9 bp genotype is associated with higher TNF-α, IL-6, and IL-8 levels and higher TLR-2 expression in OA patients. The basal and bradykinin-induced TLR-2 expressions in human OA synoviocytes were significantly reduced by specific inhibitors of p38, JNK1/2, and ERK1/2. Both the B2 receptor antagonist MEN16132 and TLR-2 silencing inhibited IL-6 and IL-8 secretion in human OA synoviocytes. Conclusion: The data suggested that the BDKRB2 +9/-9 polymorphisms influence pro-inflammatory cytokine levels in knee osteoarthritis by altering TLR-2 expression.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

scholarly journals Bakuchiol Suppresses Inflammatory Responses Via the Downregulation of the p38 MAPK/ERK Signaling Pathway

2019 ◽  
Vol 20 (14) ◽  
pp. 3574 ◽  
Author(s):  
Hye-Sun Lim ◽  
Yu Jin Kim ◽  
Bu-Yeo Kim ◽  
Soo-Jin Jeong
Keyword(s):  
Mrna Expression ◽  
P38 Mapk ◽  
Inflammatory Responses ◽  
Mitogen Activated Protein Kinase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mice Model ◽  
Tnf Α ◽  
Cox 2

The purpose of the present study was to evaluate the effects of bakuchiol on the inflammatory response and to identify the molecular mechanism of the inflammatory effects in a lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial cell line and mice model. The production of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was measured by enzyme-linked immunosorbent assay. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-6 was measured using reverse transcription–polymerase chain reaction analysis. Mitogen-activated protein kinase (MAPK) phosphorylation was determined by western blot analysis. In vitro experiments, bakuchiol significantly suppressed the production of PGE2 and IL-6 in LPS-stimulated BV-2 cells, without causing cytotoxicity. In parallel, bakuchiol significantly inhibited the LPS-stimulated expression of iNOS, COX-2, and IL-6 in BV-2 cells. However, bakuchiol had no effect on the LPS-stimulated production and mRNA expression of TNF-α or on LPS-stimulated c-Jun NH2-terminal kinase phosphorylation. In contrast, p38 MAPK and extracellular signal-regulated kinase (ERK) phosphorylation were inhibited by bakuchiol. In vivo experiments, Bakuchiol reduced microglial activation in the hippocampus and cortex tissue of LPS-injected mice. Bakuchiol significantly suppressed LPS-injected production of TNF-α and IL-6 in serum. These results indicate that the anti-neuroinflammatory effects of bakuchiol in activated microglia are mainly regulated by the inhibition of the p38 MAPK and ERK pathways. We suggest that bakuchiol may be beneficial for various neuroinflammatory diseases.

scholarly journals Differential Effects of Platelets Selectively Activated by Protease-Activated Receptors on Meniscal Cells

2019 ◽  
Vol 48 (1) ◽  
pp. 197-209 ◽  
Author(s):  
Hongyao Xu ◽  
Xiangjie Zou ◽  
Pengcheng Xia ◽  
Mohammad Ahmad Kamal Aboudi ◽  
Ran Chen ◽  
...  
Keyword(s):  
In Vivo Model ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protease Activated Receptors ◽  
Activated Platelets ◽  
Meniscal Tissue ◽  
Vitro Model ◽  
High Level ◽  
Meniscal Cells

Background: Meniscal injury is very common, and injured meniscal tissue has a limited healing ability because of poor vascularity. Platelets contain both pro- and anti-angiogenic factors, which can be released by platelet selective activation. Hypothesis: Platelets release a high level of vascular endothelial growth factor (VEGF) when they are activated by protease-activated receptor 1 (PAR1), whereas the platelets release endostatin when they are activated by protease-activated receptor 4 (PAR4). The PAR1-treated platelets enhance the proliferation of meniscal cells in vitro and promote in vivo healing of wounded meniscal tissue. Study Design: Controlled laboratory study. Method: Platelets were isolated from human blood and activated with different reagents. The released growth factors from the activated platelets were determined by immunostaining and enzyme-linked immunosorbent assay. The effects of the platelets with different treatments on meniscal cells were tested by an in vitro model of cell culture and an in vivo model of wounded meniscal healing. Results: The results indicated that platelets contained both pro- and antiangiogenic factors including VEGF and endostatin. In unactivated platelets, VEGF and endostatin were contained inside of the platelets. Both VEGF and endostatin were released from the platelets when they were activated by thrombin. However, only VEGF was released from the platelets when they were activated by PAR1, and only endostatin was released from the platelets when they were activated by PAR4. The rat meniscal cells grew much faster in the medium that contained PAR1-activated platelets than in the medium that contained either PAR4-activated platelets or unactivated platelets. The wounds treated with PAR1-activated platelets healed faster than those treated with either PAR4-activated platelets or unactivated platelets. Many blood vessel–like structures were found in the wounded menisci treated with PAR1-activated platelets. Conclusion: The PAR1-activated platelets released high levels of VEGF, which increased the proliferation of rat meniscal cells in vitro, enhanced the vascularization of menisci in vivo, and promoted healing of wounded menisci. Clinical Relevance: Our results suggested that selective activated platelets can be used clinically to enhance healing of wounded meniscal tissue.

scholarly journals Glucosamine Enhancement of BDNF Expression and Animal Cognitive Function

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3667
Author(s):  
Lien-Yu Chou ◽  
Yu-Ming Chao ◽  
Yen-Chun Peng ◽  
Hui-Ching Lin ◽  
Yuh-Lin Wu
Keyword(s):  
Cognitive Function ◽  
In Vivo Model ◽  
Mrna Levels ◽  
Bdnf Expression ◽  
Object Recognition Test ◽  
Creb Phosphorylation ◽  
Downstream Signaling ◽  
Vitro Model

Brain-derived neurotrophic factor (BDNF) is an important factor for memory consolidation and cognitive function. Protein kinase A (PKA) signaling interacts significantly with BDNF-provoked downstream signaling. Glucosamine (GLN), a common dietary supplement, has been demonstrated to perform a variety of beneficial physiological functions. In the current study, an in vivo model of 7-week-old C57BL/6 mice receiving daily intraperitoneal injection of GLN (0, 3, 10 and 30 mg/animal) was subjected to the novel object recognition test in order to determine cognitive performance. GLN significantly increased cognitive function. In the hippocampus GLN elevated tissue cAMP concentrations and CREB phosphorylation, and upregulated the expression of BDNF, CREB5 and the BDNF receptor TrkB, but it reduced PDE4B expression. With the in vitro model in the HT22 hippocampal cell line, GLN exposure significantly increased protein and mRNA levels of BDNF and CREB5 and induced cAMP responsive element (CRE) reporter activity; the GLN-mediated BDNF expression and CRE reporter induction were suppressed by PKA inhibitor H89. Our current findings suggest that GLN can exert a cognition-enhancing function and this may act at least in part by upregulating the BDNF levels via a cAMP/PKA/CREB-dependent pathway.

scholarly journals Nerolidol Mitigates Colonic Inflammation: An Experimental Study Using both In Vivo and In Vitro Models

Nutrients ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 2032
Author(s):  
Vishnu Raj ◽  
Balaji Venkataraman ◽  
Saeeda Almarzooqi ◽  
Sanjana Chandran ◽  
Shreesh K. Ojha ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
In Vitro Models ◽  
Mrna Levels ◽  
Colonic Inflammation ◽  
Tnf Α ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Ht 29

Nerolidol (NED) is a naturally occurring sesquiterpene alcohol present in various plants with potent anti-inflammatory effects. In the current study, we investigated NED as a putative anti-inflammatory compound in an experimental model of colonic inflammation. C57BL/6J male black mice (C57BL/6J) were administered 3% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colitis. Six groups received either vehicle alone or DSS alone or DSS with oral NED (50, 100, and 150 mg/kg body weight/day by oral gavage) or DSS with sulfasalazine. Disease activity index (DAI), colonic histology, and biochemical parameters were measured. TNF-α-treated HT-29 cells were used as in vitro model of colonic inflammation to study NED (25 µM and 50 µM). NED significantly decreased the DAI and reduced the inflammation-associated changes in colon length as well as macroscopic and microscopic architecture of the colon. Changes in tissue Myeloperoxidase (MPO) concentrations, neutrophil and macrophage mRNA expression (CXCL2 and CCL2), and proinflammatory cytokine content (IL-1β, IL-6, and TNF-α) both at the protein and mRNA level were significantly reduced by NED. The increase in content of the proinflammatory enzymes, COX-2 and iNOS induced by DSS were also significantly inhibited by NED along with tissue nitrate levels. NED promoted Nrf2 nuclear translocation dose dependently. NED significantly increased antioxidant enzymes activity (Superoxide dismutase (SOD) and Catalase (CAT)), Hemeoxygenase-1 (HO-1), and SOD3 mRNA levels. NED treatment in TNF-α-challenged HT-29 cells significantly decreased proinflammatory chemokines (CXCL1, IL-8, CCL2) and COX-2 mRNA levels. NED supplementation attenuates colon inflammation through its potent antioxidant and anti-inflammatory activity both in in vivo and in vitro models of colonic inflammation.

scholarly journals Use of Defined Mutants To Assess the Role of theCampylobacter rectus S-Layer in Bacterium-Epithelial Cell Interactions

2000 ◽  
Vol 68 (3) ◽  
pp. 1465-1473 ◽  
Author(s):  
Beinan Wang ◽  
Ellen Kraig ◽  
David Kolodrubetz
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Proinflammatory Cytokines ◽  
Transforming Growth Factor ◽  
Periodontal Diseases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minor Effect ◽  
Mrna Levels ◽  
Tnf Α

ABSTRACT Campylobacter rectus is a periodontal pathogen with a 150-kDa protein on its cell surface. This protein forms a paracrystalline lattice, called the S-layer, surrounding the outer membrane of this gram-negative bacterium. To initiate a genetic analysis of the possible role of the S-layer in the initial interaction of C. rectus with host epithelial cells, C. rectus strains lacking the S-layer protein gene (crsA) were constructed by allelic exchange mutagenesis. Surprisingly, the lack of the S-layer had only a minor effect on the interaction of C. rectus with HEp-2 epithelial cells; CrsA+ cells were 30 to 50% more adherent than were CrsA− bacteria. Since the host cell expression of cytokines appears to play an important role in the pathogenesis of periodontal diseases, the effect of the S-layer on the epithelial cell cytokine response was also examined by quantitative reverse transcriptase PCR and enzyme-linked immunosorbent assay. Although there were no changes in the mRNA levels for the anti-inflammatory cytokines interleukin-1 receptor agonist (IL-1ra), IL-13, and transforming growth factor β, the expression and secretion of the proinflammatory cytokines IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) were significantly induced by both wild-type C. rectus and CrsA− bacteria. Interestingly, the kinetics of cytokine induction differed for the CrsA+ and CrsA−bacteria. At early time points, the HEp-2 cells challenged with CrsA− bacteria produced higher levels of IL-6, IL-8, and TNF-α mRNA and protein than did cells challenged with CrsA+ bacteria. We conclude that C. rectus may help initiate periodontitis by increasing the expression of proinflammatory cytokines and that the S-layer may temper this response to facilitate the survival of C. rectus at the site of infection.

scholarly journals Modulation of Endotoxin- and Enterotoxin-Induced Cytokine Release by In Vivo Treatment with β-(1,6)-Branched β-(1,3)-Glucan

1999 ◽  
Vol 67 (1) ◽  
pp. 244-252 ◽  
Author(s):  
Jindrich Soltys ◽  
Mark T. Quinn
Keyword(s):  
Proinflammatory Cytokines ◽  
Necrosis Factor Alpha ◽  
Enhanced Production ◽  
Tnf Α ◽  
Host Mortality ◽  
Toxic Shock Syndrome Toxin ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Leukocytes activated by endotoxin or enterotoxins release proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the effects of in vivo administration of a soluble immunomodulator, β-(1,6)-branched β-(1,3)-glucan (soluble β-glucan), on toxin-stimulated cytokine production in monocytes and lymphocytes isolated from treated mice. In vitro stimulation of lymphocytes isolated from soluble β-glucan-treated mice with lipopolysaccharide (LPS) resulted in enhanced production of interleukin-6 (IL-6) and suppressed production of tumor necrosis factor alpha (TNF-α), while stimulation of these cells with staphylococcal enterotoxin B (SEB) or toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-γ) and suppressed production of IL-2 and TNF-α compared to that in cells isolated from untreated mice. In vitro stimulation of monocytes isolated from soluble β-glucan-treated mice with LPS also resulted in suppressed TNF-α production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-α production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from soluble β-glucan-treated mice reflects suppressed production of proinflammatory cytokines, especially TNF-α. Taken together, our results suggest that treatment with soluble β-glucan can modulate the induction cytokines during sepsis, resulting in an overall decrease in host mortality.

scholarly journals Characterization of the Plasmacytoid Dendritic Cell Response to Transmitted/Founder and Nontransmitted Variants of HIV-1

2018 ◽  
Vol 92 (19) ◽  
Author(s):  
Jordan Ari Schwartz ◽  
Hongliang Zhang ◽  
Zachary Ende ◽  
Martin J. Deymier ◽  
Terry Lee ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Plasmacytoid Dendritic Cell ◽  
Female Genital Tract ◽  
Enzyme Linked Immunosorbent Assay ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
The Impact ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection often arises from a single transmitted/founder (TF) viral variant among a large pool of viruses in the quasispecies in the transmitting partner. TF variants are typically nondominant in blood and genital secretions, indicating that they have unique traits. The plasmacytoid dendritic cell (pDC) is the primary alpha interferon (IFN-α)-producing cell in response to viral infections and is rapidly recruited to the female genital tract upon exposure to HIV-1. The impact of pDCs on transmission is unknown. We investigated whether evasion of pDC responses is a trait of TF viruses. pDCs from healthy donors were stimulated in vitro with a panel of 20 HIV-1 variants, consisting of one TF variant and three nontransmitted (NT) variants each from five transmission-linked donor pairs, and secretion of IFN-α and tumor necrosis factor alpha (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA). No significant differences in cytokine secretion in response to TF and NT viruses were observed, despite a trend toward enhanced IFN-α and TNF-α production in response to TF viruses. NT viruses demonstrated polarization toward production of either IFN-α or TNF-α, indicating possible dysregulation. Also, for NT viruses, IFN-α secretion was associated with increased resistance of the virus to inactivation by IFN-α in vitro, suggesting in vivo evolution. Thus, TF viruses do not appear to preferentially subvert pDC activation compared to that with nontransmitted HIV-1 variants. pDCs may, however, contribute to the in vivo evolution of HIV-1. IMPORTANCE The plasmacytoid dendritic cell (pDC) is the first cell type recruited to the site of HIV-1 exposure; however, its contribution to the viral bottleneck in HIV-1 transmission has not been explored previously. We hypothesized that transmitted/founder viruses are able to avoid the pDC response. In this study, we used previously established donor pair-linked transmitted/founder and nontransmitted (or chronic) variants of HIV-1 to stimulate pDCs. Transmitted/founder HIV-1, instead of suppressing pDC responses, induced IFN-α and TNF-α secretion to levels comparable to those induced by viruses from the transmitting partner. We noted several unique traits of chronic viruses, including polarization between IFN-α and TNF-α production as well as a strong relationship between IFN-α secretion and the resistance of the virus to neutralization. These data rule out the possibility that TF viruses preferentially suppress pDCs in comparison to the pDC response to nontransmitted HIV variants. pDCs may, however, be important drivers of viral evolution in vivo.

Tanshinone I Exerts Cardiovascular Protective Effects in Vivo and in Vitro Through Inhibiting Necroptosis via Akt/nrf2 Signaling Pathway

2021 ◽  
Author(s):  
Youqiong Zhuo ◽  
Renyikun Yuan ◽  
Xinxin Chen ◽  
Jia He ◽  
Yangling Chen ◽  
...  
Keyword(s):  
Protective Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heme Oxygenase 1 ◽  
Protective Agent ◽  
H9c2 Cells ◽  
Cardiovascular Protection ◽  
Tnf Α ◽  
Nrf2 Signaling Pathway

Abstract BackgroundTanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated.MethodsIn this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia-reperfusion (MI/R) model including hematoxylin-eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). All related protein expression was determined by western blotting.ResultsOur data demonstrated that TI pretreatment attenuated t-BHP- and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment.ConclusionCollectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.

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