Apocynin Pretreatment Research Articles

Glycolysis and the Pentose Phosphate Pathway Promote LPS-Induced NOX2 Oxidase- and IFN-β-Dependent Inflammation in Macrophages.

Macrophages undergo a metabolic switch from oxidative phosphorylation to glycolysis when exposed to gram-negative bacterial lipopolysaccharide (LPS), which modulates antibacterial host defence mechanisms. Here, we show that LPS treatment of macrophages increased the classical oxidative burst response via the NADPH oxidase (NOX) 2 enzyme, which was blocked by 2-deoxyglucose (2-DG) inhibition of glycolysis. The inhibition of the pentose phosphate pathway with 6-aminonicotinamide (6-AN) also suppressed the LPS-induced increase in NOX2 activity and was associated with a significant reduction in the mRNA expression of NOX2 and its organizer protein p47phox. Notably, the LPS-dependent enhancement in NOX2 oxidase activity was independent of both succinate and mitochondrial reactive oxygen species (ROS) production. LPS also increased type I IFN-β expression, which was suppressed by 2-DG and 6-AN and, therefore, is dependent on glycolysis and the pentose phosphate pathway. The type I IFN-β response to LPS was also inhibited by apocynin pre-treatment, suggesting that NOX2-derived ROS promotes the TLR4-induced response to LPS. Moreover, recombinant IFN-β increased NOX2 oxidase-dependent ROS production, as well as NOX2 and p47phox expression. Our findings identify a previously undescribed molecular mechanism where both glycolysis and the pentose phosphate pathway are required to promote LPS-induced inflammation in macrophages.

Apocynin ameliorates fumonisin b1 induced hepatotoxicity via NADPH oxidase inhibition and quantification of sphingosine and sphinganine

Previous studies have shown that hepatotoxicity and NADPH oxidase mediated oxidative stress was activated by fumonisin 1 (Fb1) exposure, which is considered to be a critical event in the Fb1-induced toxic effect. However, the detailed mechanisms underlying Fb1-induced liver toxicity remain elusive. In this study, apocynin, a specific inhibitor for NADPH oxidase, was used to test suppression of ROS by inhibititing NADPH oxidase and it can protect against Fb1 induced hepatotoxicity in mice model and using HepG2 cell lines. In this context, the toxicity of Fb1 was examined in male albino mice. Apocynin with 25,50,100mg/kg b/wt was pretreated for 7 days oral administration. Fb1 2.25mg/kg b/wt was injected subcutaneously for 3 days. In mice model Fb1 injection induced oxidative stress by significant rise in serum marker enzymes and lipid peroxidation along with the reduction of antioxidant enzymes. Pretreatment of mice with different doses of apocynin (25,50,100mg/kg b/wt) significantly lowered AST, ALT and lipid peroxidation levels against Fb1 treated mice. Hepatic enzymes like SOD, CAT, GPx, GR, GSH were significantly increased by treatment with apocynin, against Fb1 treated mice. Fb1 perturbs sphingolipid metabolism by inhibiting ceramide synthase activity hence in the present study quantification of sphingosine and sphinganine quantified by LC MS analysis. In Fb1 injected mice samples the sphingoid bases elevated than normal pretreatment with apocynin reduced the sphingoid bases. The observed data demonstrates 50 % cell survival with 50 µM challenge for 24 h, which was restored to 90 % by pre-treatment with 100µM apocynin. It also decreased the lactate dehydrogenase leakage and preserved the cellular morphology. The DNA damage and nuclear morphology were assessed by comet assay and DAPI staining. Fb1 damaged DNA and nuclear morphology protected by apocynin pretreatment. The ultra-structural change induced by the Fb1 in cell lines, vacuolation was observed pre-treated cells with apocynin showed normal cell structure without vacuolation. On the Other hand, cell cycle distribution analysis showed cell cycle arrest at G0/G1 phase induced by Fb1 and its modulations was observed in apocynin pretreted cells. The protein expression CAT, GPx downregulated with Fb1 treatment and apoptotic markers caspase-3 and caspase-8 was upregulated. Pretretment with apocynin restored the antioxidant enzymes and overexpressed apocynin markers also altered. These findings suggest that apocynin ameliorates Fb1 induced hepatic damage via its antioxidant defence mechanism and might be used to treat oxidative stress mediated hepatic disorders.

Effects of Apocynin on Liver Ischemia-Reperfusion Injury in Rats

ObjectiveIschemia-reperfusion (IR) injury is associated with various clinical conditions, such as myocardial infarction, shock, and surgery under vascular occlusion. We aimed to investigate the protective and therapeutic effects of apocynin (AP) on liver injury induced by IR in an in vivo rat model. MethodsThirty-two rats were randomly divided into 4 experimental groups with n = 8 in each group: sham, IR, AP, and IR + AP. AP (20 mg/kg) was intraperitoneally administered to rats in the AP and IR + AP groups for 30 minutes before 60 minutes of ischemia and followed by 60 minutes of reperfusion. All rats were killed on the same day to evaluate tissue levels of oxidants and antioxidants (catalase, malondialdehyde, myeloperoxidase, superoxidedismutase (SOD), and total glutathione). ResultsIR decreased SOD levels in IR group when compared with the sham group. AP supplementation to IR group significantly ameliorated SOD levels (P < .05). Also, IR caused elevation of myeloperoxidase production when compared with the sham group, whereas AP treatment prevented these hazardous effects (P < .05). However, plasma total glutathione, catalase, and malondialdehyde levels did not differ between the AP + IR and the IR rats. ConclusionThe main finding of the present study was that AP may be protective against liver IR injury. Our results suggested that AP pretreatment suppressed oxidative stress and increased antioxidant levels in an rat model of liver IR.

Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1β, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies.

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation.

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.

Apocynin protects against ethanol-induced gastric ulcer in rats by attenuating the upregulation of NADPH oxidases 1 and 4

Gastric ulcer is a common gastrointestinal disorder affecting many people all over the world. Absolute ethanol (5 ml/kg) was used to induce gastric ulceration in rats. Apocynin (50 mg/kg) was given orally one hour before the administration of absolute ethanol. Omeprazole (20 mg/kg) was used as a standard. Interestingly, apocynin pre-treatment provided 93.5% gastroprotection against ethanol-induced ulceration. Biochemically, gastric mucin content was significantly increased with apocynin pre-treatment. This finding was further supported by alcian blue staining of stomach sections obtained from the different treated groups. Also, gastric juice volume and acidity were significantly reduced. Apocynin significantly ameliorated ethanol-induced oxidative stress by replenishing reduced glutathione and superoxide dismutase levels as well as reducing elevated malondialdehyde levels in gastric tissues. Besides, ethanol-induced pro-inflammatory response was significantly decreased by apocynin pre-treatment via reducing elevated levels of pro-inflammatory markers; interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2 and inducible nitric oxide synthase. Additionally, caspase-3 tissue level was significantly reduced in apocynin pre-treated group. Interestingly, NADPH oxidase-1 (NOX-1) and NOX-4 up-regulation was shown to be partially involved in the pathogenesis of ethanol-induced gastric ulceration and was significantly reversed by apocynin pre-treatment. Gastroprotective properties of apocynin were confirmed by histopathological examination. It is worth mentioning that apocynin was superior in all aspects except gastric mucin content parameter where it was significantly increased by 13.5 folds in the omeprazole pre-treated group. This study was the first to show that apocynin is a promising gastroprotective agent against ethanol-induced gastric ulceration, partially via its anti-oxidant, anti-inflammatory, anti-apoptotic effects as well as down-regulating NOX-1 and NOX-4 expression.

Effects of Resveratrol on Oxidative Stress Injury Induced by Rapid-Pacing in Isolated Rabbit Hearts

Oxidative stress injury plays an important role in the process of atrial remodeling. The mechanisms of oxidative stress injury in atrial by rapid pacing and the protective effects of resveratrol will be explored in this study. Thirty-two isolated rabbit hearts were produced by rapid atrial pacing. They were randomly divided into 4 groups: control group (Ctrl group), Rapid Atrial Pacing group (RAP group), Apocynin Pretreatment group (APO group) and Resveratrol Pretreatment group (RES group); each with 8 rabbits. At the end, the indexs of the oxdise stress were measured take advantage of the techniques of the immunohistochemistry, western blotting and reverse transcription PCR.

Prevention of traumatic brain injury-induced neuronal death by inhibition of NADPH oxidase activation

The present study aimed to evaluate the therapeutic potential of apocynin, an NADPH oxidase assembly inhibitor, on traumatic brain injury. Rat traumatic brain injury (TBI) was performed using a weight drop model. Apocynin (100mg/kg) was injected into the intraperitoneal space 15 min before TBI. Reactive oxygen species (ROS) in the hippocampal CA3 pyramidal neurons were detected by dihydroethidium (dHEt) at 3h after TBI. Oxidative injury was detected by 4-hydroxy-2-nonenal (4HNE) at 6h after TBI. Blood-brain barrier disruption was detected by IgG extravasation and neuronal death was evaluated with Fluoro Jade-B staining 24h after TBI. Microglia activation was detected by CD11b immunohistochemistry in the hippocampus at 1 week after TBI. ROS production was inhibited by apocynin administration in the hippocampal CA3 pyramidal neurons. This pre-treatment with apocynin decreased the blood-brain barrier disruption, the number of degenerating neurons in the hippocampal CA3 region and microglial activation after TBI. The present study indicates that apocynin pre-treatment prevents TBI-induced ROS production, thus decreasing BBB disruption, neuronal death and microglial activation. Therefore, the present study suggests that inhibition of NADPH oxidase by apocynin may have a high therapeutic potential to reduce traumatic brain injury-induced neuronal death.

Rho-kinase inhibitor and Nicotinamide Adenine Dinucleotide PHosphate oxidase inhibitor prevent impairment of endothelium-dependent cerebral vasodilation by acute cigarette smoking in rats

We previously reported that acute cigarette smoking can cause a dysfunction of endothelium-dependent vasodilation in cerebral vessels, and that blocking the angiotensin II (Ang II) type 1 (AT1) receptor with valsartan prevented this impairment. Our aim was to investigate the effects of a Rho-kinase inhibitor (fasudil) and a Nicotinamide Adenine Dinucleotide PHosphate (NADPH) oxidase inhibitor (apocynin) on smoking-induced endothelial dysfunction in cerebral arterioles. In Sprague-Dawley rats, we used a closed cranial window preparation to measure changes in pial vessel diameters following topical acetylcholine (ACh) before smoking. After one-minute smoking, we again examined the arteriolar responses to ACh. Finally, after intravenous fasudil or apocynin pre-treatment we re-examined the vasodilator responses to topical ACh (before and after cigarette smoking). Under control conditions, cerebral arterioles were dose-dependently dilated by topical ACh (10(-6) M and 10(-5) M). One hour after a one-minute smoking (1 mg-nicotine cigarette), 10(-5) M ACh constricted cerebral arterioles. However, one hour after a one-minute smoking, 10(-5) M ACh dilated cerebral pial arteries both in the fasudil pre-treatment and the apocynin pre-treatment groups, responses that were significantly different from those obtained without fasudil or apocynin pre-treatment. Thus, inhibition of Rho-kinase and NADPH oxidase activities may prevent the above smoking-induced impairment of endothelium-dependent vasodilation.

NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE OXIDASE INHIBITION IMPROVES NEUROLOGIC FUNCTIONS IN SURGICALLY INDUCED BRAIN INJURY.

Purpose Neurosurgical procedures can result in brain injury by various means, including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically induced brain injury (SBI) can cause postoperative complications such as brain edema following blood-brain barrier (BBB) disruption. In this study, we tested whether NADPH oxidase, an important reactive oxygen species (ROS)-producing enzyme, is involved in SBI using transgenic mice lacking gp91phox subunit of NADPH oxidase (gp91-KO) and apocynin, a specific inhibitor of NADPH oxidase. Methods A mouse model of surgically induced brain injury (SBI) was used, which involves stereotaxic resection of a part of the right frontal lobe. gp91-KO and wild-type (WT, C57BL/6J) littermates were grouped into SBI and sham-surgery groups. Alternatively, mice were grouped into SBI and apocynin-treated (5 mg/kg, IP 30 minutess before surgery) groups. Neurologic function and brain edema (brain water content) were evaluated at 24-hour post-SBI. Oxidative stress indicated by lipid peroxidation (LPO) was measured at 3 and 24 hours post-SBI. Results Brain edema was observed 24-hour post-SBI in both gp91-KO and wild-type group as compared with the sham group; however, there was no significant difference between the gp91-KO and wild-type groups. Brain edema was also not affected by apocynin pretreatment. LPO levels were significantly higher in SBI group in both gp91-KO and wild-type groups compared with the sham group. LPO levels were significantly attenuated at 3-hour post-SBI by apocynin pretreatment but not at 24-hour post-SBI. A trend, although without statistical significance, was noted toward attenuation of LPO in the gp91-KO animals as comp. The gp91-KO mice and apocynin-treated mice, however, showed significantly improved neurologic scores 24-hour post-SBI compared with the wild-type and vehicle-treated mice, respectively. Conclusions Chronic and acute inhibition of NADPH oxidase activity does not affect brain edema; however, it improves neurologic functions after SBI. Further investigation is needed to find if other mechanisms, such as apoptosis or inflammation, are involved in improved neurologic functions after NADPH oxidase inhibition.