The efficacy and safety of a fixed-dose combination of apocynin and paeonol, APPA, in symptomatic knee OA: A double-blind, randomized, placebo-controlled, clinical trial.

OBJECTIVE: Apocynin (AP) and paeonol (PA) are low molecular weight phenolic compounds with a broad array of anti-inflammatory and immunoregulatory effects. This study assessed of a fixed-dose combination of APPA in people with symptomatic knee osteoarthritis (OA). METHODS: A multi-center, randomized, placebo-controlled, double-blind phase 2a trial enrolled participants with radiographic knee OA (Kellgren-Lawrence, KL, grades 2-3) and pain ≥40/100 on WOMAC pain subscale, and evaluated the efficacy and safety of oral APPA over a 28-day period. APPA 800 mg or matching placebo was administered twice daily in a 1:1 ratio. Post-hoc analyses explored the response to APPA in sub-groups with more severe pain and structural severity. RESULTS: The two groups were comparable at baseline; 152 subjects were enrolled and 148 completed the trial. There was no statistically significant difference between groups with respect to the primary outcome, WOMAC pain (mean difference between groups was -0.89, 95% CI: -5.62, 3.84, p = 0.71), nor WOMAC function or WOMAC total. However, predefined subgroup analyses of subjects with symptoms compatible with nociplastic/neuropathic pain features showed a statistically significant effect of APPA compared to placebo. Adverse events (mainly gastrointestinal) were mild to moderate. CONCLUSION: Treatment with APPA 800 mg twice daily for 28 days in subjects with symptomatic knee OA was not associated with significant symptom improvement compared to placebo. The treatment was well-tolerated and safe. While the study was not powered for such analysis, pre-planned subgroup analyses showed a significant effect of APPA in subjects with nociplastic pain/severe OA, indicating that further research in the effects of APPA in appropriate patients is warranted.

Apocynin alleviates thioacetamide-induced acute liver injury: Role of NOX1/NOX4/NF-κB/NLRP3 pathways.

The liver has a distinctive capacity to regenerate, yet severe acute injury can be life-threatening if not treated appropriately. Inflammation and oxidative stress are central processes implicated in the pathophysiology of acute livery injury. NOX isoforms are important enzymes for ROS generation, NF-κB and NLRP3 activation, its inhibition could be vital in alleviating acute liver injury (ALI). Here in our study, we used apocynin, a natural occurring potent NOX inhibitor, to exploreits potential protective effect against thioacetamide (TAA)-induced ALI through modulating crucial oxidative and inflammatory pathways. Rats were injected once with TAA (500 mg/kg/i.p) and treated with apocynin (10 mg/kg/i.p) twice before TAA challenge. Sera and hepatic tissues were collected for biochemical, mRNA expression, western blot analysis and histopathological assessments. Pretreatment with apocynin improved liver dysfunction evidenced by decreased levels of aminotransferases, ALP, GGT and bilirubin. Apocynin reduced mRNA expression of NOX1 and NOX4 which in turn alleviated oxidative stress, as shown by reduction in MDA and NOx levels, and elevation in GSH levels andcatalase and SOD activities. Moreover, apocynin significantly reduced MPO gene expression. We also demonstrate that apocynin ameliorated inflammation through activating IκBα and suppressing IKKα, IKKß, NF-κBp65 and p-NF-κBp65, IL-6 andTNF-α. Additionally, apocynin potentiated the gene expression of anti-inflammatory IL-10 and reduced levels of hepatic NLRP3, Caspase-1 and IL-1ß. These results suggest that apocynin protects against ALI in association with the inhibition of NOX1 and NOX4 and regulating oxidative and inflammatory pathways.

Apocynin exerts cytoprotective effects on dexamethasone-induced osteoblasts by inhibiting oxidative stress through the Nrf2 signalling pathway.

Steroid-induced femoral head necrosis (SIFHN) is a serious clinical complication that is caused by prolonged or excessive use of glucocorticoids (GCs). Osteoblast apoptosis and osteogenic differentiation dysfunction caused by GC-induced oxidative stress and mitochondrial impairment are strongly implicated in SIFHN. Apocynin (APO) is a kind of acetophenone extracted from an herb. In recent years, APO has received much attention for its antiapoptotic and antioxidant properties. This study aimed to investigate whether APO could protect against SIFHN and explore the mechanism. In our study, low-dose APO had no toxic effects on osteoblasts and restored dexamethasone (Dex)-treated osteoblasts by improving survival, inhibiting OS and restoring mitochondrial dysfunction. Mechanistically, APO alleviated Dex-induced osteoblast injury by activating the Nrf2 pathway, and the use of ML385 to block Nrf2 significantly eliminated the protective effect of APO. In addition, APO could reduce the formation of empty lacunae, restore bone mass and promote the expression of Nrf2 in SIFHN rats. In conclusion, APO protects osteoblasts from Dex-induced oxidative stress and mitochondrial dysfunction through activation of the Nrf2 pathway and may be a beneficial drug for the treatment of SIFHN.

Elicitation of Inhibitory Effects for AGE-Induced Oxidative Stress in Rotator Cuff-Derived Cells by Apocynin.

Advanced glycation end-products (AGEs) play a critical supportive role during musculoskeletal disorders via glycosylation and oxidative stress. Though apocynin, identified as a potent and selective inhibitor of NADPH oxidase, has been reported to be involved in pathogen-induced reactive oxygen species (ROS), its role in age-related rotator cuff degeneration has not been well clarified. Therefore, this study aims to evaluate the in vitro effects of apocynin on human rotator cuff-derived cells. Twelve patients with rotator cuff tears (RCTs) participated in the study. Supraspinatus tendons from patients with RCTs were collected and cultured. After the preparation of RC-derived cells, they were divided into four groups (control group, control + apocynin group, AGEs group, AGEs + apocynin group), and gene marker expression, cell viability, and intracellular ROS production were evaluated. The gene expression of NOX, IL-6, and the receptor for AGEs (RAGE) was significantly decreased by apocynin. We also examined the effect of apocynin in vitro. The results showed that ROS induction and increasing apoptotic cells after treatment of AGEs were significantly decreased, and cell viability increased considerably. These results suggest that apocynin can effectively reduce AGE-induced oxidative stress by inhibiting NOX activation. Thus, apocynin is a potential prodrug in preventing degenerative changes of the rotor cuff.

Effects of apocynin on ISO-induced delayed afterdepolarizations in rat atrial myocytes and the underlying mechanisms.

We aimed to investigate the effect of apocynin (APO) on delayed afterdepolarizations (DADs) in rat atrial myocytes and the underlying mechanisms. Rat atrial myocytes were isolated by a Langendorff perfusion apparatus. DADs were induced by isoproterenol (ISO). Action potentials (APs) and ion currents were recorded by the whole-cell clamp technique. The fluorescent indicator fluo-4 was used to visualize intracellular Ca2+ transients, and western blotting was used to measure the expression of related proteins. The incidence of DADs in rat atrial myocytes increased significantly after ISO treatment, leading to an increased incidence of triggered activity (TA). The incidence of ISO-induced DADs and TA were reduced by 100.0 µM APO from 48.89% to 25.56% and 17.78% to 5.56%, respectively. In the range of 3.0 µM-300.0 µM, the effect of APO was concentration dependent, with a half maximal inhibitory concentration (IC50) of 120.1 µM and a Hill coefficient of 1.063. APO reversed the increase in transient inward current (Iti) and Na+/Ca2+-exchange current (INCX) densities induced by ISO in atrial myocytes. The frequency of spontaneous Ca2+ transients in atrial myocytes was reduced by 100.0 µM APO. Compared with ISO, APO downregulated the expression of NOX2 and increased the phosphorylation of PLNSer16 and the sarcoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) level; however, it had little effect on ryanodine-receptor channel type-2 (RyR2). These findings showed that APO may block Iti and INCX and reduce intracellular Ca2+ levels in rat atrial myocytes, thus reducing the incidence of ISO-induced DADs and TA.

Histone deacetylase 9 exacerbates podocyte injury in hyperhomocysteinemia through epigenetic repression of Klotho.

Although hyperhomocysteinemia (hHcys) has been recognized as an important independent risk factor in the progression of end-stage renal disease and the development of cardiovascular complications related to end-stage renal disease, the mechanisms triggering pathogenic actions of hHcys are not fully understood. The present study was mainly designed to investigate the role of HDACs in renal injury induced by hHcys. Firstly, we identified the expression patterns of HDACs and found that, among zinc-dependent HDACs, HDAC9 was preferentially upregulated in the kidney from mice with hHcys. Deficiency or pharmacological inhibition of HDAC9 ameliorated renal injury in mice with hHcys. Moreover, podocyte-specific deletion of HDAC9 significantly attenuated podocyte injury and proteinuria. In vitro, gene silencing of HDAC9 attenuated podocyte injury by inhibiting apoptosis, reducing oxidative stress and maintaining the expressions of podocyte slit diaphragm proteins. Mechanically, we proved for the first time that HDAC9 reduced the acetylation level of H3K9 in the promoter of Klotho, then inhibited gene transcription of Klotho, finally aggravating podocyte injury in hHcys. In conclusion, our results indicated that targeting of HDAC9 might be an attractive therapeutic strategy for the treatment of renal injury induced by hHcys.

Combination Therapy of Niacin and Apocynin Attenuates Lung Injury During Sepsis in Rats.

INTRODUCTION: Oxidative stress contributes to tissue injury through reactive oxygen species-dependent signaling pathways during sepsis. We studied therapeutic benefits of the combination therapy of niacin, which increased reduced glutathione levels, and apocynin, which suppressed reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity, in septic rats. MATERIALS AND METHODS: Polymicrobial sepsis was induced through cecal ligation and puncture (CLP) with antibiotics in male Sprague-Dawley rats (n = 189). The rats were randomly divided into sham, CLP, CLP + niacin, CLP + apocynin, and CLP + niacin + apocynin groups. Six hours after CLP, vehicle, niacin (360 mg/kg through the orogastric tube), and/or apocynin (20 mg/kg through intraperitoneal injection) were administered. The occurrence of mortality for 72 h after CLP was observed. Next, a separate set of animals was euthanized at 24 h post-CLP for lung tissue analyses. RESULTS: Combination therapy with niacin and apocynin significantly improved survival in rats with sepsis (75.0% versus 28.8%, P = 0.006) but monotherapy with niacin or apocynin did not. Monotherapy with niacin and apocynin appeared to increase NADPH levels and decrease Nox levels and activity, respectively, but failed to show statistical significances. However, combination therapy significantly decreased Nox levels and activity, increased NADPH and glutathione levels, decreased intranuclear nuclear factor-κB (NF-κB) p65 levels, reduced inflammatory cytokine expression and malondialdehyde levels, and attenuated histological lung injuries. CONCLUSIONS: Combination therapy with niacin and apocynin synergistically attenuated lung injuries and improved survival in rats with sepsis through niacin-induced glutathione redox cycle activation and apocynin-induced Nox suppression.

Identification of a Noxo1 inhibitor by addition of a polyethylene glycol chain.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2) which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450, through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. Design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point. The initial compound was then modified by addition of a polyethylene glycol linked biotin. Both inhibitors were found to be non-toxic in human keratinocyte cells. Further in vitro experiments using isothermal calorimetric binding quantification showed the modified biotinylated compound bound Noxo1 peptide with a KD of 2 nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors. Experiments show that ultraviolet light exposure of with the lead compound was able to reduce the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. Further synthesis showed that the polyethylene glycol but not the biotin was essential for inhibition.

Apocynin inhibits compressive force-induced apoptosis and autophagy of periodontal ligament stem cells.

OBJECTIVE: This study aimed to evaluate the effect of Apocynin on compressive force-induced apoptosis and autophagy in periodontal ligament stem cells (PDLSCs). MATERIALS AND METHODS: Periodontal ligament stem cells were subjected to a uniform compressive force of 2.0 g/cm2 for 24 h, without and with addition of 50, 100 and 200 µM Apocynin. Beclin-1 was overexpressed in PDLSCs. Flow cytometry was used to assess apoptosis and transmission electron microscopy was used to evaluate ultrastructural features of PDLSCs. Immunofluorescence was used to assess the levels of microtubule-associated protein 1 light chain 3 (LC3). The protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), Caspase-3, LC3, and Beclin were determined by Western blot analysis. RESULTS: Compressive force of PDLSCs significantly increased apoptosis, Bax, Caspase-3, the number of autophagosome or autolysosomes, Beclin-1, the ratio of LC3 II/LC3 I, and decreased Bcl-2. Apocynin was shown to inhibit apoptosis and Beclin-1-mediated autophagy. Over-expression of Beclin-1 increased apoptosis and autophagy. CONCLUSIONS: Application of Apocynin attenuated long-term compressive force-induced apoptosis by regulating Beclin-1-mediated autophagy in PDLSCs. These results provide an alternative approach to improve orthodontic treatment outcomes for patients.

Apocynin alleviates weight gain and obesity-induced adipose tissue inflammation in high-fat diet-fed C57BL/6 mice.

Obesity involves chronic low-grade inflammation within adipose tissue. Apocynin (APO) is a therapeutic agent for the treatment of inflammatory diseases. Therefore, the present study aimed to investigate whether APO can reduce weight gain and obesity-induced adipose tissue inflammation. C57BL/6 mice were administered APO or orlistat (Orli) as a positive control with a high-fat diet (HFD) for 12 weeks. Lipopolysaccharide-stimulated 3T3-L1 adipocytes were used for the in vitro study. Our results showed a significantly lower white adipose tissue (WAT) mass index in 10 mg/kg APO-treated mice than in 20 mg/kg Orli-treated mice. Moreover, the protein expression of adipose triglyceride lipase, fatty acid synthase, sterol regulatory element-binding transcription factor 1, and peroxisome proliferator-activated receptor γ was reversed in the WAT of 10 mg/kg APO-treated mice. Furthermore, APO reduced the expression of the macrophage marker F4/80, decreased the mRNA levels of tumor necrosis factor-α and monocyte chemoattractant protein-1, and increased the mRNA levels of interleukin-10 in WAT. APO decreased the phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p65 in vivo and in vitro. Notably, APO had a stronger effect on the amelioration of adipose tissue inflammation than Orli did. Our findings lay the foundation for research on the use of APO as an agent to ameliorate weight gain and obesity-induced inflammatory diseases.

Does apocynin increase liver regeneration in the partial hepatectomy model?

BACKGROUND: Hepayocyte loss may develop secondary to liver surgery and at this point liver regeneration plays a significant act in terms of liver reserve. The purpose of this research was to investigate the efficacy of apocynin on liver regeneration and preservation after partial hepatectomy in rats. METHODS: A total of 32 rats, have been divided into 4 groups (n: 8) for hepatectomy model. Inflammatory and antiinflammatory parameters were measured from blood and liver tissue samples. In addition, the effects of apocynin were examined immunohistochemically and histopathologically from liver tissue. RESULTS: In liver tissue samples, a significant difference has been found in glutathione peroxidase, total nitrite, catalase, oxidative stress index, total antioxidant and total oxidant status between sham and hepatectomy groups. A significant difference has been achieved between hepatectomy and posthepatectomy-Apocynin in terms of glutathione peroxidase and oxidative stress index. Total antioxidant status, oxidative stress index, and total oxidant status were significantly different only between the sham and the hepatectomy groups. Statistical differences were found between sham and hepatectomy groups and between hepatectomy and pre+post-hepatectomy-Apocynin groups in terms of serum glutathione, malondialdehyde, total nitrite, and L-Arginine. There were significant differences between the sham and hepatectomy groups, between hepatectomy and posthepatectomy-apocynin groups, between posthepatctomy-apocynin and pre+posthepatectomy-apocynin groups in terms of sinusoidal dilatation, intracytoplasmic vacuolization and glycogen loss (p < 0.001), in all histopathologic parameters except sinusoidal dilatation (p < 0.05). However, significant Ki-67 increases have been elaborated in hepatectomy, posthepatectomy-apocynin, and pre+posthepatectomy-apocynin groups compared to sham group (p < 0.001), in pre+posthepatectomy apocynin group compared to hepatectomy and posthepatectomy-apocynin groups (p < 0.001). DISCUSSION: Histopathology, immunohistochemistry, and biochemistry results of this study revealed that apocynin has a protective effect on enhancing liver regeneration in partial hepatectomy cases in rats.

[Intervention effect of apocynin on silicosis induced by silica in rats].

Objective: To investigate the intervention effect and its mechanism of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) on silicosis induced by silica (SiO(2)) in rats. Methods: In October 2021, 24 SPF SD male rats were divided into control group, silicosis model group and apocynin intervention group according to random number table method, with 8 rats in each group. SiO(2) was exposed by one-time intratracheal instillation. The rats in the apocynin intervention group were intraperitoneally injected with apocynin 50 mg/kg, 3 times a week, on the second day after treatment. The rats were sacrificed 28 days later, and lung coefficients were calculated after lung tissues were weighed. Hematoxylin-eosin staining and Masson staining were used to observe the lung histopathological changes in each group, respectively. The levels of NOX, reactive oxygen species (ROS), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in lung tissue were detected. The expressions of interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The level of hydroxyproline (HYP) was detected by alkaline hydrolysate. The expressions of transforming growth factor beta 1 (TGF-ß1), E-cadherin (E-cad) and α-smooth muscle actin (α-SMA) in lung tissue were detected by Western blotting. Results: Compared with the control group, the body weight of silicosis model group was decreased, the lung tissue showed obvious inflammatory infiltration and fibrosis, and the levels of lung coefficient, IL-1ß, IL-6, TNF-α and TGF-ß1 were significantly increased (P<0.05). Compared with the silicosis model group, the lung tissue injury in the apocynin intervention group was significantly improved, the lung coefficient, NOX, ROS, MDA, IL-1ß, IL-6, TNF-α and TGF-ß1 levels were decreased, and the activity of GSH-Px was increased (P<0.05). Compared with the silicosis model group, the expressions of HYP and α-SMA were decreased and the level of E-cad was increased in the apocynin intervention group (P<0.05) . Conclusion: Apocynin may alleviate SiO(2)-induced fibrosis in silicosis rats by reducing oxidative stress, the release of inflammatory factors and inhibiting the process of epithelial-mesenchymal transition.

Role of toll-like receptor 4 in diabetic retinopathy.

Diabetic retinopathy (DR) is the most frequent microvascular complication of diabetes mellitus (DM) and a leading cause of blindness worldwide. Evidence has shown that DR is an inflammatory disease with hyperglycemia playing a causative role in the development of its main features, including inflammation, cellular apoptosis, neurodegeneration, oxidative stress, and neovascularization. Toll-like receptors (TLRs) are a well-known family of pattern recognition receptors (PRRs) responsible for the initiation of inflammatory and immune responses. TLR4 identifies both endogenous and exogenous ligands and is associated with various physiological and pathological pathways in the body. While the detailed pathophysiology of DR is still unclear, increasing data suggests a crucial role for TLR4 in the development of DR. Due to hyperglycemia, TLR4 expression increases in diabetic retina, which activates various pathways leading to DR. Considering the role of TLR4 in DR, several studies have focused on the association of TLR4 polymorphisms and risk of DR development. Moreover, evidence concerning the effect of microRNAs in the pathogenesis of DR, through their interaction with TLR4, indicates the determinant role of TLR4 in this disease. Of note, several agents have proven as effective in alleviating DR through the inhibition of the TLR4 pathway, suggesting new avenues in DR treatment. In this review, we provided a brief overview of the TLR4 structure and biological function and a more comprehensive discussion about the mechanisms of TLR4 activation in DR. Furthermore, we summarized the relationship between TLR4 polymorphisms and risk of DR and the relationship between microRNAs and TLR4 in DR. Finally, we discussed the current progress in designing TLR4 inhibitors, which could be helpful in DR clinical management.

Restoration of afferent arteriolar autoregulatory behavior in ischemia-reperfusion injury in rat kidneys.

Renal autoregulation is critical in maintaining stable renal blood flow (RBF) and glomerular filtration rate (GFR). Renal ischemia-reperfusion (IR)-induced kidney injury is characterized by reduced RBF and GFR. The mechanisms contributing to renal microvascular dysfunction in IR have not been fully determined. We hypothesized that increased reactive oxygen species (ROS) contributed to impaired renal autoregulatory capability in IR rats. Afferent arteriolar autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. IR was induced by 60 min of bilateral renal artery occlusion followed by 24 h of reperfusion. Afferent arterioles from sham rats exhibited normal autoregulatory behavior. Stepwise increases in perfusion pressure caused pressure-dependent vasoconstriction to 65 ± 3% of baseline diameter (13.2 ± 0.4 µm) at 170 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in IR rats. Baseline diameter averaged 11.7 ± 0.5 µm and remained between 90% and 101% of baseline over 65-170 mmHg, indicating impaired autoregulatory function. Acute antioxidant administration (tempol or apocynin) to IR kidneys for 20 min increased baseline diameter and improved autoregulatory capability, such that the pressure-diameter profiles were indistinguishable from those of sham kidneys. Furthermore, the addition of polyethylene glycol superoxide dismutase or polyethylene glycol-catalase to the perfusate blood also restored afferent arteriolar autoregulatory responsiveness in IR rats, indicating the involvement of superoxide and/or hydrogen peroxide. IR elevated mRNA expression of NADPH oxidase subunits and monocyte chemoattractant protein-1 in renal tissue homogenates, and this was prevented by tempol pretreatment. These results suggest that ROS accumulation, likely involving superoxide and/or hydrogen peroxide, impairs renal autoregulation in IR rats in a reversible fashion.NEW & NOTEWORTHY Renal ischemia-reperfusion (IR) leads to renal microvascular dysfunction manifested by impaired afferent arteriolar autoregulatory efficiency. Acute administration of scavengers of reactive oxygen species, polyethylene glycol-superoxide dismutase, or polyethylene glycol-catalase following renal IR restored afferent arteriolar autoregulatory capability in IR rats, indicating that renal IR led to reversible impairment of afferent arteriolar autoregulatory capability. Intervention with antioxidant treatment following IR may improve outcomes in patients by preserving renovascular autoregulatory function and potentially preventing the progression to chronic kidney disease after acute kidney injury.

Pharmacology of apocynin: a natural acetophenone.

Apocynin is a naturally occurring acetophenone, found in the roots of Apocynum cannabinum and Picrorhiza kurroa. Various chemical and pharmaceutical modifications have been carried out to enhance the absorption and duration of action of apocynin, like, formulation of chitosan-based apocynin-loaded solid lipid nanoparticles, chitosan-oligosaccharide based nanoparticles, and biodegradable polyanhydride nanoparticles. Apocynin has been subjected to a wide range of experimental screening and has proved to be useful for amelioration of a variety of disorders, like diabetic complications, neurodegeneration, cardiovascular disorders, lung cancer, hepatocellular cancer, pancreatic cancer, and pheochromocytoma. Apocynin has been primarily reported as an NADPH oxidase (NOX) inhibitor and prevents translocation of its p47phox subunit to the plasma membrane, observed in neurodegeneration and hypertension. However, recent studies highlight its off-target effects that it is able to function as a scavenger of non-radical oxidant species, which is relevant for its activity against NOX 4 mediated production of hydrogen peroxide. Additionally, apocynin has shown inhibition of eNOS-dependent superoxide production in diabetic cardiomyopathy, reduction of NLRP3 activation and TGFß/Smad signaling in diabetic nephropathy, diminished VEGF expression and decreased retinal NF-κB activation in diabetic retinopathy, inhibition of P38/MAPK/Caspase3 pathway in pheochromocytoma, inhibition of AKT-GSK3ß and ERK1/2 pathways in pancreatic cancer, and decreased FAK/PI3K/Akt signaling in hepatocellular cancer. This review aims to discuss the pharmacokinetics and mechanisms of the pharmacological actions of apocynin.

Apocynin ameliorates NADPH oxidase 4 (NOX4) induced oxidative damage in the hypoxic human retinal Müller cells and diabetic rat retina.

NADPH oxidase (NOX) is a main producers of reactive oxygen species (ROS) that may contribute to the early pathogenesis of diabetic retinopathy (DR). ROS has harmful effects on endogenous neuro-survival factors brain-derived neurotrophic factor (BDNF) and sirtuin 1 (SIRT1) are necessary for the growth and survival of the retina. The role of NOX isoforms NOX4 in triggering ROS in DR is not clear. Here we determine the protective effects of a plant-derived NOX inhibitor apocynin (APO) on NOX4-induced ROS production which may contribute to the depletion of survival factors BDNF/SIRT1 or cell death in the diabetic retinas. Human retinal Müller glial cells (MGCs) were treated with hypoxia mimetic agent cobalt chloride (CoCl2) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl2-treated MGCs and in the diabetic retinas. Increased NOX4 was accompanied by the downregulation of BDNF/SIRT1 expression or in the activation of apoptotic marker caspase-3. Whereas, APO treatment downregulates NOX4 and subsequently upregulates BDNF/SIRT1 or alleviate caspase-3 expression. Accordingly, in the diabetic retina we found a positive correlation in NOX4 vs ROS (p = 0.025; R2 = 0.488) and caspase-3 vs ROS (p = 0.04; R2 = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R2 = 0.596) and SIRT1 vs ROS (p = 0.0003; R2 = 0.817) respectively. Taken together, NOX4-derived ROS could be a main contributor in downregulating BDNF/SIRT1 expression or in the activation of caspase-3. Whereas, APO treatment may minimize the deleterious effects occurring due to hyperglycemia and/or diabetic mimic hypoxic condition in early pathogenesis of DR.

Apocynin prevents reduced myocardial nerve growth factor, contributing to amelioration of myocardial apoptosis and failure.

Reduced nerve growth factor (NGF) is associated with cardiac sympathetic nerve denervation in heart failure (HF) which is characterized by increased oxidative stress. Apocynin is considered an antioxidant agent which inhibits NADPH oxidase activity and improves reactive oxygen species scavenging. However, it is unclear whether apocynin prevents reduced myocardial NGF, leading to improvement of cardiac function in HF. In this study, we tested the hypothesis that apocynin prevents reduced myocardial NGF, contributing to amelioration of myocardial apoptosis and failure. Rabbits with myocardial infarction (MI) or sham operation were randomly assigned to receive apocynin or placebo for 4 weeks. MI rabbits exhibited left ventricular (LV) dysfunction, and elevation in oxidative stress, as evidenced by a decreased reduced-to-oxidized glutathione ratio and an increased 4-hydroxynonenal expression, and reduction in NGF and NGF receptor tyrosine kinase A (TrKA) expression in the remote non-infarcted myocardium. Apocynin treatment ameliorated LV dysfunction, reduced oxidative stress, prevented decreases in NGF and TrKA expression and reduced cardiomyocyte apoptosis after MI. In cultured H9C2 cardiomyocytes, hypoxia or hydrogen peroxide decreased NGF expression, and apocynin normalized hypoxia-induced reduction of NGF. Recombinant NGF attenuated hypoxia-induced apoptosis. Apocynin prevented hypoxia-induced apoptosis, and the suppressive effect of apocynin on apoptosis was abolished by NGF receptor TrKA inhibitor K252a. We concluded that apocynin prevented reduced myocardial NGF, leading to attenuation of cardiomyocyte apoptosis and LV remodelling and dysfunction in HF after MI. These findings suggest that strategies to prevent NGF reduction by inhibition of oxidative stress may be of value in amelioration of LV dysfunction in HF.

Acetovanillone prevents cyclophosphamide-induced acute lung injury by modulating PI3K/Akt/mTOR and Nrf2 signaling in rats.

Cyclophosphamide (CP) is a medication used as an anticancer drug and to suppress the immune system. However, its clinical applications are restricted because of the toxic and adverse side effects. The present study investigated the protective effect of acetovanillone (AV), a natural NADPH oxidase inhibitor, against acute lung injury (ALI) induced by CP. Rats were administered AV (100 mg/kg) for 10 days and a single injection of CP (200 mg/kg) at day 7. At the end of the experiment, the animals were sacrificed, and lung samples were collected for analyses. CP caused ALI manifested by the histopathological alterations. Lipid peroxidation and NADPH oxidase activity were increased, whereas GSH and antioxidant enzymes were decreased in the lung of CP-intoxicated rats. Oral administration of AV prevented CP-induced lung injury and oxidative stress and enhanced antioxidant defenses. AV downregulated Keap1 and upregulated Nrf2, GCLC, HO-1, and SOD3 mRNA. In addition, AV boosted the expression of PI3K, Akt, mTOR, and cytoglobin. In vitro, AV showed a synergistic anticancer effect when combined with CP. In conclusion, AV protected against CP-induced ALI by attenuating oxidative stress and boosting Nrf2/HO-1 and PI3K/Akt/mTOR signaling. Therefore, AV might represent a promising adjuvant to prevent lung injury in patients receiving CP.

The Effect of Apocynin on Fat Graft Survival.

BACKGROUND: In this study, we investigated the effect of apocynin on fat graft survival in a rat model. METHODS: Twenty-one Wistar albino male rats were included in the study. The grafts taken from the inguinal area were applied to the nape of each rat. The rats were randomly divided into three groups. Apocynin+DMSO, DMSO and saline were administered intraperitoneally once daily for 14 days. After 90 days, the animals were sacrificed and the grafts were excised. The weights and volumes were measured and sent for histopathological evaluation. The weight, volume, survival rates of the grafts, the number of viable cells with the MTT test and pathological parameters, inflammation, vascularization, fibrosis, lipogranuloma and cyst formation, were evaluated. RESULTS: Statistically significant differences were found between the groups in survival rates (p<0.05). In paired comparisons between the groups, the parameters of viable cells and survival rates were statistically significantly higher in apocynin group compared to the other groups (p<0.05). The survival rates relating to the weight and viable cell count in the DMSO group were statistically significantly higher than in the control group (p<0.05). However, no statistically significant difference was found between the DMSO group and the control group in the survival rate in terms of volume (p<0.05). There was no significant difference between the groups in the comparison of pathological parameters (p>0.05). CONCLUSIONS: Apocynin increases fat graft survival in the animal fat grafting model. Therefore, apocynin can be used as an effective medical agent to prevent the volume loss of fat grafts. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Apocynin ameliorates cognitive deficits in streptozotocin-induced diabetic rats.

AIMS: The aim was to investigate the improvement properties of apocynin and its potential mechanism on diabetes-associated cognitive decline. METHODS: In this study, the model of diabetic rat was established by STZ (50 mg/kg) and treated with apocynin (16 mg/kg/d for 12 weeks). The cognitive ability was evaluated by Morris water maze test. The indicators of oxidative stress (SOD and MDA) were analyzed by spectrophotometer. The inflammatory cytokines were measured by real time-PCR and ELISA. The protein expressions of Nrf-2, HO-1, Bcl-2 and Bax were determined by Western blot. RESULTS: Treatment with apocynin ameliorated diabetes-related learning and memory injury, as represented by decreasing escape latency and enhancement of the number of times of crossing platform, in the Morris water maze test. In hippocampus, apocynin markedly augmented SOD activity and inhibited MDA level to alleviate oxidative stress. Moreover, apocynin obviously relieved inflammatory reaction by suppressing TNF-α, IL-1ß and IL-6 concentrations. Concomitantly, apocynin also statistically enhanced Nrf-2 and HO-1 protein expression to improve DACD. Lastly, apocynin notably ameliorated Bax/Bcl-2 ratio by regulating Bax and Bcl-2 protein expression to mitigate apoptosis. CONCLUSION: Our results have shown that apocynin may be a valid therapeutic agent against DACD via modulation of antioxidant, anti-inflammatory, and anti-apoptosis (Tab. 1, Fig. 18, Ref. 35).