Neferine inhibits the progression of diabetic nephropathy by modulating the miR-17-5p/nuclear factor E2-related factor 2 axis.

OBJECTIVE: To investigate the effect of Neferine (Nef) on diabetic nephropathy (DN) and to explore the mechanism of Nef in DN based on miRNA regulation theory. METHODS: A DN mouse model was constructed and treated with Nef. Serum creatinine (Crea), blood urea (UREA) and urinary albumin were measured in mice by kits, and renal histopathological changes and fibrosis were observed by hematoxylin-eosin staining and Masson staining. Renal tissue superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was used to detect the expression of nuclear factor E2-related factor 2 (Nrf2)/ heme oxygenase 1 (HO-1) signaling pathway-related proteins in kidney tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-17-5p in kidney tissues. Subsequently, a DN in vitro model was constructed by high glucose culture of human mesangial cells (HMCs), cells were transfected with miR-17-5p mimic and/or treated with Nef, and we used qRT-PCR to detect cellular miR-17 expression, flow cytometry to detect apoptosis, ELISAs to detect cellular SOD, MDA, and GSH-Px activities, Western blots to detect Nrf2/HO-1 signaling pathway-related protein expression, and dual luciferase reporter gene assays to verify the targeting relationship between Nrf2 and miR-17-5p. RESULTS: Administration of Nef significantly reduced the levels of blood glucose, Crea, and UREA and the expression of miR-17-5p, improved renal histopathology and fibrosis, significantly reduced MDA levels, elevated SOD and GSH-Px activities, and activated Nrf2 expression in kidney tissues from mice with DN. Nrf2 is a post-transcriptional target of miR-17-5p. In HMCs transfected with miR-17-5p mimics, the mRNA and protein levels of Nrf2 were significantly suppressed. Furthermore, miR-17-5p overexpression and Nef intervention resulted in a significant increase in high glucose-induced apoptosis and MDA levels in HMCs and a significant decrease in the protein expression of HO-1 and Nrf2. CONCLUSION: Collectively, these results indicate that Nef has an ameliorative effect on DN, and the mechanism may be through the miR-17-5p/Nrf2 pathway.

Astragaloside IV mitigates cerebral ischaemia-reperfusion injury via inhibition of P62/Keap1/Nrf2 pathway-mediated ferroptosis.

Cerebral ischaemia-reperfusion injury (CIRI) is a critical component of ischaemic stroke pathogenesis. Ferroptosis contributes to and aggravates CIRI, whereas the P62/Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) pathway exerts neuroprotective effects. Astragaloside IV (AST IV) is the primary active ingredient of Astragalus, an herb with anti-CIRI properties used in traditional Chinese medicine. However, the mechanism of its anti-CIRI action is unclear. This study examined the mechanisms underlying the anti-CIRI action of AST IV using a combination of in vitro and in vivo approaches. We established an erastin-induced ferroptosis model, oxygen and glucose deprivation/reoxygenation (OGD/R)-induced model in SH-SY5Y cells, and middle cerebral artery occlusion-reperfusion (MCAO/R) model using Sprague-Dawley rats. The extent of cell damage and brain damage in rats, ferroptosis indicator changes, and expression of P62, Keap1, and Nrf2 were investigated. AST IV inhibited erastin-induced ferroptosis, attenuated OGD/R-induced cell damage, and ameliorated sensorimotor dysfunction and injury in the MCAO/R model. Further, AST IV promoted Nrf2 activation, inhibited ferroptosis, and reduced cell damage. Notably, these effects were inhibited by ML385, an Nrf2 inhibitor. AST IV increased the P62 and Nrf2 levels and decreased the Keap1 levels. P62 silencing reduced the effects of AST IV on the P62/Keap1/Nrf2 pathway and ferroptosis. Our findings suggest that AST IV mitigates CIRI by inhibiting ferroptosis via activation of the P62/Keap1/Nrf2 pathway. This study provides an important scientific basis and direction for the application and research of AST IV and provides new potential targets and ideas for the study of the pathological mechanism of CIRI.

Depleted uranium causes renal mitochondrial dysfunction through the ETHE1/Nrf2 pathway.

The kidney is the main organ affected by acute depleted uranium (DU) toxicity. The mechanism of nephrotoxicity induced by DU is complex and needs to be further explored. This study aimed to elucidate the function of mitochondrial dysfunction in nephrotoxicity generated by DU and confirm the latent mechanism. We verified that DU (2.5-10 mg/kg) caused mitochondrial dysfunction in male rat kidneys and decreased ATP content and the mitochondrial membrane potential. In addition, melatonin (20 mg/kg), as an antioxidant, alleviated DU-induced oxidative stress and mitochondrial dysfunction in male rats, further reducing kidney damage caused by DU. These results indicate that mitochondrial dysfunction plays a vital role in DU nephrotoxicity. When ethylmalonic encephalopathy 1 (ETHE1) was knocked down, DU-induced oxidative stress and mitochondrial dysfunction were increased, and renal injury was aggravated. When exogenous ETHE1 protein was applied to renal cells, the opposite changes were observed. We also found that ETHE1 knockdown increased the expression of NF-E2-related factor 2 (Nrf2), a vital oxidative stress regulator, and its downstream molecules heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO1). Nrf2 knockout also aggravated DU-induced oxidative stress, mitochondrial dysfunction, and kidney damage. In conclusion, DU causes oxidative stress and antioxidant defense imbalance in renal cells through the ETHE1/Nrf2 pathway, further causing mitochondrial dysfunction and ultimately leading to nephrotoxicity.

Role of Nrf2/GPX4 signaling pathway-mediated ferroptosis in midazolam-induced reduction of HIBD in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the role of nuclear factor-erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase-4 (GPX4) signaling pathway-mediated ferroptosis in midazolam-induced reduction of hypoxic-ischemic brain damage (HIBD) in neonatal rats.Methods:Ninety healthy 7-day-old neonatal rats, weighing 16-20 g, were divided into 6 groups ( n=15 each) using the random number table method: sham operation group (Sham group), HIBD group, low-dose midazolam (10 mg/kg) group (group L), medium-dose midazolam (20 mg/kg) group (group M), high-dose midazolam (40 mg/kg) group (group H), and Nrf2 inhibitor ML385 group (group I). The HIBD model was developed by ligating the left carotid artery and exposing to a hypoxic condition for 2 h in anesthetized animals. Starting from 2nd day after developing the model, the corresponding doses of midazolam were intraperitoneally injected in midazolam groups, the equal volume of normal saline was intraperitoneally injected in Sham and HIBD groups, midazolam 40 mg/kg and Nrf2 inhibitor ML385 30 mg/kg were intraperitoneally injected once a day for 8 consecutive days in group I. The rats were weighed and subjected to the Morris water maze test after the end of administration. Blood samples were taken from the abdominal aorta after the end of the Morris water maze test, and then the animals were sacrificed to remove the brain for determination of the concentrations of serum iron, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay), contents of iron and GSH in hippocampal tissues (by ultraviolet spectrophotometry and micro method), the number of Nrf2/neuronal nuclear antigen (NeuN) and GPX4/NeuN positive cells (by immunofluorescent staining), and expression of Nrf2, GPX4, and 4-hydroxynonaenoic acid (4-HNE) in hippocampal tissues and for microscopic examination of the pathological changes of hippocampal neurons in brain tissues (after HE staining and Nissl staining). Results:Compared with Sham group, the first time to arrival at platform was significantly prolonged, the number of crossing the origional platform was reduced, and the time of staying at the target quadrant was shortened, the iron content in the hippocampal tissues was increased, the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were decreased, the expression of Nrf2 and GPX4 was down-regulated, the expression of 4-HNE was up-regulated, the concentrations of serum iron, IL-6 and TNF-α were increased, and the injury to hippocampal neurons was marked in HIBD group ( P<0.05). Compared with HIBD group, the first time to arrival at platform was significantly shortened, the number of crossing the origional platform was increased, and the time of staying at the target quadrant was prolonged, the iron content in the hippocampus tissues was decreased, the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were increased, the expression of Nrf2 and GPX4 was up-regulated, the expression of 4-HNE was down-regulated, the concentrations of serum iron, IL-6 and TNF-α were decreased ( P<0.05), and the injury to hippocampal neurons was significantly reduced in H, M and L groups. Compared with group H, the first time to arrival at platform was significantly prolonged, the number of crossing the origional platform was reduced, and the time of staying at the target quadrant was shortened, the iron content in the hippocampus tissue was increased, the content of GSH and the number of Nrf2/NeuN and GPX4/NeuN positive cells were decreased, the expression of Nrf2 and GPX4 was down-regulated, the expression of 4-HNE was up-regulated, the concentrations of serum iron, IL-6 and TNF-α were increased ( P<0.05), and the injury to hippocampal neurons was aggravated in group I. Conclusions:The mechanism by which midazolam reduces HIBD may be related to activation of the Nrf2/GPX4 signaling pathway and inhibition of hippocampal neuronal ferroptosis in neonatal rats.

Antihepatofibrotic effect of Guizhifuling pill on carbon tetrachloride-induced liver fibrosis in mice.

OBJECTIVE: To evaluate the protective effects and the underlying mechanism of Guizhifuling pill (, GZFL) on carbon tetrachloride (CCl)-induced hepatic fibrosis in mice. METHODS: Male ICR mice by intraperitoneally administered with 20% CCl (mixed 1∶4 in soybean oil) to induce liver fibrosis. Mice that underwent CCl were orally with GZFL. Using hematoxylin and eosin and Masson staining to examine the pathological changes in liver tissue. Serum biochemical parameters, antioxidant enzyme activity and proinflammatory cytokines was assessed. Nuclear factor-kappaB (NF-κB) pathway and nuclear factor-erythroid 2-related factor 2 (Nrf2) family members were evaluated by Western blotting. RESULTS: Our findings indicated that GZFL could effectively suppress the progression of liver fibrosis in mice, which was determined based on the improvement in liver function and reduction of collagen deposition. GZFL treatment also decreased the level of cytokines and increased the activity of antioxidant enzymes in liver tissue. Moreover, GZFL exerted anti-inflammatory and antioxidant effects through regulating the Nrf2-mediated antioxidant system and inhibiting the NF-κB pathway. CONCLUSIONS: GZFL may prevent the progression of liver fibrosis by regulating the Nrf2/ heme oxygenase-1 and NF-κB signaling pathways, thereby highlighting its role in the management of liver fibrosis.

Lycium barbarum polysaccharides attenuate cardiovascular oxidative stress injury by enhancing the Keap1/Nrf2 signaling pathway in exhaustive exercise rats.

Moderate exercise is beneficial to physical and mental health. When the amount of exercise and exercise intensity exceeds a certain limit and reaches the state of exhaustion, oxidative stress levels in the body increase, which can lead to oxidative stress­associated damage. Lycium barbarum polysaccharide (LBP) is one of the primary active ingredients extracted from wolfberry. Following exhausting exercise in rats, LBP supplements decrease damage to the myocardium and blood vessels, indicating that LBP exerts a protective effect on the cardiovascular system. The Kelch­like ECH­associated protein 1 (Keap1)/NF­E2­related factor 2 (Nrf2) anti­oxidative stress signaling pathway improves total oxidizing ability; anti­apoptosis and other aspects serve a vital role. In the present study, LBP intervention was performed in vivo and in vitro to observe its effect on the Keap1/Nrf2 pathway and oxidative stress­associated indicators in order to clarify its protective mechanism. For the in vivo experiments, 60 male Sprague­Dawley rats were randomly divided into normal control and aerobic, exhaustive and exhaustive exercise + LBP (200 mg/kg/day) groups. For the in vitro experiments, a rat thoracic aortic endothelial cell (RTAEC) oxidative stress model was established using angiotensin II (AngII) and divided into blank control, LBP (3,200 µg/ml), AngII (1x10­4 mol/l) and AngII + LBP groups. For in vitro experiments, small interfering (si)RNA (50 nmol) was used to transfect RTAEC and induce gene silencing of Nrf2. ELISA, hematoxylin and eosin staining, TUNEL, immunofluorescence, western blotting, immunohistochemistry and reverse transcription­quantitative PCR were used to evaluate and verify the effect of LBP on oxidative stress indicators and the expression of Keap1/Nrf2 antioxidative stress signaling pathway. The in vivo experiments showed that LBP decreased the expression of serum malondialdehyde (MDA) and AngII, as well as apoptosis of blood vessels and cardiomyocytes and expression of TNF­α in rats following exhaustive exercise. Meanwhile, LBP enhanced expression of the Keap1/Nrf2 signaling pathway and downstream associated protein glutamyl­cysteine synthetase catalytic subunit (GCLC), quinone oxidoreductase 1 (NQO1) and glutamate­cysteine ligase modified subunit (GCLM) in the thoracic aorta and myocardium of rats following exhaustive exercise. In RTAEC in vitro, LBP decreased the expression of MDA and TNF­α in the supernatant, promoted the nuclear translocation of Nrf2 and increased expression levels of GCLC, NQO1 and GCLM. Following siNrf2 transfection into endothelial cells, the anti­inflammatory and antioxidant stress effects of LBP were decreased. LBP was found to enhance the expression of the Keap1/Nrf2 antioxidant stress signaling pathway in endothelial cells, decreasing oxidative stress and the inflammatory response. Moreover, LBP improved the antioxidant stress ability of endothelial cells and alleviated injury of myocardial vascular tissue, thereby protecting the cardiovascular system.

Porous SiO2 -coated ultrasmall selenium particles nanospheres attenuate cerulein-induce acute pancreatitis in mice by downregulating oxidative stress.

OBJECTIVE: To investigate the potential therapeutic role of porous SiO2 -coated ultrasmall selenium particles nanospheres (Se@SiO2 nanospheres) pretreatment in acute pancreatitis (AP) and to investigate the related mechanism. METHODS: C57BL/6 mice were randomized to the normal control (CON) group, the AP (induced by cerulein injection) (CAE) group, and AP pretreated with Se@SiO2 nanocomposites at 1 and 2 mg/kg (CAE + 1 or 2 mg/kg Se@SiO2 ) groups, respectively. Serum levels of amylase and lipase, inflammatory cytokines (interleukin [IL]-6, IL-1ß and tumor necrosis factor [TNF]-α), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) were measured, and histopathology was performed to examine the tissue samples of the pancreas, lungs, kidneys and liver. Immunofluorescence assay of reactive oxygen species (ROS), myeloperoxidase (MPO) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling were conducted, and levels of MPO, malondialdehyde, superoxide dismutase and glutathione were evaluated. Finally, Western blot analysis was used to evaluate protein expressions of Nrf2, HO-1, NQO1, TLR4, MyD88 and p-p65 in pancreatic tissue. RESULTS: Se@SiO2 nanospheres alleviated pathological damage to the pancreas, and reduced pancreatic enzymes and inflammatory cytokines. Injury to other organs such as the liver, lungs and kidneys was also alleviated, as indicated by decreased ALT, AST, BUN, and Cr levels as well as improved histopathology. Moreover, Se@SiO2 nanospheres reduced oxidative stress, and ultimately inhibited TLR4/ MyD88/p-p65 pathway and increased the protein expressions of NQO1, Nrf2, and HO-1. CONCLUSION: Se@SiO2 nanospheres may alleviate AP by relieving oxidative stress and targeting the TLR4/Myd88/p-p65 and NQO1/Nrf2/HO-1 pathways.

Effect of electroacupuncture pretreatment on oxidative stress response of hippocampus in rats with sepsis-associated encephalopathy and relationship with Nrf2/HO-1 signaling pathway / 中华麻醉学杂志

Objective:To evaluate the effect of electroacupuncture (EA) pretreatment on oxidative stress response of hippocampus in rats with sepsis-associated encephalopathy (SAE) and the relationship with nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (Nrf2/HO-1) signaling pathway.Methods:A total of 64 healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 4 groups ( n=16 each) using a random number table method: sham operation group (Sham group), SAE group, SAE+ EA group and SAE plus sham EA group (SAE+ SEA group). In SAE+ EA group, Baihui, Quchi and Zusanli acupoints were stimulated for 30 min once a day for 5 consecutive days.Sepsis was induced by cecal ligation and puncture immediately after the end of the last EA.At 1 and 7 days after establishment of the model, the hippocampal malondialdehyde (MDA) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay, the expression of hippocampal Nrf2 mRNA was detected using real-time reverse transcription polymerase chain reaction, and the expression of Nrf2 and HO-1 was determined by Western blot.At 3-7 days after establishment of the model, cognitive function was assessed using the Morris water maze test, and the escape latency and the target quadrant exploration time were recorded. Results:Compared with Sham group, the content of MDA was significantly increased and the activity of SOD was decreased at 1 and 7 days after establishment of the model, the expression of Nrf2 protein and mRNA and HO-1 was down-regulated at day 7 after establishment of the model, the escape latency was prolonged, and the target quadrant exploration time was shortened in SAE group ( P<0.05). Compared with SAE group, the content of MDA was significantly decreased and the activity of SOD was increased at 1 and 7 days after establishment of the model, the expression of Nrf2 protein and mRNA and HO-1 was up-regulated at day 7 after establishment of the model, the escape latency was shortened, and the target quadrant exploration time was prolonged in group SAE+ EA ( P<0.05), and no significant change was found in the parameters mentioned above in group SAE+ SEA ( P>0.05). Conclusion:EA pretreatment can reduce oxidative stress response of hippocampus in rats with SAE, and the mechanism may be related to activating Nrf2/HO-1 signaling pathway.

Cardamonin Inhibits Oxazolone-Induced Atopic Dermatitis by the Induction of NRF2 and the Inhibition of Th2 Cytokine Production.

The skin is constantly exposed to various types of chemical stresses that challenge the immune cells, leading to the activation of T cell-mediated hypersensitivity reactions including atopic dermatitis. Previous studies have demonstrated that a variety of natural compounds are effective against development of atopic dermatitis by modulating immune responses. Cardamonin is a natural compound abundantly found in cardamom spices and many other medicinal plant species. In the present study, we attempted to examine whether cardamonin could inhibit oxazolone-induced atopic dermatitis in vivo. Our results show that topical application of cardamonin onto the ear of mice suppressed oxazolone-induced inflammation in the ear and hyperplasia in the spleen. Cardamonin also inhibited oxazolone-induced destruction of connective tissues and subsequent infiltration of mast cells into the skin. In addition, we found that the production of Th2 cytokines is negatively regulated by NRF2, and the induction of NRF2 by cardamonin contributed to suppressing oxazolone-induced Th2 cytokine production and oxidative damages in vivo. Together, our results demonstrate that cardamonin is a promising natural compound, which might be effective for treatment of atopic dermatitis.

Apigenin protects human melanocytes against oxidative damage by activation of the Nrf2 pathway.

Vitiligo is a chronic, autoimmune destruction of melanocytes, resulting in progressively expanding depigmented skin patches. Severity of the disorder, which affects approximately 1% of humans, may be mitigated using topical corticosteroids combined with phototherapy; along with other clinical strategies; however, no definitive cures are currently available. Here, the capacity of apigenin, a plant-derived aglycone, to inhibit oxidative stress-mediated melanocyte depletion in vitro using a PIG3V vitiligo perilesional melanocyte cell model is evaluated. PIG3V cells, treated with selected doses of apigenin, were challenged with H2O2, then assessed for viability and the oxidative stress-related parameters: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) by enzyme-linked immunoabsorbent assay (ELISA). Additionally, expression of nuclear factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) and downstream targets was detected using Western blotting. Outcomes demonstrated that compared with negative control cultures, apigenin-treated cells exhibited enhanced viability. Likewise, apigenin enhanced expression of the cellular anti-oxidants SOD, CAT, and GSH-Px, but inhibited production of MDA, an oxidative stress biomarker. Interestingly, the expression and nuclear localization of the Nrf2 transcription factor, an important regulator oxidative stress and its downstream target genes, was significantly increased by apigenin treatment. Apigenin influence on Nrf2 was further validated by experiments demonstrating that Nrf2 knockdown cells failed to exhibit significant apigenin-mediated effects on cell viability and oxidative stress. Apigenin's non-toxicity and ability to affect multiple oxidative stress-related parameters through its effects on Nrf2 signaling in melanocytes suggests that it may prove to be a valuable therapeutic tool in long-term management of vitiligo.

Antinociceptive and neuroprotective effects of bromelain in chronic constriction injury-induced neuropathic pain in Wistar rats.

BACKGROUND: The continuous search for a novel neuropathic pain drug with few or no side effects has been a main focus of researchers for decades. This study investigated the antinociceptive and neuroprotective effects of bromelain in sciatic nerve ligation-induced neuropathic pain in Wistar rats. METHODS: Forty-eight Wistar rats randomly divided into eight groups comprised of six animals each were used for this study. Peripheral neuropathy was induced via chronic constriction of the common sciatic nerve. Thermal hyperalgesic and mechanical allodynia were assessed using a hotplate and von Frey filaments, respectively. The functional recovery and structural architecture of the ligated sciatic nerve were evaluated using the sciatic functional index test and a histological examination of the transverse section of the sciatic nerve. The neuroprotective effects of bromelain were investigated in the proximal sciatic nerve tissue after 21 days of treatment. RESULTS: Bromelain significantly (P < 0.05) attenuated both the thermal hyperalgesia and mechanical allodynic indices of neuropathic pain. There were improvements in sciatic function and structural integrity in rats treated with bromelain. These rats showed significant (P < 0.05) increases in sciatic nerve nuclear transcription factors (nuclear factor erythroid-derived-2-related factors-1 [NrF-1] and NrF-2), antioxidant enzymes (superoxide dismutase and glutathione), and reduced membrane-lipid peroxidation compared with the ligated control group. CONCLUSIONS: This study suggest that bromelain mitigated neuropathic pain by enhancing the activities of nuclear transcription factors (NrF-1 and NrF-2) which increases the antioxidant defense system that abolish neuronal stress and structural disorganization.

15-Deoxy-Δ12,14-prostaglandin J2 ameliorates dextran sulfate sodium-induced colitis in mice through heme oxygenase-1 induction.

The prostaglandin D2 metabolite, 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2), exerts an anti-inflammatory effect through peroxisome proliferator-activated receptor γ (PPARγ)-dependent and -independent anti-inflammatory actions. In the present study, we focused on heme oxygenase-1 (HO-1) induced by 15d-PGJ2, and evaluated the effects of enema treatment with 15d-PGJ2 in the development of intestinal inflammation using a murine colitis model. Acute colitis was induced with dextran sulfate sodium (DSS) in male C57BL/6 mice (8 weeks old) and NF-E2-related factor-2 (Nrf2) deficient mice. Mice were rectally administered 15d-PGJ2 (1 µM, 0.2 mL: 66.9 ng) daily during DSS administration. Intestinal expression of HO-1 mRNA and protein after rectal administration of 15d-PGJ2 was evaluated by real-time PCR and western blotting, respectively. A disease activity index (DAI) was determined on a daily basis for each animal, and consisted of a calculated score based on changes in body weight, stool consistency, and intestinal bleeding. Tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration and mRNA expression levels of TNF-α, IFN-γ, and IL-17A were measured in the colonic mucosa. In addition, we evaluated the effects of co-treatment with a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP), or a specific PPARγ antagonist, GW9662. As a result, rectal administration of 15d-PGJ2 markedly induced HO-1 protein and mRNA expression in the colonic mucosa. Treatment with 15d-PGJ2 ameliorated the increase in DAI score and MPO activity and the mRNA expression levels of TNF-α, IFN-γ, and IL-17A after DSS administration. These effects of 15d-PGJ2 against intestinal inflammation were negated by co-treatment with ZnPP, but not with GW9662. In Nrf2 deficient mice, the rectal administration of 15d-PGJ2 did not affect colonic HO-1 expression and activity of DSS-induced colitis. These results demonstrate that 15d-PGJ2 inhibits development of intestinal inflammation in mice via PPAR-independent and Nrf2-HO-1-dependent mechanisms.

Aucubin, a natural iridoid glucoside, attenuates oxidative stress-induced testis injury by inhibiting JNK and CHOP activation via Nrf2 up-regulation.

BACKGROUND: Eucommia ulmoides has been used for many years as a successful strategy to treat male infertility. Aucubin (AU) is the active ingredient extracted from Eucommia ulmoides. However, its protective action and exact mechanism on testicular injury is not yet known. PURPOSE: Here, the protective effect and the mechanism of action of AU on testis damage under oxidative stress was investigated in vivo and in vitro. METHODS: As regard the in vivo experiment, male mice were divided into five groups and testicular injury model was established by Triptolide (TP) (120 µg/kg) intraperitoneal injection for two weeks. Animals in the treatment group were pretreated with an intraperitoneal injection of AU at different doses (5, 10 and 20 mg/kg) for 1 h and subsequently treated with TP (120 µg/kg). At the end of the experimental period, the testis was collected for biochemical and histological examination. As regard the in vitro experiment, Sertoli cells (SCs) were used to investigate the protective effect and mechanism of action of AU against disruption of the blood-testis-barrier (BTB) and apoptosis induced by TP via apoptosis detection, western blot, immunofluorescence analysis, and siRNA transient transfection. RESULTS: TP-treated animals showed testicular atrophy, BTB disruption, increased ROS levels and spermatogenic dysfunction. Pre-administration of AU resulted in a significant protection on keeping a normal testicular weight, sperm morphology, BTB integrity, and a normal level of oxidative stress markers and antioxidants. Furthermore, AU prevented apoptosis through an effective inhibition of PERK/CHOP and JNK dependent apoptosis pathway, as well as protected the integrity of BTB by up-regulating the expression of tight junction proteins (ZO-1, Occludin, Claudin-11) and gap junction protein (Cx43). The mechanistic study revealed that AU significantly triggered Nrf2 translocation, thus increasing nuclear Nrf2 accumulation and then induced antioxidant enzymes expression in the testis and SCs. Furthermore, Nrf2 silencing unsuccessfully reversed the increased CHOP and p-JNK expression induced by TP, abolishing the protective effect of AU. CONCLUSION: These results indicate that AU might be considered as a potential protective agent against testicular injury.

Alleviated Oxidative Damage by Taraxacum officinale through the Induction of Nrf2-MAPK/PI3K Mediated HO-1 Activation in Murine Macrophages RAW 264.7 Cell Line.

Taraxacum officinale has been consumed as a folk remedy due to its diverse physiological activities. This study aimed to investigate the antioxidative potential of T. officinale water extract (TOWE) and ethanol extract (TOEE) against oxidative stress and compare their molecular mechanism via the induction of heme oxygenase-1 (HO-1) in RAW 264.7 cells. The antioxidative activity was evaluated through the radical scavenging assay, the cytoprotection assay against oxidative damage, and Western blot analysis. Both extracts dose-dependently induced HO-1 expression without any cytotoxicity in accordance with the activation of a transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). In addition, TOWE induced HO-1 expression through the phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and c-Jun NH2-terminal kinase (JNK), while TOEE activated HO-1 by PI3K/Akt phosphorylation. In order to identify the antioxidative potential by HO-1 induction, oxidative damage-caused cell death by tert-butyl hydroperoxide (t-BHP) was significantly attenuated by both extracts. Their antioxidative potential was confirmed by HO-1 selective inducer and inhibitor, cobalt protoporphyrin (CoPP), and tin protoporphyrin (SnPP), respectively. These results indicate that TOWE and TOEE potently alleviated oxidative damage via the induction of Nrf2/MAPK/PI3K mediated HO-1 induction in RAW 264.7 cells.

Physalin A regulates the Nrf2 pathway through ERK and p38 for induction of detoxifying enzymes.

BACKGROUND: Physalin A isolated from Physalis alkekengi var. franchetii has been known to have many pharmacological properties. However, its effect through the Nrf2 pathway has not yet been elucidated. In the present study, we determined the effects of physalin A on cancer chemoprevention via the Nrf2 pathway. METHODS: Experiments were performed in Hepa-1c1c7 and HepG2 cells. The quinone reductase (QR) activity assay was used to assess the activity of physalin A and other compounds isolated from P. alkekengi. The antioxidant response element (ARE) reporter assay was used to determine physalin A induced transcription of Nrf2 target genes, whereas the oligonucleotide pull-down assay was used to investigate Nrf2 binding to the AREs post physalin A treatment. Real-time PCR and western blotting were performed to determine the expression of Nrf2 target genes. Immunocytochemistry was used to determine Nrf2 localization after treatment with physalin A. Kinase inhibitors were used to test the involvement of Nrf2-targeting kinases and the role of ERK and p38 phosphorylation was confirmed using western blotting. RESULTS: Physalin A significantly induced QR activity. As an upstream effector of QR, Nrf2 induced genes containing the ARE, which encode various antioxidants and detoxification enzymes. We observed that physalin A increased the expression of Nrf2 and its target genes in HepG2 cells. Moreover, we observed that physalin A-induced Nrf2 activation was regulated by ERK and p38 kinase in HepG2 cells. CONCLUSIONS: Taken together, we showed that physalin A increased detoxifying enzyme expression via activation of Nrf2 and its target genes. These results imply that physalin A could be a potential chemopreventive agent for liver diseases, as well as cancer.

Dendrobium moniliforme Stem Extract Inhibits Lipoteichoic Acid-Induced Inflammatory Responses by Upregulation of Heme Oxygenase-1.

The stems of Dendrobium moniliforme have been used in traditional herbal medicine for the treatment of fever and lack of body fluid in Korea. In this study, we investigated anti-inflammatory effects of the aqueous extract of D. moniliforme stems (DM) in response to lipoteichoic acid (LTA), a major constituent of the cell wall of Gram-positive bacteria. DM inhibited LTA-induced expression of a pro-inflammatory mediator inducible nitric oxide synthase (iNOS) in the murine macrophages. And DM induced expression of heme oxygenase-1 (HO-1) at the transcriptional level. Conversely, the knockdown of HO-1 expression by siRNA markedly reversed the inhibitory effects of DM on LTA-induced iNOS expression. We also demonstrated that nuclear translocation of Nrf2 was increased following treatment with DM. In addition, DM-mediated Nrf2 activation and HO-1 expression were suppressed by PI3K/Akt and p38 inhibitors; treatment with DM also resulted in phosphorylation of Akt and p38. These results suggest that DM inhibits the expression of iNOS in LTA-stimulated macrophages, and that these effects are mediated by the upregulation of HO-1 expression via PI3K/Akt/p38-Nrf2 signaling.

Curcumin ameliorates cisplatin-induced cystopathy via activating NRF2 pathway.

OBJECTIVES: The present work evaluated preventive effect of curcumin on cisplatin-induced bladder cystopathy. METHODS: Fifteen female rats were divided into (i) Control group administered with physiological saline solution for 5 days; (ii) Cis-P group injected with cisplatin (6 mg/kg); and (iii) Cis-Cur group given cisplatin (6 mg/kg) with curcumin for 5 consecutive days. The function of bladder was measured by means of urodynamic analysis. Furthermore, hematoxylin-eosin staining and Masson trichrome staining were performed for morphological analysis. The cell apoptosis was evaluated through terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry. The expression of nerve growth factor (NGF), NF-E2-related factor 2, and hemeoxygenase-1 (HO-1) levels were measured through Western blotting. RESULTS: Urodynamic assay and histopathological manifestations revealed that curcumin ameliorated the bladder dysfunction induced by cisplatin. The level of cisplatin-induced apoptosis in the bladder decreased following curcumin treatment. Also, the increased protein expression of NGF indicated that the curcumin could offer neuroprotection for bladder against cisplatin. Curcumin also activated NRF2, and elevated the expression of HO-1, but curcumin could not rescue cisplatin-induced apoptosis in the cell lines with knockdown of NRF2. CONCLUSIONS: Taken together, the results of this paper showed that curcumin could ameliorate cisplatin-induced cystopathy and inhibit the apoptosis of bladder cell in cisplatin-treated rats. This may be attributed to curcumin's broad biological functions, particularly antioxidant effect, and to its ability to activate the NRF2 protein.

Dietary arginine affects growth, gut morphology, oxidation resistance and immunity of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂) juveniles.

An 8-week growth trial was conducted to evaluate the effects of dietary arginine (Arg) levels on growth, gut morphology, oxidation resistance and immunity of hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂) juveniles. Seven isoenergetic (1465 kJ (350 kcal)/100-g DM), isoproteic (53·5 % of DM) and isolipidic (7 % of DM) experimental diets were formulated to contain graded Arg levels ranging from 1·9 to 4·7 % (dry weight) at approximately 0·5 % increments. Each diet was randomly assigned to triplicate groups of 16 juvenile fish (average initial body weight: 11·7 (sd 0·1) g) and was administered twice daily (08.00 and 16.00 hours). After the growth trial, all remaining fish were fed their prescribed diets for 2 d and then exposed to 4·5 mg Cu2+/l water for 36 h. Results showed that growth performance and feed utilisation of experimental fish were significantly affected by different dietary Arg levels. Weight gain % (WG%) of fish was increased as dietary Arg increased, reaching a peak value at 3·8 % dietary Arg level, and when dietary Arg level increased to 4·7 % WG% was reduced. Fish fed 1·9 and 2·2 % dietary Arg levels had higher daily feed intake compared with fish fed other dietary Arg levels. Feed conversion ratios in fish fed 1·9, 2·2, 2·7 and 4·7 % dietary Arg levels were higher than those in fish fed 3·1, 3·8 and 4·1 % dietary Arg levels. Protein efficiency ratio and protein productive value (PPV) increased with an increase in dietary Arg, up to a peak value at 3·8 % dietary Arg level, above which these parameters declined. On the basis of quadratic regression analysis of weight gain % (WG%) or PPV against dietary Arg levels, the optimal dietary Arg requirement for hybrid grouper was estimated to be 3·65 %. Fish fed 3·8 % dietary Arg had higher whole-body and muscle protein contents compared with fish fed other dietary Arg levels. Fish fed 3·8 and 4·1 % dietary Arg levels had higher levels of mRNA for insulin-like growth factor-I and target of rapamycin in the liver compared with fish fed other dietary Arg levels. Hepatic S6 kinase 1 mRNA expression in fish fed 3·8 % dietary Arg level was higher than that in fish fed any of the other dietary Arg levels. Gut morphology, hepatic antioxidant indices and immune indices in serum and head kidney were significantly influenced by dietary Arg levels. In conclusion, the optimal dietary Arg requirement for hybrid grouper was estimated to be 3·65 %, and suitable dietary Arg supplementations improved gut morphology and oxidation resistance of hybrid grouper.

Role of spinal Nrf2/HO-1 signaling pathway in hydrogen-induced reduction of inflammatory pain in rats / 中华麻醉学杂志

Objective To evaluate the role of spinal nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in hydrogen-induced reduction of inflammatory pain in rats.Methods Sixty-four SPF healthy adult male Sprague-Dawley rats,weighing 200-250 g,were divided into 4 groups (n =16 each) using a random number table:control group (group C),inflammatory pain group (group IP),inflammatory pain plus hydrogen-rich saline group (group IP+H2) and inflammatory pain plus hydrogen-rich saline plus Nrf2 inhibitor all-trans retinoic acid (ATRA) group (group IP+H2+ATRA).Chronic inflammatory pain was induced by injecting complete Freund's adjuvant (CFA) 100 μl into the plantar surface of the left hind paw in IP group and IP+H2 group.The equal volume of normal saline was given instead in group C.Hydrogen-rich saline 5 ml/kg was injected intraperitoneally once a day for 7consecutive days starting from 1 day after injecting CFA in group IP+H2 and group IP+H2+ATRA,and the equal volume of normal saline was given instead in the other groups.ATRA 7 mg/kg was injected intraperitoneally once a day for 2 consecutive days starting from 2 days before injecting CFA.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before establishing the model (T0) and 1,3 and 7 days after establishing the model (T1-3).Six rats were sacrificed after the last measurement of pain threshold on day 7 after establishing the model,and the L4-6 lumbar segments of the spinal cord were removed for determination of the expression of Nrf2,HO-1 and glial fibrillary acidic protein (GFAP) by Western blot.Results Compared with group C,the MWT was significantly decreased and the TWL was shortened at T1-3,and the expression of Nrf2,HO-1 and GFAP was up-regulated in IP and IP+H2 groups (P＜0.05).Compared with group IP,the MWT was significantly increased and the TWL was prolonged at T1-3,the expression of Nrf2 and HO-1 was up-regulated,and the expression of GFAP was down-regulated in group IP+H2 (P＜0.05),and no significant change was found in the parameters mentioned above in group IP+H2+ATRA (P＞0.05).Compared with group IP+H2,the MWT was significantly decreased and the TWL was shortened at T1-3,the expression of Nrf2 and HO-1 was down-regulated,and the expression of GFAP was up-regulated in group IP+H2+ATRA (P＜0.05).Conclusion Activation of spinal Nrf2/HO-1 signaling pathway is involved in hydrogen-induced reduction of infflammatory pain in rats.

Metformin Sensitizes Non-small Cell Lung Cancer Cells to an Epigallocatechin-3-Gallate (EGCG) Treatment by Suppressing the Nrf2/HO-1 Signaling Pathway.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, is widely studied as a cancer chemopreventive agent with potential anti-cancer effects. The NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway is considered to mediate cellular resistance to EGCG. Metformin, a classical antidiabetic drug, has been shown to prevent cancer progression. Researchers have not reported whether metformin potentiates the anti-cancer efficacy of EGCG. In this study, metformin inhibited HO-1 expression and augmented the anti-tumor effect of EGCG. Metformin also enhanced ROS (reactive oxygen species) generation induced by EGCG (100 µM), subsequently resulting in apoptosis. Based on the results of the in vivo study, size of xenografts treated with the combination of metformin and EGCG was smaller than other groups. Mechanistically, metformin modulated the EGCG-activated Nrf2/HO-1 pathway through Sirtuin 1 (SIRT1)-dependent deacetylation of Nrf2. Moreover, metformin upregulated SIRT1 expression partially through the NF-kB pathway. Comparatively, the combination of EGCG and metformin showed little impact on normal lung epithelial BEAS-2B cells. Based on our findings, metformin sensitized NSCLC cells to the EGCG treatment by suppressing the Nrf2/HO-1 signaling pathway.