The antioxidant effect of preischemic dexmedetomidine in a rat model: increased expression of Nrf2/HO-1 via the PKC pathway

Abstract Background The precise underlying mechanism of antioxidant effects of dexmedetomidine-induced neuroprotection against cerebral ischemia has not yet been fully elucidated. Activation of Nuclear factor erythroid 2-related factor (Nrf2) and Heme Oxygenase-1 (HO-1) represents a major antioxidant-defense mechanism. Therefore, we determined whether dexmedetomidine increases Nrf2/HO-1 expression after global transient cerebral ischemia and assessed the involvement of Protein Kinase C (PKC) in the dexmedetomidine-related antioxidant mechanism. Methods Thirty-eight rats were randomly assigned to five groups: sham (n = 6), ischemic (n = 8), chelerythrine (a PKC inhibitor; 5 mg.kg-1 IV administered 30 min before cerebral ischemia) (n = 8), dexmedetomidine (100 µg.kg-1 IP administered 30 min before cerebral ischemia (n = 8), and dexmedetomidine + chelerythrine (n = 8). Global transient cerebral ischemia (10 min) was applied in all groups, except the sham group; histopathologic changes and levels of nuclear Nrf2 and cytoplasmic HO-1 were examined 24 hours after ischemia insult. Results We found fewer necrotic and apoptotic cells in the dexmedetomidine group relative to the ischemic group (p< 0.01) and significantly higher Nrf2 and HO-1 levels in the dexmedetomidine group than in the ischemic group (p< 0.01). Additionally, chelerythrine co-administration with dexmedetomidine attenuated the dexmedetomidine-induced increases in Nrf2 and HO-1 levels (p< 0.05 and p< 0.01, respectively) and diminished its beneficial neuroprotective effects. Conclusion Preischemic dexmedetomidine administration elicited neuroprotection against global transient cerebral ischemia in rats by increasing Nrf2/HO-1 expression partly via PKC signaling, suggesting that this is the antioxidant mechanism underlying dexmedetomidine-mediated neuroprotection.

Mechanisms mediating the inhibitory effects of quercetin against phthalates-induced testicular oxidative damage in rats / 南方医科大学学报

OBJECTIVE@#To explore the mechanism underlying the inhibitory effect of quercetin against testicular oxidative damage induced by a mixture of 3 commonly used phthalates (MPEs) in rats.@*METHODS@#Forty male Sprague-Dawley rats were randomly divided into control group, MPEs exposure group, and MPEs with low-, median- and high-dose quercetin treatment groups. For MPEs exposure, the rats were subjected to intragastric administration of MPEs at the daily dose of 900 mg/kg for 30 consecutive days; Quercetin treatments were administered in the same manner at the daily dose of 10, 30, and 90 mg/kg. After the treatments, serum levels of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH), and testicular malondialdeyhde (MDA), catalase (CAT) and superoxide dismutase (SOD) were detected, and testicular pathologies of the rats were observed with HE staining. The expressions of nuclear factor-E2-related factor 2 (Nrf2), Kelch-like ECH2 associated protein 1 (Keap1) and heme oxygenase 1 (HO-1) in the testis were detected using immunofluorescence assay and Western blotting.@*RESULTS@#Compared with the control group, the rats with MPEs exposure showed significant reductions of the anogenital distance, weight of the testis and epididymis, and the coefficients of the testis and epididymis with lowered serum testosterone, LH and FSH levels (P < 0.05). Testicular histological examination revealed atrophy of the seminiferous tubules, spermatogenic arrest, and hyperplasia of the Leydig cells in MPEs-exposed rats. MPEs exposure also caused significant increments of testicular Nrf2, MDA, SOD, CAT and HO-1 expressions and lowered testicular Keap1 expression (P < 0.05). Treatment with quercetin at the median and high doses significantly ameliorated the pathological changes induced by MPEs exposure (P < 0.05).@*CONCLUSION@#Quercetin treatment inhibits MPEs-induced oxidative testicular damage in rats possibly by direct scavenging of free radicals to lower testicular oxidative stress and restore the regulation of the Nrf2 signaling pathway.

Antidepressant mechanism of Shenling Kaixin Granules based on BDNF/TrkB/CREB pathway / 中国中药杂志

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1β), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), B-cell lymphoma-2(Bcl-2)/Bcl-2 associated X protein(Bax) and caspase-3 in hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1β and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1β and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway / 中国中药杂志

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Eucommia lignans alleviate the progression of diabetic nephropathy through mediating the AR/Nrf2/HO-1/AMPK axis in vivo and in vitro / 中国天然药物

Lignans derived from Eucommia ulmoides Oliver (Eucommia lignans) inhibit the progression of inflammatory diseases, while their effect on the progression of diabetic nephropathy (DN) remained unclear. This work was designed to assess the function of Eucommia lignans in DN. The major constituents of Eucommia lignans were analyzed by UPLC-Q-TOF-MS/MS. The binding between Eucommia lignans and aldose reductase (AR) was predicted by molecular docking. Eucommia lignans (200, 100, and 50 mg·kg-1) were used in model animals to evaluate their renal function changes. Rat glomerular mesangial cells (HBZY-1) were transfected with sh-AR, sh-AMPK, and oe-AR in the presence of high glucose (HG) or HG combined with Eucommia lignans to evaluate whether Eucommia lignans affected HG-induced cell injury and mitochondrial dysfunction through the AR/Nrf2/HO-1/AMPK axis. Eucommia lignans significantly attenuated the progression of DN in vivo. Eucommia lignans notably reversed HG-induced upregulation of inflammatory cytokines and mitochondrial injury, while downregulating the levels of Cyto c, caspase 9, AR, and NOX4 in HBZY-1 cells. In contrast, HG-induced downregulation of Nrf2, HO-1 and p-AMPKα levels were abolished by Eucommia lignans. Meanwhile, knockdown of AR exerted similar therapeutic effect of Eucommia lignans on DN progression, and AR overexpression reversed the effect of Eucommia lignans. Eucommia lignans alleviated renal injury through the AR/Nrf2/HO-1/AMPK axis. Thus, these findings might provide evidence for the use of Eucommia lignans in treating DN.

Role and mechanism of SIRT1 in regulating Nrf2/HO-1 signaling pathway in septic liver injury / 中华危重病急救医学

OBJECTIVE@#To investigate the role and mechanism of silent information regulator 1 (SIRT1) in regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in oxidative stress and inflammatory response to sepsis-induced liver injury.@*METHODS@#A total of 24 male Sprague-Dawley (SD) rats were randomly divided into sham operation (Sham) group, cecal ligation and puncture (CLP) group, SIRT1 agonist SRT1720 pretreatment (CLP+SRT1720) group and SIRT1 inhibitor EX527 pretreatment (CLP+EX527) group, with 6 rats in each group. Two hours before operation, SRT1720 (10 mg/kg) or EX527 (10 mg/kg) were intraperitoneally injected into the CLP+SRT1720 group and CLP+EX527 group, respectively. Blood was collected from the abdominal aorta at 24 hours after modeling and the rats were sacrificed for liver tissue. The serum levels of interleukins (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by microplate method. Hematoxylin-eosin (HE) staining was used to observe the pathological injury of rats in each group. The levels of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH) and superoxide dismutase (SOD) in liver tissue were detected by corresponding kits. The mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting.@*RESULTS@#Compared with the Sham group, the serum levels of IL-6, IL-1β, TNF-α, ALT and AST in the CLP group were significantly increased; histopathological results showed that liver cords were disordered, hepatocytes were swollen and necrotic, and a large number of inflammatory cells infiltrated; the contents of MDA and 8-OHdG in liver tissue increased, while the contents of GSH and SOD decreased; and the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 in liver tissues were significantly decreased. These results suggest that sepsis rats have liver dysfunction, and the levels of SIRT1, Nrf2, HO-1 and antioxidant protein in liver tissues were decreased, while the levels of oxidative stress and inflammation were increased. Compared with the CLP group, the levels of inflammatory factors and oxidative stress were significantly decreased in the CLP+SRT1720 group, the mRNA and protein expressions of SIRT1, Nrf2 and HO-1 were significantly increased [IL-6 (ng/L): 34.59±4.21 vs. 61.84±3.78, IL-1β (ng/L): 41.37±2.70 vs. 72.06±3.14, TNF-α (ng/L): 76.43±5.23 vs. 130.85±5.30, ALT (U/L): 30.71±3.63 vs. 64.23±4.59, AST (U/L): 94.57±6.08 vs. 145.15±6.86, MDA (μmol/g): 6.11±0.28 vs. 9.23±0.29, 8-OHdG (ng/L): 117.43±10.38 vs. 242.37±11.71, GSH (μmol/g): 11.93±0.88 vs. 7.66±0.47, SOD (kU/g): 121.58±5.05 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 1.20±0.13 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 1.21±0.12 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 1.71±0.06 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.89±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.87±0.08 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.93±0.14 vs. 0.54±0.12, all P < 0.05], these results indicated that SIRT1 agonist SRT1720 pretreatment could improve liver injury in sepsis rats. However, pretreatment with SIRT1 inhibitor EX527 showed the opposite effect [IL-6 (ng/L): 81.05±6.47 vs. 61.84±3.78, IL-1β (ng/L): 93.89±5.83 vs. 72.06±3.14, TNF-α (ng/L): 177.67±5.12 vs. 130.85±5.30, ALT (U/L): 89.33±9.52 vs. 64.23±4.59, AST (U/L): 179.59±6.44 vs. 145.15±6.86, MDA (μmol/g): 11.39±0.51 vs. 9.23±0.29, 8-OHdG (ng/L): 328.83±11.26 vs. 242.37±11.71, GSH (μmol/g): 5.07±0.34 vs. 7.66±0.47, SOD (kU/g): 59.37±4.28 vs. 83.57±4.84, SIRT1 mRNA (2-ΔΔCt): 0.34±0.03 vs. 0.46±0.02, Nrf2 mRNA (2-ΔΔCt): 0.46±0.04 vs. 0.58±0.03, HO-1 mRNA (2-ΔΔCt): 0.21±0.03 vs. 0.48±0.07, SIRT1 protein (SIRT1/β-actin): 0.47±0.04 vs. 0.58±0.03, Nrf2 protein (Nrf2/β-actin): 0.32±0.07 vs. 0.51±0.09, HO-1 protein (HO-1/β-actin): 0.19±0.09 vs. 0.54±0.12, all P < 0.05].@*CONCLUSIONS@#SIRT1 can inhibit the release of proinflammatory factors and alleviate the oxidative damage of hepatocytes by activating Nrf2/HO-1 signaling pathway, thus playing a protective role against CLP-induced liver injury.

Neuroprotective effect of ginsenoside Re on drosophila model of Parkinson's disease / 中国中药杂志

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 μmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 μmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.

Research advances on the role of nuclear factor-erythroid 2-related factor 2 in wound healing / 中华烧伤杂志

Wound healing is one of the common pathophysiological processes in the body. How to improve the condition of wound healing to promote rapid wound healing has always been a hotspot in research. Oxidative stress is one of the important factors affecting wound healing. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a classic antioxidant stress factor as well as a factor with great potential in facilitating wound healing. The activation of Nrf2 can regulate the downstream antioxidant stress elements and play roles of anti-apoptosis and cell homeostasis maintaining, which improves wound healing environment and promotes wound repair. This paper summarized the common agonists and inhibitors of Nrf2 and reviewed the roles of Nrf2 in promoting skin wound healing including diabetic ulcers, radiation injury, and ischemia-reperfusion injury, etc.

Dynamic Changes of ROS and Nrf2-Related Factors During Erythroid Development / 中国实验血液学杂志

OBJECTIVE@#To investigate the changes and roles of reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) related antioxidases during erythroid development.@*METHODS@#Flow cytometry was used to detect the sensibility of peripheral red blood cells of wild-type mice to a strong oxidant hydrogen peroxide (H2O2). Erythroid cells from different developmental stages in bone marrow (BM) were obtained using fluorescence-activated cell sorter and the ROS levels were detected by flow cytometry. RT-qPCR was used to detect the changes of expression levels of Nrf2 and related antioxidases in erythroid cells from different developmental stages in BM. The ROS levels of the peripheral blood and BM nucleated erythrocytes in Nrf2 knockout mice were further examined. The expression level of Nrf2 in erythroid precursors isolated from 14.5 d embryonic liver of wild-type mice during differentiation and culture in vitro was detected.@*RESULTS@#In the peripheral blood of wild-type mice, the ROS level of reticulocytes and mature erythrocytes treated with H2O2 increased about 4 times and 7 times, respectively (P<0.01). In BM erythrocytes, the ROS level gradually decreased as the cells matured (r=0.85), while the expression level of Nrf2 and its related anti-oxidative genes increased (r=0.99). The ROS levels in peripheral blood erythrocytes and BM nucleated erythrocytes of Nrf2 knockout mice were significantly increased compared with wild-type mice (P<0.01). The expression of Nrf2 increased during the early erythroid development after embryonic liver cell sorting (P<0.01).@*CONCLUSION@#The expression levels of Nrf2 and its related factors vary during erythropoiesis. Nrf2 at physiological level plays an important antioxidant role during the erythroid development.

The role of Keap1/Nrf2/HO-1 signal pathway in liver injury induced by rare earth neodymium oxide in mice / 中华劳动卫生职业病杂志

Objective: To investigate the role of Keap1/Nrf2/HO-1 signaling pathway in liver injury induced by neodymium oxide (Nd(2)O(3)) in mice. Methods: In March 2021, forty-eight SPF grade healthy male C57BL/6J mice were randomly divided into control group (0.9% NaCl), low dose group (62.5 mg/ml Nd(2)O(3)), medium dose group (125.0 mg/ml Nd(2)O(3)), and high dose group (250.0 mg/ml Nd(2)O(3)), each group consisted of 12 animals. The infected groups were treated with Nd(2)O(3) suspension by non-exposed tracheal drip and were killed 35 days after dust exposure. The liver weight of each group was weighed and the organ coefficient was calculated. The content of Nd(3+) in liver tissue was detected by inductively coupled plasma mass spectrometry (ICP-MS). HE staining and immunofluorescence was used to observe the changes of inflammation and nuclear entry. The mRNA expression levels of Keap1, Nrf2 and HO-1 in mice liver tissue were detected by qRT-PCR. Western blotting was used to detect the protein expression levels of Keap1 and HO-1. The contents of catalase (CAT), glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were detected by colorimetric method. The contents of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. The data was expressed in Mean±SD. Two-independent sample t-test was used for inter-group comparison, and one-way analysis of variance was used for multi-group comparison. Results: Compared with the control group, the liver organ coefficient of mice in medium and high dose groups were increased, and the Nd(3+) accumulation in liver of mice in all dose groups were significantly increased (P<0.05). Pathology showed that the structure of liver lobules in the high dose group was slightly disordered, the liver cells showed balloon-like lesions, the arrangement of liver cell cords was disordered, and the inflammatory exudation was obvious. Compared with the control group, the levels of IL-1β and IL-6 in liver tissue of mice in all dose groups were increased, and the levels of TNF-α in liver tissue of mice in high dose group were increased (P<0.05). Compared with the control group, the mRNA and protein expression levels of Keap1 in high dose group were significantly decreased, while the mRNA expression level of Nrf2, the mRNA and protein expression levels of HO-1 were significantly increased (P<0.05), and Nrf2 was successfully activated into the nucleus. Compared with the control group, the activities of CAT, GSH-Px and T-SOD in high dose group were significantly decreased (P<0.05) . Conclusion: A large amount of Nd(2)O(3) accumulates in the liver of male mice, which may lead to oxidative stress and inflammatory response through activation of Keap1/Nrf2/HO-1 signal pathway. It is suggested that Keap1/Nrf2/HO-1 signal pathway may be one of the mechanisms of Nd(2)O(3) expose-induced liver injury in mice.

The role of Nrf2 in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated MMP9 / 中华劳动卫生职业病杂志

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.

B7-H3 confers stemness characteristics to gastric cancer cells by promoting glutathione metabolism through AKT/pAKT/Nrf2 pathway / 中华医学杂志(英文版)

BACKGROUND@#Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.@*METHODS@#GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis.@*RESULTS@#B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02).@*CONCLUSIONS@#B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.

Lyciumbarbarum polysaccharides ameliorate canine acute liver injury by reducing oxidative stress, protecting mitochondrial function, and regulating metabolic pathways / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The development of acute liver injury can result in liver cirrhosis, liver failure, and even liver cancer, yet there is currently no effective therapy for it. The purpose of this study was to investigate the protective effect and therapeutic mechanism of Lyciumbarbarum polysaccharides (LBPs) on acute liver injury induced by carbon tetrachloride (CCl4). To create a model of acute liver injury, experimental canines received an intraperitoneal injection of 1 mL/kg of CCl4 solution. The experimental canines in the therapy group were then fed LBPs (20 mg/kg). CCl4-induced liver structural damage, excessive fibrosis, and reduced mitochondrial density were all improved by LBPs, according to microstructure data. By suppressing Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), promoting the production of sequestosome 1 (SQSTM1)/p62, nuclear factor erythroid 2-related factor 2 (Nrf2), and phase II detoxification genes and proteins downstream of Nrf2, and restoring the activity of anti-oxidant enzymes like catalase (CAT), LBPs can restore and increase the antioxidant capacity of liver. To lessen mitochondrial damage, LBPs can also enhance mitochondrial respiration, raise tissue adenosine triphosphate (ATP) levels, and reactivate the respiratory chain complexes I‒V. According to serum metabolomics, the therapeutic impact of LBPs on acute liver damage is accomplished mostly by controlling the pathways to lipid metabolism. 9-Hydroxyoctadecadienoic acid (9-HODE), lysophosphatidylcholine (LysoPC/LPC), and phosphatidylethanolamine (PE) may be potential indicators of acute liver injury. This study confirmed that LBPs, an effective hepatoprotective drug, may cure acute liver injury by lowering oxidative stress, repairing mitochondrial damage, and regulating metabolic pathways.

Immature rat testis sustained long-term development using an integrative model

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model. METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis. RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05). CONCLUSION: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Berberine alleviates programmed necrosis of metabolic-associated fatty liver disease via activating Nrf2 pathway in mice / 中华肝脏病杂志

Objective: To investigate the effect of berberine on programmed necrosis of hepatocytes induced by metabolic-associated fatty liver disease (MAFLD) in mice and its related molecular mechanism. Methods: Twenty male C57BL/6N mice were randomly divided into four groups (n=5 in each group): control group (S), fatty liver group (H), berberine group(B), nuclear factor erythroid 2-related factor 2 inhibitor group (Nrf2), and all-trans-retinoic acid (ATRA) group (A). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) concentrations were detected at the end of week 12 to calculate fatty liver index (liver mass/body mass ratio). Liver tissue was stained with HE, Masson and Oil Red O, and SAF score was used to evaluate the degree of liver injury. The expression levels of hepatic programmed necrosis-related proteins, namely receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed series protease-like domain (p-MLKL) and Nrf2 were detected by Western blot method. One-way ANOVA was used for intragroup comparisons and LSD-t tests were used for intergroup comparisons. Results: Compared with S group, H group serum ALT, AST, LDH, TG, TC, TNF-α, IL-1β levels and fatty liver index were significantly increased. The liver tissue was filled with vacuolar-like changes and inflammatory cell infiltration. Numerous red lipid droplets were observed with oil red O staining. Collagen fiber hyperplasia was evident with Masson staining. SAF scores (6.60 ± 0.55 and 0.80 ± 0.45) were significantly increased. The expressions of RIPK3 and p-MLKL were up-regulated. Nrf2 level was relatively increased, and the differences were statistically significant (P < 0.05). Compared with H group, berberine intervention group liver biochemical indexes, lipid levels, pro-inflammatory mediator expression, fatty liver index, and SAF score were significantly reduced, and the expression of RIPK3 and p-MLKL were down-regulated, while Nrf2 levels were further increased, and the differences were statistically significant (P<0.05). Compared with B group, treatment with Nrf2 inhibitor had antagonized the protective effect of berberine on fatty liver. Serum ALT, AST, LDH, TG, TC and TNF-α, IL-1β levels, fatty liver index, and SAF scores were significantly increased and the expressions of RIPK3 and p-MLKL were relatively increased, and the differences were statistically significant (P < 0.05). Conclusion: Berberine can significantly improve the metabolic-associated fatty liver disease injury in mice, and its mechanism is related to activation of Nrf2 and inhibition of programmed necrosis of hepatocytes.

Berberine inhibits erastin-induced ferroptosis of mouse hippocampal neuronal cells possibly by activating the Nrf2-HO-1/GPX4 pathway / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which berberine inhibits ferroptosis of mouse hippocampal neuronal cells (HT22).@*METHODS@#Cultured HT22 cells were pretreated with 30 or 60 μmol/L berberine for 2 h before exposure to 0.5 μmol/L erastin for 8 h, and the cell proliferation, intracellular ferric iron level, changes in intracellular reactive oxygen species (ROS) and cell apoptosis were detected using CCK-8, Fe2+ fluorescent probe, fluorescent dye (DAPI) and fluorescent probe (H2DCFH-DA). RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of Nrf2, HO-1 and GPX4 in the cells. We further tested the effects of treatments with 2 μmol/L ML385 (a Nrf2 inhibitor), 60 μmol/L berberine and erastin in the cells to explore the protective mechanism of berberine against erastin-induced ferroptosis in the neuronal cells.@*RESULTS@#Treatment with 0.5 μmol/L erastin significantly lowered the viability of HT22 cells (P < 0.05) and increased the production of ROS, cell apoptosis rate and ferric iron level (P < 0.05). Pretreatment with 30 and 60 μmol/L berberine both significantly increased the vitality of erastin-exposed cells (P < 0.05) and lowered the levels of intracellular ROS and ferric iron content (P < 0.05). RT-qPCR and Western blotting showed that berberine obviously promoted the expressions of Nrf2, HO-1 and GPX4 in the cells (P < 0.05), and treatment with ML385 significantly inhibited the Nrf2-HO-1/GPX4 pathway, increased intracellular ROS and ferric iron contents and mitigated the protective effect of berberine against erastin-induced ferroptosis (P < 0.05).@*CONCLUSION@#Berberine can inhibit erastin-induced ferroptosis in HT22 cells possibly by activating the Nrf2-HO-1/ GPX4 pathway.

Effects of butyphthalide on microglia polarization after intracerebral hemorrhage and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Because intracerebral hemorrhage (ICH) has high morbidity, disability and mortality, it is significant to find new and effective treatments for ICH. This study aims to explore the effect of butyphthalide (NBP) on neuroinflammation secondary to ICH and microglia polarization.@*METHODS@#A total of 48 healthy male SD rats were randomly divided into 6 groups: a sham 24 h group, a sham 72 h group, an ICH 24 h group, an ICH 72 h group, an ICH+NBP 24 h group, and an ICH+NBP 72 h group (8 rats per group). After operation, the neurological deficiencies were assessed based on improved Garcia scores and corner test. The expressions of Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), aquaporin-4 (AQP4), zonula occludens-1 (ZO-1), occludin, CD68, CD86, and CD206 were observed by Western blotting. Inflammatory cytokines were detected by ELISA. The immunofluorescence was to detect the polarization of microglia.@*RESULTS@#1) Compared with the sham groups, the expression of TLR4 (24 h: P<0.05; 72 h: P<0.01), NF-κB (both P<0.01) and Nrf2 (both P<0.01) in the perihematoma of the ICH group was increased, leading to microglia activation (P<0.01). The expressions of IL-6 (24 h: P<0.05; 72 h: P<0.01) and TNF-α (both P<0.01), the pro-inflammatory cytokines were up-regulated, and the expression of anti-inflammatory cytokine IL-4 was down-regulated (both P<0.01). Besides, the expression of AQP4 was enhanced (both P<0.01). The protein level of tightly connected proteins (including ZO-1, occludin) was decreased (all P<0.01). The neurological function of the rats in the ICH group was impaired in the 2 time points (both P<0.01). 2) Compared with the sham group at 24 h and 72 h after the intervention of NBP, the expressions of TLR4 (both P<0.05) and NF-κB (both P<0.01) were significantly declined, and the expression of Nrf2 was further enhanced (both P<0.05) in the perihematoma of the ICH+NBP group. Furthermore, the expression of M1 microglia marker was inhibited (P<0.05), and the polarization of microglia to the M2 phenotype was promoted (P<0.01). 3) In terms of inflammation after ICH, the IL-4 expression in the ICH+NBP group was increased compared with the ICH group (24 h: P<0.05; 72 h: P<0.01); the expression of IL-6 was decreased significantly in the ICH+NBP 72 h group (P<0.01); the level of AQP4 was declined significantly in the ICH+NBP 24 h group (P<0.05), there was a downward trend in the 72-hour intervention group but without significant statistical difference. 4) Compared with the ICH group, the ZO-1 protein levels were increased (24 h: P<0.05; 72 h: P<0.01), and the symptoms of nerve defect were improved eventually (both P<0.05) in the ICH+NBP groups.@*CONCLUSIONS@#After ICH, the TLR4/NF-κB pathway is activated. The M1 microglia is up-regulated along with the release of detrimental cytokines, while the anti-inflammatory cytokines are down-regulated. The expression of AQP4 is increased, the tight junction proteins from the blood-brain barrier (BBB) is damaged, and the neurological function of rats is impaired. On the contrary, NBP may regulate microglia polarization to M2 phenotype and play a role in the neuroprotective effect mediated via inhibiting TLR4/NF-κB and enhancing Nrf2 pathways, which relieves the neuroinflammation, inhibits the expression of AQP4, repairs BBB, and improves neurological functional defects.

Protective Effects of Anthocyanins Extracted from Vaccinium Uliginosum on 661W Cells Against Microwave-Induced Retinal Damage / 中国结合医学杂志

OBJECTIVE@#To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum (VU) on retinal 661W cells against microwave radiation induced retinal injury.@*METHODS@#661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave (30 mW/cm2, 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU (25, 50, 100 and 200 µg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm2, 1 h]. After treatment with different interventions, the cell apoptosis index (AI) was determined using Heochst staining; contents of malonaldehyde (MDA), glutataione (GSH), and activity of superoxide dismutase (SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis.@*RESULTS@#There was significant difference in AI among the groups (F=322.83, P<;0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups (P<;0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment (r=0.8419, P<;0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group (P<;0.05). Compared with the model group, the SOD activity in the VU-treated groups (50, 100 and 200 µg/mL) was significantly higher (all P<;0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 µg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased (P<;0.05), and the changes in cytoplasm were not obvious, whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups.@*CONCLUSIONS@#Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation.

Fucoxanthin regulates Nrf2/Keap1 signaling to alleviate myocardial hypertrophy in diabetic rats / 南方医科大学学报

OBJECTIVE@#To investigate the protective effect of fucoxanthin (FX) against diabetic cardiomyopathy and explore the underlying mechanism.@*METHODS@#Rat models of diabetes mellitus (DM) induced by intraperitoneal injection of streptozotocin (60 mg/kg) were randomized into DM model group, fucoxanthin treatment (DM+FX) group and metformin treatment (DM+ Met) group, and normal rats with normal feeding served as the control group. In the two treatment groups, fucoxanthin and metformin were administered after modeling by gavage at the daily dose of 200 mg/kg and 230 mg/kg, respectively for 12 weeks, and the rats in the DM model group were given saline only. HE staining was used to examine the area of cardiac myocyte hypertrophy in each group. The expression levels of fibrotic proteins TGF-β1 and FN proteins in rat hearts were detected with Western blotting. In the cell experiment, the effect of 1 μmol/L FX on H9C2 cell hypertrophy induced by exposure to high glucose (HG, 45 mmol/L) was evaluated using FITC-labeled phalloidin. The mRNA expression levels of the hypertrophic factors ANP, BNP and β-MHC in H9C2 cells were detected using qRT-PCR. The protein expressions of Nrf2, Keap1, HO-1 and SOD1 proteins in rat heart tissues and H9C2 cells were determined using Western blotting. The DCFH-DA probe was used to detect the intracellular production of reactive oxygen species (ROS).@*RESULTS@#In the diabetic rats, fucoxanthin treatment obviously alleviated cardiomyocyte hypertrophy and myocardial fibrosis, increased the protein expressions of Nrf2 and HO-1, and decreased the protein expressions of Keap1 in the heart tissue (P < 0.05). In H9C2 cells with HG exposure, fucoxanthin significantly inhibited the enlargement of cell surface area, lowered the mRNA expression levels of ANP, BNP and β-MHC (P < 0.05), promoted Nrf2 translocation from the cytoplasm to the nucleus, and up-regulated the protein expressions its downstream targets SOD1 and HO-1 (P < 0.05) to enhance cellular antioxidant capacity and reduce intracellular ROS production.@*CONCLUSION@#Fucoxanthin possesses strong inhibitory activities against diabetic cardiomyocyte hypertrophy and myocardial fibrosis and is capable of up-regulating Nrf2 signaling to promote the expression of its downstream antioxidant proteins SOD1 and HO-1 to reduce the level of ROS.

Hyperoside protects mouse spermatocytes GC-2 cells from oxidative damage by activating the Keap1/Nrf2/HO-1 pathway / 南方医科大学学报

OBJECTIVE@#To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.@*METHODS@#GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.@*RESULTS@#Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).@*CONCLUSION@#Hyperoside protects GC-2 cells against H2O2- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.