Keap1 Protein Expression Research Articles

The role of MOTS-c-mediated antioxidant defense in aerobic exercise alleviating diabetic myocardial injury.

Myocardial remodeling and dysfunction are commonly observed in type 2 diabetes mellitus (T2DM). Aerobic exercise can partly alleviate diabetes-induced myocardial dysfunction through its antioxidant actions. MOTS-c is a potential exercise mimic. This study aimed to investigate the effects of MOTS-c on improving diabetic heart function and its mechanism and to identify whether MOTS-c improved antioxidant defenses due to aerobic exercise. Herein, we established a rat model of T2DM induced by high-fat diet combined with a low-dose streptozotocin injection. Interventions were performed using intraperitoneal injections of MOTS-c (i.p. 0.5mg/kg/day, 7days/week) or aerobic exercise training (treadmill, 20 m/min, 60 min/day, 5days/week) for 8 weeks. Myocardial ultrastructure was assessed using transmission electron microscopy (TEM), myocardial lipid peroxidation levels (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) levels were assessed using colorimetric methods, and molecular analyses including MOTS-c, Kelch-like ECH-associated protein 1 (Keap1), Nuclear factor E2-related factor 2 (Nrf2), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)and phospho-AMPK (p-AMPK) were examined using Western blot. The results showed that MOTS-c, with or without exercise, reduced myocardial ultrastructural damage and improved glucolipid metabolism and cardiac function in T2DM. Furthermore, MOTS-c increased antioxidant markers such as SOD, CAT, and the protein expression of myocardial MOTS-c, Keap1, Nrf2, and p-AMPK. MOTS-c with exercise treatment reduced myocardial MDA and increased p-AMPK significantly comparing to only exercise or MOTS-c alone. Our findings suggest that MOTS-c may be a helpful supplement for overcoming exercise insufficiency and improving myocardial structure and function in diabetes.

KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability

To investigate the toxic effects and potential mechanisms of tri(1, 3-dichloro-2-propyl) phosphate(TDCIPP) exposure on the mouse testicular supporting cell line(TM4 cells). TM4 cells were treated with different concentrations of TDCIPP(0, 12.5, 25 and 50 μmol/L), or 50 μmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC) for 24 h. Cell viability was assessed using the CCK8 assay, intracellular ROS levels were detected using the DCFH-DA probe, and the protein levels of oxeiptosis-related proteins, such as KEAP1, PGAM5, AIFM1 and phosphorylated AIFM1(p-AIFM1), were detected using Western blot. TDCIPP dose-dependently reduced TM4 cell viability(P<0.05). ROS levels in TM4 cells treated with 12.5, 25 and 50 μmol/L TDCIPP were 9.44±1.42, 17.25±1.81 and 18.38±2.66, respectively, significantly higher than the control group's 5.08±0.90(P<0.05). ROS levels in the 5 mmol/L NAC+50 μmol/L TDCIPP group were 14.70±0.50, significantly lower than the corresponding TDCIPP group's 26.44±0.73(P<0.05). The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53, respectively, significantly higher than TDCIPP group 68.67±1.53(P<0.05). The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50 μmol/L TDCIPP were 0.77±0.04 and 0.82±0.02, respectively, significantly higher than the control group's 0.57±0.01(P<0.05). The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04, 1.38±0.03 and 1.41±0.03, respectively, significantly higher than the control group's 0.81±0.02(P<0.05). The relative expression of AIFM1 protein were 0.42±0.01, 0.63±0.01 and 0.68±0.02, respectively, significantly higher than the control group's 0.34±0.02(P<0.05). The relative expression of p-AIFM1 protein were 1.73±0.02, 1.52±0.02 and 0.73±0.01, respectively, significantly lower than the control group's 2.25±0.02(P<0.05). In the 5 mmol/L NAC+50 μmol/L TDCIPP group, the relative expression of KEAP1, PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01, 0.58±0.01 and 0.48±0.03, respectively, significantly lower than the TDCIPP group's 0.86±0.12(P<0.05), 0.74±0.02(P<0.05) and 0.92±0.01(P<0.05). The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50 μmol/L TDCIPP group was 0.45±0.11, significantly higher than the TDCIPP group's 0.23±0.01(P<0.05). The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway, leading to oxeiptosis.

Antioxidant enzymes and Nrf2/Keap1 in human skeletal muscle: Influence of age, sex, adiposity and aerobic fitness

Ageing, a sedentary lifestyle, and obesity are associated with increased oxidative stress, while regular exercise is associated with an increased antioxidant capacity in trained skeletal muscles. Whether a higher aerobic fitness is associated with increased expression of antioxidant enzymes and their regulatory factors in skeletal muscle remains unknown. Although oestrogens could promote a higher antioxidant capacity in females, it remains unknown whether a sex dimorphism exists in humans regarding the antioxidant capacity of skeletal muscle. Thus, the aim was to determine the protein expression levels of the antioxidant enzymes SOD1, SOD2, catalase and glutathione reductase (GR) and their regulatory factors Nrf2 and Keap1 in 189 volunteers (120 males and 69 females) to establish whether sex differences exist and how age, VO2max and adiposity influence these. For this purpose, vastus lateralis muscle biopsies were obtained in all participants under resting and unstressed conditions. No significant sex differences in Nrf2, Keap1, SOD1, SOD2, catalase and GR protein expression levels were observed after accounting for VO2max, age and adiposity differences. Multiple regression analysis indicates that the VO2max in mL.kg LLM−1.min−1can be predicted from the levels of SOD2, Total Nrf2 and Keap1 (R = 0.58, P < 0.001), with SOD2 being the main predictor explaining 28 % of variance in VO2max, while Nrf2 and Keap1 explained each around 3 % of the variance. SOD1 protein expression increased with ageing in the whole group after accounting for differences in VO2max and body fat percentage. Overweight and obesity were associated with increased pSer40-Nrf2, pSer40-Nrf2/Total Nrf2 ratio and SOD1 protein expression levels after accounting for differences in age and VO2max. Overall, at the population level, higher aerobic fitness is associated with increased basal expression of muscle antioxidant enzymes, which may explain some of the benefits of regular exercise.

The Regulatory Effects of JAK2/STAT3 on Spermatogenesis and the Redox Keap1/Nrf2 Axis in an Animal Model of Testicular Ischemia Reperfusion Injury.

The male reproductive system requires the pleiotropic activity of JAK/STAT to maintain its function, especially spermatogenesis. The study aims to investigate the effect of JAK2 signaling on the expression of the Keap1/Nrf2 axis, spermatogenesis, and the Sertoli cells (Sc) junctions in an animal model of testicular ischemia reperfusion injury (tIRI). Testes subjected to tIRI exhibited increased JAK2/STAT3 activity associated with spermatogenic arrest and reduced expression of the Sc junctions. In addition, there was an increased protein expression of Keap1 and decreased Nrf2., which was coupled with the downregulation of gene expression of antioxidant enzymes. Reduced SOD and CAT activities were accompanied by increased lipid peroxidation and protein carbonylation during tIRI. Increased caspase 9 activity and Bax/Bcl2 ratio indicated initiation of apoptosis. Inhibition of JAK2 activity by AG490 maintained the integrity of spermatogenesis and SC junctions, normalized the expression of the Keap1/Nrf2 axis and its downstream antioxidant enzymes, and prevented germ cell apoptosis. The results further emphasized the regulatory role of JAK2/STAT3 on spermatogenesis, Keap1/Nrf2 signaling, and maintenance of the testicular redox balance to combat testicular dysfunction and male infertility.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice

The protective effects of remote ischemic conditioning (RIC) on acute ischemic stroke have been reported. However, the protective mechanisms of RIC have not been fully elucidated. This study aimed to investigate whether RIC could reduce oxidative stress and inflammatory responses in middle cerebral artery occlusion (MCAO)-reperfusion mice via the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. C57BL/6 mice were subjected to MCAO and underwent RIC twice daily at 1, 3, and 7 days after MCAO. ML385 was used to specifically inhibit Nrf2 in MCAO mice. Neurological deficit scores, infarct volume, and hematoxylin-eosin (HE) staining were assessed. Oxidative stress levels were assessed based on total antioxidant capacity (TAC), malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione/glutathione disulfide (GSH/GSSG). mRNA levels were detected using real-time polymerase chain reaction (PCR), and protein levels were detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Protein localization was investigated using immunofluorescence staining. RIC significantly reduced infarct volume and improved neurological function and histological changes after MCAO. RIC significantly increased TAC, SOD, and GSH/GSSG levels and decreased MDA levels. RIC significantly increased Nrf2 and HO-1 mRNA levels and decreased Keap1, NLRP3, and Cleaved Caspase-1 mRNA levels. RIC significantly increased Nrf2, HO-1, and NQO1 protein expression and decreased Keap1, NLRP3, Cleaved Caspase-1, Cleaved IL-1β, IL-6, and TNF-α protein expression. RIC promoted the activation and translocation of Nrf2 into the nucleus. The protective effects of RIC were abolished by ML385 treatment. In conclusion, our findings suggest that RIC alleviates oxidative stress and inflammatory responses via the Nrf2/HO-1 pathway, which in turn improves neurobehavioral function. RIC may provide novel therapeutic options for acute ischemic stroke.

Therapeutic role of boron on acrylamide-induced nephrotoxicity, cardiotoxicity, neurotoxicity, and testicular toxicity in rats: Effects on Nrf2/Keap-1 signaling pathway and oxidative stress

BackgroundAcrylamide (ACR) is a heat-related carcinogen used in cooking some foods as well as in other thermal treatments. The present study aims to investigate the possible protective effect of boron (BA) against ACR-induced toxicity of kidney, brain, heart, testis, and bladder tissues in rats. MethodsRats have been divided into 5 equal groups: Control (saline), ACR (38.27 mg/kg), BA (20 mg/kg), BA+ ACR (10 mg/kg + ACR), and BA+ ACR (20 mg/kg BA+ACR). Kidney tissue from rats was collected and the levels of malondialdehyde (MDA), glutathione (GSH), and the activity of superoxide dismutase (SOD) were measured. In addition, the kidneys of these animals, as well as the brain, heart, testes, and bladder tissues were examined for possible histological changes. Total Nrf2 and Keap-1 protein expression in kidney, heart, and testis tissues was examined by immunohistochemistry. ResultsWhile significant increases in MDA levels were observed in the kidneys of rats receiving ACR alone, significant decreases in antioxidant markers (SOD and GSH) were observed. Besides, kidney, brain, heart, and testicular tissues were analyzed and damage was observed in the groups receiving ACR. However, no significant histologic changes were noted in the bladder tissue. Both dosages of BA in combination with ACR improved the changes in ACR-induced antioxidant tissue parameters. Despite the fact that MDA levels were decreased with these two dosages, histological structural abnormalities were found to be greatly improved. ConclusionOur results show that BA has a strong protective effect on ACR-induced multi-organ toxicity. The study results show that BA could be a potential element to reduce ACR toxicity to which we are often exposed.

Adipose-derived stem cell exosomes regulate Nrf2/Keap1 in diabetic nephropathy by targeting FAM129B

BackgroundExosomes from adipose-derived stem cells (ADSCs-Exos) have exhibited a therapeutic role in diabetic nephropathy (DN). Further studies are needed to investigate how ADSCs-Exos regulate oxidative stress and inflammation in high glucose-induced podocyte injury.MethodsAn enzyme-linked immunosorbent assay (ELISA) was used to detect cellular inflammation. Reactive oxygen species (ROS) levels were assessed using flow cytometry in podocytes with different treatments. A malondialdehyde (MDA) kit was used to evaluate the lipid peroxidation levels in podocytes and kidney tissues of mice. Western blotting and co-immunoprecipitation were performed to detect protein expression and protein-protein interactions.ResultsADSCs-Exos reversed oxidative stress and inflammation in podocytes and kidney tissues of DN mice induced by high glucose levels in vitro and in vivo. Interference with heme oxygenase-1 expression could reverse the improvement effect of ADSCs-Exos on oxidative stress induced by high glucose levels. Furthermore, high glucose inhibited nuclear factor erythroid 2-related factor 2 (Nrf2) protein expression and promoted Kelch-like ECH-associated protein 1 (Keap1) protein expression in podocytes, as well as their binding ability. As a potential target for Nrf2/Keap1 pathway regulation, FAM129B expression in podocytes is regulated by high glucose and ADSCs-Exos. Moreover, FAM129B siRNA blocked the inhibitory effect of ADSCs-Exos on intracellular ROS and MDA upregulation induced by high glucose in podocytes.ConclusionADSCs-Exos regulate the Nrf2/Keap1 pathway to alleviate inflammation and oxidative stress in DN by targeting FAM129B, which may provide a potential therapeutic strategy for DN.

Impact of Nrf2 overexpression on cholangiocarcinoma treatment and clinical prognosis.

Nrf2 regulates antioxidant protein expression and protects against drug toxicity and oxidative stress, whereas Keap1 controls Nrf2 activity. The Keap1-Nrf2 pathway affects the prognosis of various cancers, however, its effect on cholangiocarcinoma chemoresistance and prognosis remains unclear. This study aimed to determine whether the Keap1-Nrf2 pathway affects chemoresistance and prognosis of distal cholangiocarcinoma. We investigated the correlation between Nrf2 and Keap1 expression and clinical characteristics and prognosis in 91 patients with distal cholangiocarcinoma who underwent curative surgery. Immunohistochemical staining was performed on paraffin blocks using primary antibodies against Nrf2 and Keap1. The relationship between Keap1 and Nrf2 protein expression levels, and clinical characteristics and prognosis was examined. Nrf2 expression was not associated with overall survival in patients who did not receive adjuvant chemotherapy (P=0.994). Among patients receiving adjuvant chemotherapy, the Nrf2 low expression group had a significantly longer median overall survival than the Nrf2 high expression group in Kaplan-Meier survival analysis (P=0.019). In multivariate analysis, high expression of Nrf2 was confirmed as an independent poor prognostic factor in the group receiving adjuvant chemotherapy (P=0.041). This study suggests that Nrf2 overexpression reduces the efficacy of adjuvant chemotherapy in distal cholangiocarcinoma.

The Mitochondrial-Derived Peptide (MOTS-c) Interacted with Nrf2 to Defend the Antioxidant System to Protect Dopaminergic Neurons Against Rotenone Exposure.

MOTS-c is a 16-amino acid mitochondrial-derived peptide reported to be involved in regulating energy metabolism. However, few studies have reported the role of MOTS-c on neuron degeneration. In this study, it was aimed to explore the action of MOTS-c in rotenone-induced dopaminergic neurotoxicity. In an in vitro study, it was observed that rotenone could influence the expression and localization of MOTS-c significantly in PC12 cells, with more MOTS-c translocating into the nucleus from mitochondria. Further study showed that the translocation of MOTS-c from the mitochondria into the nucleus could directly interact with Nrf2 to regulate HO-1 and NQO1 expression in PC12 cells exposed to rotenone, which had been suggested to be involved in the antioxidant defense system. In vivo and in vitro experiments demonstrated that exogenous MOTS-c pretreatment could protect PC12 cells and rats from mitochondrial dysfunction and oxidative stress induced by rotenone. Moreover, MOTS-c pretreatment significantly decreased the loss of TH, PSD95, and SYP protein expression in the striatum of rats exposed to rotenone. In addition, MOTS-c pretreatment could clearly alleviate the downregulated expression of Nrf2, HO-1, and NQO1, as well as the upregulated Keap1 protein expression in the striatum of rotenone-treated rats. Taken together, these findings suggested that MOTS-c could directly interact with Nrf2 to activate the Nrf2/HO-1/NQO1 signal pathway to defend the antioxidant system to prevent dopaminergic neurons from rotenone-induced oxidative stress and neurotoxicity in vitro and in vivo.

Network pharmacology-based study on the mechanism of ShenKang injection in diabetic kidney disease through Keap1/Nrf2/Ho-1 signaling pathway

ObjectiveTo study the effect of ShenKang Injection (SKI) on the kidneys of DKD rats and its effect on oxidative stress mediated by the Keap1/Nrf2/Ho-1 signaling pathway through network pharmacology and in vivo and in vitro experiments. MethodsSKI drug targets were screened by TCMSP, DKD targets were screened by GenGards, OMIM, Drugbank, TTD, and Disgenet databases, and the two intersected for PPI network analysis and target prediction was performed by GO and KEGG. A total of 40 SD rats were randomly divided into 10 in the control group and 30 in the model group. After the model group was fed 8 W with high-sugar and high-fat diets, a DKD model was constructed by one-time intraperitoneal injection of streptozotocin (35 mg/kg). According to the weight, the model animals were randomly divided into three groups: 8 for model validation group, 8 for Irbesartan (25 mg/kg daily) group, and 8 for SKI group (5 ml/kg). Gavaged deionized water was given to the control group and the model validation group equally. The general conditions of the rats were observed, their body weights measured and their urine volumes recorded for 24 h. After the intervention of 16 W, serum was collected to detect Urea, Scr, blood lipids, and oxidative stress and lipid peroxidation indicators; Transmission electron microscopy, HE and Mallory staining were used to observe the pathological morphology of renal tissue. Immunohistochemistry and RT-PCR were used to detect the expression of Keap1, Nrf2, Ho-1, Gpx4 proteins and mRNA in rat kidney tissues. HK-2 cells were cultured in vitro and divided into: the control group, AGEs (200 μg/ml) group and AGEs + SKI group. The cell activity of the groups was detected using CCK-8 after 48 h of cell culture, and ROS were detected using fluorescent probes. Gpx4 expression was detected by immunofluorescence, while Keap1, Nrf2, Ho-1, and Gpx4 were detected by Western Blot. ResultsNetwork pharmacological analysis predicted that SKI may delay DKD kidney injury by affecting redox-related signaling pathways and mitigating AGEs-induced oxidative stress. In the animal experiment, compared with the model validation group, the general state of rats in the SKI group was improved, and 24-hour urine protein levels were significantly reduced, and the Scr in the serum was reduced. A decreasing trend was seen in Urea, and TC, TG, and LDL levels significantly decreased and the levels of ROS, LPO and MDA were significantly lowered. Pathological staining showed that renal interstitial fibrosis was significantly improved, and electron microscopy showed that foot process effacement was alleviated. Immunohistochemistry and RT-PCR showed decreased expression of Keap1 protein and mRNA in kidney tissues of the SKI group. Additionally, Nrf2, Ho-1, and Gpx4 proteins and mRNA were expressed significantly. In the cell experiment, after 48 h treatment with AGEs, ROS in HK-2 cells increased significantly and cell activity decreased significantly, while cell activity in AGEs + SKI group increased significantly and ROS decreased. The expression of Keap1 protein in HK-2 cells in the AGEs + SKI group decreased, while the expression of Nrf2, Ho-1 and Gpx4 proteins increased significantly. ConclusionSKI can protect kidney function in DKD rats, delay DKD progression, inhibit AGEs-induced oxidative stress damage in HK-2 cells, and the mechanism of SKI to improve DKD may be achieved by activating the Keap1/Nrf2/Ho-1 signal transduction pathway.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli (ETEC) K88-infected piglet.

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)‍-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‍‒‍14 and all infused with ETEC K88 1×1010 CFU on Days 15‍‒‍17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

The hepatoprotective effect of Sophora viciifolia fruit extract against acetaminophen-induced liver injury in mice.

This research demonstrated the protective effect and possible mechanism of the Sophora viciifolia extract (SVE) against acetaminophen-induced liver injury in mice. The levels of ALT and AST in the serum and antioxidant enzyme activity in the liver were measured. We used immunohistochemistry to detect CYP2E1, Nrf2, and Keap1 protein expression in the liver. The mRNA expression in the liver of TNF-α, NF-κB, and IL-6, Nrf2 and its downstream genes HO-1 and GCLC were measured by qRT-PCR. We found that SVE could decrease the ALT and AST levels, promote the activities of SOD, CAT, GSH-Px, and GSH, and ameliorate pathological liver lesions. SVE could down-regulate the mRNA expression of inflammatory factors and up-regulate Nrf2, HO-1 and GCLC. SVE reduced the protein expression of the CYP2E1 and increased the Nrf2 and Keap1. SVE has been shown to have a protective effect against APAP-induced liver injury, possibly through activation of the Keap1-Nrf2 pathway.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2

BackgroundPanax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs).MethodsThe present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured.ResultsTreatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS.ConclusionTaken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.

Angelica Sinensis polysaccharide antagonizes 5-Fluorouracil-induced spleen injury and dysfunction by suppressing oxidative stress and apoptosis

Angelica Sinensis polysaccharide (ASP), the main active component of Angelica sinensis, possesses antioxidative and anti-apoptotic properties. In this study, we have investigated the antagonistic effect of ASP on 5-FU-induced injury of mouse spleen in vivo and splenocytes in vitro, and its possible mechanism. Our results showed that ASP inhibited 5-FU-induced decreases in spleen weight and organ index in mice, restored the number of peripheral blood leukocytes and lymphocytes, repaired spleen structure disorder and functional impairment, rescued serum IL-2, IL-6, and IFN-γ levels, and relieved 5-FU-induced mitochondrial swelling， reduced the oxidant accumulation including MDA and ROS, whereas increasing the activities of GSH, SOD and CAT. The mechanism may be related to ASP downregulation of Keap1 protein expression thus motivating the nuclear translocation of Nrf2. Furthermore, ASP alleviated the apoptosis of spleens in vivo and splenocytes in vitro, and reactivated PI3K / AKT signalling. In conclusion, the protective effect of ASP on spleens and splenocytes may be related to the reduction of oxidative stress and apoptosis via reactivation of Nrf2 and PI3K/AKT pathways. This study has provided a new protective agent for minimizing the spleen injury caused by 5-FU and a new idea for improving the prognosis of chemotherapy patients.

Inflammatory and oxidative endophenotypes in borderline personality disorder: A biomarker cluster analysis

Objectives This study is designed to search for aggrupation of inflammatory/oxidative biomarker alterations in borderline personality disorder (BPD) and their association with phenotypic features. Methodology Inflammatory/nitrosative proteins were measures in plasma and peripheral blood mononuclear cells obtained from BPD patients. Patients were assessed on different clinical dimensions of BPD. Oxidative damage was tested by measuring TBARS, nitrites, catalase, GPx and SOD. Protein expression of IκBα, NFκB, iNOS, COX-2, PPARγ, Keap1, NQO1, Nrf2 and α7nAChR was also determined. Western blot and ELISA were used for measurements and a cluster analysis of inflammatory/oxidative biomarkers alterations was performed to investigate subgroups of patients with similar alterations and its relationship with clinical features of BPD. Results 69 patients were included in the study. Two inflammatory/nitrosative clusters of patients were found: Cluster 1 patients showed significantly higher levels of GPx, IκBα, keap1, NQO1, PPARγ, α7nAChR and Nrf2 than cluster 2 patients. These patients had significantly longer duration of illness, milder anxiety symptoms and lower prescription of antipsychotic drugs than cluster 2. Conclusions Two clusters of BPD patients according to the inflammatory/nitrosative profiles were identified. Cluster 1 had increased antioxidant and anti-inflammatory biomarkers and was characterised by greater chronicity of illness but less acute symptomatic severity.

Deoxynivalenol induces intestinal injury: insights from oxidative stress and intestinal stem cells.

Mycotoxins are fungal secondary metabolites that frequently occur in human and animal diets. Deoxynivalenol (DON) is one of the most widely occurring mycotoxins globally and poses significant harm to the animal husbandry industry and human health. People are increasingly aware of the adverse effects of DON on vulnerable structures and functions in the intestine, especially in the field of intestinal stem cells (ISCs). In this review, we present insights into DON that induces oxidative stress and affects the expansion of ISCs. Related studies of strategies for reducing its harm are summarized. We also discussed promising approaches such as regulation of microbiota, molecular docking, and modulation of the redox status via reducing the expression of Keap1 protein and single-cell sequencing, which may be critical for further revealing the mechanism of DON that induces oxidative stress and affects the expansion of ISCs.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense.

To determine whether an antisense RNA corresponding to the human Alu transposable element (Aluas RNA) can protect human lens epithelial cells (HLECs) from methylglyoxal-induced apoptosis. Cell counting kit-8 (CCK-8) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to assess HLEC viability. HLEC viability/death was detected using a Calcein-AM/PI double staining kit; the annexin V-FITC method was used to detect HLEC apoptosis. The cytosolic reactive oxygen species (ROS) levels in HLECs were determined using a reactive species assay kit. The levels of malondialdehyde (MDA) and the antioxidant activities of total-superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were assessed in HLECs using their respective kits. RT-qPCR and Western blotting were used to measure mRNA and protein expression levels of the genes. Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxal-induced decrease in Bcl-2 mRNA and the methylglyoxal-induced increase in Bax mRNA. In addition, Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression. Aluas RNA inhibited the production of ROS induced by methylglyoxal, restored T-SOD and GSH-Px activity, and moderated the increase in MDA content after treatment with methylglyoxal. Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes, including glutathione peroxidase, heme oxygenase 1, γ-glutamylcysteine synthetase and quinone oxidoreductase 1. Aluas RNA ameliorated methylglyoxal-induced increases of the mRNA and protein expression of Keap1 that is the negative regulator of Nrf2. Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Untargeted serum and liver metabolomics analyses reveal the gastroprotective effect of polysaccharide from Evodiae fructus on ethanol-induced gastric ulcer in mice

This study aimed at investigating the gastroprotective effect of Evodiae fructus polysaccharide (EFP) against ethanol-induced gastric ulcer in mice. Biochemical indexes along with untargeted serum and liver metabolomics were determined. Results showed that pre-treatment of EFP alleviated ethanol-induced gastric ulcer in mice. EFP lessened oxidative stress and inflammation levels of stomachs, showing as increments of SOD and GSH-Px activities, GSH content and IL-10 level, and reductions of MDA and IL-6 levels. Meanwhile, EFP activated the Keap1/Nrf2/HO-1 signaling pathway through increasing Nrf2 and HO-1 protein expressions, and decreasing Keap1 protein expression. Serum and liver metabolomics analyses indicated that 10 metabolic potential biomarkers were identified among normal control, ulcer control and 200 mg/kg·bw of EFP groups, which were related to 5 enriched metabolic pathways including vitamin B6 metabolism, nicotinate and nicotinamide metabolism, pentose phosphate pathway, bile secretion and ascorbate and aldarate metabolism. Further pearson's correlation analysis indicated that there were some positive and negative correlations between the biomarkers and the biochemical indexes. It could be concluded that the gastroprotection of EFP might be related to anti-oxidative stress, anti-inflammation, activation of Keap1/Nrf2/HO-1 signaling pathway and alteration of metabolic pathways. This study supports the potential application of EFP in preventing ethanol-induced gastric ulcer.