Keap1 Protein Levels Research Articles

Tile: Construction of a specific nanoprobe for scavenging ROS in hypobaric hypoxia induced brain injury of mice

The prevention and treatment of hypobaric hypoxia brain injury (HHBI) remains an unprecedented challenge due to the complex oxidative stress response at the damage site. In this study, RuCO phthalocyanine compound (RuPc) and bovine serum albumin (BSA) were self-assembled to obtain RuPc-BSA nanoparticles for HHBI therapy. As a nanoprobe carrying and storing carbon monoxide (CO), RuPc-BSA delivers CO to pathologically damaged areas of the brain. CO specifically attaches itself to the heme functional groups on mitochondria and restricts the source of reactive oxygen species (ROS) generation. RuPc-BSA nanoparticles have been demonstrated in vitro to exhibit amazing stability as well as remarkable scavenging activity on hydroxyl radical, superoxide anion, and hydrogen peroxide. In vivo experiments showed that ROS levels in the brain of HHBI rats pretreated with RuPc-BSA decreased significantly, and neuronal function and oxidative stress levels were alleviated. Western blot and qRT-RCR results indicated that RuPc-BSA restricted the protein levels of Keap1, whereas enhanced the gene and protein levels of Nrf2. This study demonstrated that RuPc-BSA can ameliorate HHBI of mice by scavenging ROS partly via activating Keap1/Nrf2 signaling pathway.

The KEAP1/PGAM5/AIFM1-Mediated oxeiptosis pathway in Alzheimer’s disease

BackgroundAlzheimer’s Disease (AD) is a neurodegenerative disease with mitochondrial dysfunction and oxidative stress. Oxeiptosis is a cell death pathway sensitive to reactive oxygen species (ROS). This study investigates the role of oxeiptosis pathway and mitochondrial damage in AD. MethodsAn AD model was developed in C57BL/6 mice by injecting Aβ1-42 oligomers into the brain. Cognitive function was tested using the Morris water maze. Exposure of HT22 mouse hippocampal neurons to H2O2 induces oxidative stress. Protein levels of KEAP1, PGAM5 and AIFM1 were analyzed by western blot, and mitochondrial damage was observed with electron microscopy. Cell survival rates were using the CCK8 assay and flow cytometry after knocking down KEAP1, PGAM5 and AIFM1. ResultsThe protein concentrations of KEAP1, PGAM5 and AIFM1 were found to be elevated in the hippocampal tissues of AD mice compared to control group, accompanied by mitochondrial damage in the hippocampal neurons of the AD group. Similarly, in the HT22 oxidative stress model, there was an increase in the protein levels of KEAP1, PGAM5 and AIFM1, along with observed mitochondrial damage. Following individual and combined knockdown of KEAP1, PGAM5 and AIFM1, cell survival rates under oxidative stress conditions were higher compared to H2O2 group, with no significant difference in cell survival rates among the knockdown groups. ConclusionThis research underscores the critical role of the KEAP1/PGAM5/AIFM1-mediated oxeiptosis pathway in neuronal cell death, offering insights into potential therapeutic targets for mitigating neurodegeneration in AD.

Nrf-2-dependent antioxidant and anti-inflammatory effects underlie the protective effect of esculeoside A against retinal damage in streptozotocin-induced diabetic rats

Esculeoside A (ESA) is a tomato-derived glycoside with antioxidant and anti-inflammatory properties. The protective effect of ESA against diabetic retinopathy is not well-investigated and was the core objective of this study. In addition, we tested if such protection involves the activation of Nrf2 signaling. Type 1 diabetes mellitus (T1DM) was induced in adult Wistar male rats by an intraperitoneal injection of streptozotocin (65 mg/kg). Non-diabetic and T1DM rats were divided into two subgroup groups given either the vehicle or ESA (100 mg)/kg. An additional T1DM group was given ESA (100 mg/kg) and an Nrf2 inhibitor (2 mg/kg) (n=8 rats/group). Treatments continued for 12 weeks. In this study, according to the histological features, ESA improved the structure of ganglionic cells and increased the number of cells of the inner nuclear and plexiform layers in the retinas of T1DM rats. Concomitantly, it reduced the retina levels of malondialdehyde (lipid peroxides), vascular endothelial growth factor, interleukin-6, tumor necrosis factor-α, Bax, and caspase-3. In the retinas of the control and diabetic rats, ESA boosted the levels of total glutathione, superoxide dismutase, heme-oxygenase-1, and Bcl2, reduced the mRNA levels of REDD1, and enhanced cytoplasmic and nuclear levels of Nrf2. However, ESA failed to alter the mRNA levels of Nrf2 and keap1, protein levels of keap1, plasma glucose, plasma insulin, serum triglycerides, cholesterol, and LDL-c in both the control and T1DM rats. In conclusion, ESA alleviates retinopathy in T1DM rats by suppressing REDD1-associated degradation and inhibiting the Nrf2/antioxidant axis.

The gut-liver axis perspective: Exploring the protective potential of polysaccharides from Cistanche deserticola against alcoholic liver disease

The primary objective of this study is to investigate the potential mechanism behind the protective effect of Cistanche deserticola polysaccharides (CP) against alcoholic liver disease (ALD). Multiple chromography techniques were employed to characterize CP from polysaccharide, the molecular weight distribution of polysaccharides, monosaccharide composition, isomeric hydrogen and isomeric carbon, in order to clarify the material basis of CP. To create the ALD mouse model, we utilized the well-established Lieber-DeCarli alcoholic liquid feed method. Findings from the study revealed that CP administration resulted in significant improvements in intestinal permeability, upregulation of barrier proteins expression, and reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse liver and serum. Additionally, CP treatment reduced the presence of inflammatory cytokines both in serum and liver while enhancing the activity of antioxidant enzymes in the liver. Furthermore, CP effectively reduced alcohol-induced oxidative damage by downregulating Keap1 protein levels in the liver, leading to increased expression of Nrf2 protein. The 16S rDNA sequencing results revealed that CP significantly restored the intestinal microbiota composition in ALD mice. These findings establish a strong association between gut microbiota and liver injury indicators, highlighting the potential of CP in preventing and treating ALD by modulating the gut-liver axis.

Abstract 14638: NPA7: A Novel Designed Bispecific Peptide That Protects Human Cardiomyocytes Against Oxidative Stress Through the GC-A/Mas Receptor-KEAP1-NRF2 Pathway

Introduction: Excess cardiomyocyte oxidative stress is a major metabolic abnormality in HTN and its progression to HF. Thus, therapies that reduce oxidative stress in the heart may lead to a reduction in CVD related adverse outcomes such as HF and death. Recently, we bioengineered a bispecific peptide NPA7 that co-activates the protective GC-A/cGMP and MasR/cAMP pathways. Previously we showed that NPA7 possesses cardiorenal protective properties. However, the antioxidant effects of NPA7 within the heart is unknown. Hypothesis: NPA7 protects human cardiomyocytes against oxidative stress through the GC-A/Mas Receptor-KEAP1-NRF2 pathway. Methods: HCMs were pretreated with PBS or NPA7 (10 uM) followed by 400 uM hydrogen peroxide (H2O2) to induce oxidative stress. Intracellular ROS was measured by dihydroethidium (DHE) staining and protein levels of KEAP1 and NRF2 via western blot. The ratio of GSH/GSSG were determined by using commercial kit. mRNA levels of G6PD, GCLC, GCLM and GSS in HCMs, which represents antioxidant capacity, were determined by real-time PCR. For knockdown experiments, HCMs were transfected with the siRNA target GC-A and MasR for 24 h and then treated with NPA7 and H2O2 for another 20 h. Results: H2O2 increased DHE staining level, which indicated elevated intracellular ROS levels within HCMs. Notably, treatment with NPA7 decreased DHE level and increased the ratio of GSH/GSSG in HCMs with H2O2. Furthermore, NPA7 significantly increased the mRNA level of NRF2 target genes (G6PD, GCLC, GCLM, GSS) through NRF2 activation and KEAP1 degradation, indicating that NPA7 activates KEAP1-NRF2 pathway, the upstream pathway that regulate antioxidant gene expression, in HCMs. Moreover, these effects were eliminated if GC-A and MasR were silenced by siRNA, thus supporting the protective antioxidant role of both GC-A and MasR. Conclusions: The present study establishes an antioxidant action of NPA7, a novel bispecific designer peptide, on H2O2-induced oxidative stress by the dual targeting of GC-A and MasR through the KEAP1-NRF2 signaling pathway in HCMs. These results advance NPA7 as an innovative therapeutic strategy targeting oxidative stress related to CVD.

Total triterpenes of Euphorbium alleviates rheumatoid arthritis via Nrf2/HO-1/GPX4 pathway

This study aims to investigate the effect and mechanism of total triterpenes of Euphorbium in treating rheumatoid arthritis(RA). The rat model of RA was established with Freund's complete adjuvant(FCA). Male rats were randomly assigned into control, model, Tripterygium glycosides(7.5 mg·kg~(-1)), and low-, medium-, and high-dose total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1), respectively) groups, with 10 rats in each group. In other groups except the control group, 0.2 mL FCA was injected into the right hind toe. Rats in the intervention groups were administrated with corresponding drugs by gavage, and the control group and the model group with the same volume of 0.5% CMC-Na solution once a day. During the treatment period, the swelling degree of the hind paw was measured and the arthritis was scored until day 30. At the end of drug administration, the pathological changes of the joint tissue were observed by hematoxylin-eosin staining. The content of malondialdehyde(MDA), glutathione(GSH), and Fe~(2+) and the activity of superoxide dismutase(SOD) in the joint tissue were measured by biochemical colorimetry. RT-PCR was performed to determine the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2), glutathione peroxidase 4(GPX4), and acyl-CoA synthetase long chain family member 4(ACSL4) in the joint tissue. Western blot was employed to determine the protein levels of Nrf2, Kelch-like ECH-associated protein 1(Keap1), heme oxygenase-1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), SOD2, GPX4, and ACSL4 in the joint tissue. The results showed that the treatment with Tripterygium glycosides(7.5 mg·kg~(-1)) and total triterpenes of Euphorbium(32, 64, and 128 mg·kg~(-1)) alleviated the swelling degree of bilateral hind limbs, decreased the arthritis score, reduced joint tissue lesions and the content of MDA and Fe~(2+) in the joint tissue, and increased GSH content and SOD activity. Furthermore, the interventions up-regulated the protein and mRNA levels of Nrf2 and GPX4, down-regulated the protein and mRNA levels of ACSL4, and up-regulated the protein levels of Keap1, NQO1, HO-1, and SOD2 in the Nrf2/HO-1/GPX4. In summary, the total triterpenes of Euphorbium can treat RA by inhibiting lipid peroxidation and abnormal ferroptosis, which may involve the Nrf2/HO-1/GPX4 signaling pathway.

Protective effect of ginsenoside Rg1 on 661W cells exposed to oxygen-glucose deprivation/reperfusion via keap1/nrf2 pathway.

To construct an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) induced injury to the optic nerve and to study the oxidative damage mechanism of ischemia-reperfusion (I/R) injury in 661W cells and the protective effect of ginsenoside Rg1. The 661W cells were treated with different concentrations of Na2S2O4 to establish OGD/R model in vitro. Apoptosis, intracellular reactive oxygen species (ROS) levels and superoxide dismutase (SOD) levels were measured at different time points during the reperfusion injury process. The injury model was pretreated with graded concentrations of ginsenoside Rg1. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of cytochrome C (cyt C)/B-cell lymphoma-2 (Bcl2)/Bcl2 associated protein X (Bax), heme oxygenase-1 (HO-1), caspase9, nuclear factor erythroid 2-related factor 2 (nrf2), kelch-like ECH-associated protein 1 (keap1) and other genes. Western blot was used to detect the expression of nrf2, phosphorylated nrf2 (pnrf2) and keap1 protein levels. Compared to the untreated group, the cell activity of 661W cells treated with Na2S2O4 for 6 and 8h decreased (P<0.01). Additionally, the ROS content increased and SOD levels decreased significantly (P<0.01). In contrast, treatment with ginsenoside Rg1 reversed the cell viability and SOD levels in comparison to the Na2S2O4 treated group (P<0.01). Moreover, Rg1 reduced the levels of caspase3, caspase9, and cytC, while increasing the Bcl2/Bax level. These differences were all statistically significant (P<0.05). Western blot analysis showed no significant difference in the protein expression levels of keap1 and nrf2 with Rg1 treatment, however, Rg1 significantly increased the ratio of pnrf2/nrf2 protein expression compared to the Na2S2O4 treated group (P<0.001). The OGD/R process is induced in 661W cells using Na2S2O4. Rg1 inhibits OGD/R-induced oxidative damage and alleviates the extent of apoptosis in 661W cells through the keap1/nrf2 pathway. These results suggest a potential protective effect of Rg1 against retinal I/R injury.

Bisphenol S induces oxidative stress-mediated impairment of testosterone synthesis by inhibiting the Nrf2/HO-1 signaling pathway.

Bisphenol S (BPS) is an environmental endocrine disruptor widely used in industrial production. BPS induces oxidative stress and exhibits male reproductive toxicity in mice, but the mechanisms by which BPS impairs steroid hormone synthesis are not fully understood. Nuclear factor erythroid 2-related factor 2(Nrf2)/HO-1 signaling is a key pathway in improving cellular antioxidant defense capacities. Therefore, this study explored the effects of exposure to BPS on testosterone synthesis in adult male mice and its mechanisms with regard to the Nrf2/HO-1 signaling pathway. Adult male C57BL/6 mice were orally exposed to BPS (2, 20, and 200 mg/kg BW) with sesame oil as a vehicle (0.1 ml/10 g BW) per day for 28 consecutive days. The results showed that compared with the control group, serum testosterone levels were substantially reduced in the 20 and 200 mg/kg BPS treatment groups, and testicular testosterone levels were reduced in all BPS treatment groups. These changes were accompanied by a prominent decrease in the expression levels of testosterone synthesis-related enzymes (STAR, CYP11A1, CYP17A1, HSD3B1, and HSD17B3) in the mouse testis. In addition, BPS induced oxidative stress in the testis by upregulating the messenger RNA and protein levels of Keap1 and downregulating the levels of Nrf2, HO-1, and downstream antioxidant enzymes (CAT, SOD1, and Gpx4). In summary, our results indicate that exposure of adult male mice to BPS can inhibit Nrf2/HO-1 signaling and antioxidant enzyme activity, which induces oxidative stress and thereby may impair testosterone synthesis in testicular tissues, leading to reproductive damage.

Salvianolactone acid A isolated from Salvia miltiorrhiza ameliorates lipopolysaccharide-induced acute lung injury in mice by regulating PPAR-γ

BackgroundSevere inflammation of the lungs results from acute lung injury (ALI), a common life-threatening lung disease with a high mortality rate. The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR) γ plays essential roles in diverse biological processes including inflammation, metabolism, development, and immune response. Salvianolactone acid A (SA) is a terpenoid derived from the herb Salvia miltiorrhiza. However, there is a scarcity of experimental evidence indicating whether the effect of SA on ALI occurs via PPAR-γ. MethodsSA (20 or 40 mg/kg, i.g., 1 time/day) was administered to mice for 3 d, followed by the induction of ALI by intranasal lipopolysaccharide (LPS, 10 mg/kg). The lung function and levels of inflammation, reactive oxygen species (ROS), immune cells, apoptosis, and PPAR-γ were examined. The antagonistic activity of GW9662 (GW, 1 µM, specific PPAR-γ blocker) and PPAR-γ transfection silencing against SA (10 μM) in BEAS-2B cells induced by LPS (10 μg/ml, 24 h) was also investigated to assess whether the observed effects caused by SA were mediated by PPAR-γ. ResultsThe results showed that lung histopathological injury, the B-line, the fluorescence intensity of live small animal, and the biomarkers in BALF or lung in the treatment of SA could regulate significantly. In addition, SA obviously decreased the levels of ROS and apoptosis in the primary lung cells, and MDA, increased the levels of GSH-Px and SOD. SA reduced levels of macrophages and neutrophils. Furthermore, SA reduced the protein levels of Keap-1, Cleaved-caspase-3, Cleaved-caspase-9, p-p65/p65, NLRP3, IL-1β, and upregulated the levels of p-Nrf2/Nrf2, HO-1, Bcl-2/Bax, PPAR-γ, p-AMPK/AMPK in lung tissue. In addition, silencing and inhibition of PPAR-γ effectively decreased the protective effects of SA in BEAS-2B cells induced by LPS, which might indicate that the active molecules of SA regulate ALI via mediation by PPAR-γ, which exhibited that the effect of SA related to PPAR-γ. ConclusionsThe anti-ALI effects of SA were partially mediated through PPAR-γ signaling. These data provide the molecular justification for the usage of SA in treating ALI and can assist in increasing the comprehensive utilization rate of Salvia miltiorrhiza.

Tert-Butylhydroquinone-induced formation of high-molecular-weight p62: A novel mechanism in the activation of Nrf2-Keap1.

The respiratory system is always exposed to air and is most vulnerable to attack by environmental free radicals. The nuclear factor E2-related factor 2-Kelch-like ECH-associated protein 1-antioxidant response element (Nrf2-Keap1-ARE) pathway and p62 are both involved in the oxidative stress response. However, the interplay between these two systems remains largely unknown. This study shows that treatment of L2 cells with tert-Butylhydroquinone (tBHQ) generates a high-molecular-weight (HMW) form of p62, leading to activation of the Nrf2-Keap1-ARE pathway. The levels of HMW-p62 increased as the tBHQ concentration increased, with concomitant decreases seen in the classical form of p62. Moreover, small interfering RNA targeting p62 increases Keap1 protein levels and inactivates the Nrf2-Keap1-ARE pathway. These results demonstrate that the Nrf2-Keap1 pathway is partially regulated by p62. tBHQ-induced HMW-p62 production may be a novel mechanism in the activation of the Nrf2-Keap1-ARE pathway.

Effect of dietary L-theanine supplementation on skeletal muscle fiber type transformation in weaning piglets

The aim of this study was to explore the effect of dietary L-theanine (LT) supplementation on skeletal muscle fiber type transformation in weaning piglets. Our data showed that LT significantly increased the slow-twitch fiber-related genes expression and the percentage of slow oxidative fiber, and decreased the MyHC IIb mRNA expression and the percentage of fast glycolytic fiber. In addition, LT significantly increased the succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities and increased the LDH activities. In addition, LT significantly affected mitochondrial biogenesis and function and antioxidative related genes expression, and increased the protein expression of p-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear factor E2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) and decreased the Keap1 protein levels. Furthermore, our data indicated that LT significantly increased the mRNA and protein expression of prospero-related homeobox 1 (Prox1), calcineurin A (CnA), and NFATc1, suggesting that dietary LT supplementation promoted skeletal muscle fiber transition from types II to I might be via activation of calcineurin signaling pathway. Taken together, these findings suggested that LT promoted the transformation of muscle fiber types from slow oxidative to fast glycolytic by increasing antioxidant capacity and improving mitochondrial biogenesis and function and activation of calcineurin signaling pathway.

Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Kaempferol prevents cadmium chloride-induced liver damage by upregulating Nrf2 and suppressing NF-κB and keap1.

This study evaluated the protective effect of kaempferol, a natural flavonoid, against cadmium chloride (CdCl2)-induced liver damage and examined the possible anti-inflammatory and antioxidant mechanisms of protection. Adult male rats were divided into 4 groups (each of 8 rats) as control, kaempferol (50 mg/kg/day orally), CdCl2 (15 ppm/day), and CdCl2 (15 ppm/day) + kaempferol (50 mg/kg/day). All treatments were given for 30 days. With no effect on attenuating the reduced food intake, kaempferol significantly increased body weight and lowered serum levels of liver injury markers including bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase 1 (γ-GTT1) in the CdCl2-treated rats. It also restored normal liver architectures, prevented hepatocyte, loss, and swelling and reduced inflammatory cell infiltration. These effects were associated with a reduction in mitochondrial permeability transition pore, as well as in the expression of cytochrome-c and cleaved caspase-3, markers of mitochondrial damage, and intrinsic cell death. In both the control positive and CdCl2-treated rats, kaempferol significantly lowered the hepatic levels of reactive oxygen species, malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), Interleukine-6 (IL-6), and the nuclear activity and localization of NF-κB p65. Besides, kaempferol significantly increased the hepatic total and nuclear levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1, as well as levels of superoxide dismutase (SOD) and reduced glutathione (GSH) but reduced the cytoplasmic protein levels of keap1. In conclusion, the protective effect of kaempferol against CdCl2-induced hepatic damage is mediated by antioxidant and anti-inflammatory effects driven by upregulating Nrf2/HO-1 axis and suppressing the NF-κB p65 and keap1.

Chrysosplenol D protects mice against LPS-induced acute lung injury by inhibiting oxidative stress, inflammation, and apoptosis via TLR4-MAPKs/NF-κB signaling pathways.

This study investigated the effect and mechanism of chrysosplenol D (CD) on LPS-induced acute lung injury in mice. Histological changes in the lungs were measured by hematoxylin-eosin staining. The levels of IL-6, IL-1β, and TNF-α in the bronchoalveolar lavage fluid were detected by ELISA. The levels of oxidative stress were detected by the cuvette assay. Immune cells in peripheral blood, the levels of reactive oxygen species, and apoptosis of primary lung cells were detected by flow cytometry. The mRNA levels of TLR4, MyD88, IL-1β, and NLRP3 were measured by quantitative real-time polymerase chain reaction. The levels of proteins in apoptosis and the TLR4-MAPKs/NF-κB signaling pathways were detected by Western blot. Hematoxylin-eosin staining showed that CD could improve lung injury; decrease the levels of inflammatory factors, oxidative stress, reactive oxygen species, and cell apoptosis; and regulate the immune system. Moreover, CD could down-regulate the mRNA levels of TLR4, MyD88, NLRP3, and IL-1β in lung, and the protein levels of Keap-1, Cleaved-Caspase-3/Caspase-3, Cleaved-Caspase-9/Caspase-9, TLR4, MyD88, p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-p65/p65, NLRP3, and IL-1β, and up-regulated the levels of Bcl-2/Bax, p-Nrf2/Nrf2, and HO-1. The results suggested that CD could protect mice against LPS-induced acute lung injury by inhibiting oxidative stress, inflammation, and apoptosis via the TLR4-MAPKs/NF-κB signaling pathways.

Dahuang Danshen Decoction Inhibits Pancreatic Fibrosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress.

Background In Traditional Chinese Medicine (TCM), Dahuang Danshen decoction (DD) is used to treat pancreatic fibrosis. Pancreatic fibrosis is a typical manifestation of chronic pancreatitis (CP), which affects the digestive system. The therapeutic mechanisms of DD in pancreatic fibrosis are unclear. Aim This study aimed to investigate the regulatory mechanisms of DD on oxidative stress and endoplasmic reticulum stress in CP. Materials and Methods Experimental rats were intraperitoneally injected with 500 mg/kg BW of diethyldithiocarbamate (DDC) twice a week for six weeks to induce CP. At the same time, DD was administered orally at daily doses of 1.37 g/kg BW, 2.74 g/kg BW, and 5.48 g/kg BW to evaluate its treatment effects on CP. After all treatments, pancreatic tissues were harvested and subjected to H&E staining. Transmission electron microscopy (TEM) was also performed to show the endoplasmic reticulum structure in the pancreatic tissues. Immunohistochemistry was used to detect the α-SMA expression level in the pancreatic tissues. Metabolomics analysis of the serum and proteomics analysis of the pancreatic tissues were performed to reveal the changes of endogenous metabolites and proteins, respectively. Concentrations of GSH, MDA, SOD, ROS, col-1, and col-3 were determined using corresponding kits. The western blotting method was used to determine the protein levels of Keap-1, HO-1, NQO1, Nrf2, GRP, JNK, and caspase 12. The pancreatic mRNA levels of NQO1, GPX1, HO-1, GST-π, GRP, JNK, and caspase 12 were also determined by quantitative PCR. The interactions between TCM components and Keap-1 were investigated by molecular docking modeling. Results The pathohistological results demonstrated that DD could ameliorate DDC-induced CP in vivo, indicated by reduction of α-SMA, col-1, col-3, TNF-α, and IL-6. DD increased serum levels of GSH and SOD but reduced pancreatic ROS. DD decreased cytoplasmic Keap-1 and increased Nrf2 nuclear localization. Correspondingly, DD increased the expression levels of Nrf2 downstream antioxidant genes NQO1, GPX1, HO-1, and GST-π. DD also decreased ERS hallmarks caspase 12 cleavage and GRP expression. Eventually, DD inhibited PSC activation by reducing JNK phosphorylation and MMK-3/p38 expression. Molecular docking analysis showed that salvianolic acid B and emodin had a good binding affinity toward Keap-1. Conclusions These results demonstrated that DD could ameliorate the oxidative and endoplasmic reticulum stress through releasing Nrf2 from Keap-1 binding and inducing the downstream antioxidant enzymes. As a result, DD could thwart pancreatic fibrosis by inhibiting PSCs activation, which was induced by OS and ERS through JNK and MMK3/p38 pathways.

Puerarin induces Nrf2 as a cytoprotective mechanism to prevent cadmium-induced autophagy inhibition and NLRP3 inflammasome activation in AML12 hepatic cells

Liver is the main target organ of cadmium (Cd) toxicity and puerarin (PU) has been shown to prevent Cd-induced hepatic cell damage via its antioxidant activity. Nrf2 acts as a critical regulator of cellular defense against various oxidative insults, but its role in the protection of PU against Cd-induced hepatic damage has not yet been clarified. Hereby, this study was designed to investigate the underlying mechanism using mouse hepatocyte line AML-12. Data firstly showed that Cd-inhibited Nrf2 pathway was markedly restored by PU treatment, assessed by Nrf2 nuclear translocation, protein levels of Keap1 and Nrf2 downstream target genes. Accordingly, Cd-reduced protein levels of antioxidant enzymes were significantly up-regulated by PU. Next, Nrf2 silencing cellular model was established to further elucidate the role of Nrf2 in the protection of PU against Cd-induced hepatic damage. Attenuation of Cd-induced autophagy inhibition and autophagosome accumulation by PU was remarkably countered by Nrf2 silencing. Moreover, alleviation of Cd-induced NLRP3 inflammasome activation by PU was distinctly prevented by Nrf2 knockdown, assessed by protein levels of NLRP3 inflammosome complex and downstream IL-18 and IL-1β production. Collectively, our data suggest that PU restores Cd-induced Nrf2 inhibition to prevent autophagy inhibition and NLRP3 inflammasome activation, providing novel insights into the protection of PU against Cd-induced hepatic cell damage.

Kahweol activates the Nrf2/HO-1 pathway by decreasing Keap1 expression independently of p62 and autophagy pathways.

Kahweol is a diterpene found in coffee beans and unfiltered coffee drinks. Several studies have demonstrated that kahweol induces the nuclear factor erythroid-2 related factor 2/ hemeoxygenase-1 (Nrf2/HO-1) pathway; however, the mechanisms involved are currently unknown. Kelch-like ECH-associated protein 1 (Keap1) is a major regulator of Nrf2 expression and is degraded mostly by autophagy. The p62 protein enhances binding to Keap1 and contributes to the activation of Nrf2. Here, we examined the role of Keap1 regulation in the effect of kahweol on the Nrf2/HO-1 pathway in hepatocytes. In AML12 cells and primary mouse hepatocytes, kahweol increased the levels of Nrf2 and HO-1 protein without increasing expression of the Nrf2 mRNA. In addition, kahweol reduced Keap1 protein levels significantly without decreasing Keap1 mRNA levels. Although regulation of the Keap1-Nrf2-pathway by p62-dependent autophagy is well known, we confirmed here that the reduction of Keap1 protein levels by kahweol does not involve p62-dependent autophagy degradation or ubiquitination. In conclusion, kahweol increases the expression of Nrf2 in hepatocytes by inhibiting translation of the Keap1 mRNA.

Protective effect of carnosic acid on acrylamide-induced liver toxicity in rats: Mechanistic approach over Nrf2-Keap1 pathway.

Acrylamide is a food contaminant with a range of toxic effects. Carnosic acid (C20 H28 O4 ) is a phenolic compound found in plants and has many beneficial effects. In this study, we aimed at investigating the effect of carnosic acid on acrylamide-induced liver damage. Rats (n = 7) were allotted to control, carnosic acid, acrylamide, acrylamide + carnosic acid groups. Animals were euthanized. Their blood was taken for biochemical analysis, and liver tissue was excised for morphological, immunohistochemical, and immunoblotting analyses. As a result, acrylamide reduced bodyweight, liver weight, catalase, and total antioxidant capacity levels but increased alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, malondialdehyde, total oxidant status, oxidative stress index levels, Nrf2, and Keap1 protein levels. In addition, acrylamide disrupted liver histology leading to vascular congestion, cellular infiltration, necrotic cells, and so forth. Carnosic acid cotreatment ameliorated the altered biochemical parameters, liver histology, Nrf2, and Keap1 enzyme levels. In conclusion, carnosic acid has the potential to be used as a protective agent against acrylamide-induced liver damage.

Associations between the Nrf2/Keap1 pathway and mitochondrial functions in colorectal cancer are affected by metastasis.

Both mitochondria and the Nrf2/Keap1 pathway are targets of cancer therapy. Reactive oxygen species released from mitochondria can activate Nrf2, and the Nrf2/Keap1 pathway affects glycolysis, oxidative phosphorylation, mitochondrial biogenesis and mitophagy. This study investigates the associations between the expressions of proteins in the Nrf2/Keap1 pathway and those related to mitochondrial function and glycolysis in colorectal cancer (CRC) with or without metastasis. The protein levels of HO1, Nrf2, Keap1, Bach1, p21, p62, NRF1, LC3, ATP5B, HSP60 and GAPDH in the normal and tumor tissues of 60 CRC subjects were determined by Western blot. The Keap1 protein levels, the ATP5B/HSP60 ratio and the BEC index were higher in the tumor than in the normal tissues of CRC with or without metastasis. The following clusters were found in the dendrogram: Nrf2 and p21 with ATP5B and GADPH in all the tissues and with NRF1 in all except the tumor tissues with metastasis; Bach1 with ATP5B and GAPDH in the tumor tissues; Keap1 with p62 in all the tissues, with LC3 in the tumor tissues and with NRF1 and HO1 in the tumor tissues with metastasis. Nrf2, Keap1, Bach1 and p21 have the association with the proteins related to mitochondrial functions different among the tissues of CRC with or without metastasis.

Ellagic acid ameliorates oxidative stress and insulin resistance in high glucose-treated HepG2 cells via miR-223/keap1-Nrf2 pathway

As a promising new target, miR-233 may regulate oxidative stress by targeting keap1-Nrf2 system to affect the pathological process of liver injury in T2DM. Ellagic acid (EA) is versatile for protecting oxidative stress damage and metabolic disorders. In the present study, we investigated the effect of EA on oxidative stress and insulin resistance in high glucose-induced T2DM HepG2 cells and examined the role of miR-223/keap1-Nrf2 pathway in system. HepG2 cells were incubated in 30 mM of glucose, with or without EA (15 and 30 μM) or metformin (Met, 150 μM) for 12 h. Glucose consumption, phosphorylation of IRS1, Akt and ERK under insulin stimulation, ROS and O2− production, MDA level, SOD activity and miR-223 expression, as well as protein levels of keap1, Nrf2, HO-1, SOD1 and SOD2 were analyzed. Furthermore, dual luciferase reporter assay, miR-223 mimic and inhibitor were implemented in cellular studies to explore the possible mechanism. EA upregulated glucose consumption, IRS1, Akt and ERK phosphorylation under insulin stimulation, reduced ROS and O2− production and MDA level, and increased SOD activity in high glucose-exposed HepG2 cells. In addition, EA elevated miR-223 expression level, downregulated mRNA and protein levels of keap1, and upregulated Nrf2, HO-1, SOD1 and SOD2 protein levels in this cell model. What’s more, dual luciferase reporter assay, miR-223 mimic and inhibitor transfection confirmed that EA activated keap1-Nrf2 system via elevating miR-223. The miR-223, a negative regulator of keap1, represents an attractive therapeutic target in hepatic injury in T2DM. EA ameliorates oxidative stress and insulin resistance via miR-223-mediated keap1-Nrf2 activation in high glucose-induced T2DM HepG2 cells.