Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats.
ABSTRACT: Puerarin is an isoflavone isolated from Radix puerariae. Emerging evidence shown that puerarin possesses therapeutic benefits that aid in the prevention of cardiovascular diseases. In this study, we evaluated the effects of puerarin on oxidative stress and cardiac fibrosis induced by abdominal aortic banding (AB) and angiotensin II (AngII). We also investigated the mechanisms underlying this phenomenon. The results of histopathological analysis, as well as measurements of collagen expression and cardiac fibroblast proliferation indicated that puerarin administration significantly inhibited cardiac fibrosis induced by AB and AngII. These effects of puerarin may reflect activation of Nrf2/ROS pathway. This hypothesis is supported by observed decreases of reactive oxygen species (ROS), decreases Keap 1, increases Nrf2 expression and nuclear translocation, and decreases of collagen expressions in cardiac fibroblasts treated with a combination of puerarin and AngII. Inhibition of Nrf2 with specific Nrf2 siRNA or Nrf2 inhibitor brusatol attenuated anti-fibrotic and anti-oxidant effects of puerarin. The metabolic effects of puerarin were mediated by Nrf2 through upregulation of UDP-glucuronosyltransferase (UGT) 1A1. The Nrf2 agonist tBHQ upregulated protein expression of UGT1A1 over time in cardiac fibroblasts. Treatment with Nrf2 siRNA or brusatol dramatically decreased UGT1A1 expression in puerarin-treated fibroblasts. The results of chromatin immunoprecipitation-qPCR further confirmed that puerarin significantly increased binding of Nrf2 to the promoter region of Ugt1a1. Western blot analysis showed that puerarin significantly inhibited AngII-induced phosphorylation of p38-MAPK. A specific inhibitor of p38-MAPK, SB203580, decreased collagen expression, and ROS generation induced by AngII in cardiac fibroblast. Together, these results suggest that puerarin prevents cardiac fibrosis via activation of Nrf2 and inactivation of p38-MAPK. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1. Autoregulatory circuits between puerarin and Nrf2-regulated UGT1A1 attenuates side effects associated with treatment, but it does not weaken puerarin's pharmacological effects.
Project description:BACKGROUND:Neomorphic IDH1 mutations disrupt the redox balance by promoting reactive oxygen species (ROS) production. However, the mechanism by which IDH1-mutant cells maintain ROS homeostasis remains elusive. It is also not known whether reprogrammed ROS homeostasis establishes targetable vulnerability in IDH1-mutated cancers. METHODS:We investigated ROS homeostasis in wild-type (GSC827, GSC923, GSC627, and GSC711) and IDH1-mutated cells (IDH1R132C- and IDH1R132H-transduced U87, U251; MGG152, and TS603 cells). We analyzed the stability and transcriptional activity of NRF2 in IDH1-mutated cells. The oxidative DNA damage was analyzed using NRF2-targeting small interfering RNA. Moreover, we evaluated the effect of the NRF2 inhibitor brusatol in an IDH1-mutated subcutaneous xenograft nude mouse model (control group, n?=?5; brusatol-treated group, n?=?6). All statistical tests were two-sided. RESULTS:We showed that IDH1-mutated cells develop a dependency on the NRF2 antioxidative pathway. Genetic or pharmacologic blockade of NRF2 not only disrupted ROS homeostasis (mean [SD] ROS levels increased by 317 [42.1]%, P?=?.001, in IDH1R132C and by 286. 5 [48.7]%, P?=?.003, in IDH1R132H cells) but also enhanced oxidative DNA damage and decreased proliferation of IDH1-mutated cells. Brusatol selectively suppressed IDH1-mutated cancer progression in vivo (mean [SD] final tumor volume was 761.6 [391.6] mm3 in the control and 246.2  mm3 in the brusatol-treated group, P?=?.02). CONCLUSIONS:IDH1 mutation reprograms ROS homeostasis in cancer cells, which leads to dependency on the NRF2 antioxidant pathway for ROS scavenging. NRF2 blockade might be a novel therapeutic approach to treat malignancies with IDH1 mutation.
Project description:Endothelial cells (ECs) express a Nox2 enzyme, which, by generating reactive oxygen species (ROS), contributes to EC redox signaling and angiotensin II (AngII)-induced endothelial dysfunction. ECs also express abundantly an adenosine A(2A) receptor (A(2A)R), but its role in EC ROS production remains unknown. In this study, we investigated the role of A(2A)R in the regulation of Nox2 activity and signaling in ECs with or without acute AngII stimulation. In cultured ECs (SVEC4-10), AngII (100 nm, 30 min) significantly increased Nox2 membrane translocation and association with A(2A)R. These were accompanied by p47(phox), ERK1/2, p38 MAPK, and Akt phosphorylation and an increased ROS production (169 ± 0.04%). These AngII effects were inhibited back to the control levels by a specific A(2A)R antagonist (SCH58261), or adenosine deaminase, or by knockdown of A(2A)R or Nox2 using specific siRNAs. Knockdown of A(2A)R, as determined by Western blotting, decreased Nox2 and p47(phox) expression. In wild-type mouse aorta, SCH58261 significantly reduced acute AngII-induced ROS production and preserved endothelium-dependent vessel relaxation to acetylcholine. These results were further confirmed by using aortas from A(2A)R knock-out mice. In conclusion, A(2A)R is involved in the regulation of EC ROS production by Nox2. Inhibition or blockade of A(2A)R protects ECs from acute AngII-induced oxidative stress, MAPK activation, and endothelium dysfunction.
Project description:The HER2-targeting antibody trastuzumab has shown effectiveness in treating HER2-positive breast and gastric cancers; however, its responses are limited. Currently, Nrf2 has been deemed as a key transcription factor in promoting cancer progression and resistance by crosstalk with other proliferative signaling pathways. Brusatol as a novel Nrf2 inhibitor has been deemed as an efficacious and safe drug candidate in cancer therapy. In this study, we firstly reported that brusatol exerted the growth-inhibitory effects on HER2-positive cancer cells by regressing Nrf2/HO-1 and HER2-AKT/ERK1/2 signaling pathways in these cells. More importantly, we found that brusatol synergistically enhanced the antitumor activity of trastuzumab against HER2-positive SK-OV-3 and BT-474 cells, which may be attributed to the inhibition of Nrf2/HO-1 and HER2-AKT/ERK1/2 signaling pathways. Furthermore, the synergistic effects were also observed in BT-474 and SK-OV-3 tumor xenografts. In addition, our results showed that trastuzumab markedly enhanced brusatol-induced ROS accumulation and apoptosis level, which could further explain the synergistic effects. To conclude, the study provided a new insight on exploring Nrf2 inhibition in combination with HER2-targeted trastuzumab as a potential clinical treatment regimen in treating HER2-positive cancers.
Project description:Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high ex-vivo IC50 to Ara-C, ATO, Dnr had significantly high NRF2 RNA expression. Gene-specific knockdown of NRF2 improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell's ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing NRF2 showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.
Project description:Brusatol is a natural quassinoid that shows a potential therapeutic use in cancer models by the inhibition of Nuclear factor erythroid 2-related factor 2 (Nrf2) and is capable of inducing a variety of biological effects. The effects of Brusatol on oocyte meiosis has not been addressed. In this study, we investigated the impact of Brusatol treatment on mouse oocyte maturation and its possible mechanism. Our data demonstrated that Brusatol treatment disrupted oocyte maturation and spindle/chromosome organization by modulating Nrf2-Cyclin B1 pathway, as the influence of Brusatol was compensated by the addition of Nrf2 activation plasmid, and the mRNA and protein levels of Cyclin B1 were severely reduced in oocytes following Nrf2 decline. In summary, our data support a model that Brusatol, through the inhibition of Nrf2, modulate Cyclin B1 levels, consequently disturbing proper spindle assembly and chromosome condensation in meiotic oocytes.
Project description:Abstract: Connexin 43 (Cx43) is essential for cardiac electrical coupling, but its effects on myocardial fibrosis is controversial. Here, we analyzed the role of Cx43 in myocardial fibrosis caused by angiotensin II (AngII) using Cx43fl/fl and Cx43Cre-ER(T)/fl inducible knock-out (Cx43 content: 50%) mice treated with vehicle or 4-hydroxytamoxifen (4-OHT) to induce a Cre-ER(T)-mediated global deletion of the Cx43 floxed allele. Myocardial collagen content was enhanced by AngII in all groups (n = 8-10/group, p < 0.05). However, animals with partial Cx43 deficiency (vehicle-treated Cx43Cre-ER(T)/fl) had a significantly higher AngII-induced collagen accumulation that reverted when treated with 4-OHT, which abolished Cx43 expression. The exaggerated fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice was associated with enhanced p38 MAPK activation and was not evident in Cx43 heterozygous (Cx43+/-) mice. In contrast, normalization of interstitial collagen in 4-OHT-treated Cx43Cre-ER(T)/fl animals correlated with enhanced MMP-9 activity, IL-6 and NOX2 mRNA expression, and macrophage content, and with reduced ?-SMA and SM22? in isolated fibroblasts. In conclusion, our data demonstrates an exaggerated, p38 MAPK-dependent, fibrotic response to AngII in partially deficient Cx43Cre-ER(T)/fl mice, and a paradoxical normalization of collagen deposition in animals with an almost complete Cx43 ablation, an effect associated with increased MMP-9 activity and inflammatory response and reduced fibroblasts differentiation.
Project description:Mycobacterium abscessus (Mabs), a non-tuberculous mycobacterium, is an emerging and rapidly growing opportunistic pathogen that is frequently found in patients with cystic fibrosis and in immunosuppressed patients. Its high tolerance to antibiotics is of great concern for public health. In this study, our results showed that human THP-1-derived macrophages infected with M. abscessus presented an increase in ROS production and cell necrosis. In addition, M. abscessus infection triggered activation of the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and the induction of HO-1 and NQO1 expression levels. Interestingly, pretreatment of macrophages with sulforaphane (SFN), an activator of the antioxidant key regulator Nrf2, followed by M. abscessus infection significantly decreased mycobacterial burden. We demonstrated that this reduction in mycobacterial growth was due to an activation in cell apoptosis in SFN-pretreated and M. abscessus-infected macrophages. Pretreatment with specific MAPK inhibitors, PD98059, SP600125, and SB203580 to ERK, JNK, and p38 respectively, failed to inhibit induction of Nrf2 expression, suggesting that Nrf2 signaling pathway was upstream of MAPK signaling. Activation of cell apoptosis was caspase 3/7 independent but p38 MAPK dependent. Moreover, p38 MAPK induction was abolished in macrophages transfected with Nrf2 siRNA. In addition, p38 inhibitor abolished Nrf2-dependent apoptosis in infected macrophages. Taken together, our results indicate that modulation of the Nrf2 signaling using Nrf2 activators may help potentiate the actual drug therapies used to treat mycobacterial infection.
Project description:BACKGROUND:We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS:The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS:Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS:Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.
Project description:Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.
Project description:Lasting activations of toll-like receptors (TLRs), MAPK and NF-?B pathways can support influenza A virus (IAV) infection and promote pneumonia. In this study, we have investigated the effect and mechanism of action of emodin on IAV infection using qRT-PCR, western blotting, ELISA, Nrf2 luciferase reporter, siRNA and plaque inhibition assays. The results showed that emodin could significantly inhibit IAV (ST169, H1N1) replication, reduce IAV-induced expressions of TLR2/3/4/7, MyD88 and TRAF6, decrease IAV-induced phosphorylations of p38/JNK MAPK and nuclear translocation of NF-?B p65. Emodin also activated the Nrf2 pathway, decreased ROS levels, increased GSH levelss and GSH/GSSG ratio, and upregulated the activities of SOD, GR, CAT and GSH-Px after IAV infection. Suppression of Nrf2 via siRNA markedly blocked the inhibitory effects of emodin on IAV-induced activations of TLR4, p38/JNK, and NF-?B pathways and on IAV-induced production of IL-1?, IL-6 and expression of IAV M2 protein. Emodin also dramatically increased the survival rate of mice, reduced lung edema, pulmonary viral titer and inflammatory cytokines, and improved lung histopathological changes. In conclusion, emodin can inhibit IAV replication and influenza viral pneumonia, at least in part, by activating Nrf2 signaling and inhibiting IAV-induced activations of the TLR4, p38/JNK MAPK and NF-?B pathways.