Yuchen Sheng

The involvement of p62–Keap1–Nrf2 antioxidative signaling pathway and JNK in the protection of natural flavonoid quercetin against hepatotoxicity

Quercetin, one of the most abundant dietary flavonoids, is reported to have protective function against various hepatotoxicant-induced hepatotoxicity. The present study aims to investigate the critical role of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidative signaling pathway in the protection of quercetin against hepatotoxicity. Quercetin prevented the cytotoxicity induced by a variety of hepatotoxicants including clivorine (Cliv), acetaminophen (APAP), ethanol, carbon tetrachloride (CCl4), and toosendanin (TSN) in human normal liver L-02 cells. Quercetin induced the nuclear translocation of Nrf2, along with the increased expression of the antioxidant responsive element (ARE)-dependent genes like catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), and heme oxygenase-1 (HO-1). In addition, the HO-1 inhibitor zinc protoporphyrin (ZnPP) and the GCL inhibitor L-buthionine-(S,R)-sulfoximine (BSO) both reduced the hepatoprotection induced by quercetin. Quercetin had no effect on kelch-like ECH-associated protein-1(Keap1) expression, but molecular docking results indicated the potential interaction of quercetin with the Nrf2-binding site in Keap1 protein. Quercetin increased the expression of p62, and p62 siRNA decreased quercetin-induced hepatoprotection. Quercetin induced the activation of c-Jun N-terminal kinase (JNK) in hepatocytes. JNK inhibitor SP600125 and JNK siRNA both reduced quercetin-induced hepatoprotection. SP600125 and JNK siRNA decreased the increased p62 expression induced by quercetin. In addition, SP600125 also decreased the increased mRNA and protein expression of GCLC, GCLM, and HO-1 induced by quercetin. Taken together, our present study demonstrates that quercetin prevents hepatotoxicity by inducing p62 expression, inhibiting the binding of Keap1 to Nrf2, and thus leading to the increased expression of antioxidative genes dependent on Nrf2. Meanwhile, our study indicates that JNK plays some regulation in this process.

Chlorogenic acid, a dietary polyphenol, protects acetaminophen-induced liver injury and its mechanism☆☆☆

Chlorogenic acid (CGA) is one of the most abundant dietary polyphenols, possessing well-known antioxidant capacity. The present study is designed to observe the protection provided by CGA against acetaminophen (AP)-induced liver injury in mice in vivo and the underlying mechanisms engaged in this process. Serum transaminases analysis and liver histological evaluation demonstrated the protection of CGA against AP-induced liver injury. CGA treatment decreased the increased number of liver apoptotic cells induced by AP in a dose-dependent manner. CGA also inhibited AP-induced cleaved activation of caspase-3, 7. Moreover, CGA reversed AP-decreased liver reduced glutathione (GSH) levels, glutamate-cysteine ligase (GCL) and glutathione reductase activity. Further results showed that CGA increased mRNA and protein expression of the catalytic subunit of GCL (GCLC), thioredoxin (Trx) 1/2 and thioredoxin reductase (TrxR) 1. Furthermore, CGA abrogated AP-induced phospholyated activation of ERK1/2, c-Jun N-terminal kinase (JNK), p38 kinases and molecular signals upstream. The results of this study demonstrate that CGA counteracts AP-induced liver injury at various levels by preventing apoptosis and oxidative stress damage, and more specifically, both the GSH and Trx antioxidant systems and the mitogen-activated protein kinase (MAPK) signaling cascade appear to be engaged in this protective mechanism.

Caffeic acid prevents acetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense system

UNLABELLED Acute liver failure induced by acetaminophen (APAP) overdose is the main cause of drug-induced liver injury (DILI). Caffeic acid (CA) is a phenolic compound from many natural products. This study aims to investigate the protective mechanism of CA in APAP-induced liver injury. The results of serum alanine/aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, liver glutathione (GSH) and reactive oxygen species (ROS) levels demonstrated the protection of CA against APAP-induced liver injury. Liver histological observation provided further evidences of CA-induced protection. CA was found to reverse the APAP-induced decreased cell viability in human normal liver L-02 cells and HepG2 cells. CA also reduced the increased cellular ROS level induced by APAP in hepatocytes. The results of luciferase assay and Western-blot analysis showed that CA increased the transcriptional activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the presence of APAP. Nrf2 siRNA reduced the protection of CA against APAP-induced hepatotoxicity. CA also reversed the APAP-induced decreased mRNA and protein expression of heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1(NQO1). In addition, HO-1 inhibitor zinc protoporphyrin (ZnPP) and NQO1 inhibitor diminutol (Dim) reduced the protection of CA against APAP-induced hepatotoxicity. CA also decreased the expression of kelch-like ECH-associated protein-1(Keap1). Molecular docking indicated the potential interacting of CA with Nrf2 binding site in the Keap1 protein. CA had little effect on the enzymatic activity of cytochrome P450 (CYP) 3A4 and CYP2E1 in vitro. In conclusion, we demonstrated that CA prevented APAP-induced hepatotoxicity by decreasing Keap1 expression, inhibiting binding of Keap1 to Nrf2, and thus activating Nrf2 and leading to increased expression of antioxidative signals including HO-1 and NQO1.

The gender-dependent difference of liver GSH antioxidant system in mice and its influence on isoline-induced liver injury

Intracellular reduced glutathione (GSH) antioxidant system is crucial for counteracting oxidative stress-induced liver injury. The present study was designed to observe the gender-dependent difference of GSH antioxidant system and its influence on hepatotoxic pyrrolizidine alkaloid (HPA) isoline-induced liver injury. Lower activities and protein expressions of glutamate-cysteine ligase (GCL) and glutathione peroxidase (GPx) were found in male mice livers than in female. Isoline is a natural HPA, our further results showed that male mice demonstrated more higher serum ALT/AST levels, less GSH amounts, lower GCL and GPx activities and proteins induced by isoline as compared to female. N-acetyl-l-cysteine (NAC), which is the precursor of cellular GSH biosynthesis, ameliorated liver injury induced by isoline. l-Buthionine-(S, R)-sulfoximine (BSO) and mercaptosuccinic acid (MA), inhibitors of GCL and GPx, both augmented isoline-induced cytotoxicity in cultured mice hepatocytes. BSO and MA also increased other natural HPAs clivorine and senecionine-induced cytotoxicity. Taken together, our results demonstrated the higher GCL and GPx activities in female mice, which indicated their crucial roles in regulating the resistance of liver injury induced by hepatotoxins in female. Meanwhile, our results also revealed the female-resistant liver injury induced by HPAs for the first time.

Andrographolide ameliorates diabetic retinopathy by inhibiting retinal angiogenesis and inflammation.

BACKGROUND Andrographolide (Andro) is the main compound distributed in medicinal herb Andrographis paniculata. This study aims to observe the amelioration of Andro on streptozotocin (STZ)-induced diabetic retinopathy (DR) in mice. METHODS STZ-induced non-proliferative DR (NPDR) for 2 months and proliferative DR (PDR) for 5 month in C57BL/6 mice were used in this study, respectively. Retinal vessels were observed by immunofluorescence staining for cluster of differentiation 31 (CD31). Evans blue permeation assay was used to detect the breakdown of blood-retinal barrier (BRB). Real-time PCR and immune-blot were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β. RESULTS Retinal immunofluorescence staining with CD31 showed that Andro reduced the increased retinal vessels in STZ-induced PDR mice. Evans blue permeation results demonstrated that Andro attenuated the breakdown of BRB in STZ-induced NPDR mice. In STZ-induced PDR mice, Andro decreased the increased vascular endothelial growth factor (VEGF) in serum and vitreous cavity, and reduced the increased retinal mRNA expression of VEGF and its receptors. In STZ-induced NPDR mice, Andro abrogated the nuclear translocation of nuclear factor κB (NF-κB) p65 and early growth response-1 (Egr-1), and reduced the increased phospho-NF-κBp65, -inhibitor of kappa B (IκB), and -IκB kinase (IKK). Andro also decreased the increased serum and retinal mRNA expression of TNF-α, IL-6, IL-1β, serpine1, and tissue factor (TF). CONCLUSIONS Andro ameliorates DR via attenuating retinal angiogenesis and inflammation, and VEGF, NF-κB, and Egr1 signaling pathways all play important roles in this process.

Scutellarin protects against the liver injury induced by diosbulbin B in mice and its mechanism

ETHNOPHARMACOLOGICAL RELEVANCE Diosbulbin B (DB) is the main hepatotoxic compound distributed in Dioscorea bulbifera L., which is widely used for the treatment of cancer and thyroid disorders in Asia. Scutellarin (SC) is the main compound in medicinal herb Scutellaria barbata D. Don, which is usually combined with Dioscorea bulbifera used for cancer therapy in clinic. AIM OF THE STUDY This study aims to investigate the protection of SC against the liver injury induced by DB and its engaged mechanism. In addition, the anti-tumor effect of DB and SC is further observed in vivo. MATERIALS AND METHODS The protection of SC against DB-induced liver injury was evaluated by detecting serum alanine/aspartate aminotransferases (ALT/AST) and alkaline phosphatase (ALP) activities, and further liver histological observation. The inflammatory response was assessed by detecting liver myeloperoxidase (MPO) activity, and serum levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon-γ (IFN-γ). Western-blot analysis was used to detect the protein expression. The oxidative liver injury was evaluated by detecting liver malondialdehyde (MDA) and glutathione (GSH) contents, and glutathione peroxidase (GPx) enzymatic activity. In vivo anti-tumor activity was analyzed in S180 tumor-bearing mice. RESULTS SC significantly decreased the increased serum ALT/AST, and ALP activities induced by DB. Liver histological observation evidenced the protection of SC against DB-induced liver injury. SC obviously reduced the increased liver MPO activity and the number of MPO-positive staining cells induced by DB. SC also reversed the decreased expression of inhibitor of κB (IκB) and the translocation of nuclear factor κB (NF-κB) p65 from cytoplasm to nucleus induced by DB. In addition, SC significantly abrogated the increased serum levels of TNF-α, IL-6, and IFN-γ induced by DB. SC decreased the increased liver MDA content induced by DB significantly, and it also increased liver GSH level. The decreased GPx protein expression and its enzymatic activity induced by DB were both obviously reversed after SC treatment. The results in S180 tumor-bearing mice showed that SC combined with DB significantly inhibited tumor growth in vivo. CONCLUSIONS Our results demonstrate that SC prevents DB-induced liver injury by attenuating NF-κB-mediated hepatic inflammation and ameliorating liver oxidative stress injury. Meanwhile, DB plus SC has significant anti-tumor activity in vivo. This study indicates the potential combination of DB with SC for the treatment of cancer in clinic.

Ethanol extract of Dendrobium chrysotoxum Lindl ameliorates diabetic retinopathy and its mechanism.

Diabetic retinopathy (DR) is the most common and serious complication of diabetes mellitus (DM). The present study investigates the amelioration of ethanol extract of Dendrobium chrysotoxum Lindl (DC) on streptozotocin (STZ)-induced DR and its engaged mechanism. Retinal immunofluorescence staining with cluster of differentiation 31 (CD31) demonstrated that DC (30-300 mg/kg) decreased the increased retinal vessels in STZ-induced diabetic rats. Retinal histopathological observation also showed that retinal vessels were decreased in DC-treated diabetic rats. DC decreased the increased retinal mRNA expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in diabetic rats, and DC also decreased the elevated serum VEGF level. Immunohistochemical staining further evidenced that DC decreased VEGF and VEGFR2 expression in retinas. Retinal mRNA expression of matrix metalloproteinase (MMP) 2/9 was decreased in DC (300 mg/kg)-treated diabetic rats. Serum levels of MMP 2/9, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A/B, insulin-like growth factor 1 (IGF-1), interleukin 1β (IL-1β), and IL-6 were all decreased in DC-treated diabetic rats. In addition, DC decreased the increased phosphorylation of p65 and the increased expression of intercellular adhesion molecule-1 (ICAM-1). In conclusion, DC can alleviate retinal angiogenesis during the process of DR via inhibiting the expression of VEGF/VEGFR2, and some other pro-angiogenic factors such as MMP 2/9, PDGF A/B, bFGF, IGF-1. In addition, DC can also ameliorate retinal inflammation via inhibiting NFκB signaling pathway.

The difference of glutathione antioxidant system in newly weaned and young mice liver and its involvement in isoline-induced hepatotoxicity

Cellular glutathione antioxidant system plays important roles in counteracting hepatotoxins-induced oxidative stress injury. The present study was designed to observe the differences of this system in newly weaned and young mice liver and its involvement in the susceptibility to isoline-induced liver injury. Our results showed that liver reduced glutathione (GSH) amounts were higher in newly weaned mice than young mice. Glutamate-cysteine ligase (GCL) activity was higher in newly weaned mice due to the higher expression of catalytic subunit of GCL (GCLC) protein and mRNA. However, the activities of glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) were higher in young mice liver, which might be due to the higher expression of GR, GPx-1, and GST-Pi proteins. Next, the results of AST analysis and histopathological evaluation showed that newly weaned mice demonstrated more severe liver injury induced by isoline. Furthermore, liver GSH amounts and the activities of GR, GPx, and GST were all lower in newly weaned mice than young mice after treated with isoline. Depletion of cellular GSH by d,l-buthionine-(S, R)-sulfoximine (BSO) aggravated isoline-induced cytotoxicity, while N-acetyl-l cysteine (NAC) ameliorated such cytotoxicity. Furthermore, the inhibitors of GR, GPx, and GST all aggravated isoline-induced cytotoxicity. In conclusion, our results demonstrated the differences of glutathione antioxidant system between newly weaned and young mice liver. Meanwhile, our results also revealed age-dependent liver injury induced by isoline for the first time, which might be due to the different responses of glutathione antioxidant system to isoline between newly weaned and young mice.

Erianin inhibits high glucose-induced retinal angiogenesis via blocking ERK1/2-regulated HIF-1α-VEGF/VEGFR2 signaling pathway

Erianin is a natural compound found in Dendrobium chrysotoxum Lindl. Diabetic retinopathy (DR) is a serious and common microvascular complication of diabetes. This study aims to investigate the inhibitory mechanism of erianin on retinal neoangiogenesis and its contribution to the amelioration of DR. Erianin blocked high glucose (HG)-induced tube formation and migration in choroid-retinal endothelial RF/6A cells. Erianin inhibited HG-induced vascular endothelial growth factor (VEGF) expression, hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus and ERK1/2 activation in RF/6A and microglia BV-2 cells. MEK1/2 inhibitor U0126 blocked HG-induced HIF-1α and ERK1/2 activation in both above two cells. In addition, erianin abrogated VEGF-induced angiogenesis in vitro and in vivo, and also inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and its downstream cRaf-MEK1/2-ERK1/2 and PI3K-AKT signaling pathways in RF/6A cells. Furthermore, erianin reduced the increased retinal vessels, VEGF expression and microglia activation in streptozotocin (STZ)-induced hyperglycemic and oxygen-induced retinopathy (OIR) mice. In conclusion, our results demonstrate that erianin inhibits retinal neoangiogenesis by abrogating HG-induced VEGF expression by blocking ERK1/2-mediated HIF-1α activation in retinal endothelial and microglial cells, and further suppressing VEGF-induced activation of VEGFR2 and its downstream signals in retinal endothelial cells.

Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals

Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury.總結目 的本研究旨在观察绿原酸对乙酰氨基酚诱导肝损伤的解毒作用及其机理。创新点发现CYP450 代谢酶和一些重要的抗氧化信号分子(如Prx 家族蛋白等)参与调控了绿原酸抑制对乙酰氨基酚的肝毒性。方 法检测小鼠血清转氨酶含量,检测体外CYP2E1、 CYP3A4 和CYP1A2 酶活性,检测肝组织中丙二醛(MDA)、谷胱甘肽(GSH)和活性氧(ROS)含量,用实时聚合酶链反应(real-time PCR)检测肝组织中Prx1-6、Ephx2、Polr2k、Fmo5、Nrf2 等的mRNA 表达情况。结 论绿原酸可以明显抑制对乙酰氨基酚造成的急性肝损伤。给药组小鼠血清中的转氨酶与模型组相比均有显著下降,绿原酸在体外可以微弱抑制 CYP2E1 和CYP1A2 代谢酶的活性,通过MDA、 GSH 和ROS 分析发现绿原酸可以抑制对乙酰氨基酚造成的氧化应激损伤。通过real-time PCR 分析发现对乙酰氨基酚降低了抗氧化酶Prx 家族、 Ephx2、Polr2k 和Nrf2 的基因表达,而绿原酸可以逆转对乙酰氨基酚降低的这些基因的表达。