Xiaomei Fan

Hepato-protective effect of resveratrol against acetaminophen-induced liver injury is associated with inhibition of CYP-mediated bioactivation and regulation of SIRT1-p53 signaling pathways.

Resveratrol (RES) has been shown to possess many pharmacological activities including protective effect against liver damage induced by hepatotoxins. In the present study, the hepato-protective effect of RES against acetaminophen (APAP)-induced liver injury in mice and the involved mechanisms was investigated. This study clearly demonstrated that administration of RES three days before APAP treatment significantly alleviated APAP-induced hepatotoxicity, as evidenced by morphological, histopathological, and biochemical assessments such as GSH content and serum ALT/AST activity. Treatment with RES resulted in significant inhibition of CYP2E1, CYP3A11, and CYP1A2 activities, and then caused significant inhibition of the bioactivation of APAP into toxic metabolite NAPQI. Pretreatment with RES significantly reduced APAP-induced JNK activation to protect against mitochondrial injury. Additionally, RES treatment significantly induced SIRT1 and then negatively regulated p53 signaling to induce cell proliferation-associated proteins including cyclin D1, CDK4, and PCNA to promote hepatocyte proliferation. This study demonstrated that RES prevents APAP-induced hepatotoxicity by inhibition of CYP-mediated APAP bioactivation and regulation of SIRT1, p53, cyclin D1 and PCNA to facilitate liver regeneration following APAP-induced liver injury.

Low Dose of Oleanolic Acid Protects against Lithocholic Acid–Induced Cholestasis in Mice: Potential Involvement of Nuclear Factor-E2-Related Factor 2-Mediated Upregulation of Multidrug Resistance-Associated Proteins

Oleanolic acid (OA) is a natural triterpenoid and has been demonstrated to protect against varieties of hepatotoxicants. Recently, however, OA at high doses was reported to produce apparent cholestasis in mice. In this study, we characterized the protective effect of OA at low doses against lithocholic acid (LCA)–induced cholestasis in mice and explored further mechanisms. OA cotreatment (5, 10, and 20 mg/kg, i.p.) significantly improved mouse survival rate, attenuated liver necrosis, and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; more importantly, serum total bile acids and bilirubin, as well as hepatic total bile acids were also remarkably reduced. Gene and protein expression analysis showed that hepatic expression of multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4 was significantly increased by OA cotreatment, whereas other bile acid metabolism- and transport-related genes, including Na+/taurocholate cotransporter, organic anion transporter 1b2, bile salt export pump, multidrug resistance protein 3, Cyp3a11, Cyp2b10, Sulfotransferase 2a1 (Sult2a1), and UDP-glucuronosyltransferase 1a1 (Ugt1a1), were only slightly changed. OA also caused increased nuclear factor-E2–related factor (Nrf2) mRNA expression and nuclear protein accumulation, whereas nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor were not significantly influenced by OA. Luciferase (Luc) assays performed in HepG2 cells illustrated that OA was a strong Nrf2 agonist with moderate PXR and weak FXR agonism. Finally, in mouse primary cultured hepatocytes, OA dose- and time-dependently induced expression of Mrp2, Mrp3, and Mrp4; however, this upregulation was abrogated when Nrf2 was silenced. In conclusion, OA produces a protective effect against LCA-induced hepatotoxicity and cholestasis, possibly due to Nrf2-mediated upregulation of Mrp2, Mrp3, and Mrp4.

Hepato-protective effects of six schisandra lignans on acetaminophen-induced liver injury are partially associated with the inhibition of CYP-mediated bioactivation.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra fructus is widely-used traditional Chinese medicine which possesses hepato-protective potential. Schisandrin A (SinA), Schisandrin B (SinB), Schisandrin C (SinC), Schisandrol A (SolA), Schisandrol B (SolB), and Schisantherin A (SthA) are the major bioactive lignans. Most recently, we found SolB exerts significant hepato-protection against APAP-induced liver injury. In this study, the protective effects of the other five schisandra lignans against APAP-induced acute hepatotoxicity in mice were investigated and compared with that of SolB. The results of morphological and biochemical assessment clearly demonstrated significant protective effects of SinA, SinB, SinC, SolA, SolB, and SthA against APAP-induced liver injury. Among these schisandra lignans, SinC and SolB exerted the strongest hepato-protective effects against APAP-induced hepatotoxicity. Six lignans pretreatment before APAP dosing could prevent the depletions of total liver glutathione (GSH) and mitochondrial GSH caused by APAP. Additionally, the lignans treatment inhibited the enzymatic activities of three CYP450 isoforms (CYP2E1, CYP1A2, and CYP3A11) related to APAP bioactivation, and further decreased the formation of APAP toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI) in mouse microsomal incubation system. This study demonstrated that SinA, SinB, SinC, SolA, SolB and SthA exhibited significant protective actions toward APAP-induced liver injury, which was partially associated with the inhibition of CYP-mediated APAP bioactivation.

Wuzhi tablet (Schisandra Sphenanthera extract) protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of NRF2-ARE and p53/p21 pathways.

Schisandra sphenanthera is widely used as a tonic and restorative in many countries to enhance the function of liver and other organs. Wuzhi tablet (WZ) is a preparation of an ethanol extract of Schisandra sphenanthera. Our previous study demonstrated that WZ exerted a protective effect toward acetaminophen (APAP)-induced hepatotoxicity. However, the molecular mechanisms of this protection remain unclear. This study aimed to determine what molecular pathways contributed to the hepatoprotective effects of WZ against APAP toxicity. Administration of WZ 3 days before APAP treatment significantly attenuated APAP hepatotoxicity in a dose-dependent manner and reduced APAP-induced JNK activation. Treatment with WZ resulted in potent inhibition of CYP2E1, CYP3A11, and CYP1A2 activities and then caused significant inhibition of the formation of the oxidized APAP metabolite N-acetyl-p-benzoquinone imine–reduced glutathione. The expression of NRF2 was increased after APAP and/or WZ treatment, whereas KEAP1 levels were decreased. The protein expression of NRF2 target genes including Gclc, Gclm, Ho-1, and Nqo1 was significantly increased by WZ treatment. Furthermore, APAP increased the levels of p53 and its downstream gene p21 to trigger cell cycle arrest and apoptosis, whereas WZ pretreatment could inhibit p53/p21 signaling to induce cell proliferation-associated proteins including cyclin D1, CDK4, PCNA, and ALR to promote hepatocyte proliferation. This study demonstrated that WZ prevented APAP-induced liver injury by inhibition of cytochrome P450–mediated APAP bioactivation, activation of the NRF2–antioxidant response element pathway to induce detoxification and antioxidation, and regulation of the p53, p21, cyclin D1, CDK4, PCNA, and ALR to facilitate liver regeneration after APAP-induced liver injury.

Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.

Dynamic and Coordinated Regulation of KEAP1-NRF2-ARE and p53/ p21 Signaling Pathways Is Associated with Acetaminophen Injury Responsive Liver Regeneration

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury. Compensatory liver regeneration is crucial for the final outcome of toxicant-induced injury. However, the molecular mechanisms underlying compensatory liver regeneration in mice after APAP-induced liver injury are not completely understood. This study aimed to investigate the role of dynamic and coordinated regulation of Kelch-like ECH-associated protein 1 (KEAP1)–nuclear factor erythroid 2–related factor 2 (NRF2)– antioxidant response element (ARE) and p53/p21 pathways in APAP injury-responsive liver regeneration. We found that mice exhibited massive hepatic toxicity during the first 12 hours after 400 mg/kg APAP treatment, but responsive liver recovery occurred beyond 24 hours as demonstrated by histopathological and biochemical assessments. The expression and nuclear accumulation of NRF2 was increased after APAP treatment. The expression of NAD(P)H:quinone oxidoreductase 1, glutamate-cysteine ligase modifier subunit, and heme oxygenase-1 was inhibited during the first 24 hours and then induced to limit oxidative damage. The content of p53 and its downstream target p21 were significantly increased upon APAP exposure and subsequently decreased to normal levels at 48 hours. Furthermore, levels of cyclin D1, cyclin D–dependent kinase 4, proliferating cell nuclear antigen, and augmenter of liver regeneration at 48 hours were enhanced, suggesting initiation of hepatocyte proliferation and tissue repair. These results demonstrated that dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways was associated with compensatory liver regeneration after APAP-induced acute liver injury.

Schisandrol B protects against acetaminophen-induced acute hepatotoxicity in mice via activation of the NRF2/ARE signaling pathway

Aim:The nuclear factor erythroid 2-related factor 2 (NRF2) acts through the antioxidant response element (ARE) to regulate the expression of many detoxifying and antioxidant genes responsible for cytoprotective processes. We previously reported that Schisandrol B (SolB) isolated from Schisandra sphenanthera produced a protective effect against acetaminophen (APAP)-induced liver injury. In this study we investigated whether the NRF2/ARE signaling pathway was involved in this hepato-protective effect.Methods:Male C57BL/6 mice were treated with SolB (200 mg·kg−1·d−1, ig) for 3 d before injection of APAP (400 mg/kg, ip). Serum and liver tissue samples were collected 6 h later. The mRNA and protein expression were measured using qRT-PCR and Western blot assay, respectively. The activation of NRF2 was examined in HepG2 cells using luciferase reporter gene assay.Results:SolB pretreatment significantly alleviated the hepatic injury (large patchy necrosis and hyperemia of the hepatic sinus), the increase of serum AST, ALT levels and hepatic MDA contents, and the decrease of liver and mitochondrial glutathione levels in APAP-treated mice. Furthermore, SolB pretreatment significantly increased nuclear accumulation of NRF2 and increased hepatic expression of NRF2 downstream proteins, including GCLC, GSR, NQO1, GSTs, MRP2, MRP3 and MRP4 in APAP-treated mice. Moreover, treatment with SolB (2.5–20 μmol/L) dose-dependently increased the activity of NRF2 reporter gene in HepG2 cells.Conclusion:SolB exhibits a remarkable protective effect against APAP-induced hepatotoxicity, partially via activation of the NRF2/ARE pathway and regulation of NRF2 target genes, which induce detoxification and increase antioxidant capacity.

Oleanolic acid attenuates obstructive cholestasis in bile duct-ligated mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Obstructive cholestasis is characterized by impairment of hepatic canalicular bile efflux and there are no clinically effective drugs to cure except surgeries. Previously we revealed that oleanolic acid (OA) protected against lithocholic acid (LCA)-induced intrahepatic cholestasis in mice. Cholestasis caused by LCA is characterized by segmental bile duct obstruction, whether OA possesses the beneficial effect on completed obstructive cholestasis induced by bile duct ligation (BDL) remains unknown. In this study, we demonstrated that BDL-induced mice liver pathological change, and increase in serum levels of ALT, AST and ALP were all significantly reduced by OA (20 mg/kg, i.p.). Meanwhile, OA also lowered total bilirubin and total bile acids levels in serum, as well as total bile acids level in liver, in contrast, urinary total bile acids output was remarkably up-regulated by OA. Gene expression analysis showed that OA caused significant increased mRNA expression of MRP3 and MRP4 located at hepatic basolateral membrane, and restoration of MRP2 and BSEP located at hepatic cannalicular membrane. Furthermore, significant NRF2 protein accumulation in nucleus was also observed in OA treated mice. In mice primary cultured hepatocytes, the effects of OA on MRP2, MRP3 and MRP4 expression were directly proved to be mediated via NRF2 activation, and BSEP downregulation induced by OA was in part due to FXR antagonism. Luciferase assay performed in Hep G2 cells also illustrated that OA was a partial FXR antagonist. Taken together, we conclude that OA attenuates obstructive cholestasis in BDL mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Hepatoprotective Effects of Schisandra sphenanthera Extract against Lithocholic Acid–Induced Cholestasis in Male Mice Are Associated with Activation of the Pregnane X Receptor Pathway and Promotion of Liver Regeneration

We previously reported that the ethanol extract of Schisandra sphenanthera [Wuzhi (WZ) tablet] significantly protects against acetaminophen-induced hepatoxicity. However, whether WZ exerts a protective effect against cholestasis remains unclear. In this study, the protective effect of WZ on lithocholic acid (LCA)–induced intrahepatic cholestasis in mice was characterized and the involved mechanisms were investigated. WZ pretreatment (350 mg/kg) with LCA significantly reversed liver necrosis and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity. More importantly, serum total bile acids and total bilirubin were also remarkably reduced. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis showed that hepatic expression of pregnane X receptor (PXR) target genes such as CYP3A11 and UDP-glucuronosyltransferase (UGT) 1A1 were significantly increased by WZ treatment. Luciferase assays performed in LS174T cells illustrated that WZ extract and its six bioactive lignans could all activate human PXR. In addition, WZ treatment significantly promoted liver regeneration via inhibition of p53/p21 to induce cell proliferation–associated proteins such as cyclin D1 and proliferating cell nuclear antigen. In conclusion, WZ has a protective effect against LCA-induced intrahepatic cholestasis, partially owing to activation of the PXR pathway and promotion of liver regeneration.