Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters.

Triptolide (TP), the main bioactive component of Tripterygium wilfordii Hook F, can cause severe hepatotoxicity. Isoliquiritigenin (ISL) has been reported to be able to protect against TP-induced liver injury, but the mechanisms are not fully elucidated. This study aims to explore the role of nuclear transcription factor E2-related factor 2 (Nrf2) and hepatic transporters in TP-induced hepatotoxicity and the reversal protective effect of ISL. TP treatment caused both cytotoxicity in L02 hepatocytes and acute liver injury in mice. Particularly, TP led to the disorder of bile acid (BA) profiles in mice livers. Combined treatment of TP with ISL effectively alleviated TP-induced hepatotoxicity. Furthermore, ISL pretreatment enhanced Nrf2 expressions and nuclear accumulations and its downstream NAD(P)H: quinine oxidoreductase 1 (NQO1) expression. Expressions of hepatic P-gp, MRP2, MRP4, bile salt export pump, and OATP2 were also induced. In addition, in vitro transport assays identified that neither was TP exported by MRP2, OATP1B1, or OATP1B3, nor did TP influence the transport activities of P-gp or MRP2. All these results indicate that ISL may reduce the hepatic oxidative stress and hepatic accumulations of both endogenous BAs and exogenous TP as well as its metabolites by enhancing the expressions of Nrf2, NQO1, and hepatic influx and efflux transporters. Effects of TP on hepatic transporters are mainly at the transcriptional levels, and changes of hepatic BA profiles are very important in the mechanisms of TP-induced hepatotoxicity.

The Ethanol Extract of Licorice (Glycyrrhiza uralensis) Protects against Triptolide-Induced Oxidative Stress through Activation of Nrf2.

To investigate the potential role of nuclear factor erythroid 2-related factor 2 (Nrf2) in licorice ethanol extract (LEE) against triptolide- (TP-) induced hepatotoxicity, HepG2 cells were exposed to LEE (30, 60, and 90 mg·L-1) for 12 h and then treated with TP (50 nM) for 24 h. Besides, an acute liver injury model was established in ICR mice by a single dose of TP (1.0 mg·kg-1, i.p.). Relevant oxidant and antioxidant mediators were analyzed. TP led to an obvious oxidative stress as evidenced by increasing levels of ROS and decreasing GSH contents in HepG2 cells. In vitro results were likely to hold true in in vivo experiments. LEE protected against TP-induced oxidative stress in both in vitro and in vivo conditions. Furthermore, the decreased level of Nrf2 in the TP-treated group was observed. The mRNA levels of downstream genes decreased as well in ICR mice liver, whereas they increased in HepG2 cells. In contrast, LEE pretreatment significantly increased the level of Nrf2 and its downstream genes. LEE protects against TP-induced oxidative stress partly via the activation of Nrf2 pathway.

Glycyrrhetinic Acid Accelerates the Clearance of Triptolide through P-gp In Vitro.

Triptolide (TP) is an active ingredient isolated from Tripterygium wilfordii Hook. f. (TWHF), which is a traditional herbal medicine widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, its adverse reactions of hepatotoxicity and nephrotoxicity have been frequently reported which limited its clinical application. The aim of this study was to investigate the mechanism of glycyrrhetinic acid (GA) effecting on the elimination of TP in HK-2 cells and the role of the efflux transporters of P-gp and multidrug resistance-associated proteins (MRPs) in this process. An ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) analytical method was established to determine the intracellular concentration of TP. In order to study the role of efflux transporters of P-gp and MRPs in GA impacting on the accumulation of TP, the inhibitors of efflux transporters (P-gp: verapamil; MRPs: MK571) were used in this study. The results showed that GA could enhance the elimination of TP and reduce the TP accumulation in HK-2 cells. Verapamil and MK571 could increase the intracellular concentration of TP; in addition, GA co-incubation with verapamil significantly increased the TP cellular concentration compared with the control group. In conclusion, GA could reduce the accumulation of TP in HK-2 cells, which was related to P-gp. This is probably one of the mechanisms that TP combined with GA to detoxify its toxicity. Copyright © 2017 John Wiley & Sons, Ltd.

Maternal diet of polyunsaturated fatty acid altered the cell proliferation in the dentate gyrus of hippocampus and influenced glutamatergic and serotoninergic systems of neonatal female rats.

BACKGROUND: Long-chain polyunsaturated fatty acids (PUFAs) are major components of the phospholipids that forming the cell membrane. Insufficient availability of PUFAs during prenatal period decreases accretion of docosahexaenoic acid (DHA) in the developing brain. DHA deficiency is associated with impaired attention and cognition, and would precipitate psychiatric symptoms. However, clinical studies on the potential benefits of dietary DHA supplementation to neural development have yielded conflicting results. METHODS: To further investigate the neurochemical influence of maternal PUFAs levels, we assessed the functioning of various neurotransmitter systems including glutamatergic, dopaminergic, norepinephrinergic and serotoninergic systems in the brain of neonatal female rats by HPLC-MS/MS. Meanwhile, the cell proliferation of neonatal rats was investigated using immunefluorescence. RESULTS: Different maternal n-3 PUFAs dietary influenced the FA composition, cell proliferation in the dentate gyrus of hippocampus and the contents of γ-aminobutyric acid (GABA), glutamine (GLN), dopamine (DA) and its metabolites [3,4- dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA)], norepinephrine (NE), vanilmandelic acid (VMA) and 5-HT turnover in the brain of neonatal rats. However, the mRNA expression of key synthase of neurotransmitters remains stable. CONCLUSIONS: Our study showed that maternal deficiency of n-3 PUFAs might play an important role in central nervous system of neonatal female rats mainly through impairing the normal neurogenesis and influencing glutamatergic system and 5-HT turnover.

Long Chain Omega-3 Polyunsaturated Fatty Acid Supplementation Alleviates Doxorubicin-Induced Depressive-Like Behaviors and Neurotoxicity in Rats: Involvement of Oxidative Stress and Neuroinflammation.

Doxorubicin (DOX) is a chemotherapeutic agent widely used in human malignancies. Its long-term use can cause neurobiological side-effects associated with depression. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), the essential fatty acids found in fish oil, possess neuroprotecitve and antidepressant activities. Thus, the aim of this study was to explore the potential protective effects of ω-3 PUFAs against DOX-induced behavioral changes and neurotoxicity. ω-3 PUFAs were given daily by gavage (1.5 g/kg) over three weeks starting seven days before DOX administration (2.5 mg/kg). Open-field test (OFT) and forced swimming test (FST) were conducted to assess exploratory activity and despair behavior, respectively. Our data showed that ω-3 PUFAs supplementation significantly mitigated the behavioral changes induced by DOX. ω-3 PUFAs pretreatment also alleviated the DOX-induced neural apoptosis. Meanwhile, ω-3 PUFAs treatment ameliorated DOX-induced oxidative stress in the prefrontal cortex and hippocampus. Additionally, gene expression of pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, and the protein levels of NF-κB and iNOS were significantly increased in brain tissues of DOX-treated group, whereas ω-3 PUFAs supplementation significantly attenuated DOX-induced neuroinflammation. In conclusion, ω-3 PUFAs can effectively protect against DOX-induced depressive-like behaviors, and the mechanisms underlying the neuroprotective effect are potentially associated with its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.

The Protective Effects of Isoliquiritigenin and Glycyrrhetinic Acid against Triptolide-Induced Oxidative Stress in HepG2 Cells Involve Nrf2 Activation.

Triptolide (TP), an active ingredient of Tripterygium wilfordii Hook f., possesses a wide range of biological activities. Oxidative stress likely plays a role in TP-induced hepatotoxicity. Isoliquiritigenin (ISL) and glycyrrhetinic acid (GA) are potent hepatoprotection agents. The aim of the present study was to investigate whether Nrf2 pathway is associated with the protective effects of ISL and GA against TP-induced oxidative stress or not. HepG2 cells were treated with TP (50 nM) for 24 h after pretreatment with ISL and GA (5, 10, and 20 µM) for 12 h and 24 h, respectively. The results demonstrated that TP treatment significantly increased ROS levels and decreased GSH levels. Both ISL and GA pretreatment decreased ROS and meanwhile enhanced intracellular GSH content. Additionally, TP treatment obviously decreased the protein expression of Nrf2 and its target genes including HO-1 and MRP2 except NQO1. Moreover, both ISL and GA displayed activities as inducers of Nrf2 and increased the expression of HO-1, NQO1, and MRP2. Taken together the current data confirmed that ISL and GA could activate the Nrf2 antioxidant response in HepG2 cells, increasing the expression of its target genes which may be partly associated with their protective effects in TP-induced oxidative stress.

Contribution of NRG1 Gene Polymorphisms in Temporal Lobe Epilepsy.

The purpose of the present study was to investigate the possible association between temporal lobe epilepsy and NRG1 gene polymorphisms. A total of 73 patients and 69 controls were involved in this study. Genomic DNAs from the patients and controls were genotyped by polymerase chain reaction-ligase detection reaction method. There was an association of rs35753505 (T>C) with temporal lobe epilepsy (χ(2) = 6.730, P = .035). The frequency of risk allele C of rs35753505 was significantly higher (69.9%) in patients compared to controls (55.8%) (χ(2) = 6.023, P = .014). Interestingly, the significant difference of NRG1 genotype and allele frequency only existed among males, but not females. In addition, no statistically significant association was found between rs6994992, rs62510682 polymorphisms, and temporal lobe epilepsy. These data indicate that rs35753505 of NRG1 plays an important role in conferring susceptibility to the temporal lobe epilepsy in a Chinese Han population.

Progress on mechanism of Tripterygium wilfordii-induced liver injury and detoxification mechanism of licorice.

Tripterygium wilfordii has exihibited multiple pharmacological activities, such as anti-inflammatory, immune modulation, anti-tumor and anti-fertility. T. wilfordii have been used for the therapy of inflammation and autoimmune diseases including rheumatoid arthritis, immune complex nephritis and systemic lupus erythematosus clinically. However, it is well known that T. wilfordii has small margin between the therapeutic and toxic doses and could cause serious injury on digestive, reproductive and urogenital systems. Among all the organs, liver is one of the most remarkable targets of T. wilfordii-induced toxicities, and the damage is more serious than others. It is generally accepted that T. wilfordii-induced liver injury is a result of the combined effects of toxic elements of T. wilfordii. It is reported in several studies that the mechanism of T. wilfordii-induced liver injury may be related to lipid peroxidation, cell apoptosis and immune damage, and so on. Licorice is one of the most commonly used Chinese herbal medicine, with effects of heat- clearing and detoxicating, anti-inflammatory and hepatoprotective, reconciling various drugs, and so on. Licorice often accompany T. wilfordii in clinical application which can significantly reduce the liver injury induced by T. wilfordii. The attenuated effect is exact, but the mechanism is still a lack of in-depth study. This paper reviews the studies on T. wilfordii-induced liver injury and the related mechanism as well as licorice and other traditional Chinese medicine accompany T. wilfordii to reduce the injury in recent years, so as to provide reference for related research in the future.

Association between Vitamin D Receptor Gene Polymorphisms with Childhood Temporal Lobe Epilepsy.

Vitamin D (VD) is implicated in multiple aspects of human physiology and vitamin D receptor (VDR) polymorphisms are associated with a variety of neuropsychiatric disorders. Although VD deficiency is highly prevalent in epilepsy patients and converging evidence indicates a role for VD in the development of epilepsy, no data is available on the possible relationship between epilepsy and genetic variations of VDR. In this study, 150 controls and 82 patients with temporal lobe epilepsy (TLE) were genotyped for five common VDR polymorphisms (Cdx-2, FokI, BsmI, ApaI and TaqI) by the polymerase chain reaction-ligase detection reaction method. Our results revealed that the frequency of FokI AC genotype was significantly higher in the control group than in the patients (p = 0.003, OR = 0.39, 95% CI = 0.21-0.73), whereas the AA genotype of ApaI SNP was more frequent in patients than in controls (p = 0.018, OR = 2.92, 95% CI = 1.2-7.1). However, no statistically significant association was found between Cdx-2, BsmI and TaqI polymorphisms and epilepsy. Additionally, in haplotype analysis, we found the haplotype GAT (BsmI/ApaI/TaqI) conferred significantly increased risk for developing TLE (p = 0.039, OR = 1.62, 95% CI = 1.02-2.56). As far as we know, these results firstly underline the importance of VDR polymorphisms for the genetic susceptibility to epilepsy.

Fish oil supplementation alleviates depressant-like behaviors and modulates lipid profiles in rats exposed to chronic unpredictable mild stress.

BACKGROUND: Patients with major depressive disorder have a higher prevalence and incidence of dyslipidemia. However, clinical studies concerning the association between lipid levels and depression are inconsistent. Adipokines (like leptin and adiponectin) and ghrelin are strongly associated with lipid metabolism. Fish oil, which is reported to possess antidepressant effect, also have beneficial effects on lipid metabolism and the cardiovascular system. In the present study, we investigated lipid metabolism in rats exposed to chronic unpredictable mild stress (CUMS) and the effect of fish oil on lipid profiles, aforementioned adipokines and ghrelin. METHODS: Sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST) were used to evaluate the antidepressant-like effects of fish oil. After the behavior tests, peripheral blood were collected. Serum parameters, including fasting triglyceride (TG), total cholesterol (TCH), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), free fatty acid (FFA), glucose (GLU), adipokines (leptin, adiponectin) and ghrelin were assayed. RESULTS: After 5 weeks of CUMS procedures, rats were induced to depressive-like state, and exhibited increased serum levels of TCH, HDL-c, FFA and decreased serum levels of leptin and ghrelin, whereas the serum status of adiponectin, GLU, TG and LDL-c remained stable. Fish oil treatment showed robust antidepressant effect and reversed the stress-induced lipid disturbance and decrease in serum concentration of ghrelin. CONCLUSIONS: Our results suggested that CUMS altered the serum levels of lipid profiles, leptin and ghrelin in rats. Fish oil supplementation not only provided antidepressant-like effects, but also reversed the altered lipid profiles and ghrelin level in serum. Our data indicated that fish oil treatment exerts anti-depressant effect and regulates lipid disturbance simultaneously.