Zhen-yan Hou

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

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.

Broad targeting of triptolide to resistance and sensitization for cancer therapy

Cancer cell resistance to current anticancer therapeutics as well as the side effects are still obstacles to successful cancer therapy. Hence, the development of novel anticancer agents or therapeutics is of vital significance, and especially rational adjuvant therapies containing low-cost natural products with multiple targets have attracted great interests. Triptolide, the main biocomponent of Tripterygium wilfordii Hook F, is restricted in clinical applications mainly due to its severe systemic toxicities, although it has shown strong antitumor activities in preclinical studies. Mounting evidence suggests that triptolide at low doses as an adjuvant therapeutic agent circumvents resistance to current anticancer therapies, enhances the anticancer effectiveness, and relieves toxicities of both triptolide and anticancer therapies. Furthermore, several unique antitumor targets of triptolide make it superior to other therapeutics. The molecular mechanisms of triptolide-induced anti-resistance and sensitization effects include changes in ATP-binding cassette transporters, induction of apoptosis pathways, increase in tumor suppressors and decrease in oncogenic factors, and interactions with the RNA polymerase II complex; targeting cancer stem cells and tumor-microenvironment-mediated resistance are also involved. Besides, some synthetic derivatives and novel delivery systems of triptolide are also developed to enhance the water-solubility and reduce the toxicity, which will also be discussed.

Nrf2‑dependent antioxidant response mediated the protective effect of tanshinone IIA on doxorubicin‑induced cardiotoxicity

Doxorubicin (DOX), a potent and widely used anticancer agent, can give rise to severe cardiotoxicity that limits its clinical use by inducing oxidative stress. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the central regulator of cellular responses to electrophilic/oxidative stress, which serves a critical role in maintenance of normal cardiac function. Tanshinone IIA (Tan IIA) has previously been reported to protect against DOX-induced cardiotoxicity. The aim of the present study was to elucidate whether Nrf2 signaling serves a role in the underlying mechanism. In the animal model, DOX induced acute cardiotoxicity, whereas Tan IIA pretreatment reduced the activity of myocardial enzymes, and increased activity of the antioxidant enzymes superoxide dismutase, catalase and glutathione (GSH). Furthermore, Tan IIA pretreatment (3–10 µM) significantly increased the cell viability and markedly restored morphological changes in DOX-injured H9c2 cells, decreased the generation of reactive oxygen species, and increased the level of intracellular GSH. Additionally, Tan IIA pretreatment also induced the nuclear accumulation of Nrf2 and its downstream genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone) 1, and glutamate-cysteine ligase catalytic subunit in both the mice cardiac tissues and H9c2 cells. Nrf2 knockdown by small interfering RNA downregulated Tan IIA-induced Nrf2 activation and reversed the effect of Tan IIA on the DOX-induced inhibition of cell viability. These results suggest that the Nrf2-dependent antioxidant response mediates the protective effect of Tan IIA on DOX-induced cardiotoxicity.

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.

Danshen modulates Nrf2-mediated signaling pathway in cisplatin-induced renal injury

SummaryDanshen, an efficacious agent for cardiovascular diseases, has been found to play an essential role in kidney injury. In the present study, the effect of Danshen on cisplatin-induced renal dysfunction was investigated in a mouse model. Danshen was administered to mice at a dose of 3 g/kg 4 days before and 3 days after cisplatin treatment. A single intraperitoneal injection of 20 mg/kg cisplatin was used to induce nephrotoxicity. The mice were sacrificed 72 h after cisplatin intoxication. Biochemical parameters including serum creatinine and blood urea nitrogen were analyzed. Histopathological changes of kidney tissues were detected using HE staining. Antioxidant enzymes (GSH-Px and SOD) and peroxidative product (MDA) were detected. Protein expressions of Nrf2 and its target genes including HO-1 and NQO1 were measured by Western blotting. The results showed that pretreatment with Danshen significantly reduced serum creatinine and blood urea nitrogen in the cisplatin-treated mice. Histopathological examination showed that Danshen mitigated the renal damage induced by cisplatin. Moreover, Danshen restored the activities of antioxidant enzymes (GSH-Px and SOD) and normalized the MDA contents in renal tissues. Western blotting revealed that Danshen enhanced the expressions of Nrf2 and its target genes in cisplatin-exposed mice. It was suggested that Danshen protects against the cisplatin-induced renal impairment in the mice, which is potentially associated with the upregulation of Nrf2-mediated signaling pathway.Danshen, an efficacious agent for cardiovascular diseases, has been found to play an essential role in kidney injury. In the present study, the effect of Danshen on cisplatin-induced renal dysfunction was investigated in a mouse model. Danshen was administered to mice at a dose of 3 g/kg 4 days before and 3 days after cisplatin treatment. A single intraperitoneal injection of 20 mg/kg cisplatin was used to induce nephrotoxicity. The mice were sacrificed 72 h after cisplatin intoxication. Biochemical parameters including serum creatinine and blood urea nitrogen were analyzed. Histopathological changes of kidney tissues were detected using HE staining. Antioxidant enzymes (GSH-Px and SOD) and peroxidative product (MDA) were detected. Protein expressions of Nrf2 and its target genes including HO-1 and NQO1 were measured by Western blotting. The results showed that pretreatment with Danshen significantly reduced serum creatinine and blood urea nitrogen in the cisplatin-treated mice. Histopathological examination showed that Danshen mitigated the renal damage induced by cisplatin. Moreover, Danshen restored the activities of antioxidant enzymes (GSH-Px and SOD) and normalized the MDA contents in renal tissues. Western blotting revealed that Danshen enhanced the expressions of Nrf2 and its target genes in cisplatin-exposed mice. It was suggested that Danshen protects against the cisplatin-induced renal impairment in the mice, which is potentially associated with the upregulation of Nrf2-mediated signaling pathway.

Pharmacokinetic properties and bioequivalence of spironolactone tablets in fasting and fed healthy Chinese male subjects.

BACKGROUND AND PURPOSE Spironolactone is a potassium-sparing diuretic and is a competitive antagonist of aldosterone, which is widely used in the treatment of primary aldosteronism, essential hypertension, congestive cardiac failure, and various edematous states. The purpose of this study was to compare the pharmacokinetic properties and bioequivalence of the two formulations of spironolactone tablets in healthy Chinese male subjects under fasting and fed condition. METHODS A total of 40 male subjects were enrolled in this randomized, open-label, two-period crossover study, subjects in 2 groups (20 individuals in each group) received a single 100-mg dose of test or reference spironolactone tablet formulations with a 2-week washout period under both fasting and fed condition. The plasma concentrations of canrenone, a major active metabolite of spironolactone, were quantified by a validated high performance liquid chromatography-tandem mass spectrometry method. The pharmacokinetic parameters including AUC0-tlast, AUC0-∞, tmax, and Cmax were employed to test bioequivalence. RESULTS The relative bioavailability was 99.2 ± 11.6% and 97.6 ± 7.4% under fasting and fed condition, respectively. The 90% confidence intervals of the adjusted geometric mean ratio (test/reference) of Cmax, AUC0-tlast, and AUC0-∞ were 89.7-113.8%, 93.9-103.3%, and 90.0-103.0% in fasting study and 87.7-102.3%, 95.1-99.5%, and 94.1-98.9% in fed study, respectively. CONCLUSIONS Based on pharmacokinetic parameters and the Chinese Food and Drug Administrations guidance and regulatory criteria for bioequivalence, the test and reference formulations of spironolactone were bioequivalent under both fasting and fed condition. Both formulations were generally well tolerated, with no adverse reaction reported.