Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation.

BACKGROUND: Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF). AIMS: To investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation. MATERIALS AND METHODS: In vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2-/- were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection. RESULTS: Our results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2-/- HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2-/- mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2-/- mice. CONCLUSIONS: These investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.

Beneficial effects of estrogens in obstructive sleep apnea hypopnea syndrome.

Epidemiological studies showing the higher frequency of obstructive sleep apnea hypopnea syndrome in men, polycystic ovary syndrome (PCOS), and in post-menopausal women suggest the beneficial role of estrogen. These findings are well supported by the pre-clinical studies (ten research studies described in this review) showing that estrogen and phytoestrogens attenuate the deleterious effects of chronic intermittent hypoxia (obstructive apnea in animals) on the genioglossal muscles and on other organs (co-morbidities) in ovariectomized rodents. Moreover, clinical studies (four research studies described in this review) have also shown the beneficial role of estrogen therapy on the parameters of obstructive apnea in post-menopausal women. The beneficial effects of estrogen and phytoestrogens on obstructive sleep apnea and its co morbidities have been attributed to increase in thioredoxin, Nrf-2, activation of p38 MAP kinases, inhibition of vagal C fibers, and attenuation of HIF-1α. It is possible that estrogen-mediated activation of p38 MAP kinase may inhibit HIF-1α to attenuate lung inflammation, which may inhibit the activation of vagal C fibers to attenuate bronchoconstriction and prevent obstruction during sleep. Moreover, estrogen-mediated increase in thioredoxin and Nrf-2 may also contribute in increasing antioxidant defense and attenuating inflammation.

Beneficial Effects of Electroacupuncture on Neuropathic Pain Evoked by Spinal Cord Injury and Involvement of PI3K-mTOR Mechanisms.

The purpose of this study was to examine the beneficial effects of electroacupuncture (EA) on neuropathic pain evoked by spinal cord injury (SCI) and determine the underlying molecular mechanisms of these effects. SCI was induced in rats. Behavioral tests were performed to examine pain responses induced by mechanical and thermal stimulation. Western blot analysis was used to measure the protein expression of phosphorylated mammalian target of rapamycin (p-mTOR), mTOR-mediated phosphorylated ribosomal protein S6 kinase beta-1 (p-S6K1), and phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1) in the superficial dorsal horn of the spinal cord. We showed that SCI increased the expression of p-mTOR, p-S6K1, and p-4E-BP1. The EA intervention attenuated the upregulation of mTOR signaling and alleviated mechanical and thermal pain responses in SCI rats. Blocking spinal mTOR by intrathecal injection of rapamycin also inhibited mechanical and thermal pain. In addition, blocking spinal phosphorylated phosphatidylinositide 3-kinase (p-PI3K) pathway attenuated p-mTOR pathways and mechanical and thermal hyperalgesia in SCI rats. EA also decreased the enhanced p-PI3K in the superficial dorsal horn of SCI rats. In conclusion, findings revealed specific signaling pathways that lead to neuropathic pain in response to SCI, including activation of PI3K-mTOR signaling. Further, results link the beneficial role of EA in alleviating SCI-induced neuropathic pain to its effect on these molecular mechanisms.

Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression.

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti-inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical-induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real-time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn-induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose-dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn-induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348-1355, 2017. © 2017 Wiley Periodicals, Inc.

[Effect of ginsenoside Rh2 on transplanted-tumor and expression of JAM in mice].

OBJECTIVE: To discuss the anti-tumor activity of ginsenoside Rh2, we observed the expressions of the junction adhesion molecul (JAM) in transplanted-tumor in mice. METHOD: The models of 40 transplanted-tumor mice that were established by subsequently injecting cancer ascite of mice (S180) with 0.2 mL per mouse into the preepipodite skin were divided into two groups. Experiment group was drenched with 2 mL ginsenoside Rh2 per mouse, equating to a dose 20 mg x kg(-1). Control group was drenched with 2 mL normal saline per mouse. The expression of JAM-1, JAM-2 in the lymphatics, blood vessels and tumours were observed by immunohistochemical staining. RESULT: The expression of JAM-1 on the cancer cells was significantly decreased in experiment group (IA 340.55) as compared with control group (IA 549.90, P<0.05). However, JAM-2 weakly expressed in both two groups. The density of blood vessels in which JAM-1, JAM-2 expressed showed 2.33 and 1.34 in control group, and 1.09 and 0.9 in experiment group respectively. Moreove, the density of lymph vessels were respectively 2.23 and 1.88 in control group compared with 0.99 and 0.79 in experiment group. The expression in blood vessels and lymph vessels in control group were significantly higher than those in experiment group, respectively (P<0.05). CONCLUSION: Ginsenoside Rh2 can affect the tumor growth, further angiogenesis and lymphangiogenesis by down-regulating JAM expression in tumor.