Neuroprotective effects of Ginkgolide B in focal cerebral ischemia through selective activation of prostaglandin E2 receptor EP4 and the downstream transactivation of epidermal growth factor receptor.

The present study was undertaken to identify whether prostaglandin E2 receptor is the potential receptor/binding site for Ginkgolide A, Ginkgolide B, Ginkgolide K, and Bilobalide, the four main ingredients of the Ginkgo biloba L., leaves. Using functional assays, we identified EP4, coupled with Gs protein, as a target of Ginkgolide B. In human neuroblastoma SH-SY5Y cells suffered from oxygen-glucose deprivation/reperfusion, Ginkgolide B-activated PKA, Akt, and ERK1/2 as well as Src-mediated transactivation of epidermal growth factor receptor. These resulted in downstream signaling pathways, which enhanced cell survival and inhibited apoptosis. Knockdown of EP4 prevented Ginkgolide B-mediated Src, epidermal growth factor receptor (EGFR), Akt, and ERK1/2 phosphorylation and neuroprotective effects. Moreover, Src inhibitor prevented Ginkgolide B-mediated EGFR transactivation and the downstream Akt and ERK1/2 activation, while the phosphorylation of PKA induced by Ginkgolide B was not affected, indicating Ginkgolide B might transactivate EGFR in a ligand-independent manner. EP4 knockdown in a rat middle cerebral artery occlusion (MCAO) model prevented Ginkgolide B-mediated infarct size reduction and neurological assessment improvement. At the same time, the increased expressions of p-Akt, p-ERK1/2, p-PKA, p-Src, and p-EGFR and the deceased expression of cleaved capases-3 induced by Ginkgolide B in cerebral cortex were blocked due to EP4 knockdown. In conclusion, Ginkgolide B exerts neuroprotective effects in rat MCAO model through the activation of EP4 and the downstream transactivation of EGFR.

Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo.

Ginkgolide terpenoid lactones, including ginkgolides and bilobalide, are two crucial bioactive constituents of extract of Ginkgo biloba (EGb) which was used in the treatment of cardiovascular and cerebrovascular diseases. The aims of this study were to investigate the antioxidant effects and mechanism of ginkgolides (ginkgolide A (GA), ginkgolide B (GB), ginkgolide K (GK)) and bilobalide (BB) against oxidative stress induced by transient focal cerebral ischemia. In vitro, SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) for 4 h followed by reoxygenation with ginkgolides and BB treatments for 6 h, and then cell viability, superoxide dismutase (SOD), and ROS were respectively detected using kit. Western blot was used to confirm the protein levels of hemeoxygenase-1 (HO-1), quinone oxidoreductase l (Nqo1), Akt, phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2), and phosphorylated Nrf2 (p-Nrf2). GB combined with different concentrations of LY294002 (PI3K inhibitor) were administrated to SH-SY5Y cells for 1 h after OGD, and then p-Akt and p-Nrf2 levels were detected by western blot. In vivo, 2 h of middle cerebral artery occlusion (MCAO) model was established, followed with reperfusion and GB treatments for 24 and 72 h. The infarct volume ratios were confirmed by TTC staining. The protein levels of HO-1, Nqo1, SOD1, Akt, p-Akt, Nrf2, and p-Nrf2 were detected using western blot and immunohistochemistry (IHC). Experimental data in vitro confirm that GA, GB, GK, and BB resulted in significant decrease of ROS and increase of SOD activities and protein levels of HO-1 and Nqo1; however, GB group had a significant advantage in comparison with the GA and GK groups. Moreover, after ginkgolides and BB treatments, p-Akt and p-Nrf2 were significantly upregulated, which could be inhibited by LY294002 in a dose-dependent manner, meanwhile, GB exhibited more effective than GA and GK. In vivo, TTC staining indicated that the infarct volume ratios in MCAO rats were dramatically decreased by GB in a dose-dependent manner. Furthermore, GB significantly upregulated the protein levels of HO-1, Nqo1, SOD, p-Akt, p-Nrf2, and Nrf2. In conclusion, GA, GB, GK, and BB significantly inhibited oxidative stress damage caused by cerebral ischemia reperfusion. Compared with GA, GK, and BB, GB exerts the strongest antioxidant stress effects against ischemic stroke. Moreover, ginkgolides and BB upregulated the levels of antioxidant proteins through mediating the Akt/Nrf2 signaling pathway to protect neurons from oxidative stress injury.

The essential oil from the twigs of Cinnamomum cassia Presl inhibits oxytocin-induced uterine contraction in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: The twigs and bark of Cinnamomum cassia Presl (Lauraceae) are widely used in traditional Chinese medicine in the treatment of tumor, abdominal pain, dysmenorrhea, digestive system disease and inflammatory diseases. The aim of this study was to determine the inhibitory effect of the essential oil from the twigs of Cinnamomum cassia Presl (EOCC) on uterine contraction in vitro and in vivo. MATERIALS AND METHODS: The Institute of Cancer Research (ICR) mouse uterine contraction was induced by oxytocin (OT) exposure following estradiol benzoate pretreatment. Mice were given the EOCC (60, 30, and 15mg/kg) by gavage. The level of prostaglandin F2α (PGF2α) in uterine tissue were determined according to specification of enzyme linked immunosorbent assay (ELISA) kit. Uterine tissue was collected for histopathological analysis (H&E). Myosin light chain 20 (MLC20), phosphorylation of myosin light chain 20 (p-MLC20) and cyclooxygenase-2 (COX-2) proteins in uterine tissue were assessed by Western Blot. Mouse isolated uterus strips were mounted in tissue organ baths containing Locke's solution. The contractile responses were recorded with Power Lab recording system. The effect of the EOCC on uterine contraction induced by OT, PGF2α, and acetylcholine (Ach) was observed. Myometrial cells were exposed to OT (7µM) to induce Ca2+ release, and the effect of the EOCC (100, 50, and 25µg/ml) on intracellular Ca2+ was analysed with fluorometry imaging. RESULTS: In vivo study demonstrated that the EOCC significantly reduced OT-induced writhing responses with a maximal inhibition of 66.5%. It also decreased the level of PGF2α in OT-induced mice uterine tissue. Moreover, Western blot analysis showed that COX-2 and p-MLC20 expressions in uterine tissue of dysmenorrhea mice were significantly reduced. EOCC inhibited spontaneous uterus contractions in a dose-dependent manner, and the concentration of the EOCC giving 50% of maximal contraction (IC50) value was 61.3µg/ml. The IC50 values of the EOCC on OT, PGF2α, and Ach-induced contractions were 113.0µg/ml, 94.7µg/ml, and 61.5µg/ml, respectively. Further in vitro studies indicated that the EOCC could restrain intracellular Ca2+ levels in favour of uterine relaxation. CONCLUSION: Both in vivo and in vitro results suggest that the EOCC possesses significant spasmolytic effect on uterine contraction. Thus, the EOCC yields a possible therapeutic choice for the prevention and treatment of primary dysmenorrhea.

The essential oil from the twigs of Cinnamomum cassia Presl alleviates pain and inflammation in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cinnamomum cassia Presl (Lauraceae) can be found southern China and its bark is commonly used for centuries as ingredient in food and cosmetic industry. The twigs of Cinnamomum cassia Presl is popularly used in China to treat inflammatory processes, pain, menstrual disorders, hypertension, fever etc. The aim of this study is to evaluate the antinociceptive and anti-inflammatory properties of the essential oil (EO) from the twigs of Cinnamomum cassia Presl. MATERIAL AND METHODS: The chemical characterization of the EO was performed by gas chromatography coupled with mass spectrometry (GC-MS). The EO doses of 15, 30, and 60mg/kg were employed in the biological assays. The antinociceptive effects of the EO were evaluated using the models of acetic acid-induced writhing, oxytocin-induced writhing, and formalin and complete Freund's adjuvant (CFA) -induced overt pain tests. we also investigated the effect of the EO in pain intensity to a mechanical stimulus (mechanical hyperalgesia) after carrageenan by using an electronic version of von Frey filaments. Evaluation of anti-inflammatory activity was based on paw edema induced by carrageenan (300µg/25µL/paw) in mice. The levels of cytokines, NO, and PGE2 in paw skin tissue were determined according to instructions. COX-2 and iNOS proteins in paw skin tissue were assessed by Western Blot. RESULTS: The EO (15, 30, and 60mg/kg) reduced the number of abdominal writhings induced by acetic acid with inhibition of 38.0%, 55.4% and 58.7%, respectively. The EO (15, 30, and 60mg/kg) also reduced the number of abdominal writhings induced by oxytocin with inhibition of 27.3%, 51.7% and 69.0%, respectively. The EO significant inhibited the inflammatory (second phase: 10-30min) phase of the formalin-induced paw flinching and licking at the doses of 15, 30, and 60mg/kg. The EO at the tested doses of 15, 30, and 60mg/kg showed inhibited CFA-induced paw flinching and licking. The EO (15, 30, and 60mg/kg) also inhibited carrageenan-induced mechanical hyperalgesia and paw edema. It also decreased the levels of cytokines (TNF-α, and IL-1ß), NO, and PGE2 in carrageenan-induced mice paw skin tissue. Moreover, Western blot analysis showed that COX-2 and iNOS expressions in paw skin tissue of mice were significantly reduced. CONCLUSIONS: These results demonstrate that the antinociceptive and anti-inflammatory properties of the EO from the twigs of Cinnamomum cassia Presl, corroborating its use in folk medicine.

Traditional Chinese medicine Guizhi Fuling capsule used for therapy of dysmenorrhea via attenuating uterus contraction.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling formula, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, is composed of Cinnamomum cassia (L.) J.Presl (Cassia bark), Poria cocos (Schw.) Wolf (Poria), Paeonia suffruticosa andrews (Moutan Cortex), Paeonia lactiflora Pall (Herbaceous peony), and Amygdalus persica L.(Persicae Semen). It has clinical efficacy of activating blood circulation to dissipate blood stasis and is commonly used for the treatment of primary dysmenorrhea. However, its therapeutic mechanism has not been clearly elucidated. The aim of this study is to reveal molecular mechanisms of action using in vivo and in vitro experimental models. MATERIAL AND METHODS: The ICR mouse uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment. Mice were given GZFLC (0.54, 1.08g/kg) by gavage. The levels of NO, PGF2α and Ca(2+) in uterine tissue were determined according to instructions. Cyclooxygenase-2 (COX-2) and oxytocin receptor (OTR) proteins in uterine tissue were assessed by Western Blot. Mouse isolated uterus strips were mounted in tissue organ baths containing Locke's solution. The contractile responses were recorded with Power Lab recording system. The effect of GZFLC on spontaneous uterine contraction, and uterine contraction induced by oxytocin, PGF2α was observed. Myometrial cells were exposed to oxytocin (5U/L) to induce calcium release, and the effect of GZFLC and its components (PL, PGG, CA) on intracellular Ca(2+) was analyzed with fluorometry imaging. RESULTS: In vivo study demonstrated that GZFLC significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 55%. It also decreased the levels of NO, PGF2α and Ca(2+) in oxytocin-induced mice uterine tissue. Moreover, Western blot analysis showed that COX-2 and OTR expressions in uterine tissue of dysmenorrhea mice were significantly reduced. GZFLC inhibited spontaneous uterus contractions in a dose-dependent manner, and the IC50 value was 0.99mg/ml. The IC50 values of GZFLC on PGF2α, oxytocin-induced contractions were 1.45mg/ml, 3.53mg/ml, respectively. Further in vitro studies indicated that GZFLC and its components (PL, PGG, CA) could restrain intracellular calcium levels in favour of uteri relaxation. CONCLUSIONS: Both in vivo and in vitro results indicated that GZFLC possessed a significant spasmolytic effect on uterine tetanic contraction. The present study provides in vivo and in vitro experimental evidence to support the use of GZFLC for the clinical treatment of primary dysmenorrheal (PD).

[Analysis on content of serum monoamine neurotransmitters in macaques with anger-in-induced premenstrual syndrome and liver-qi depression syndrome].

OBJECTIVE: To observe the changes in content of monoamine neurotransmitters in the serum of rhesus macaques, and explore the role of serum monoamine neurotransmitters in premenstrual syndrome (PMS) and liver-qi depression induced by anger-in emotion. METHODS: Social level pressure was applied on 24 female macaques to induce the angry emotional reaction, and then nine of the low-status macaques with anger-in emotional reaction were screened out and were divided into anger-in emotion group, PMS and liver-qi depression group (model group) and Jingqianshu Granule group. Macaques in the last two groups were suffered extruding in a pack cage for inducing PMS liver-qi depression. After 5 d of extruding, experimental animals were evaluated according to the emotional evaluation scale, meanwhile, macaque serum of follicular phase and middle-late luteal phase was collected to analyze the content of serum norepinephrine, dopamine, and 5-hydroxytryptamine. RESULTS: Compared with the normal control group, the scores of depression of the model group and the anger-in emotion group evaluated with emotional evaluation scale were significantly increased (P<0.01, P<0.05); while the score of the model group was significantly higher than that of the anger-in emotion group (P<0.05), and it returned to normal after Jingqianshu Granule treatment. As compared to the normal control group, serum monoamine neurotransmitter levels of the model group and the anger-in emotion group were increased (P<0.05, P<0.01), and the serum monoamine neurotransmitter levels of the model group were significantly higher than those of the anger-in emotion group (P<0.05), while there was no significant difference when compared with the normal control group after the treatment. CONCLUSION: Anger-in emotion can induce liver-qi depression syndrome which is related to the changes in monoamine neurotransmitters.