Lihua Qin

Multiple effects of 2ME2 and D609 on the cortical expression of HIF-1α and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model

Despite 2‐methoxyestradiol (2ME2) and tricyclodecan‐9‐yl‐xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down‐regulate hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF). We hypothesize HIF‐1α plays an essential role in cerebral ischemia as a pro‐apoptosis regulator; 2ME2 and D609 decrease the levels of HIF‐1α and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague–Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF‐1α and VEGF (enzyme‐linked immunosorbent assay), depress the expression of HIF‐1α, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF‐1α positive immunoreactive materials are co‐localized with BNIP3 and terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7‐triphenyltetrazolium chloride) and blood–brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF‐1α expression, attenuating the superfluous expression of VEGF to avoid blood–brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.

The role of p53 in brain edema after 24 h of experimental subarachnoid hemorrhage in a rat model

Our previous study demonstrated that p53 plays an orchestrating role in the vasospasm and apoptotic cell death after subarachnoid hemorrhage (SAH). We now hypothesize that p53 also plays an important role in brain edema by up-regulating the expression of MMP-9 via the NF-kappaB molecular signaling pathway. Adult male rats (300-350 g) were divided into five groups (n=20 each): Sham, SAH treatment with DMSO or PFT-alpha (0.2 mg/kg and 2.0 mg/kg), intraperitoneally. The monofilament puncture model was used to induce SAH and animals were subsequently sacrificed at 24 h. The blood-brain barrier (BBB) disruption, brain water content, MMP-9 activity, immunohistochemistry, treble fluorescence labeling, Western blot, and ultra-structural observations were performed. Evans blue extravagation, BBB diffuse leakage of IgG protein and brain water content were significantly reduced by PFT-alpha treatment; and the expression of p53, NF-kappaB and MMP-9 were significantly increased. The tight junction protein (Occludin) in endothelia cells and Collage IV in basal lamina were decreased in the brain of SAH rats, and were also modified by PFT-alpha treatment. Ultra-structural changes included disruption of endothelial tight junction and widening of the inter-endothelial spaces. Treble labeling showed p53 colocalized with NF-kappaB and MMP-9 in cerebral endothelia cells. We thus conclude that the level of p53 in cerebral microvasculature significantly affects the BBB permeability and brain edema after 24 h of SAH in rats. This can be at least partially ascribed to p53 inducing a significant up-regulation of MMP-9 via NF-kappaB in the endothelium, which in turn opened the tight junction by degrading Occludin and disrupting the basal lamina by degrading collagen IV.

Effects of an isopropanolic-aqueous black cohosh extract on central body temperature of ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE Black cohosh (Cimicifuga racemosa) is widely used in menopause symptoms strategy. AIM OF THE STUDY The aim of this study was to examine the effect of isopropanolic black cohosh extract (iCR) on the central body temperature (CBT) of ovariectomized rats (OVX) and elaborate its possible effects in alleviating menopause related hot flushes. MATERIALS AND METHODS 64 female Sprague-Dawley rats, weighing 230 ± 10g and aged 6-8 weeks, were divided into four groups: ovariectomy (OVX), sham, ovariectomy plus estradiol valerate (OVX+E), and ovariectomy plus iCR (OVX+ICR). The sham group underwent a sham surgery without ovariectomies, while the other three groups underwent bilateral ovariectomies under sterile conditions and a temperature implant was embedded in the abdominal cavity of all four groups. After 2-week recovery period, the temperature of all animals was monitored for 6 weeks. RESULTS CBT of four groups maintained a normal circadian rhythm, with a low day CBT and a high night CBT. CBTs of the sham group were lower than that of the other three groups. The day CBTs of the (OVX+E) group and (OVX+ICR) group were lower than that of the OVX group from day 2 and day 22 respectively. For the difference between day and night CBT, the sham group was smallest, while (OVX+E) and (OVX+ICR) groups were higher than that of OVX group. The amplitude of day and night CBT, CBT fluctuation frequency at 5 min intervals, were higher for the OVX group than the sham group; the amplitude of day and night CBT of (OVX+E) group and the amplitude of night CBT of (OVX+ICR) group were higher than those of OVX group; while the amplitude of day CBT of (OVX+ICR) group was lower than that of OVX group; CBT fluctuation frequency at 5 min intervals was higher for the (OVX+E) and (OVX+ICR) groups than the OVX group. CONCLUSIONS Ovariectomized rats had abnormal thermoregulation, demonstrating an increase in day and night CBT, greater difference between day and night CBT, higher amplitude of day and night CBT, and more CBT fluctuation frequency. For the herbal extract iCR, the onset of affecting abnormal thermoregulation took longer than that of estradiol valerate. ICR had a significant effect on day CBT but was only little effective on night CBT of ovariectomized rats.

Effects of Estradiol Valerate and Remifemin on Norepinephrine Signaling in the Brain of Ovariectomized Rats

Aims: We investigated the norepinephrine pathway changes from the locus coeruleus (LC) to the preoptic area of the hypothalamus (POAH) in the brain of ovariectomized rats under low estrogen levels and explored the therapeutic effects of estradiol valerate (E2) and Remifemin (ICR) on these changes. Methods: 40 female Sprague-Dawley rats were randomly divided into the following groups: surgery with vehicle (SHAM), ovariectomy surgery with vehicle (OVX), ovariectomy with E2 treatment (OVX + E2), and ovariectomy with Remifemin (OVX + ICR). After 4 weeks of treatment, we observed the changes by immunohistochemistry. Results: (1) The average optical density of DBH-ir fibers and the number of α1-adrenoreceptor- and estrogen receptor (ER)α-positive neurons in the main nuclei of POAH were all reduced in OVX rats compared with the SHAM group. The above changes were normalized in all nuclei of the POAH in the E2 group, while they were normalized in some nuclei in the ICR group. Coexpression of ERα and α1-adrenoreceptor was observed in the POAH. (2) The number of DBH- and ERα-positive neurons in the LC decreased in the OVX group compared with the SHAM group and increased after treatment with E2 and ICR. Coexpression of ERα and DBH was observed in the LC. Conclusion: Low estrogen (OVX) altered norepinephrine synthesis in the LC, the projection of norepinephrine fibers and α1-adrenoreceptor expression in the POAH. Both E2 and ICR normalized the norepinephrine pathway, but E2 achieved greater effects than ICR. ICR had different effects in different nuclei in the POAH and its therapeutic effect was better in the LC.

Effects of remifemin treatment on bone integrity and remodeling in rats with ovariectomy-induced osteoporosis.

This study aims to evaluate the effects of Remifemin (isopropanolic extract of Cimicifuga Racemosa) on postmenopausal osteoporosis. 120 female Sprague-Dawley rats were randomly assigned to four groups: sham surgery with vehicle, ovariectomy with vehicle, ovariectomy with estradiol valerate, or ovariectomy with Remifemin. Daily oral administrations of the vehicle, estradiol valerate, or Remifemin began 2 weeks after surgery and lasted to 4, 8, or 12 weeks. Ten rats in each group were sacrificed at each timestep with assessment of bone mineral density, trabecular bone structure, and biomechanical parameters of the femur and lumbar vertebra. Bone turnover markers were evaluated 12 weeks after surgery. Both drugs prevented bone density loss in the distal end of the femur and preserved the trabecular bone structure in both the lumbar vertebra and distal end of the femur following ovariectomy. Both drugs protected bone stiffness at the tested regions and reduced bone reabsorption in ovariectomized rats. The preventive effects of Remifemin against bone-loss can rival those of estradiol valerate if treatment duration is adequately extended. In conclusion, Remifemin may demonstrate equivalent effects to estradiol valerate in terms of preventing postmenopausal osteoporosis.

A Comparison of the Effects of Estrogen and Cimicifuga racemosa on the Lacrimal Gland and Submandibular Gland in Ovariectomized Rats

This study aims to observe the effects of estradiol and Cimicifuga racemosa on the lacrimal gland and submandibular gland of ovariectomized rats. We randomly divided 20 adult female SD rats into four groups—a sham-operated group (SHAM), ovariectomized (OVX) group, ovariectomized group treated with estradiol (OVX+ E), and ovariectomized group treated with the isopropanolic extract of Cimicifuga racemosa (OVX+ iCR). The SHAM group and OVX group used distilled water to instead the drugs. Two weeks after ovariectomy, the estradiol and iCR were administered for 4 weeks. Next, we used H&E staining and electron microscopy to observe any histological changes in the lacrimal and submandibular glands and immunohistochemical staining to observe the expressions of cleaved caspase-3 (Casp-3) and Cu-Zn SOD (superoxide dismutase). The H&E staining find that both drugs can prevent the cells of area from shrinkage in the two kinds of gland. But under the electron microscopy, estradiol and iCR have different efficacy. Estradiol is more effective at protecting mitochondria in lacrimal gland acinar cells than iCR, and iCR is more effective at suppressing endoplasmic reticulum expansion than estradiol. Both estradiol and iCR have a similar protective function on mitochondria in the submandibular gland. The protective function of the two glands may inhibit apoptosis by suppressing the expression of Casp-3. In addition, iCR increases the expression of Cu-Zn SOD in duct system of submandibular gland. The results suggest that both estradiol and iCR confer a protective effect on the lacrimal and submandibular glands of ovariectomized rats via different mechanisms.

Estradiol Valerate and Remifemin ameliorate ovariectomy-induced decrease in a serotonin dorsal raphe–preoptic hypothalamus pathway in rats

Perimenopausal syndromes begin as ovarian function ceases and the most common symptoms are hot flushes. Data indicate that the projections of serotonin to hypothalamus may be involved in the mechanism of hot flushes. Therefore, the aim of this study is to investigate the potential role of the serotonin dorsal raphe-preoptic hypothalamus pathway for hot flushes in an animal model of menopause. We determined the changes in serotonin expression in the dorsal raphe (DR) and preoptic anterior hypothalamus (POAH) in ovariectomized rats. We also explored the therapeutical effects of estradiol valerate and Remifemin in this model. Eighty female Sprague-Dawley rats were randomly assigned to sham-operated (SHAM) group, ovariectomy (OVX) group with vehicle, ovariectomy with estradiol valerate treatment (OVX+E) group and ovariectomy with Remifemin (OVX+ICR) group. Serotonin expression was evaluated in the DR and POAH using immunofluorescence and quantified in the DR using an enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed in the DR by TUNEL assay. The number of serotonin immunoreactive neurons and the level of serotonin expression in the DR decreased significantly following OVX compared to the SHAM group. No TUNEL-positive cells were detected in the DR in any group. In addition, following OVX, the number of serotonin-positive fibers decreased significantly in the ventromedial preoptic nucleus (VMPO), especially in the ventrolateral preoptic nucleus (VLPO). Treatment with either estradiol or Remifemin for 4 weeks countered the OVX-induced decreases in serotonin levels in both the DR and the hypothalamus, with levels in the treated rats similar to those in the SHAM group. A fluorescently labeled retrograde tracer was injected into the VLPO at the 4-week time point. A significantly lower percentage of serotonin with CTB double-labeled neurons in CTB-labeled neurons was demonstrated after ovariectomy, and both estradiol and Remifemin countered this OVX-induced decrease. We conclude that serotonin pathway is changed after ovariectomy, including the serotonin synthesis in DR and serotonin fibers in PO/AH, both E and Remifemin have an equivalent therapeutic effect on it.

Post-hypoxic and ischemic neuroprotection of BMP-7 in the cerebral cortex and caudate-putamen tissue of rat.

Previous reports have indicated that exogenous bone morphogenetic protein-7 (BMP-7) has a neuroprotective effect after cerebral ischemia injury and promotes motor function recovery, but the appropriate BMP-7 concentration and time course are unclear. Here, we assessed endogenous BMP-7 expression in hypoxia and ischemia-damaged brain tissues and investigated the effects of different BMP-7 concentrations in pre- and post-hypoxic primary rat neurons. The results showed that BMP-7 expression was significantly higher in the ischemic hemisphere. The expressions of BMP-7 and caspase-3 were localized in the cytoplasm of the primary cerebral cortical and caudate-putamen neurons 24h after hypoxia/reoxygenation. After BMP-7 treatment, the number of caspase-3 positive neurons began to decrease with increasing BMP-7 concentrations up to 80ng/ml, but not beyond. Although the numbers of caspase-3-positive neurons between pre- and post-hypoxia/reoxygenation were not significantly different, more dendrites were observed in the groups treated prior to hypoxia/reoxygenation. These results suggest that increased BMP-7 expression can be induced in the cerebral cortex and caudate-putamen both in vivo and in vitro in hypoxic-ischemic states. The neuroprotective mechanism of BMP-7 may include apoptosis suppression, and its effect was enhanced from 40 to 80ng/ml. Pre-hypoxic BMP-7 treatment may be useful to stimulate dendrite sprouting in non-injured neurons.

Mechanisms underlying alterations in norepinephrine levels in the locus coeruleus of ovariectomized rats: Modulation by estradiol valerate and black cohosh

Hot flushes are common in menopause. Norepinephrine (NE), primarily synthesized in the locus coeruleus (LC), plays a major role in central thermoregulation. Furthermore, we previously observed decreased dopamine beta hydroxylase (DβH), a key enzyme in NE synthesis, in LC neurons following ovariectomy. In this study, we explore the mechanisms underlying decreased NE levels in the LC (LC-NE) in ovariectomized (OVX) rats, and the modulating effects of estradiol valerate (E2) and black cohosh (ICR). We used high-performance liquid chromatography to detect LC-NE in SHAM, OVX, OVX-E2, and OVX-ICR groups. Western blotting and immunohistochemistry were performed to investigate the expression of NE metabolic enzymes, the NE reuptake transporter (NET), and estrogen receptors (ERs) in the LC. We observed significant LC-NE decreases in the OVX group. E2 and ICR enhanced LC-NE but did not restore them to SHAM levels. Ovariectomy affected NE synthesis, degradation, and reuptake. Levels of NE catabolic enzymes monoamine oxidase A (MAOA) and catechol-O-methyltransferase (COMT) decreased, while NET expression increased. E2 restored MAOA and COMT to SHAM levels but had no effect on NET. ICR restored COMT and NET to SHAM levels but had no effect on MAOA. Moreover, the OVX group also exhibited decreased expression of ERα and ERβ. E2 enhanced the expression of ERα and ERβ, while ICR only enhanced ERβexpression. Taken together, reduced NE in OVX rats resulted from reduced synthesis and increased degradation and reuptake. E2 and ICR may regulate these processes in different ways through various ERs.

Effect of low estrogen on neurons in the preoptic area of hypothalamus of ovariectomized rats.

The purpose of this study was to investigate the difference in neuronal activity in the preoptic area of the hypothalamus (POAH) under low estrogen condition induced by ovariectomy. One hundred and twenty sham-operated (SHAM) and ovariectomized (OVX) rats were placed in different temperatures for 2h. Twelve rats from each group were stimulated by 4°C, 10°C, 25°C, 33°C and 38°C, respectively. c-Fos expression in the POAH was detected by immunohistochemistry. Following exposure to warm and cold stimuli, there were markedly lower c-Fos-positive cell densities in the OVX group compared with the SHAM group in the median preoptic nucleus (MnPO) at 4°C, 10°C, 33°C and 38°C, in the medial preoptic area (MPA) at 25°C and 38°C, in the ventromedial preoptic nucleus (VMPO) at 4°C, 10°C and 38°C and in the ventrolateral preoptic nucleus (VLPO) at 4°C and 38°C. Both temperature and surgery had an impact on c-Fos expression by two-way ANOVA method except in the lateral preoptic area (LPO). c-Fos expression differed within different nuclei of the two groups in the same and different temperature stimuli. This indicated that the temperature-sensitive nuclei in the POAH exhibited lower and different activities during temperature stimuli following ovariectomy, which possibly resulted in abnormal thermoregulation and menopausal symptoms.