Jing Jia

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.

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.

Proteomics in plasma of ovariectomized rats and those exposed to estradiol valerate

The menopausal period, an inevitable physiological process for women, is frequently associated with physiological and psychological dysfunction attributable to substantial fluctuation and gradual decrease in female hormones induced by ovarian failure, leading to corresponding symptoms and diseases that impact multiple systems in the body to varying degrees. As prior studies have focused primarily on menopausal syndrome-related pathophysiological changes and hormone replacement therapy effects, here we approached menopausal disease incidence risk and pathogenesis through systemic plasma proteomics analysis. Female Sprague-Dawley rats were randomly divided into sham, ovariectomized, and estrogen treatment after ovariectomy groups (n=9 per group). Tandem Mass Tag quantitative proteomics analysis of their plasma identified over 900 proteins by MS. Between group fold change of >1.2 and p<0.05 (Students t-test) identified 121 (including 36 up-regulated and 85 down-regulated), 117 (69 up-regulated and 48 down-regulated), and 109 (41 up-regulated and 68 down-regulated) differentially expressed proteins between groups, respectively. Of these, 5 (GHR, LIFR, apoA IV, RTN, and Lin28b) were verified by parallel reaction monitoring to be reliable. Further application of optimized screening criteria and performance of a series of bioinformatics analyses allowed the selection of 35 optimal differentially expressed proteins. Gene ontology annotation results suggested that the differentially expressed proteins are mainly annotated as protein binding, cell, and single organism process in terms of molecular function, cell composition, and biological process, respectively. KEGG pathway analysis indicated that the PI3-Akt pathway has the highest aggregation degree of differentially expressed proteins. Protein-protein interaction analysis noted GLUT4 as an important node protein. This research is the first to comprehensively analyze plasma protein changes, together with estrogen efficacy, in ovariectomized rats. The findings facilitate our understanding of the molecular mechanism of systemic menopausal changes and provide valuable clues for developing diagnostic biomarkers for menopausal dysfunctions and selecting clinical therapeutic strategies.

Changes in cardiovascular function based on adrenalin and norepinephrine metabolism in ovariectomized rats

ABSTRACT Menopause is a cardiovascular risk factor in women, and cardiovascular changes during perimenopause can increase the risk. We observed the influence of plasma adrenalin and norepinephrine and their metabolites on the cardiovascular system and the rectification effect of estrogen in ovariectomized rats. Fifty‐four adult female Sprague‐Dawley rats were randomly divided into sham (Sham), ovariectomized (OVX), or ovariectomized + estrogen treatment groups (OVX + E), with 18 rats in each. The Sham and OVX groups were given normal saline and the OVX + E group was given estradiol valerate beginning 2 weeks after ovariectomy and continuing for 4 weeks. Radioimmunoassay, high‐performance liquid chromatography, high‐performance liquid chromatography‐tandem mass spectrometry, and chromatography‐spectrophotometry were used to detect estradiol, adrenalin, norepinephrine, metanephrine, and normetanephrine in plasma and vanillylmandelic acid in urine. Echocardiography, Doppler blood flow detection technologyand hamnatodynamometer were applied to assess cardiovascular function. After ovariectomy, levels of estrogen reduced, adrenalin and metanephrine increased, and norepinephrine and normetanephrine in the plasma and vanillylmandelic acid in urine decreased. Symptoms indicative of cardiac diastolic dysfunction, including decreased diastolic left ventricular cavity capacity, increased wall thickness and decreased cardiac rate were observed. Different degrees of vasomotor dysfunction appeared in different peripheral positions, and the tail vessels were in relatively systolic conditions. However, the claw pad vessels were diastolic. Besides, blood pressure also increased. After ovariectomy, estrogen levels reduced and the metabolic processes of adrenalin and norepinephrine changed, which impacted cardiovascular functions. Changes of adrenalin and norepinephrine and its metabolites were correlated with the cardiovascular function. Cardiovascular disease occurred during the perimenopausal period. Estrogen replacement therapy can mitigate, rectify, and improve menopause‐related conditions such as hot flash. HighlightsWe first observed that after ovariectomy, the metabolic processes of adrenalin and norepinephrine have changed.Changes of adrenalin and norepinephrine and its metabolites were correlated with the changes of cardiovascular function.Estrogen replacement therapy can mitigate, rectify, and improve menopause‐related conditions such as hot flash.This study provides a theoretical basis for the clinical work of menopause‐related diseases.

Effects of standardized isopropanolic black cohosh and estrogen on salivary function in ovariectomized rats

Oral dryness is a common feature in menopausal women. Estrogen therapy can relieve this symptom; however, the underlying mechanism was not clear. Standardized isopropanolic black cohosh (Actaea racemosa; Remifemin) can also relieve menopausal symptoms, such as hot flashes and sweating. Our previous study showed that standardized isopropanolic black cohosh could protect the submandibular gland structure. To investigate the effects and possible mechanisms of action of estrogen and standardized isopropanolic black cohosh on submandibular gland function in ovariectomized (OVX) rats, we measured body weight, daily water consumption, and blood flow in the submandibular glands. Immunohistochemistry and western blotting were used to detect the expression of muscarinic acetylcholine receptors 1 (M1) and 3 (M3), and aquaporin 5 (AQP5) in the submandibular gland. OVX increased daily water consumption and reduced vasodilation in the submandibular gland. It suggested that ovariectomy could damage the salviary function. Moreover, the expression of M1 and M3 receptors decreased, whereas that of AQP5 increased. These changes may explain the dysfunction of saliva secretion in menopause. Estrogen and standardized isopropanolic black cohosh treatment had the same effect on daily water consumption and vasodilation in the submandibular gland. It indicated that estrogen and standardized isopropanolic black cohosh could relieve oral dryness in menopause. However, the mechanism of the two treatments may differ because standardized isopropanolic black cohosh only protected against changes in M1 expression, whereas estrogen protected against variations in M1, M3, and AQP5 expression.