Marantodes pumilum (Kacip Fatimah) Aqueous Extract Enhances Osteoblast and Suppresses Osteoclast Activities in Cancellous Bone of a Rat Model of Postmenopause.

Evidence pointed towards the benefits of Marantodes pumilum in treating osteoporosis after menopause; however, the detailed mechanisms still have not been explored. Therefore, this study aims to identify the molecular mechanisms underlying M. pumilum's bone-protective effect via the involvement of RANK/RANKL/OPG and Wnt/ß-catenin signaling pathways. Ovariectomized adult female rats were given M. pumilum leaf aqueous extract (MPLA) (50 and 100 mg/kg/day) and estrogen (positive control) orally for twenty-eight consecutive days. Following the treatment, rats were sacrificed, and femur bones were harvested. Blood was withdrawn for analysis of serum Ca2+, PO43-, and bone alkaline phosphatase (BALP) levels. The bone microarchitectural changes were observed by H&E and PAS staining and distribution and expression of RANK/RANKL/OPG and Wnt3a/ß-catenin and its downstream proteins were determined by immunohistochemistry, immunofluorescence, Western blot, and real-time PCR. MPLA treatment increased serum Ca2+ and PO43- levels and reduced serum BALP levels (p < 0.05). Besides, deterioration in cancellous bone microarchitecture and the loss of bone glycogen and collagen content were mitigated by MPLA treatment. Levels of RANKL, Traf6, and NF-kB but not RANK in bone were decreased; however, levels of OPG, Wnt3a, LRP-5, Frizzled, Dvl, ß-catenin, RUNX, and Bmp-2 in bone were increased following treatment with MPLA. In conclusion, MPLA helps to protect against bone deterioration in estrogen deficiency state and thus, this herb could potentially be used to ameliorate osteoporosis in women after menopause.

Marantodes pumilum Var Alata (Kacip Fatimah) ameliorates derangement in RANK/RANKL/OPG pathway and reduces inflammation and oxidative stress in the bone of estrogen-deficient female rats with type-2 diabetes.

BACKGROUND: M. pumilum has been claimed to protect the bone against the adverse effect of estrogen deficiency. Additionally, it also exhibits anti-diabetic activity. In view of these, this study aims to identify the mechanisms underlying the bone protective effect of M. pumilum in the presence of both estrogen deficiency and diabetes mellitus (DM). METHODS: Ovariectomized, diabetic female rats were given M. pumilum leave aqueous extract (MPLA) (50 and 100 mg/kg/day), estrogen, glibenclamide and estrogen plus glibenclamide for 28 consecutive days. At the end of the treatment, fasting blood glucose (FBG), serum insulin, Ca2+, PO43- and bone alkaline phosphatase (BALP) levels were measured. Rats were sacrificed and femur bones were harvested for determination of expression level and distribution of RANK, RANKL, OPG and oxidative stress and inflammatory proteins by molecular biological techniques. RESULTS: 100 mg/kg/day MPLA treatment decreased the FBG and BALP levels but increased the serum insulin, Ca2+ and PO43- levels in estrogen deficient, diabetic rats. Expression and distribution of RANKL, NF-κB p65, IKKß, IL-6, IL-1ß and Keap-1 decreased however expression and distribution of RANK, OPG, BMP-2, Type-1 collagen, Runx2, TRAF6, Nrf2, NQO-1, HO-1, SOD and CAT increased in the bone of estrogen deficient, diabetic rats which received 100 mg/kg/day MPLA with greater effects than estrogen-only, glibenclamide-only and estrogen plus glibenclamide treatments. CONCLUSION: MPLA helps to overcome the adverse effect of estrogen deficiency and DM on the bone and thus this herb could potentially be used for the treatment and prevention of osteoporosis in postmenopausal women with diabetes.

Marantodes pumilum (blume) Kuntze (Kacip Fatimah) leaves aqueous extract prevents downregulation of Wnt/ß-catenin pathway and upregulation of apoptosis in osteoblasts of estrogen-deficient, diabetes-induced rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Marantodes pumilum (Blume) Kuntze has been claimed to be beneficial in protecting the bone against loss in post-menopausal women. In view of increased incidence of diabetes mellitus (DM) in post-menopausal period, M. pumilum ability to overcome the detrimental effect of estrogen-deficiency and DM on the bones were identified. AIM OF THE STUDY: To identify the mechanisms underlying protective effect of MPLA on the bone in estrogen-deficient, diabetic condition. METHODS: Adult female, estrogen-deficient, diabetic rats (225 ± 10 g) were divided into untreated group and treated with M. pumilum leaf aqueous extract (MPLA) (50 mg/kg/day and 100 mg/kg/day) and estrogen for 28 days (n = 6 per group). Fasting blood glucose (FBG) levels were weekly monitored and at the end of treatment, rats were sacrificed and femur bones were harvested. Bone collagen distribution was observed by Masson's trichome staining. Levels of bone osteoblastogenesis, apoptosis and proliferative markers were evaluated by Realtime PCR, Western blotting, immunofluorescence and immunohistochemistry. RESULTS: MPLA treatment was able to ameliorate the increased in FBG levels in estrogen deficient, diabetic rats. In these rats, decreased bone collagen content, expression level of osteoblastogenesis markers (Wnt3a, ß-catenin, Frizzled, Dvl and LRP-5) and proliferative markers (PCNA and c-Myc) and increased expression of anti-osteoblastogenesis marker (Gsk-3ß) and apoptosis markers (Caspase-3, Caspase-9 and Bax) but not Bcl-2 were ameliorated. Effects of 100 mg/kg/day MPLA were greater than estrogen. CONCLUSION: MPLA was able to protect against bone loss, thus making it a promising agent for the treatment of osteoporosis in women with estrogen deficient, diabetic condition.

Oral administration of Centella asiatica (L.) Urb leave aqueous extract ameliorates cerebral oxidative stress, inflammation, and apoptosis in male rats with type-2 diabetes.

Centella asiatica is claimed to have a neuroprotective effect; however, its ability to protect the cerebrum against damage in diabetes has never been identified. The aims were to identify the possibility that C. asiatica ameliorates inflammation, oxidative stress, and apoptosis in the cerebrum in diabetes. C. asiatica leave aqueous extract (C. asiatica) (50, 100, and 200 mg/kg/b.w.) were given to diabetic rats for 28 days. Changes in rats' body weight, food and water intakes, and insulin and FBG levels were monitored. Following sacrificed, cerebrum was harvested and subjected for histological, biochemical, and molecular biological analyses. The results revealed treatment with C. asiatica was able to ameliorate the loss in body weight, the increase in food and water intakes, the decrease in insulin, and the increase in FBG levels in diabetic rats. Additionally, histopathological changes in the cerebrum and levels of p38, ERK, JNK, cytosolic Nrf2, Keap-1, LPO, RAGE, and AGE levels decreased; however, PI3K, AKT, IR, IRS, GLUT-1, nuclear Nrf2, Nqo-1, Ho-1, and anti-oxidative enzymes (SOD, CAT, and GPx) levels increased in diabetic rats receiving C. asiatica. Furthermore, C. asiatica treatment also caused cerebral inflammation and apoptosis to decrease as indicated by decreased inflammatory markers (cytosolic NF-κB p65, p-Ikkß, Ikkß, iNOS, COX-2, TNF-α, IL-6, and IL-1ß), decreased pro-apoptosis markers (Casp-3, 9, and Bax), but increased anti-apoptosis marker, Bcl-2. Activity level of Na+/K+, Mg2+, and Ca2+-ATPases in the cerebrum also increased by C. asiatica treatment. Conclusions: C. asiatica treatment helps to prevent cerebral damage and maintain near normal cerebral function in diabetes.

Marantodes pumilum (Blume) Kuntze (Kacip Fatimah) stimulates uterine contraction in rats in post-partum period.

ETHNOPHARMACOLOGICAL RELEVANCE: Marantodes pumilum (Blume) Kuntze has traditionally been used to firm the uterus after delivery, however scientific evidences behind this claim is still lacking. AIMS OF STUDY: To demonstrate Marantodes pumilum leaves aqueous extract (MPE) has an effect on uterine contraction after delivery and to elucidate the molecular mechanisms involved. METHODS: Day-1 post-delivery female rats were given MPE (100, 250 and 500 mg/kg/day) orally for seven consecutive days. A day after the last treatment (day-8), rats were sacrificed and uteri were harvested and subjected for ex-vivo contraction study using organ bath followed by protein expression and distribution study by Western blotting and immunohistochemistry techniques, respectively. The proteins of interest include calmodulin-CaM, myosin light chain kinase-MLCK, sarcoplasmic reticulum Ca2+-ATPase (SERCA), G-protein α and ß (Gα and Gß), inositol-triphosphate 3-kinase (IP3K), oxytocin receptor-OTR, prostaglandin (PGF)2α receptor-PGFR, muscarinic receptor-MAChR and estrogen receptor (ER) isoforms α and ß. Levels of estradiol and progesterone in serum were determined by enzyme-linked immunoassay (ELISA). RESULTS: Ex-vivo contraction study revealed the force of uterine contraction increased with increasing doses of MPE. In addition, expression of CaM, MLCK, SERCA, Gα, Gß, IP3K, OTR, PGF2α, MAChR, Erα and ERß in the uterus increased with increasing doses of MPE. Serum analysis indicate that estradiol levels decreased while progesterone levels remained low at day-8 post-partum in rats receiving 250 and 500 mg/kg/day MPE. CONCLUSIONS: These findings support the claims that MPE help to firm the uterus and pave the way for its use as a uterotonic agent after delivery.

Intravaginal treatment with Marantodes pumilum (Kacip Fatimah) ameliorates vaginal atrophy in rats with post-menopausal condition.

ETHNOPHARMACOLOGICAL RELEVANCE: Marantodes pumilum (MP) (Kacip Fatimah) is used to maintain the well-being of post-menopausal women. However, its role in ameliorating post menopause-related vaginal atrophy (VA) is unknown. AIMS: To investigate the ability of intravaginal MP gel treatment to ameliorate VA in sex-steroid deficient condition, mimicking post-menopause. METHODS: Ovariectomized female Sprague-Dawley rats received MP (100 µg/ml, 250 µg/ml and 500 µg/ml) and estriol (E) gels intravaginally for seven consecutive days. Rats were then euthanized and vagina was harvested and subjected for histological and protein expression and distribution analyses. Vaginal ultrastructure was observed by transmission electron microscopy (TEM). RESULTS: Thickness of vaginal epithelium increased with increasing intravaginal MP doses. Additionally, increased in expression and distribution of proliferative protein i.e. PCNA, tight junction protein i.e. occludin, water channel proteins i.e. AQP-1 and AQP-2 and proton extruder protein i.e. V-ATPase A1 were observed in the vagina following intravaginal MP and E gels treatment. Intravaginal MP and E gels also induced desmosome formation and approximation of the intercellular spaces between the vaginal epithelium. CONCLUSIONS: Intravaginal MP was able to ameliorate features associated with VA; thus, it has potential to be used as an agent to treat this condition.

In vivo administration of quercetin ameliorates sperm oxidative stress, inflammation, preserves sperm morphology and functions in streptozotocin-nicotinamide induced adult male diabetic rats.

INTRODUCTION: Diabetes mellitus (DM) has been associated with sperm damage. In view of the fact that quercetin possesses antioxidant and anti-inflammatory activities, this compound may help to protect sperm against damage in DM. In this study, in-vivo effects of quercetin on sperm parameters in DM were investigated. MATERIAL AND METHODS: Quercetin (10, 25 and 50 mg/kg/b.w.) was given orally to streptozotocin-nicotinamide induced adult male diabetic rats for 28 days. Following treatment completion, rats were sacrificed and sperm were harvested from the cauda epididymis. Sperm count, motility, viability, hyperosmotic swelling (HOS) tail-coiled sperm and morphology were assessed. Levels of lipid peroxidation (LPO) and anti-oxidative enzymes (SOD, CAT and GPx) in sperm with and without H2O2 incubation were determined by biochemical assays. Expression levels of SOD, CAT and GPx mRNAs in sperm were evaluated by qPCR. Sperm DNA integrity was estimated by flow cytometry while expression levels of the inflammatory markers NF-κß and TNF-α in sperm were determined by Western blotting. RESULTS: In diabetic rats receiving quercetin, sperm count and motility, viability and HOS tail-coiled sperm increased (p < 0.05) while sperm with abnormal morphology decreased. Moreover, sperm SOD, CAT, GPx activities and their mRNA expression levels increased while sperm LPO, NF-κß and TNF-α levels decreased. In normal and diabetic rat sperm incubated with H2O2, a further increase in MDA and further decreases in SOD, CAT and GPx were observed, and these were ameliorated by quercetin treatment. CONCLUSIONS: In-vivo administration of quercetin to diabetic rats helps to ameliorate sperm damage and improves sperm morphology and functions in DM.

Hormonal control of vas deferens fluid volume and aquaporin expression in rats.

Precise regulation of vas deferens fluid volume which is important for sperm survival might be influenced by testosterone. In order to investigate changes in vas deferens fluid volume and aquoporins (AQP) isoforms expression under testosterone influence, orchidectomized Sprague-Dawley rats were given 125 and 250 µg/kg/day testosterone with or without flutamide, an androgen receptor blocker or finasteride, a 5alpha-reductase inhibitor for seven consecutive days. Following treatment completion, vas deferens was perfused and changes in the fluid secretion rate and osmolality were determined in the presence of acetazolamide. Rats were then sacrificed and vas deferens was harvested for histology, tissue expression and distribution analyses of AQP-1, AQP-2, AQP-5, AQP-7 and AQP-9 proteins by Western blotting and immunohistochemistry, respectively. Our findings indicate that testosterone causes vas deferens fluid secretion rate to increase, which was antagonized by acetazolamide. Fluid osmolality increased following testosterone treatment and further increased when acetazolamide was given. Co-administration of flutamide or finasteride with testosterone causing both fluid secretion rate and osmolality to decrease. Histology revealed increased size of vas deferens lumen with increased thickness of vas deferens stroma. Expression of AQP-1, AQP-2 and AQP-9 were detected in vas deferens but not AQP-5 and AQP-7, and the levels of these proteins were increased by testosterone treatment mainly at the apical membrane of vas deferens epithelium. In conclusion, increased in vas deferens fluid secretion rate under testosterone influence mediated via the up-regulation of AQP-1, 2 and 9 might be important for vas deferens fluid homeostasis in order to ensure normal male fertility.

Effects of Marantodes pumilum (Kacip Fatimah) on vaginal pH and expression of vacoular ATPase and carbonic anhydrase in the vagina of sex-steroid deficient female rats.

BACKGROUND: In sex-steroid deficiency, increased in the pH of vaginal fluid is due to low estrogen levels. HYPOTHESIS: Consumption of Marantodes pumilum leaves helps to ameliorate increased in vaginal fluid pH in sex-steroid deficient condition. PURPOSE: To investigate changes in vaginal fluid pH and expression of proteins that participate in pH changes i.e vacoular (V)-ATPases and carbonic anhydrases (CA) in the vagina following M. pumilum leaves consumption. METHODS: Ovariectomized adult female rats were treated orally with M. pumilum leaves extract (MPE) at 100, 250 and 500 mg/kg.b.w and estradiol at 0.2 µg/kg/b.w for 28 days. At the end of the treatment, vaginal fluid pH was measured in anesthetised rats by using micropH probe. Following sacrificed, levels of V-ATPase and CA proteins and mRNAs in the vagina were identified by Western blotting and real-time PCR, respectively. Protein distribution was visualized by immunohistochemistry. RESULTS: Administration of MPE causes the pH of vaginal fluid to decrease and expression and distribution of vaginal V-ATPase A & B and CA II, III, IX, XII and XIII to increase. CONCLUSIONS: The decrease in vaginal fluid pH following MPE treatment suggested that this herb has potential to be used to ameliorate vaginal fluid pH changes in sex-steroid deficient condition.

Differential expression of the receptors for thyroid hormone, thyroid stimulating hormone, vitamin D and retinoic acid and extracellular signal-regulated kinase in uterus of rats under influence of sex-steroids.

Sex-steroids play important role in modulating uterine functions. We hypothesized that these hormones affect expression of proteins in the uterus related to thyroid hormone action. Therefore, changes in expression levels of receptors for thyroid hormone (TRα-1 and TRß-1), thyroid stimulating hormone (TSHR), vitamin D (VDR) and retinoid acid (RAR) as well as extracellular signal-regulated kinase (ERK1/2) in uterus were investigated under sex-steroid influence. METHODS: Two rat models were used: (i) ovariectomised, sex-steroid replaced and (ii) intact, at different phases of oestrous cycle. A day after completion of sex-steroid treatment or following identification of oestrous cycle phases, rats were sacrificed and expression and distribution of these proteins in uterus were identified by Western blotting and immunohistochemistry, respectively. RESULTS: Expression of TRα-1, TRß-1, TSHR, VDR, RAR and ERK1/2 in uterus was higher following estradiol (E2) treatment and at estrus phase of oestrous cycle when E2 levels were high. A relatively lower expression was observed following progesterone (P) treatment and at diestrus phases of oestrous cycle when P levels were high. Under E2 influence, TRα, TRß, TSHR, VDR, RAR and ERK1/2 were distributed in luminal and glandular epithelia while under P influence, TSHR, VDR abn RAR were distributed in the stroma. CONCLUSIONS: Differential expression and distribution of TRα-1, TRß-1, TSHR, VDR, RAR and ERK1/2 in different uterine compartments could explain differential action of thyroid hormone, TSH, vitamin D, and retinoic acid in uterus under different sex-steroid conditions.

Anti-Inflammatory, Antiapoptotic and Proproliferative Effects of Vitis vinifera Seed Ethanolic Extract in the Liver of Streptozotocin-Nicotinamide-Induced Type 2 Diabetes in Male Rats.

OBJECTIVES: Consumption of Vitis vinifera seed has been reported to ameliorate liver pathology in diabetes mellitus; however, the mechanisms underlying its effects remain unknown. In this study, the anti-inflammatory, anti-apoptotic and pro-proliferative effects of the ethanolic seed extract of V. vinifera (VVSEE) in the liver in cases of diabetes were identified. METHODS: Adult male rats with streptozotocin-nicotinamide-induced diabetes were given 50, 100 or 200 mg/kg body weight VVSEE orally for 28 days. At the end of the treatment, body weights were determined, and the blood was collected for analyses of fasting blood glucose, insulin and liver enzyme levels. Following sacrifice, livers were harvested and their wet weights and glycogen contents were measured. Histologic appearances of the livers were observed under light microscopy, and the expression and distribution of inflammatory, apoptosis and proliferative markers in the livers were identified by molecular biologic techniques. RESULTS: Treatment of rats with diabetes by VVSEE attenuates decreased body weight, liver weight and liver glycogen content. Additionally, increases in fasting blood glucose levels and liver enzyme levels and decreases in serum insulin levels were ameliorated. Lesser histopathologic changes were also observed: decreased inflammation and apoptosis, as indicated by decreased levels of inflammatory markers (TNF-α, NF-Kß, IKK-ß, IL-6, IL-1ß) and apoptosis markers (caspase-3, caspase-9 and Bax). VVSEE treatment induces increase in hepatocyte regeneration, as indicated by increased PCNA and Ki-67 distribution in the livers of rats with diabetes. Several molecules identified in VVSEE via gas chromatography mass spectrometry might contribute to these effects. CONCLUSIONS: The anti-inflammatory, anti-apoptotic and pro-proliferative effects of VVSEE could account for its hepatoprotective actions in diabetes.

Phyllanthus niruri leaves aqueous extract improves kidney functions, ameliorates kidney oxidative stress, inflammation, fibrosis and apoptosis and enhances kidney cell proliferation in adult male rats with diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Phylanthus niruri has been used to treat ailments related to the urogenital organs. In this study, this herb was hypothesized to help to ameliorate kidney disease in diabetes mellitus (DM). AIMS: To investigate P. niruri leaves aqueous extract (PN) effects on kidney functions, histopathological changes and levels of oxidative stress, inflammation, fibrosis, apoptosis and proliferation in DM. METHODS: PN was orally administered to streptozotocin-nicotinamide-induced male diabetic rats for 28 days. At the end of the treatment, fasting blood glucose (FBG) and kidney functions were measured. Kidney somatic index, histopathological changes and levels of RAGE, Nrf2, oxidative stress markers (TBARS, SOD, CAT and GPx), inflammatory markers (NFkß-p65, Ikk-ß, TNF-α, IL-1ß and IL-6), apoptosis markers (caspase-3, caspase-9 and Bax), fibrosis markers (TGF-ß1, VEGF and FGF-1) and proliferative markers (PCNA and Ki-67) were determined by biochemical assays, qPCR, Western blotting, immunohistochemistry or immunofluorescence. RESULTS: Administration of PN helps to maintain near normal FBG, creatinine clearance (CCr), blood urea nitrogen (BUN), BUN/Cr ratio, serum electrolytes, uric acid and urine protein levels in DM. Decreased RAGE, TBARS and increased Nrf2, SOD-1, CAT and GPx-1 were observed in PN-treated diabetic rat kidneys. Expression of inflammatory, fibrosis and apoptosis markers in the kidney reduced but expression of proliferative markers increased following PN treatment. Lesser histopathological changes were observed in the kidney of PN-treated diabetic rats. CONCLUSION: PN helps to preserve near normal kidney function and prevents histopathological changes via ameliorating oxidative stress, inflammation, fibrosis and apoptosis while enhancing proliferation of the kidney in DM.

Quercetin interferes with the fluid volume and receptivity development of the uterus in rats during the peri-implantation period.

HYPOTHESIS: Quercetin could induce changes to the fluid volume and receptivity development of the uterus during peri-implantation period. METHODS: Female rats were treated with quercetin (10, 25 and 50mg/kg/day) subcutaneously beginning from day-1 pregnancy. Uterus was harvested at day-4 (following three days quercetin treatment) for morphological, ultra-structural, protein and mRNA expressional changes and plasma sex-steroid levels analyses. In another cohort of rats, implantation rate was determined at day-6 (following five days quercetin treatment). RESULTS: Administration of 50mg/kg/day quercetin causes increased in uterine fluid volume and CFTR expression but decreased in γ-ENaC, AQP-5, AQP-9 claudin-4, occludin, E-cadherin, integrin αnßЗ, FGF, Ihh and Msx-1expression in the uterus. Pinopodes were poorly develop, tight junctions appear less complex and implantation rate decreased. Serum estradiol levels increased but serum progesterone levels decreased. CONCLUSIONS: Interference in the fluid volume and receptivity development of the uterus during peri-implantation period by quercetin could adversely affect embryo implantation.

Combinatorial effects of quercetin and sex-steroids on fluid and electrolytes' (Na+, Cl-, HCO3-) secretory mechanisms in the uterus of ovariectomised female Sprague-Dawley rats.

Dysregulation of uterine fluid environment could impair successful reproduction and this could be due to the effect of environmental estrogens. Therefore, in this study, effect of quercetin, an environmental estrogen on uterine fluid and electrolytes concentrations were investigated under sex-steroid influence. Ovariectomised adult female Sprague-Dawley rats were given 10, 50 or 100mg/kg/day quercetin subcutaneously with 17-ß estradiol (E) for seven days or three days E, then three days E plus progesterone (P) (E+P) treatment. Uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations were determined by in-vivo perfusion. Following sacrifice, uteri were harvested and levels of the proteins of interest were identified by Western blotting and Realtime PCR. Distribution of these proteins in the uterus was observed by immunofluorescence. Levels of uterine cAMP were measured by enzyme-linked immunoassay (EIA). Administration of quercetin at increasing doses increased uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations, but to the levels lesser than that of E. In concordant, levels of CFTR, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP in the uterus increased following increased in the doses of quercetin. Co-administration of quercetin with E caused uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations to decrease. In concordant, uterine CFTR, SLC26A6, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP decreased. Greatest effects were observed following co-administration of 10mg/kg/day quercetin with E. Co-administration of quercetin with E+P caused uterine fluid Na+ and HCO3- concentrations to increase but no changes in fluid secretion rate and Cl- concentration were observed. Co-administration of high dose quercetin (100 mg/kg/day) with E+P caused uterine CFTR, SLC26A6, AC, GPα/ß and ENaC (α, ß and γ) to increase. Quercetin-induced changes in the uterine fluid secretion rate and electrolytes concentrations could potentially affect the uterine reproductive functions under female sex-steroid influence.

Quercetin alters uterine fluid volume and aquaporin (AQP) subunits (AQP-1, 2, 5 & 7) expression in the uterus in the presence of sex-steroids in rats.

Effects of quercetin on uterine fluid volume and aquaporin (AQP) expression in the uterus were investigated. Estradiol (E) or estradiol followed by progesterone (E+P) were given to ovariectomised rats with or without quercetin (10, 50 or 100mg/kg/day) treatment. Uteri were harvested and its inner/outer circumference ratio was determined. AQP-1, 2, 5 and 7 mRNA and protein levels in uterus were quantified by Real-time PCR and Western blotting respectively. Protein distribution was observed by immunohistochemistry. Administration of quercetin in E-treated rats decreased the uterine fluid volume and uterine AQP-2 expression. In E+P-treated rats, administration of 100mg/kg/day quercetin increased uterine fluid volume, AQP-1 and 2 expression but decreased AQP-7 expression in uterus. AQP-1 was distributed in stromal blood vessels while AQP-2, 5 and 7 were distributed in uterine epithelium. CONCLUSIONS: Quercetin-induced changes in uterine fluid volume and AQP subunits expression in uterus could affect the uterine reproductive functions under different sex-steroid influence.

Quercetin ameliorates oxidative stress, inflammation and apoptosis in the heart of streptozotocin-nicotinamide-induced adult male diabetic rats.

INTRODUCTION: Quercetin is known to possess beneficial effects in ameliorating diabetic complications, however the mechanisms underlying cardioprotective effect of this compound in diabetes is not fully revealed. In this study, quercetin effect on oxidative stress, inflammation and apoptosis in the heart in diabetes were investigated. Normal and streptozotocin-nicotinamide induced adult male diabetic rats received quercetin (10, 25 and 50mg/kg/bw) orally for 28days were anesthetized and hemodynamic parameters i.e. systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were measured. Blood was collected for analyses of fasting glucose (FBG), insulin and cardiac injury marker levels (troponin-C, CK-MB and LDH). Following sacrificed, heart was harvested and histopathological changes were observed. Heart was subjected for analyses of oxidative stress marker i.e. lipid peroxidation and activity and expression levels of anti-oxidative enzymes i.e. SOD, CAT and GPx. Levels of inflammation in the heart were determined by measuring nuclear factor (p65-NF-κB), tumor necrosis factor (TNF-α), interleukins (IL)-1ß and IL-6 levels by using enzyme-linked immunoassay (ELISA). Distribution and expression levels of TNF-α and Ikk-ß (inflammatory markers), caspase-3, caspase-9, Blc-2 and Bax (apoptosis markers) in the heart were identified by immunohistochemistry and Western blotting respectively. RESULTS: Administration of quercetin to diabetic rats caused significant decrease in FBG and cardiac injury marker levels with increased in insulin levels. In diabetic rat heart, lesser histopathological changes were observed with oxidative stress, inflammation and apoptosis levels markedly decreased. CONCLUSIONS: Quercetin could potentially be used to ameliorate myocardial damage due to oxidative stress, inflammation and apoptosis in diabetes.

Isoflavone genistein inhibits estrogen-induced chloride and bicarbonate secretory mechanisms in the uterus in rats.

We hypothesized that genistein could affect the chloride (Cl- ) and bicarbonate (HCO3- ) secretory mechanisms in uterus. Ovariectomized female rats were given estradiol or estradiol plus progesterone with 25, 50, or 100 mg/kg/day genistein. Following completion of the treatment, uterine fluid Cl- and HCO3- concentrations were determined by in vivo uterine perfusion. Uteri were subjected for molecular biological analysis (Western blot, qPCR, and immunohistochemistry) to detect levels of expression of Cystic Fibrosis transmembrane regulator (CFTR), Cl- /HCO3- exchanger (SLC26a6), Na+ /HCO3- cotransporter (SLC4a4), and estrogen receptor (ER)-α and ß. Coadministration of genistein resulted in decrease in Cl- and HCO3- concentrations and expression of CFTR, SLC26a6, SLC4a4, and ER-α and ER-ß in the uteri of estradiol-treated rats. In estradiol plus progesterone-treated rats, a significant increase in the above parameters were observed following high-dose genistein treatment except for the SLC24a4 level. In conclusion, genistein-induced changes in the uterus could affect the reproductive processes that might result in infertility.

Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17ß (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17ß followed by 3 days vehicle, progesterone, or estradiol-17ß plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence.

Estrogen and progesterone differentially regulate carbonic anhydrase II, III, IX, XII, and XIII in ovariectomized rat uteri.

Changes in the uterus expression of carbonic anhydrase (CA) II, III, IX, XII, and XIII were investigated under the influence of sex-steroids in order to elucidate mechanisms underlying differential effects of these hormones on uterine pH. Uteri of ovariectomised rats receiving over three days either vehicle, estrogen, or progesterone or three days estrogen followed by three days either vehicle or progesterone were harvested. Messenger RNA (mRNA) and protein levels were quantified by real-time PCR and Western blotting, respectively. The distribution of CA isoenzymes proteins were examined by immunohistochemistry. The levels of CAII, III, XII, and XIII mRNAs and proteins were elevated while levels of CAIX mRNA and protein were reduced following progesterone-only and estrogen plus progesterone treatment, compared to the control and estrogen plus vehicle, respectively. Following estrogen treatment, expression of CAII, IX, XII, and CAXIII mRNAs and proteins were reduced, but remained at a level higher than control, except for CAIX, where its level was higher than the control and following progesterone treatment. Under progesterone-only and estrogen plus progesterone influences, high levels of CAII, III, XII, and XIII were observed in uterine lumenal and glandular epithelia and myometrium. However, a high level of CAIX was observed only under the influence of estrogen at the similar locations. In conclusion, high expression of CAII, III, XII, and XIII under the influence of progesterone and estrogen plus progesterone could result in the reduction of uterine tissue and fluid pH; however, the significance of high levels of CAIX expression under the influence of estrogen remains unclear.