Mitigation of letrozole induced polycystic ovarian syndrome associated inflammatory response and endocrinal dysfunction by Vitex negundo seeds.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder in women that necessitates effective and safe treatment alternatives. This study aimed to evaluate the therapeutic efficacy of Vitex negundo seed in a letrozole-induced PCOS rat model. RESULTS: Findings of the present study demonstrated that administration of hydro-ethanolic extract of Vitex negundo (VNE) effectively restored endocrino-metabolic imbalances associated with PCOS, along with correction of antioxidant enzymes level, proinflammatory cytokines, and apoptotic bio-markers. LC-MS analysis confirmed the presence of cinnamic acid, plumbagin and nigundin B as the prominent phytochemicals in VNE. The observed beneficial effects could be attributed to the active compounds in Vitex negundo extract, which exhibited hypoglycemic, hypolipidemic, and catabolic effects on body weight. Additionally, the extract contributed to hormonal balance regulation by modulating the steroidogenic enzymes, specifically by tuning gonadotropins level and correcting the LH:FSH ratio, through the modulation of ERα signalling and downregulation of NR3C4 expression. The antioxidant properties of phytochemicals in Vitex negundo seed were apparent through the correction of SOD and catalase activity. While it's anti-inflammatory and antiapoptotic action were associated with the regulation of mRNA expression of TNF-α, IL-6, BAX, Bcl2. Molecular docking study further indicated the molecular interaction of above mentioned active phytocompounds of VNE with ERα, NR3C4 and with TNFα that plays a critical mechanistic gateway to the regulation of hormone signalling as well as synchronizing the inflammation cascade. Furthermore, the histomorphological improvement of the ovaries supported the ameliorative action of Vitex negundo extract in the letrozole-induced PCOS model. CONCLUSIONS: This study indicates the potential of Vitex negundo seed as a multifaceted therapeutic option for PCOS. VNE offers a holistic strategy for PCOS with antiandrogenic, anti-inflammatory, and antioxidant properties, driven by its major compounds like cinnamic acid, plumbagine, and nigundin B.

n-Butanol fraction of moringa seed attenuates arsenic intoxication by regulating the uterine inflammatory and apoptotic pathways.

The adverse effects of arsenic-chelating drugs make it essential to replace invasive chelating therapy with non-invasive oral therapy for arsenic poisoning. The goal of the current investigation was to determine whether the uterine damage caused by arsenization could be repaired by the n-butanol fraction of Moringa oleifera seed (NB). The rats were orally administered with arsenic (10 mg/kg BW) for the initial 8 days, followed by NB (50 mg/kg) for the next 8 days without arsenic. The probable existence of different components in NB was evaluated by HPLC-MS. Pro and anti-inflammatory indicators were assessed by RT-PCR and western blot. ESR-α was detected via immunostaining. Arsenic-exposed rats had significantly increased lipid peroxidation and decreased antioxidant enzyme activity, which were markedly reduced after NB treatment. Weaker ESR-α expression and distorted uterine histomorphology following arsenication were retrieved significantly by NB. Meaningful restoration by NB was also achieved for altered mRNA and protein expression of various inflammatory and apoptotic indicators. Molecular interaction predicted that glucomoringin and methyl glucosinolate of moringa interact with the catalytic site of caspase-3 in a way that limits its activity. However, NB was successful in restoring the arsenic-mediated uterine hypofunction. The glucomoringin and methyl glucosinolate present in n-butanol fraction may play a critical role in limiting apoptotic event in the arsenicated uterus.

Arsenic-induced uterine apoptotic damage is protected by ethyl acetate fraction of Camellia sinensis (green tea) via Bcl-2-BAX through NF-κB regulations in Wistar rats.

The non-invasive treatment strategy is indispensable to overcome the side effects of conventional treatment with chelating agents against arsenic. Presence of catechins and flavonoids in Camellia sinensis have potential antioxidant properties and other beneficial effects. The aim of the study was to explore the curative potential role of Camellia sinensis against uterine damages produced by sodium arsenite in mature albino rats. A dose of 10 mg of Camellia sinensis ethyl acetate (CS-EA) fraction/100 gm body weight was provided to the sodium arsenite-treated rats (10 mg/Kg body weight). LC-MS analysis was used for the detection of active component in CS-EA fraction. Enzymatic antioxidants analysis carried out by reproducible native gel technique. Hormones and some pro and anti-inflammatory markers were detected by ELISA, PCR, and western blot techniques respectively. Immunostaining was performed for the detection of estradiol receptor alpha. LC-MS analysis of CS-EA fraction ensured the presence of active tea polyphenol and tea catechin of which highest peak of epigallocatechin-3 gallate (EGCG) was obtained in this study. Significant elevations of lipid peroxidation end products followed by the diminution of antioxidant enzymes activities were noted in arsenicated rats which were capably retrieved by the treatment of CS-EA fraction. Post-treatment with CS-EA fraction meaningfully improved gonadotrophins and estradiol signalling in association with a highly expressing estradiol receptor-α (ERα) in the ovary and uterus followed by the maintenance of normal utero-ovarian histoarchitecture in arsenic fed rats. CS-EA fractioned treated group overturned the sodium arsenite driven higher expression of pro-inflammatory cytokines and proapoptotic markers along with a low level of anti apoptotic Bcl-2 expression and comparatively lower NF-κB signalling in the uterus via regulating IKK ß kinase mostly by EGCG of CS-EA fraction. However, ethyl acetate fraction of Camellia sinensis played a critical role in minimizing arsenic-mediated uterine hypo-function.

Mitigation of arsenic driven utero-ovarian malfunction and changes of apoptotic gene expression by dietary NAC.

An oral painless dietary therapy is also indispensable in the management of arsenic toxicity despite of its conventional painful therapeutic management. The present study focused on the management of arsenic mediated female reproductive dysfunctions by dietary therapy of N-acetyl cysteine (NAC). Here, sodium arsenite was given at the dose of 10 mg/kg body weight orally for the first 8 day. Day 9 onwards up to day 16 these arsenicated rats were provided with NAC (250 mg/kg body weight) enriched basal diet once daily. Arsenic intoxicated group exhibited a comparable inactivation of antioxidant enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) due to oxidative stress in reproductive organs along with a simultaneous elevation of lipid peroxidation state and decline in non-protein soluble thiols (NPSH) level in female reproductive organs. Arsenic intoxication also accomplished with the up-regulation of inflammatory markers tumour necrosis factor (TNF α) and nuclear factor κB (NF κB). Pro-apoptotic Bax gene and p53 gene expressions were also raised due to arsenic intoxication while anti-apoptotic Bcl-2 gene expression was suppressed. In fact, arsenication decreased the circulating level of vitamin B12 and folic acid. Dietary NAC supplementation significantly reversed back the activity of antioxidant enzymes in arsenite fed rats towards normalcy and also sustained the normal reproductive cyclicity, utero-ovarian histo-morphology and estradiol receptor α (ER-α) expression in these reproductive organs. Dietary NAC exerted its positive action against arsenic intoxication by up-regulation of Bcl-2 gene expression along with the suppression of pro-apoptotic Bax gene and p53 gene. Thus, dietary NAC also plays anti-apoptotic, anti-inflammatory, and anti-oxidative role against arsenic toxicity. NAC also regulates the components (vitamin B12 and folic acid) of S-adenosylmethionine pool in the way of probable removal of arsenic from the system.

Involvement of metallothionein, homocysteine and B-vitamins in the attenuation of arsenic-induced uterine disorders in response to the oral application of hydro-ethanolic extract of Moringa oleifera seed: a preliminary study.

The painful invasive chelation therapy makes it challenging to continue the prolonged treatment against arsenic toxicity. Hence, the significance of the present preliminary investigation was to explore a noninvasive treatment strategy against sodium arsenite (As3+) by the use of a hydroethanolic extract of Moringa oleifera (MO) seed. Arsenic treatment (10 mg/kg body-weight) in animals showed significant level of oxidative stress as evidenced by increased serum levels of malondialdehyde (MDA), conjugated dienes (CD) and reduced level of non-protein thiol (NPSH). A significant diminution in the activities of enzymatic antioxidants was noted in As3+-treated rats. As3+ treatment showed a lengthy phase of metestrous in animals followed by significantly diminished ovarian steroidogenesis, increased ovarian follicular degeneration and distortion of uterine tissue histomorphology. In addition, there was a significant depletion of Vitamin-B9 (folate) and B12 following As3+ ingestion. The levels of circulating TNF-α, homocysteine (Hcy), uterine-IL-6, and liver metallothionein (MT-1) were significantly elevated in arsenic treated rats. MO at a dose of 100 mg/kg body-weight could successfully mitigate the uterine ROS generation by maintaining the uterine antioxidant status in As3+- treated rats. This seed extract prevented the deterioration of As3+-mediated ovarian-steroidogenesis and ovarian and uterine histoarchitecture significantly. B9 and B12 levels were also improved following the ingestion of the MO extract in arsenicated animals. Elevation of Hcy, TNF-α and IL-6 was also prevented by this MO seed extract in As3+-treated rats. A further increase of MT-1 level was achieved after MO ingestion in As3+-treated rats. Here, the alleviation of arsenic toxicity might involve via the regulation of the components of S-adenosine methionine (SAM) pool and MT-1.

Dietary CCPS from bitter gourd attenuates sodium arsenite induced female reproductive ailments cum infertility in wistar rats: anti-inflammatory and anti-apoptotic role.

This investigation explored a dietary therapy of pectic polysaccharide (CCPS) (2 mg/ Kg BW) against female repro-toxicity and infertility triggered by sodium arsenite (As3+) (10 mg/ Kg BW) in Wistar rats. The isolated CCPS consists of D-galactose and D-methyl galacturonate with a molar ratio of 1: 4. FTIR spectral analysis of CCPS and CCPS- sodium arsenite (As3+) complex indicated a possible chelating property of CCPS in presence of binding sites (OH-/COOH) for As3+. Series of negatively charged galacturonate residues in CCPS provide better potential for cation chelation. CCPS significantly mitigated As3+ induced ovarian, uterine lipid peroxidation, and reactive oxygen species (ROS) generation by the restoration of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities. CCPS post-treatment enhanced ovarian steroidogenesis along with a restoration of normal tissue histoarchitecture in As3+ fed rats by regulating the estradiol receptor alpha (ER-α). CCPS suppressed anti-inflammatory properties effectively found since a down-regulation of NF-kappa B (NF-қB), pro-inflammatory tumor necrosis-α (TNF-α) and interleukin-6 (IL-6) were observed in arsenicated rats with CCPS. This study confirmed the up-regulation of uterine pro-apoptotic/ apoptotic proteins caspase-3, poly ADP ribose polymerase (PARP), proliferating cell nuclear antigen (PCNA), phospho p53 and Bax, followed by down-regulation of Bcl-2 and protein Kinase B (AKT) signaling pathway along with uterine tissue regeneration in As3+ exposed rats. Oral CCPS attenuated the above apoptotic expressional changes significantly and dietary CCPS ensured successful fertility with the birth of healthy pups in lieu of infertile condition in As3+ fed rats. Moreover, this study also supports that CCPS treatment attenuated the As3+ toxicity by modulating the S-adenosine methionine (SAM) pool components, B12, folate and homocysteine.

Arsenic-Induced Antioxidant Depletion, Oxidative DNA Breakage, and Tissue Damages are Prevented by the Combined Action of Folate and Vitamin B12.

Arsenic is a grade I human carcinogen. It acts by disrupting one-carbon (1C) metabolism and cellular methyl (-CH3) pool. The -CH3 group helps in arsenic disposition and detoxification of the biological systems. Vitamin B12 and folate, the key promoters of 1C metabolism were tested recently (daily 0.07 and 4.0 µg, respectively/100 g b.w. of rat for 28 days) to evaluate their combined efficacy in the protection from mutagenic DNA-breakage and tissue damages. The selected tissues like intestine (first-pass site), liver (major xenobiotic metabolizer) and lung (major arsenic accumulator) were collected from arsenic-ingested (0.6 ppm/same schedule) female rats. The hemo-toxicity and liver and kidney functions were monitored. Our earlier studies on arsenic-exposed humans can correlate carcinogenesis with DNA damage. Here, we demonstrate that the supplementation of physiological/therapeutic dose of vitamin B12 and folate protected the rodents significantly from arsenic-induced DNA damage (DNA fragmentation and comet assay) and hepatic and renal tissue degeneration (histo-architecture, HE staining). The level of arsenic-induced free-radical products (TBARS and conjugated diene) was significantly declined by the restored actions of several antioxidants viz. urate, thiol, catalase, xanthine oxidase, lactoperoxidase, and superoxide dismutase in the tissues of vitamin-supplemented group. The alkaline phosphatase, transaminases, urea and creatinine (hepatic and kidney toxicity marker), and lactate dehydrogenase (tissue degeneration marker) were significantly impaired in the arsenic-fed group. But a significant protection was evident in the vitamin-supplemented group. In conclusion, the combined action of folate and B12 results in the restitution in the 1C metabolic pathway and cellular methyl pool. The cumulative outcome from the enhanced arsenic methylation and antioxidative capacity was protective against arsenic induced mutagenic DNA breakages and tissue damages.

Green tea (Camellia sinensis) alleviates arsenic-induced damages to DNA and intestinal tissues in rat and in situ intestinal loop by reinforcing antioxidant system.

This study elucidates the protective role of Green tea (Camellia sinensis or CS) against arsenic-induced mutagenic DNA-breakage/intestinal (small) damages in female rats. Intestinal epithelial cells receive ingested arsenic initially. Though, the possibility of damages in this tissue is immense and the therapeutic strategies against this damage are of great concern, reports on either issue are scanty. Our earlier study on arsenic-exposed human unveils a link between carcinogenesis and mutagenic DNA damage. Here, we demonstrate that supplementation of CS-extract (10 mg/mL water) with NaAsO2 (0.6 ppm)/100 g b.w. for 28 days to rats offered a significant protection against arsenic-induced oxidative damages to DNA and intestinal (small) tissues by buttressing antioxidant systems. Necrotic and apoptotic damages and their CS-protection are shown in DNA-fragmentation, comet-assay, and histoarchitecture (hematoxylin and eosin and periodic acid-schiff staining) results. Only arsenic exposure significantly decreased intestinal superoxide dismutase, catalase activities, and level of soluble thiol with a concomitant increase in malondialdehyde/conjugated dienes. Alteration of serum necrotic marker lactate dehydrogenase and the metabolic inflammatory marker c-reactive protein also indicate the impairment may be occurring at transcription and/or cellular signal transduction level. In addition, in situ incubation in rat intestinal loop filled for 24 h with NaAsO2 alone (250 µM) or with aqueous CS-extract (250 mg/mL) suggests that small intestinal epithelial cells are significantly protected by CS against arsenic-associated necrotic/mutagenic damages, which is observed in DNA-breakage studies. In conclusion, besides intensifying endogenous antioxidant system, CS polyphenols also offer a direct role on free radical scavenging activity that is associated to the protection from mutagenic DNA-breakages and prevention of tissue necrosis/carcinogenesis generated by arsenic.

Chemoprevention against arsenic-induced mutagenic DNA breakage and apoptotic liver damage in rat via antioxidant and SOD1 upregulation by green tea (Camellia sinensis) which recovers broken DNA resulted from arsenic-H2O2 related in vitro oxidant stress.

Green tea (Camellia sinensis; CS) strongly reverses/prevents arsenic-induced apoptotic hepatic degeneration/micronecrosis and mutagenic DNA damage in in vitro oxidant stress model and in rat as shown by comet assay and histoarchitecture (HE and PAS staining) results. Earlier, we demonstrated a link between carcinogenesis and impaired antioxidant system-associated mutagenic DNA damage in arsenic-exposed human. In this study, arsenic-induced (0.6 ppm/100 g body weight/day for 28 days) impairment of cytosolic superoxide-dismutase (SOD1), catalase, xanthine-oxidase, thiol, and urate activities/levels led to increase in tissue levels of damaging malondialdehyde, conjugated dienes, serum necrotic-marker lactate-dehydrogenase, and metabolic inflammatory-marker c-reactive protein suggesting dysregulation at the transcriptional/signal-transduction level. These are decisively restrained by CS-extract (≥10 mg/ml aqueous) with a restoration of DNA/tissue structure. The structural/functional impairment of dialyzed and centrifugally concentrated (6-8 kd cutoff) hepatic SOD1 via its important Cys modifications by H2O2/arsenite redox-stress and that protection by CS/2-mercaptoethanol are shown in in vitro/in situ studies paralleling the present Swiss-Model-generated rSOD1 structural data. Here, arsenite(3+) incubation (≥10(-8) µM + 10 mM H2O2, 2 hr) is shown for the first time with this low-concentration to initiate breakage in rat hepatic-DNA in vitro whereas, arsenite/H2O2/UV-radiation does not affect DNA separately. Arsenic initiates Fe and Cu ion-associated free-radical reaction cascade in vivo. Here, 10 µM of Cu(2+)/Fe(3+)/As(3+) +H2O2-induced in vitro DNA fragmentation is prevented by CS (≥1 mg/ml), greater than the prevention of ascorbate or tocopherol or DMSO or their combination. Moreover, CS incubation for various time with differentially and already degraded DNA resulted from pre-incubation in 10 µM As(3+)-H2O2 system markedly recovers broken DNA. Present results decisively suggest for the first time that CS and its mixed polyphenols have potent SOD1 protecting, diverse radical-scavenging and antimutagenic activities furthering to DNA protection/therapy in arsenic-induced tissue necrosis/apoptosis.

Hepatoprotective role of vitamin B(12) and folic acid in arsenic intoxicated rats.

The present study elucidated the protective role of vitamin B(12) with folic acid against arsenic-induced hepatotoxicity in female rats. Ingestion of sodium-arsenite- contaminated water [0.4 ppm/100 g body weight (b.w.)/day] in combination with vitamin B(12) plus folic acid (0.07 and 4.0 µg, respectively/100 g b.w./day) for 24 days to Wistar rats offered a significant protection against alone arsenic-induced distorted liver function, damaged histoarchitecture, elevated oxidative stress, and DNA fragmentation of hepatic tissues. Arsenic only exposure decreased hepatic superoxide dismutase (SOD), catalase activities, and the level of nonprotein-soluble thiol (NPSH), with a concomitant increase in thiobarbituric acid reactive substances (TBARS) and conjugated dienes (CDs) in the liver. Vitamin supplementation restrained the increase of TBARS and CDs by restoring catalase, SOD, and NPSH levels. Restricted generation of free radicals may be correlated to the protection of DNA stability and hepatic morphology. This study explains the decisive role of vitamin B(12) with folic acid to ameliorate arsenic-mediated liver injuries.

Protective role of Moringa oleifera (Sajina) seed on arsenic-induced hepatocellular degeneration in female albino rats.

In an attempt to develop new herbal therapy, an aqueous extract of the seed of Moringa oleifera was used to screen the effect on arsenic-induced hepatic toxicity in female rat of Wistar strain. Subchronic exposure to sodium arsenite (0.4 ppm/100 g body weight/day via drinking water for a period of 24 days) significantly increased activities of hepatic and lipid function markers such as alanine transaminase, aspartate transaminase, cholesterol, triglycerides, LDL along with a decrease in total protein and HDL. A notable distortion of hepatocellular histoarchitecture was prominent with a concomitant increase in DNA fragmentation following arsenic exposure. A marked elevation of lipid peroxidation in hepatic tissue was also evident from the hepatic accumulation of malondialdehyde and conjugated dienes along with suppressed activities in the antioxidant enzymes such as superoxide dismutase and catalase. However, co-administration of aqueous seed extract of M. oleifera (500 mg/100 g body weight/day for a period of 24 days) was found to significantly prevent the arsenic-induced alteration of hepatic function markers and lipid profile. Moreover, the degeneration of histoarchitecture of liver found in arsenic-treated rats was protected along with partial but definite prevention against DNA fragmentation induction. Similarly, generation of reactive oxygen species and free radicals were found to be significantly less along with restored activities of antioxidant enzymes in M. oleifera co-administered group with comparison to arsenic alone treatment group. The present investigation offers strong evidence for the hepato-protective and antioxidative efficiencies of M. oleifera seed extract against oxidative stress induced by arsenic.

Effect of dietary co-administration of sodium selenite on sodium arsenite-induced ovarian and uterine disorders in mature albino rats.

The subchronic treatment of mature female Wistar-strain albino rats in diestrous phase with sodium arsenite at a dose of 0.4 ppm/100 g body weight/rat/day via drinking water for period of 28 days (seven estrous cycles) caused a significant reduction in the plasma levels of leutinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol along with a significant decrease in ovarian activities of delta five, 3 beta-hydroxysteroid dehydrogenase (Delta5,3beta-HSD), and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD) followed by a reduction in ovarian and uterine peroxidase activities. A significant weight loss of the ovary and uterus was also observed after this treatment, along with a prolonged diestrous phase and a high accumulation of arsenic in the plasma and these organs. Moreover, sodium arsenite was also responsible for ovarian follicular and uterine cell degeneration characterized by a high number of regressing follicles and a reduction in the uterine luminal diameter, respectively, in comparison with the controls. A dietary supplementation of sodium selenite at the dose of 0.6 mg/100 g body weight/rat/day for a period of 28 days along with arsenic treatment minimized the gonadal weight loss significantly and increased the activities of the ovarian steroidogenic enzymes as well as the ovarian and uterine peroxidase at the control level. Selenium was also able to increase the plasma levels of LH, FSH, and estradiol toward the control level. Vaginal smears showed normal estrous cyclicity in sodium selenite-supplemented arsenic-treated rats along with lower arsenic levels in the plasma and gonadal tissue in comparison with arsenic-only-treated rats. Histological sections of ovary and uterine tissues in the control and experimental groups confirmed that sodium selenite supplementation was able to prevent arsenic-induced histopathological changes in the ovary and uterus. Plasma levels of norepinephrine and dopamine in the midbrain and diencephalon decreased significantly, whereas the serotonin level was increased significantly after 28 days of sodium arsenite treatment. All of these parameters were, in most cases, unchanged from the control level when sodium selenite was co-administered with sodium arsenite. Arsenic intoxication was also associated with increased liver weight and elevation in the activities of hepatic and renal acid phosphatase, alkaline phosphatase, and transaminases, but selenium co-administration was not able to change these toxic effects of arsenic. The results of our experiments indicate the significant protective action of sodium selenite on arsenic-induced toxicity in the female reproductive system, while there was no significant protective effect of selenium on arsenic-induced toxicity in other organs.