Crosstalk between follicular fluid (FF) and granulosa cells (GCs) plays a vital role in the regulation of folliculogenesis, ensuring regular reproductive cycle in mammals. This crosstalk is primarily ...mediated by hormones and signaling molecules, such as cytokines and chemokines. Recently, extracellular microRNAs (miRNAs) have gained a lot of attention in cell-to-cell communication. Extracellular miRNA transportation occurs through exosomes, a kind of micro-vesicles produced from almost all cells. However, the mode of non-exosomal miRNA internalization is not much studied. In the present study, we explored the role of neuropilin
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1 (NRP-1) as a receptor in internalizing FF non-exosomal miRNAs in GCs. We first confirmed the expression of NRP-1 in GCs during follicular development followed by its role in the internalization of miR-210, a non-exosomal miRNA. This study showed that incubation of GCs with a non-exosomal fraction of FF increased the content of miR-210 in GCs as compared to their control. To illustrate the role of NRP-1 as a receptor, NRP-1 was knockdown using siRNA. Silencing experimental results showed a significant decrease in uptake of miR-210 in NRP-1 knockdown GCs. Furthermore, downstream expression analysis of miR-210 target genes (CYP19A1, PCNA, and EFNA3) also confirmed the NRP-1 mediated miR-210 internalization. Results of the present study clearly demonstrated that FF non-exosomal miR-210 can be internalized through the NRP-1 receptor. Furthermore, differential expression of NRP-1 in GCs suggests its role in follicular development. Overall, these findings suggest that FF non-exosomal miRNA plays an important role in GC functions and female reproduction.
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•MiR-326 target CYP19A1 and CREB gene.•MiR-326 downregulates CYP19A1 expression and inhibits estradiol-17b synthesis.•ChIP showed increased binding of C/EBP-β to CYP19A1 promoter II ...in miR-326 transfected cells.•ChIP showed decreased binding of RNA polymerase II to CYP19A1 promoter II in miR-326 transfected cells.
Ovarian granulosa cells, known to be endocrine cells, have well active TLR4-/NFKB signalling mediated innate immune capabilities. We have previously shown that endotoxin not only transiently regulates proinflammatory cytokines but cells become tolerant on repeated exposure to endotoxin and impaired granulosa cells functions, which includes downregulation of CYP19A1 gene. To understand further endotoxin tolerance and impaired granulosa cells function, genome-wide transcriptomic profiling in endotoxin tolerant buffalo granulosa cells (bGCs) identified miR-326 as upregulated amongst top 5 DE miRNAs unpublished data and qPCR validation confirmed its upregulation during endotoxin tolerance. In silico analyses showed that miR-326 targets CYP19A1 gene. Therefore, in the present study, we elucidated the role of miR-326 in buffalo granulosa cells (bGCs). We first validated its expression vis-à-vis CYP19A1 gene expression in bGCs, both in vivo and in vitro. Results showed an inverse relationship between miR-326 and CYP19A1 expression. Similarly, transcription factors, known to be involved in CYP19A1 gene regulation, CREB and C/EBP-β expression was also found to be decreased in granulosa cells mimicking pre-ovulatory follicular stage. Further, miR-326 mimic was transfected to bGCs in culture and expression of CYP19A1 and CREB & C/EBP-β and genes encoding other enzymes of steroidogenesis pathway were also analyzed. The present study results showed that miR-326 significantly inhibits the expression of CYP19A1 gene while expression of transcription factors CREB and C/EBP-β was found to be upregulated. The expression of STAR and CYP11A1 was found to be unaffected. To elucidate the molecular mechanism of miR-326 mediated downregulation of CYP19A1, binding analyses of RNA polymerase II and CEBP-β to CYP19A1 gene promoter II was analyzed. The result also showed decreased binding of RNA polymerase II with increased binding of CEBP-β to CYP19A1 gene promoter II in bGCs, transfected with miR-326 as compared to control. In summary, our results suggest that miR-326 upregulate CREB and CREB may activate C/EBP-β and later inhibited the transcription of CYP19A1 and decreased estradiol-17b production. The miR-326 mediated down-regulation of the CYP19A1 gene involving CREB-C/EBP-β can be exploited in developing strategies to attenuate endotoxin-mediated tolerance induced impaired granulosa cells function to ensure proper fertility in females.
A genome-wide profiling of microRNA (miRNA) in endotoxin tolerant buffalo granulosa cells identified miR-326 amongst top-10 upregulated miRNAs. In this study, we have elucidated the role of miR-326 ...in granulosa cells in vitro. In-silico analysis revealed that miR-326 have binding site for 3′UTR of TLR-4 (Toll-like receptor 4). Transfection experiments showed that there was a significant inhibition of TLR-4 when miR-326 mimic was transfected to buffalo granulosa cells. Furthermore, when miR-326 transfected granulosa cells were exposed to LPS, followed by expression analysis of TLR-4 complex genes (TLR-4 and MyD88) and pro-inflammatory cytokines, we found a decreased expression of both TLR-4 complex and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β). We also found that the expression of anti-inflammatory (IL-10) gene was upregulated. To the best of our knowledge, this is the first study that showed the regulation of TLR-4 using miR-326. The present findings on regulation of TLR-4 are important and would help in understanding innate immunity regulation under different patho-physiology.
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Adipose tissue (AT) expansion through hyperplasia or hypertrophy requires vascular remodeling that involves angiogenesis. There is quite some evidence that obese white AT (WAT) ...displays altered vasculature. Some studies suggest that this is associated with hypoxia, which is thought to play a role in inducing inflammatory activation of the excessively expanding WAT. Increasing evidence, based on genetic manipulations or treatments with inhibitory or activator pharmaceuticals, demonstrates that AT angiogenesis is crucial for AT metabolic function, and thereby for whole body metabolism and metabolic health. Despite some contradiction between studies, disturbance of WAT angiogenesis in obesity could be an important factor driving WAT dysfunction and the comorbidities of obesity. Endothelial cells (ECs) contribute to healthy WAT metabolism via transport of fatty acids and other plasma components, secretory signaling molecules, and extracellular vesicles (EVs). This communication is crucial for adipocyte metabolism and underscores the key role that the AT endothelium plays in systemic energy homeostasis and healthy metabolism. Adipocytes communicate towards the neighboring endothelium through several mechanisms. The pro-inflammatory status of hypertrophic adipocytes in obesity is reflected in ECs activation, which promotes chronic inflammation. On the other hand, adiponectin secreted by the adipocytes is important for healthy endothelial function, and adipocytes also secrete other pro- or anti-angiogenic effector molecules and a wealth of EVs – however, their detailed roles in signaling towards the endothelium are yet poorly understood. To conclude, targeting AT angiogenesis and promoting the healthy communication between adipocytes and ECs represent potentially promising strategies to treat obesity and its comorbidities.
Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in ...detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.
HPLC was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of
B. caapi. The extracts were simultaneously tested
in vitro for inhibition of human MAO-A and ...antioxidant activity in order to correlate phytochemical composition of the extracts and bioactivities.
Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a medicinal tea for healing and spiritual exploration. The usefulness of
Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinson's disease.
Primary objective of this study was to develop the process for preparing standardized extracts of
Banisteriopsis caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties. The aqueous extracts prepared from different parts of the plant collected from different geographical locations and seasons were analyzed by HPLC for principal bioactive markers. The extracts were simultaneously tested
in vitro for inhibition of human MAOs and antioxidant activity for analysis of correlation between phytochemical composition of the extracts and bioactivities.
Reversed-phase HPLC with photodiode array detection was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of
Banisteriopsis caapi. The
Banisteriopsis caapi extracts and standardized compositions were tested
in vitro for inhibition of recombinant preparations of human MAO-A and MAO-B.
In vitro cell-based assays were employed for evaluation of antioxidant property and mammalian cell cytotoxicity of these preparations.
Among the different aerial parts, leaves, stems/large branches and stem bark of
Banisteriopsis caapi, HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7–9) were present in high concentrations in dried bark of large branch. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied
Banisteriopsis caapi species. The correlation between potency of MAO inhibition and antioxidant activity with the content of the main active constituents of the aqueous
Banisteriopsis caapi extracts and standardized compositions was established. Phytochemical analysis of regular/commercial
Banisteriopsis caapi dried stems, obtained from different sources, showed a similar qualitative HPLC profile, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency compared to
Banisteriopsis caapi Da Vine.
The ethnopharmacological use of bark of matured stem/large branch of
Banisteriopsis caapi as well as whole matured stem is supported by the results obtained in this investigation. Among various constituents of
Banisteriopsis caapi, harmine (7), harmaline (6) and tetrahydroharmine (5) are responsible for MAO-A inhibition, while two major proanthocyanidines, epicatechin (8) and procyanidine B2 (9) produce antioxidant effects. The compounds 1–9 can serve as reliable markers for identification and standardization of
Banisteriopsis caapi aerial parts, collected in different seasons and/or from different geographical regions.
•Cohort effect shows that obesity for recent cohort has narrowed down.•Our study shows as age increases the risk of obesity also increases.•Cohort relative risk is higher among women in rural area ...than women in urban area.
Ethnopharmacological relevance - Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a ...medicinal tea for healing and spiritual exploration. The usefulness of Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinson's disease. Aim of the study - Primary objective of this study was to develop the process for preparing standardized extracts of Banisteriopsis caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties. The aqueous extracts prepared from different parts of the plant collected from different geographical locations and seasons were analyzed by HPLC for principal bioactive markers. The extracts were simultaneously tested in vitro for inhibition of human MAOs and antioxidant activity for analysis of correlation between phytochemical composition of the extracts and bioactivities. Materials and methods - Reversed-phase HPLC with photodiode array detection was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of Banisteriopsis caapi. The Banisteriopsis caapi extracts and standardized compositions were tested in vitro for inhibition of recombinant preparations of human MAO-A and MAO-B. In vitro cell-based assays were employed for evaluation of antioxidant property and mammalian cell cytotoxicity of these preparations. Results - Among the different aerial parts, leaves, stems/large branches and stem bark of Banisteriopsis caapi, HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7-9) were present in high concentrations in dried bark of large branch. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied Banisteriopsis caapi species. The correlation between potency of MAO inhibition and antioxidant activity with the content of the main active constituents of the aqueous Banisteriopsis caapi extracts and standardized compositions was established. Phytochemical analysis of regular/commercial Banisteriopsis caapi dried stems, obtained from different sources, showed a similar qualitative HPLC profile, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency compared to Banisteriopsis caapi da Vine. Conclusion - The ethnopharmacological use of bark of matured stem/large branch of Banisteriopsis caapi as well as whole matured stem is supported by the results obtained in this investigation. Among various constituents of Banisteriopsis caapi, harmine (7), harmaline (6) and tetrahydroharmine (5) are responsible for MAO-A inhibition, while two major proanthocyanidines, epicatechin (8) and procyanidine B2 (9) produce antioxidant effects. The compounds 1-9 can serve as reliable markers for identification and standardization of Banisteriopsis caapi aerial parts, collected in different seasons and/or from different geographical regions.
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