Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1 and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.

Curcumin, a natural phenolic compound, exhibits poor absorption and extensive first pass metabolism after oral administration. In the present study, curcumin-chitosan nanoparticles (cur-cs-np) were prepared and incorporated into ethyl cellulose patches for the management of inflammation via skin delivery. Ionic gelation method was used for the preparation of nanoparticles. The prepared nanoparticles were evaluated for size, zetapotential, surface morphology, drug content, and % encapsulation efficiency. The nanoparticles were then incorporated into ethyl cellulose-based patches using solvent evaporation technique. ATR-FTIR was used to study/assess incompatibility between drug and excipients. The prepared patches were evaluated physiochemically. The in vitro release, ex vivo permeation, and skin drug retention studies were carried out using Franz diffusion cells and rat skin as permeable membrane. The prepared nanoparticles were spherical, with particle size in the range of 203–229 nm, zetapotential 25–36 mV, and PDI 0.27–0.29 Mw/Mn. The drug content and %EE were 53% and 59%. Nanoparticles incorporated patches are smooth, flexible, and homogenous. The in vitro release and ex vivo permeation of curcumin from nanoparticles were higher than the patches, whereas the skin retention of curcumin was significantly higher in case of patches. The developed patches deliver cur-cs-np into the skin, where nanoparticles interact with skin negative charges and hence result in higher and prolonged retention in the skin. The higher concentration of drug in the skin helps in better management of inflammation. This was shown by anti-inflammatory activity. The inflammation (volume of paw) was significantly reduced when using patches as compared to nanoparticles. It was concluded that the incorporation of cur-cs-np into ethyl cellulose-based patches results in controlled release and hence enhanced anti-inflammatory activity.

MicroRNAs are small noncoding RNAs that play critical roles in regulation of gene expression in wide array of tissues including the ovary through sequence complementarity at post-transcriptional level. Tight regulation of multitude of genes involved in ovarian development and folliculogenesis could be regulated at transcription level by these miRNAs. Therefore, tissue specific miRNAs identification is considered a key step towards understanding the role of miRNAs in biological processes. To investigate the role of microRNAs during ovarian development and folliculogenesis we sequenced eight different libraries using Illumina deep sequencing technology. Different developmental stages were selected to explore miRNAs expression pattern at different stages of gonadal maturation with/without treatment of PMSG/hCG for superovulation. From massive sequencing reads, clean reads of 16–26 bp were selected for further analysis of differential expression analysis and novel microRNA annotation. Expression analysis of all miRNAs at different developmental stages showed that some miRNAs were present ubiquitously while others were differentially expressed at different stages. Among differentially expressed miRNAs we reported 61 miRNAs with a fold change of more than 2 at different developmental stages among all libraries. Among the up-regulated miRNAs, mmu-mir-1298 had the highest fold change with 4.025 while mmu-mir-150 was down-regulated more than 3 fold. Furthermore, we found 2659 target genes for 20 differentially expressed microRNAs using seven different target predictions programs (DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR5, TargetScan). Analysis of the predicted targets showed certain ovary specific genes targeted by single or multiple microRNAs. Furthermore, pathway annotation and Gene ontology showed involvement of these microRNAs in basic cellular process. These results suggest the presence of different miRNAs at different stages of ovarian development and superovulation. Potential role of these microRNAs was elucidated using bioinformatics tools in regulation of different pathways, biological functions and cellular components underlying ovarian development and superovulation. These results provide a framework for extended analysis of miRNAs and their roles during ovarian development and superovulation. Furthermore, this study provides a base for characterization of individual miRNAs to discover their role in ovarian development and female fertility.

The current study aims to assess the aqueous extract of Pinus wallichiana stem for the synthesis of small spherical-shaped (10–30 nm) silver nanoparticles (AgNPs) and their in-vitro and in-vivo biomedical applications. The biosynthesized AgNPs were nonmutagenic and safe at all test doses as per Ames and acute toxicity assay (20, 40, 60, and 80 mg/kg). The percent writhing inhibitory effect generated by AgNPs was 42.51, 50.84, and 59.06 at test doses of 10, 20, and 30 mg/kg, respectively. The percent decreased in gastrointestinal tract motility observed was 41.34%, 32.69%, and 28.48% at 10, 20, and 30 mg/kg, respectively. They also showed a significant antipyretic effect after 1, 2, and 3 h in comparison to normal saline. The AgNPs of P. wallichiana showed good antibacterial activity against Acinetobacter baumannii (60% with MIC₅₀ = 2.36 mg/ml and MBC = 5.0 mg/ml). These nanoparticles also possessed good antioxidant activity of 61.77 ± 0.828% and 70.25 ± 0.56% at 400 and 500 µg/ml, respectively and lack phytoagglutinin potential. Because of their high potency as biomedical agents, these nanoparticles can be a good alternative to the currently available drugs and approaches. GRAPHICAL ABSTRACT