Molecular cues to the anti-implantation effect of nano-puerarin in rats.

Puerarin, a selective oestrogen receptor modulator, intercepts implantation in rats, albeit at unacceptably higher doses. We developed poly lactic-co-glycolic acid-encapsulated nano-puerarin (PN) and mapped the molecular pathway underlying its anti-implantation effects. Smooth-surfaced and spherical PN having a mean diameter of ∼147nm was obtained with good yield, efficient encapsulation, and optimum drug loading. In culture, PN slowly and steadily released puerarin, which was readily taken up by the decidual cells. PN exerted a dose-dependent anti-implantation effect. As marked by attenuated expression of stromal cell desmin, alkaline phosphatase, IGFBP1, and decidual prolactin-related protein, the anti-implantation effect of PN seemed secondary to compromised decidualization. Using in vivo (pregnant and pseudopregnant rats) and in vitro (endometrial stromal cell culture) treatment models, we document that PN enforced inhibition of uterine expression of Hbegf and Hoxa10 and their downstream signalling molecules, Cyclin D3 (CCND3)/CDK4. PN also efficiently ablated the Ihh-Nr2f2-Bmp2 signalling pathway and invited the loss of uterine potential for decidualization. There was a dose-dependent up-regulation of RHOA and its effector protein kinase, ROCK1, leading to the promotion of MLC phosphorylation and actin-myosin interaction. PN also down-regulated the stromal cell activation of ERK½ and expression of MMP9. These effects acting together stabilized the stroma and inhibited the stromal cell migration. Central to this array of events was the adversely altered endometrial expression of oestrogen receptor subtypes and repression of progesterone receptor that indulged endless proliferation of luminal epithelia and distorted the precisely choreographed stroma-epithelia crosstalk. Thus, PN dismantles the endometrial bed preparation and prevents implantation.

Female reproductive aging is master-planned at the level of ovary.

The ovary receives a finite pool of follicles during fetal life. Atresia remains the major form of follicular expenditure at all stages since development of ovary. The follicular reserve, however, declines at an exponential rate leading to accelerated rate of decay during the years preceding menopause. We examined if diminished follicle reserve that characterizes ovarian aging impacts the attrition rate. Premature ovarian aging was induced in rats by intra-embryonic injection of galactosyltransferase-antibody on embryonic day 10. On post-natal day 35 of the female litters, either a wedge of fat (sham control) or a wild type ovary collected from 25-day old control rats, was transplanted under the ovarian bursa in both sides. Follicular growth and atresia, and ovarian microenvironment were evaluated in the follicle-deficient host ovary and transplanted ovary by real time RT-PCR analysis of growth differentiation factor-9, bone morphogenetic protein 15, and kit ligand, biochemical evaluation of ovarian lipid peroxidation, superoxide dismutase (SOD) and catalase activity, and western blot analysis of ovarian pro- and anti-apoptotic factors including p53, bax, bcl2, and caspase 3. Results demonstrated that the rate of follicular atresia, which was highly preponderant in the follicle-deficient ovary of the sham-operated group, was significantly prevented in the presence of the transplanted ovary. As against the follicle-deficient ovary of the sham-operated group, the follicle-deficient host ovary as well as the transplanted ovary in the ovary-transplanted group exhibited stimulated follicle growth with increased expression of anti-apoptotic factors and down regulation of pro-apoptotic factors. Both the host and transplanted ovaries also had significantly lower rate of lipid peroxidation with increased SOD and catalase activity. We conclude that the declining follicular reserve is perhaps the immediate thrust that increases the rate of follicle depletion during the final phase of ovarian life when the follicle reserve wanes below certain threshold size.

α-Dihydroxychalcone-glycoside (α-DHC) isolated from the heartwood of Pterocarpus marsupium inhibits LPS induced MAPK activation and up regulates HO-1 expression in murine RAW 264.7 macrophage.

Three phenolic glycosides isolated from the heartwood of Pterocarpus marsupium showed significant free radical and superoxide ion scavenging activity and antioxidant potential that were comparable to, or several folds higher than those of standard antioxidants, trolox and ascorbic acid. The effective concentrations of these compounds were far below their cytotoxic levels. Compound 3, which was characterized to be α-dihydroxychalcone-glycoside (α-DHC), was the most potent one. Subsequent studies demonstrated that α-DHC effectively reduced nitric oxide and cytokine production by the LPS stimulated RAW 264.7 mouse macrophage cell line. The compound effectively attenuated the expression of inflammation-mediating enzymes COX-2 and iNOS at the mRNA as well as protein levels in a concentration dependent manner. It prevented phosphorylation of all the three MAPKs (JNK, ERK, p38) and eventually blocked the activation of downstream elements contributing to inflammation. Phosphorylation of IκB-α and subsequent translocation of NF-κB into the nucleus were restricted, while the expression of stress responsive gene HO-1 was up-regulated. α-DHC targeted Keap-1 by modifying its cysteine thiols, dissociating it from Nrf-2 and facilitating nuclear entry of the latter; and this in turn induced HO-1 expression. Thus α-DHC exerts its anti-inflammatory activity in a dual manner: by down regulating MAPKs and restricting nuclear stabilization of NF-κB at one end, and by disrupting Nrf-2-Keap-1 complex on the other. In conclusion, the anti-inflammatory potential together with its high therapeutic index envisages α-DHC as a prospective candidate molecule for the development of therapeutic strategy against inflammatory disorders.

Puerarin, a selective oestrogen receptor modulator, disrupts pregnancy in rats at pre-implantation stage.

The tubers of Pueraria tuberosa have folkloric repute as emmenagogue. The n-BuOH fraction of the ethanolic extract of tubers exhibits significant antifertility activity in laboratory animals. The present investigation explored the active principle(s) of the tuber extract with reference to contragestive effects in rats and probed the possible mechanism of action. Bioactivity-guided fractionation identified puerarin as the major constituent that exerted pregnancy-terminating effects. Oral administration of puerarin at ≥300  mg/kg per day for days (D) 1-2 post-coitus resulted in complete implantation failure. Serum oestradiol levels during D2-D5 and progesterone (P(4)) level on D5 remained unaffected, but the endometrial expression of oestrogen receptor α (ERα) and ERß was adversely modulated that disrupted the implantation-specific characteristic endometrial oestrogenic milieu. The eventual consequence was loss of endometrial receptivity characterised by down-regulation of the uterine expression of P(4) receptor (PR) and attenuation of endometrial expression of leukaemia inhibitory factor, vascular endothelial growth factor and cyclo-oxygenase-2, the three important signalling molecules involved in the process of implantation. Light microscopic examination of the embryos demonstrated no untoward effect of puerarin on the development of embryos until D4, but D5 blastocysts underwent gross morphological distortion. The findings taken together are interpreted to suggest that puerarin adversely impacts the uterine expression of ER and PR that disrupts the implantation-conducive uterine milieu and prevents implantation. In conclusion, puerarin may be envisaged as a prospective molecule that merits further exploration for the development of non-steroidal post-coital contraceptive for women.