Pharmacological Evaluation of Polygoni Multiflori Radix Praeparata Extract: Inhibition of PANoptosis in Alleviating Premature Ovarian Insufficiency

Abstract

Polygoni Multiflori Radix Praeparata (PMRP), a processed root of Polygonum multiflorum Thunb. (known as Zhiheshouwu in Chinese medicine), exhibits anti-aging properties and is used to improve ovarian aging. However, its therapeutic mechanism against premature ovarian insufficiency (POI) remains unclear. This study investigates whether PMRP alleviates POI by inhibiting PANoptosis-a cell death pathway characterized by the concurrent occurrence and interplay of pyroptosis, apoptosis, and necroptosis. POI was induced in rats using tripterygium glycosides. We evaluated the estrous cycle, serum hormone levels (follicle-stimulating hormone [FSH], estrogen [E₂], anti-Müllerian hormone [AMH]), follicular development, and the ultrastructure of granulosa cells. PANoptosome assembly (apoptosis-associated speck-like protein containing a CARD [ASC]/caspase-8/receptor-interacting protein kinase 3 [RIPK3] co-localization) and key effectors of PANoptosis (caspase 3, cleaved caspase 3, gasdermin D [GSDMD], cleaved GSDMD, GSDME, RIPK1, mixed-lineage kinase domain-like protein [MLKL], and p-MLKL) were analyzed. PMRP restored the estrous cycle, lowered FSH levels, and increased E₂ and AMH levels in POI rats. It reduced follicular atresia, preserved primordial follicles, and suppressed PANoptosis-like death in granulosa cells. Mechanistically, PMRP disrupted PANoptosome assembly and downregulated key effectors of PANoptosis. PMRP alleviates POI by inhibiting PANoptosis in granulosa cells, overcoming the previous limitations of targeting single death pathways and providing novel insights into the pathogenesis and treatment strategies for POI.

Keywords: PANoptosis; PANoptosome; polygoni multiflori radix praeparata; premature ovarian insufficiency.

Figure 1

LC–MS analysis of PMRP components. (A) Total ion chromatograms showing negative (black) and positive (red) ion modes. (B–D) Chromatograms and mass spectrums for TSG, Emodin, and Emodin-3-methyl ether.

Figure 2

PMRP improves the estrous cycle and ovarian index in POI rats. (A) Representative estrous cycle phases (×200): (a) Proestrus, (b) Estrus, (c) Metestrus, (d) Diestrus. Blue arrows indicate nucleated epithelial cells, red arrows represent keratinized epithelial cells, and black arrows denote leukocytes. (B) Estrous cycle counts. (C) Body weight. (D) Ovarian index comparison. (Mean ± SEM, n = 6, * p < 0.05 and ** p < 0.01 vs. model group, NS means not significant).

Figure 3

PMRP regulates the serum sex hormone levels in POI rats. (A–C) Serum concentrations of FSH, E₂, and AMH. (Mean ± SEM, n = 6, * p < 0.05 and ** p < 0.01 vs. model group, NS means not significant).

Figure 4

PMRP improves follicular development in POI rats (HE staining). (A) Histomorphological alterations in follicles. Red arrows indicate primordial follicles, blue arrows represent primary follicles, green arrows denote secondary follicles, yellow arrows point to atretic follicles, black arrows indicate mature follicles, and CL denotes corpus luteum. (B) Numbers of primordial follicles, growing follicles (including primary and secondary follicles), atretic follicles, mature follicles, and corpora lutea. (Mean ± SEM, n = 6, * p < 0.05 and ** p < 0.01, NS means not significant vs. model group).

Figure 5

PMRP mitigates the PANoptosis-like death of granulosa cells in POI rats (TEM). Nuclear (N), mitochondria (red arrows), cytoplasmic vacuolization (yellow arrows), and cell membrane rupture with content release (green arrows) are indicated.

Figure 6

PMRP inhibits the PANoptosome assembly in POI rat follicles. (A) Immunofluorescence images of ASC (red), caspase 8 (green), and RIPK3 (rose red) co-localization with nuclear DAPI staining (blue). (B–D) Average fluorescence intensity of ASC, caspase 8, and RIPK3. (Mean ± SEM, n = 3, * p < 0.05 and ** p < 0.01 vs. model group). Dashed box shows the zoomed area.

Figure 7

PMRP suppresses PANoptosis effector proteins in POI rat ovaries. (A) Western blot images. (B–I) Relative protein expression levels for caspase 3, cleaved caspase 3, GSDMD, cleaved GSDMD, GSDME, RIPK1, MLKL, and p-MLKL. (Mean ± SEM, n = 3, * p < 0.05 and ** p < 0.01, NS means not significant vs. model group).

Figure 8

PMRP alleviates POI by inhibiting PANoptosis in follicular granulosa cells.