BuShen HuoXue decoction improves fertility through intestinal hsp-16.2-mediated heat-shock signaling pathway in Caenorhabditis elegans

Abstract

Introduction: BuShen HuoXue (BSHX) decoction is commonly used in the clinical treatment of premature ovarian failure because it can increase estradiol level and decrease follicle-stimulating hormone level. In this study, we determined the potential therapeutic effects of BSHX decoction via anti-stress pathway and the underlying mechanism by using the nematode Caenorhabditis elegans as an assay system. Methods: Bisphenol A (BPA, 175 μg/mL) was used to establish a fertility-defective C. elegans model. Nematodes were cultivated according to standard methods. Brood size, DTC, the number of apoptotic cells and oocytes were used to evaluate the fertility of nematodes. Nematodes were cultivated at 35°C as heat stress. RNA isolation and RT-qPCR were used to detect the mRNA expression level of genes. Intestinal ROS and intestinal permeability were used to evaluate the function of intestinal barrier. BSHX decoction was extracted with water and analyzed by LC/Q-TOF. Results and Discussion: In BPA-treated N2 nematodes, 62.5 mg/mL BSHX decoction significantly improved the brood size and the oocytes quality at different developmental stages. BSHX decoction improved resistance to heat stress through the hsf-1-mediated heat-shock signaling pathway. Further analysis showed that the decoction significantly improved the transcriptional levels of hsf-1 downstream target genes, such as hsp-16.1, hsp-16.2, hsp-16.41, and hsp-16.48. Other than hsp-16.2 expression in the gonad, the decoction also affected intestinal hsp-16.2 expression and significantly reversed the adverse effects induced by BPA. Moreover, the decoction ameliorated intestinal ROS and permeability. Thus, BSHX decoction can improve fertility by increasing intestinal barrier function via hsp-16.2-mediated heat-shock signaling pathway in C. elegans. These findings reveal the underlying regulatory mechanisms of hsp-16.2-mediated heat resistance against fertility defect.

Keywords: Bushen Huoxue decoction; C. elegans; HSP-16.2; fertility; intestinal barrier.

FIGURE 1

Extracted ion chromatograms (EICs) of the representative active ingredients identified in BSHX decoction and sample of BSHX-treated nematodes by LC/Q-TOF operated in the negative ionization mode. (A–D) The different components are as follows: I, catalpol; II, loganin; III, paeoniflorin; and IV, verbascoside; Upper panel: Standards, lower panel: BSHX decoction. (E–G) The different components are as follows: Ⅴ, salvianolic acid B; VI, quercetin; VII, asperosaponin VI; Upper panel: Standards, middle panel: BSHX decoction, and lower panel: BSHX-treated nematodes. BSHX, Bushen Huoxue.

FIGURE 2

Effects of BSHX decoction administration on brood size of the fertility-defective nematodes. (A) Diagram of BPA and BSHX administration to the nematodes. (B) Effects of BPA exposure on brood size. (C) Effects of BSHX on fertility-defective nematodes. (D) Effects of BSHX on fertility-defective nematodes on each spawning day. (E) Left panel: Representative images of DTC in JK2868 and JK2868 nematodes treated with BPA and BPA as well as BSHX (scale bar, 10 μm); right panel: Histogram of the fluorescence intensity of DTC. (F) Left panel: Representative images of apoptotic cells in N2 nematodes and N2 nematodes treated with BPA and BPA as well as BSHX (scale bar, 20 μm), with arrows pointing to apoptotic cells; right panel: Histogram of the number of apoptotic cells. (G) Left panel: Representative images of oocytes in the diakinesis stage in OD95 and OD95 nematodes treated with BPA and BPA as well as BSHX (scale bar, 20 μm); the drawn frame is the observed gonad arm; right panel: Histogram of the number of oocytes in the diakinesis stage. BPA, Bisphenol A. BSHX, BuShen HuoXue. Bars represent means ± SEM. *p < 0.05 vs. the control group. #p < 0.05 vs. the BPA-treated group. BSHX, Bushen Huoxue. BPA, Bisphenol A.

FIGURE 3

BSHX decoction improves resistance to heat stress through hsf-1-mediated heat-shock signaling pathway in fertility-defective nematodes. (A) Time course of survival in N2 nematodes treated with/without BSHX and a BPA after heat stress (B) Time course of survival in glp-1 nematodes treated with/without BSHX after heat stress (C) Time course of survival in hsf-1 nematodes treated with/without BSHX after heat stress (D) Relative mRNA levels of genes relevant to the heat-shock signaling pathway in nematodes treated with BPA and BSHX. (E) Left panel: Representative images of hsp-16.2 _p ::GFP expression in TJ375 and TJ375 nematodes treated with BPA and BPA as well as BSHX (scale bar, 100 μm). Right panel: Histogram of hsp-16.2 _P ::GFP relative fluorescence intensity/body area of the nematodes. Bars represent means SEM. *p < 0.05 vs. the control group. #p < 0.05 vs. the BPA-treated group. BSHX, Bushen Huoxue. BPA, Bisphenol A.

FIGURE 4

BSHX decoction improves the fertility of fertility-defective nematodes through the combined effects of hsp-16.2 in the gonad and intestinal Tissue-specific activity of hsp-16.2 in response to the effects of BSHX on BPA-treated nematodes in brood size, (A) N2 nematodes, (B) DCL569 nematodes (germline-specific RNAi), (C) VP303 nematodes (intestine-specific RNAi), and (D) NR350 nematodes (muscle-specific RNAi). Tissue-specific activity of hsp-16.2 in response to the effects of BSHX on BPA-treated nematodes in heat-stress resistance, (E) N2 nematodes, (F) DCL569 nematodes (germline-specific RNAi), (G) VP303 nematodes (intestine-specific RNAi) and (H) NR350 nematodes (muscle-specific RNAi). Bars represent means ± SEM. *p < 0.05 vs. the control group. #p < 0.05 vs. the BPA-treated group. + p < 0.05 vs. the BPA and BSHX co-treated group. BSHX, Bushen Huoxue. BPA, Bisphenol A.

FIGURE 5

BSHX decoction improves the intestinal barrier by decreasing intestinal ROS and permeability. (A) Relative mRNA levels of genes relevant to intestinal function in fertility-defective nematodes treated with BSHX. (B) Relative mRNA levels of genes relevant to intestinal function in nematodes and hsp-16.2 (RNAi) nematodes treated with BSHX and BPA. (C) Left panel: Representative images of intestinal ROS content in N2 nematodes and N2 nematodes treated with BPA, BSHX, and BPA as well as BSHX with/without hsp-16.2 RNAi (scale bar, 100 μm); right panel: Histogram of the fluorescence intensity of intestinal ROS. (D) Representative images of intestinal permeability in N2 nematodes and N2 nematodes treated with BPA, BSHX, and BPA as well as BSHX with/without hsp-16.2 RNAi (scale bar, 1 mm). Bars represent means ± SEM. *p < 0.05 vs. the control group. #p < 0.05 vs. the BPA-treated group. BSHX, Bushen Huoxue. BPA, Bisphenol A.