The imbalance in the aortic ceramide/sphingosine-1-phosphate rheostat in ovariectomized rats and the preventive effect of estrogen

Abstract

Background: The prevalence of hypertension in young women is lower than that in age-matched men while the prevalence of hypertension in women is significantly increased after the age of 50 (menopause) and is greater than that in men. It is already known that sphingosine-1-phosphate (S1P) and ceramide regulate vascular tone with opposing effects. This study aimed to explore the effects of ovariectomy and estrogen supplementation on the ceramide/S1P rheostat of the aorta in rats, and to explore a potential mechanism for perimenopausal hypertension and a brand-new target for menopausal hormone therapy to protect vessels.

Methods: In total, 30 female adult SD rats were randomly divided into three groups: The sham operation group (SHAM), ovariectomy group (OVX) and ovariectomy plus estrogen group (OVX + E). After 4 weeks of treatment, the blood pressure (BP) of the rats was monitored by a noninvasive system; the sphingolipid content (e.g., ceramide and S1P) was detected by liquid chromatography-mass spectrometry (LC-MS); the expression of the key enzymes involved in ceramide anabolism and catabolism was measured by real-time fluorescence quantitative polymerase chain reaction (qPCR); and the expression of key enzymes and proteins in the sphingosine kinase 1/2 (SphK1/2)-S1P-S1P receptor 1/2/3 (S1P1/2/3) signaling pathway was detected by qPCR and western blotting.

Results: In the OVX group compared with the SHAM group, the systolic BP (SBP), diastolic BP (DBP) and pulse pressure (PP) increased significantly, especially the SBP and PP (P < 0.001). For aortic ceramide metabolism, the mRNA level of key enzymes involved in anabolism and catabolism decreased in parallel 2-3 times, while the contents of total ceramide and certain long-chain subtypes increased significantly (P < 0.05). As for the S1P signaling pathway, SphK1/2, the key enzymes involved in S1P synthesis, decreased significantly, and the content of S1P decreased accordingly (P < 0.01). The S1P receptors showed various trends: S1P1 was significantly down-regulated, S1P2 was significantly up-regulated, and S1P3 showed no significant difference. No significant difference existed between the SHAM and OVX + E groups for most of the above parameters (P > 0.05).

Conclusions: Ovariectomy resulted in the imbalance of the aortic ceramide/S1P rheostat in rats, which may be a potential mechanism underlying the increase in SBP and PP among perimenopausal women. Besides, the ceramide/S1P rheostat may be a novel mechanism by which estrogen protects vessels.

Keywords: Aorta; Ceramide; Estrogen; Ovariectomized rats; Sphingosine 1-phosphate.

Fig. 1

Comparative analysis of S1P signaling between the SHAM group, the OVX group, and the OVX + E group. a and b: expression of SphK at mRNA (n = 4) and protein (n = 4 for SphK1, n = 3 for SphK2) levels respectively; c: the content of aortic S1P between groups (n = 6); d content of aortic C1P between groups (n = 6); e and f: expression of S1P receptors at the mRNA (n = 4) and protein (n = 4 for S1P1, n = 5 for S1P2, n = 3 for S1P3) levels respectively. SphK: sphingosine kinase; S1P: sphingosine-1-phosphate; S1P1/2/3: sphingosine-1-phosphate receptor 1/2/3; SHAM group: sham-operated group; OVX group: ovariectomized group; OVX + E group: OVX group treated with estradiol valerate. Data presented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 2

Relative mRNA levels of key enzymes involved in ceramide metabolism. Y-axis represents the expression in each group relative to the mean value in the Sham group. a, b and c represent the anabolic pathways, while catabolic pathways are illustrated in d, e, and f. Cerk, ceramide kinase; Asah, acid ceramidase; Sgms, sphingomyelin synthase; LASS, ceramide synthase; SPTLC, serine palmitoyltransferase long chain base subunit; nSMase, neutral sphingomyelinase; aSMase, acid sphingomyelinase; n = 4 for SPTLC1, LASS4, aSMase, Sgms2,and n = 3 for the rest of the genes. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 3

Comparison of total ceramide (a), individual ceramide subtypes (b), and BP (c) among three groups. SBP, systolic BP; DBP, diastolic BP; PP, pulse pressure; SHAM group: sham-operated group; OVX group: ovariectomized group; OVX + E group: OVX group treated with estradiol valerate. n = 6 for ceramide and n = 10 for BP, * P < 0.05, ** P < 0.01, *** P < 0.001