Application of a quantitative 1H-NMR method for the determination of amygdalin in Persicae semen, Armeniacae semen, and Mume fructus.

A quantitative (1)H-NMR method (qHNMR) was used to measure the amygdalin content of Persicae semen, Armeniacae semen, and Mume fructus, in each of which amygdalin constitutes a major component. The purity of amygdalin was calculated from the ratio of the intensity of the amygdalin H-2 signal at δ 6.50 ppm in pyridine-d 5 to that of the hexamethyldisilane (HMD) signal at 0 ppm. The HMD concentration was corrected by the International System of Units (SI) traceability with certified reference material (CRM)-grade bisphenol A. qHNMR revealed the amygdalin contents to be 2.72 and 3.13% in 2 lots of Persicae semen, 3.62 and 5.19% in 2 lots of Armeniacae semen, and 0.23% in Mume fructus. Thus, we demonstrated the utility of this method for the quantitative analysis of crude drugs.

Downregulation of lymphatic vessel formation factors in PGF2α-induced luteolysis in the cow.

Prostaglandin F2α (PGF2α) induces luteolysis in cows and causes infiltration of immune cells, which resembles inflammatory immune response. Since the general immune response is mediated by the lymphatic system, we hypothesized that luteolysis is associated with generation of an immune response that involves lymphatic vessels in the bovine corpus luteum (CL). The CL was obtained from Holstein cows at the mid-luteal phase (days 10-12, ovulation = day 0) by ovariectomy at various time points after PGF2α injection. Lymphatic endothelial cell (LyEC) marker, endothelial hyaluronan receptor 1 (LYVE1), levels decreased significantly 12 h after PGF2α injection. Podoplanin, another LyEC marker, decreased from 15 min after PGF2α injection. PGF2α also diminished mRNA expression of lymphangiogenic factors, such as vascular endothelial growth factor (VEGF) C, VEGFD and VEGF receptor 3 (VEGFR3). During PGF2α-induced luteolysis, the levels of mRNA expression of tumor necrosis factor α (TNFα; the major pro-inflammatory cytokine) and chemokine (C-X-C motif) ligand 1 (neutrophil chemokine) were increased. On the other hand, chemokine (C-C motif) ligand 21, which regulates outflow of immune cells from tissues via the lymphatic vessels during an immune response, was decreased. This study demonstrated that the lymphatic network in the CL is disrupted during luteolysis and suggests that during luteolysis, immune cells can induce a local immune response in the CL without using the lymphatic vessels.

Possible action of vasohibin-1 as an inhibitor in the regulation of vascularization of the bovine corpus luteum.

The development of the corpus luteum (CL), which secretes large amounts of progesterone to establish pregnancy, is accompanied by active angiogenesis, vascularization, and lymphangiogenesis. Negative feedback regulation is a critical physiological mechanism. Vasohibin-1 (VASH1) was recently discovered as a novel endothelium-derived negative feedback regulator of vascularization. We therefore investigated the expression of VASH1 in the bovine CL. Expression of VASH1 mRNA and protein was predominantly localized to luteal endothelial cells (LECs). VASH1 expression in the CL was constant through the early to late luteal phases and decreased during CL regression relating with the action of luteolytic prostaglandin F(2)(α) in vivo. To investigate the role of VASH1, we determined whether VASH1 treatment affects angiogenesis and/or lymphangiogenesis using LECs and lymphatic endothelial cells (LyECs) in vitro. Vascular endothelial growth factor A (VEGFA) stimulated the expression of VASH1 in LECs but not in LyECs, and VASH1 completely blocked VEGFA-induced formation of capillary-like tube structures of LECs and LyECs in vitro. In summary, VASH1 is predominantly located on LECs in the bovine CL and inhibits the angiogenic and lymphangiogenic actions of VEGFA. Bovine CL therefore has a VEGFA-VASH1 system that may be involved in regulation of luteal function, especially in the development of the CL. The results indicate that VASH1 has the potential to act as a negative feedback regulator of angiogenesis and lymphangiogenesis in the CL in cows.

Rapid accumulation of polymorphonuclear neutrophils in the Corpus luteum during prostaglandin F(2α)-induced luteolysis in the cow.

Prostaglandin F(2α) (PGF(2α)) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF(2α) administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF(2α) injection. The number of PMNs was increased at 5 min after PGF(2α) administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF(2α) directly stimulated P-selectin protein expression at 5-30 min in luteal endothelial cells (LECs). Moreover, PGF(2α) enhanced PMN adhesion to LECs, and this enhancement by PGF(2α) was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF(2α) is crucial in PMN migration. In conclusion, PGF(2α) rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF(2α) may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.

Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows.

In cows, interferon-tau (IFNT) regulates maternal recognition around days 15-19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12-21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP.

Possible involvement of IFNT in lymphangiogenesis in the corpus luteum during the maternal recognition period in the cow.

The corpus luteum (CL), which secretes large amounts of progesterone and is thus essential for establishing pregnancy, contains various types of immune cells that may play essential roles in CL function by generating immune responses. The lymphatic system is the second circulation system and is necessary for immune function, but the lymphatic system of the bovine CL has not been characterized in detail. We collected bovine CLs on days 12 and 16 of the estrous cycle (C12 and C16) and days 16 and 40 of early pregnancy (P16 and P40). Lymphatic endothelial hyaluronan receptor 1 (LYVE1) protein was detected in the CL by immunohistochemistry and western blotting and increased at P40 compared with C16. The mRNA expression levels of lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGFC), VEGFD, and their common receptor VEGFR3, as well as the lymphatic endothelial cell (LyEC) marker podoplanin, increased in P16 and P40 CLs. Thus, it is suggested that the lymphatic system of the bovine CL reconstitutes during early pregnancy. Interferon tau (IFNT) from the conceptus in the uterus is a candidate for activating luteal lymphangiogenesis during the maternal recognition period (MRP). We found that treatment of LyECs isolated from internal iliac lymphatic vessels with IFNT stimulated LyEC proliferation and significantly increased mRNA expression of VEGFC and IFN-stimulated gene 15. Moreover, both IFNT and VEGFC induced LyECs to form capillary-like tubes in vitro. In conclusion, it is suggested that new lymphangiogenesis in the bovine CL begins during the MRP and that IFNT may mediate this novel phenomenon.

Evidence that polymorphonuclear neutrophils infiltrate into the developing corpus luteum and promote angiogenesis with interleukin-8 in the cow.

BACKGROUND: After ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL. METHODS AND RESULTS: Polymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs. CONCLUSION: PMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.