Effects of progesterone on hippocampal ultrastructure and expression of inflammatory mediators in neonatal rats with hypoxic-ischemic brain injury.

Progesterone (PROG) has been shown to exhibit a protective function against hypoxic-ischemic brain damage. The aim of the present study was to study the effects of PROG in a neonatal rat model of hypoxic-ischemic brain injury. A total of 30 Wistar rats, aged 7 days, were randomly divided into three groups: Sham, model and PROG. The rats in the model and PROG groups underwent a left common carotid artery ligation and were placed in a sealed container at 37°C with 8% O2 and 92% N2 gas mixtures for 2.5 h to establish animal models of hypoxic-ischemic encephalopathy. The rats in the PROG group were intraperitoneally treated with 8 mg/kg PROG solution 30 min prior to the induction of hypoxia-ischemia. All animals were sacrificed after 24 h and neuronal changes were observed with electron microscopy to investigate the hypoxic-ischemic brain damage. The protein and mRNA expression levels of tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) in the hippocampus were detected by immunohistochemistry and quantitative polymerase chain reaction, respectively. The results revealed that the neuronal structures in the sham group were normal. The neuronal structures in the model group exhibited cavitation changes, but these were reduced following PROG administration. The protein and mRNA expression levels of TNF-α and NF-κB in the hippocampal neurons were increased in the model group, and pretreatment with 8 mg/kg PROG was shown to reduce the expression levels of these inflammatory mediators. Therefore, PROG was shown to exert an important protective function in hypoxic-ischemic brain injury by inhibiting the cascade of inflammatory injury induced by TNF-α and NF-κB.

Downregulated RASD1 and upregulated miR-375 are involved in protective effects of calycosin on cerebral ischemia/reperfusion rats.

Isoflavone calycosin is a typical phytoestrogen extracted from Chinese medical herb Radix Astragali. It has been reported that estrogens could provide neuroprotective effects, and dietary intake of phytoestrogens could reduce stroke injury in cerebral ischemia/reperfusion (I/R) animal models. In the present study, we investigate the molecular mechanisms underlying the neuroprotective effects of calycosin on middle cerebral artery occlusion (MCAO) rats. Focal cerebral ischemia was induced in male rats by MCAO, neurological deficits and brain edema was evaluated after 24h of reperfusion. The results shown calycosin significantly reduced the infarcted volume and the brain water content, and improved the neurological deficit. To provide insight into the functions of estrogen receptor (ER)-mediated signaling pathway in neuroprotection by calycosin, the expression of miR-375, ER-α, RASD1 (Dexamethasone-induced Ras-related protein 1) and Bcl-2 was determined by RT-PCR or western blot assay. Calycosin exhibited a downregulation of RASD1, and an upregulation of ER-α, miR-375 and Bcl-2. Our finding illustrated that calycosin had been shown neuroprotective effects in cerebral ischemia/reperfusion rats, and the molecular mechanisms may correlate with the positive feedback between ER-α and miR-375, along with the regulation of downstream targets.

Formononetin protects TBI rats against neurological lesions and the underlying mechanism.

Traumatic brain injury (TBI) is a major cause of disability or death worldwide, especially in the young. Thus, effective medication with few side effects needs to be developed. This work aimed to explore the potential benefits of formononetin (FN) on TBI rodent model and to discuss the regarding mechanism. These findings showed that FN effectively increased the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in brain tissue of TBI rats (P<0.01), while it reduced intracephalic malonaldehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations (P<0.01). Meanwhile, the hydrocephalus in the TBI rat was alleviated, and the injured nerve cell of the lesioned brain was reduced as showed in hematoxylin-eosin (HE) staining assay. In addition, the endogenous mRNA level of cyclooxygenase-2 (COX-2) in the brain of the TBI rat was significantly down-regulated (P<0.01). Furthermore, the protein expression of nuclear factor E2-related factor 2 (Nrf2) was effectively up-regulated (P<0.01). Taken together, we conclude that formononetin mediates the promising anti-TBI effects against neurocyte damage, which the underlying mechanisms are associated with inhibiting intracephalic inflammatory response and oxidative stress for neuroprotection.

Estradiol inhibits the activity of proton-coupled amino acid transporter PAT1 expressed in Xenopus oocytes.

Estrogen has great potential as a therapeutic agent in focal ischemic brain injury. Amino acids as energy resources and neurotransmitters in the central nervous system are crucial for proper neuronal function and excitability. The proton-coupled amino acid transporter PAT1 has clear potential in drug absorption. In this paper, human brain PAT1 was cloned and expressed in Xenopus oocytes. The effects of estradiol on the activity of PAT1 were investigated. Glycine-induced membrane currents mediated by PAT1 were measured using the two-electrode voltage clamp technique. The amplitude of the glycine-elicited current was decreased progressively with increasing concentrations of ß-estradiol. A concentration-dependent outwards current of PAT1 was also detected by the presence of ß-estradiol. We conclude that estrogen attenuates the activity of PAT1 by directly closing PAT1 channel. Our results may provide an additional mechanism for estrogen on neurotransmission and neuronal metabolism during ischemic injury.

[Effects of peptides originated from the proadrenomedullin on the production of nitric oxide in rat aorta].

OBJECTIVE: To observe the effect of adrenomedullin (ADM) on the production of nitric oxide (NO) in rat aorta and the effect of Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenotensin (ADT) on the ADM-induced NO production. METHODS: Isolated aortic tissues were exposed to ADM, PAMP and ADT for 2 h. The NO production, indicated by nitrite content in the incubated media, and the nitric oxide synthase (NOS) activity in the incubated tissues were assayed. RESULTS: Nitrite productions and NOS activities of the aortic tissues were significantly increased by ADM in a concentration-dependent manner. The nitrite production and NOS activity of the aortic tissues stimulated by ADM (10(-8) mol.L-1) incubation were (0.282 +/- 0.046) mumol per mg protein and (0.323 +/- 0.056) pmol.min-1 per mg protein, respectively, which were greater than those of the control (0.173 +/- 0.026) mumol per mg protein and (0.110 +/- 0.028) pmol.min-1 per mg protein (P < 0.01), respectively. The nitrite production and NOS activity were (0.204 +/- 0.049) mumol per mg protein and(0.178 +/- 0.023) pmol.min-1 per mg protein when the tissues were treated with ADM (10(-8) mol.L-1) and PAMP (10(-8) mol.L-1) in combination, and were (0.150 +/- 0.036) mumol per mg protein and (0.123 +/- 0.031) pmol.min-1 per mg protein when ADM (10(-8) mol.L-1) and ADT (10(-8) mol.L-1) were used in combination, which were significantly less than those in ADM (10(-8) mol.L-1) group. After incubation of the aortic tissues with the same concentrations(10(-8) mol.L-1) of ADM, PAMP and ADT in combination, the nitrite production and NOS activity were (0.162 +/- 0.029) mumol per mg protein and (0.110 +/- 0.024) pmol.min-1 per mg protein, which were also greatly reduced as compared with those of the ADM group (10(-8) mol.L-1, P < 0.01). However, neither PAMP nor ADT had effect on the production of nitrite and NOS activity in the aortic tissues. CONCLUSION: ADM enhanced the NO production in rat aorta, which was antagonized by PAMP and ADT alone or in combination through influencing the NOS activity.

Roles of different peptide fragments derived from proadrenomedullin in the regulation of vascular tone in isolated rat aorta.

The effects of proadrenomedullin N-terminal 20 peptide (PAMP) and adrenotensin (ADT) on adrenomedullin (ADM)-induced vasodilation were investigated in aortic rings from rat. ADM (10(-9) to 10(-7)M) relaxed the aorta preconstricted with phenylephrine in a concentration-dependent manner. Denudation of endothelium or pretreatment with nitric oxide synthase (NOS) inhibitor, L-NAME, attenuated the vasodilatory action of ADM. ADM-induced vasorelaxation in the aortic rings with endothelium was converted to contraction by PAMP, but not by ADT. The ADM-induced vasodilation was not affected by PAMP in aorta rings without endothelium or in intact aortic rings pretreated with L-NAME. ADM-stimulated nitrite production and NOS activity of the aortas, which was inhibited by PAMP, ADT or PAMP plus ADT. ADM, PAMP, and ADT increased the cyclic adenosine monophosphate (cAMP) contents in vascular tissue. The combination of ADM with PAMP or ADT caused a smaller increase in cAMP level as compared with that of PAMP or ADT alone. These results show that ADM-induced endothelium-dependent vasodilation could be converted to vasoconstriction in the presence of PAMP, probably through a NO-dependent pathway. There was no indication that cAMP was involved in the converting effect of PAMP on ADM vasodilator action.