| ObjectiveRecently, vascular cognitive impairment (VCI) has been paid more and more attention by human. Vascular dementia (VD) is one of the major types of senile dementia. And it has a certain proportion in classification of the senile dementia. Cerebral ischemia especially the multi-cerebral infarction has been proved to be the main cause of VD, its pathogenesis needs further study. According to clinical observation,it showed that VD were present abnormal thyroid hormone(TH)levels in serum and no low performance of thyroid function.Most researchers thought this phenomenon belonged to nonthyroidal illness syndrome(NTIS). As an important target tissue of thyroid hormone action, the mammalian central nervous system(CNS) is highly dependent on proper thyroid hormone supply. In rodents, deprivation of thyroid hormone during critical periods of brain development affects processes such as neuronal migration, differentiation, synaptogenesis, myelination and glial cell proliferation. Always a number of studies have proved that the brain exists relatively independent of thyroid hormone metabolic system. Biological activity of thyroid hormone depends largely on concentration of the T3combining with the receptors in nucleus.It was used to think TH can pass the cell through free diffusion, but nearly30years research of thyroid hormone molecular biology shows that:TH mainly depend on thyroid hormone transporters transfering in and out of the cell. Organic anion transfer peptide1C1(Organic anion transporting polypeptidelCl, OATP1C1),and single carboxylic acid transporters8(Monocarboxylate transporters, MCT8) are currently believed to have high affinity to thyroid hormone. In the passed years we made the chronic cerebral ischemia animal model of bilateral carotid artery ligation permanent (2-vessel occlusion,2VO), finding that2VO5weeks rats could show abnormal central thyroid hormone levels and at the same time combine with cognitive impairment,but added exogenous thyroid hormone,the water maze and study memory disorders of chronic cerebral ischemia in rats significantly improved. The study and memory impairment of rats with chronic cerebral ischemia were associated with levels dropped of central thyroid hormone,but2VO rats central thyroid hormone levels dropped by whether thyroid hormone transporters OATP1C1and MCT8regulate TH, there is no research report.So in this experiment we used bilateral common carotid artery ligation to make adult2VO rat model, to observe thyroid hormone transporters OATP1C1and MCT8in normal rats brain and discussed changing rule of gene and protein levels in different ischemia time point and provided new ideas for prevention and treating VCI patients.Methods1.30adult male Sprague-Dawley (SD) rats were randomly divided into control group,2VO3days group,2VO2weeks group,2VO5weeks group and2VO8weeks g.roup.2.Using immunofluorescence staining to observe the expression of OATP1C1and MCT8in the lateral ventricle of the control rats.3.In the end of experiment transcription levels of OATP1C1and MCT8in the brain tissue of each2VOgroup rats were detected using fluorescent quantitation PCR.4. In the end of experiment protein levels of OATP1C1and MCT8in the brain tissue of each2VOgroup rats were detected using Western blotting.5.All the numerical data have been statistically analyzed by SPSS13.0for windows statistical package. Measurement data was said in(mean±standard deviation), independent samples comparison use single-factor analysis of variance(one-way ANOVA), multiple comparison use Dunnetts3. P<O.05was regarded as statistical significance.Results1.OATP1C1and MCT8are concentrated on the capillary endothelial cell membranes in the lateral ventricle of the normal rats;.2. OATP1C1mRNA levels in2VO3days group and2VO2weeks group had no significance when compared with the control group (P=1.000,P=0.604). OATP1C1mRNA level was increased in2VO5weeks when compared with the control group,2VO3days group and2VO2weeks group (P=0.011, P=0.012, P=0.013). There was no significance between the2VO5weeks group and2VO8weeks group (P=0.414).3. MCT8mRNA level in2VO3days group and2VO2weeks group had no significance when compared with the control group (P=1.000,P=0.984). MCT8mRNA level was increased in2VO5weeks when compared with the control group and2VO3days group(P=0.046, P=0.042). MCT8mRNA level in2VO2weeks group and2VO8weeks group had no significance when compared with2VO5weeks group (P=0.054, P=0.135).4. OATP1C1and MCT8protein levels showed trend of increase and in2VO2weeks reached the peak value.ConclusionAs is known to all.the biological activity of thyroid hormone is decised by T3concentration in cell. A model of the two routes of T3supply to central neurons. One consists of1) T4transport across the blood-brain barrier (BBB) involving OATP1C1,2) T4uptake in astrocytes via an unknown transporter,3) T4conversion to T3by D2,4) T3release from astrocytes via an unknown transporter, and5) T3uptake neurons via MCT8or another transporter. The second route consists of1) T3transport across the BBB by MCT8and2) uptake into neurons by MCT8or another transporter. Neuronal T3uptake through other transporters than MCT8.MCT8may be more important in mice than in humans. The human MCT8(SLC16A2) gene is located in Xql3.2including6explicit the son, and five introns. Mature mRNA has approximately4.4KB, including two translation starting sites (TLSs). Due to the different TLS, human MCT8genetic codes two kinds of protein:one kind contains613amino acids, and the other contains539amino acids. The lack of the first primates MCT8gene1TLS, but the second has the same homologousto human. MCT8has12transmembrane area, the N end and C end locating in cells. N praline is rich in the glutamic acid, serine, and threonine residual (hereinafter referred to as PEST), N praline of539amino acids contains one PEST; N praline of613amino acids contains two PEST. So MCT8is also called x-linked transport body containing PEST (X-linkedPEST-containingtransporter. XPCT). MCT8in different species, different organization widely distributed, such as brain, heart, liver, kidney, adrenal, thyroid,andplacenta. Mutations in theMCT8gene cause a syndrome of severe psychomotor retardation and high serum T3levels in affected male patients, known as the Allan-Herndon-Dudley syndrome. So far we think of thyroid hormone transporter-MCT8is the most specific T3transporter. We used immunofluorescence to observe central thyroid hormone transporters OATPICI and MCT8are rich in the capillary endothelial cell membranes of lateral ventricle. of Highly expression of OATP1C1and MCT8in adult rats showed that they played an important role in the transporting thyroid hormone across the blood-brain-barrier or the choroid plexus-cerebrospinal fluid barrier.Protophase work done by our colleagues showed that rats with merely chronic cerebral isehemia showed central thyroid dysfunction and cognitive impairment. But when the rats suffered from chronic cerebral isehemia were supplied exogenous thyroxin in the begin of experiment, their water maze results were normal, which suggested that study and memory impairment of rats with chronic cerebral isehemia were associated with lthyroid dysfunction in brain. There is no research reported that whether dropping of the2VO rats central thyroid hormone levels is associated with OATP1C1and MCT8regulation. In this experiment transcription levels of OATP1C1and MCT8in the brain tissue of each2VO group rats were detected using fluorescent quantitation PCR found that as extension of ischemia time OATP1C1, MCT8gene levels come out of decompensated trend of the different degree i ncreasing, and peak in2VO5weeks.It showed that rats of cerebral ischemia in5weeks when brain demand thyroid hormone increasing significantly. It was suggested that OATP1C1and MCT8play an important role in the transporting thyroid hormone across the blood-brain-barrier or the choroid plexus-cerebrospinal fluid barrier. Protein levels of OATP1C1and MCT8in the brain tissue of each2VOgroup rats were detected using Western blotting showd the similar trend with the Q-PCR.In short, from this experiment, we see, central thyroid hormone transporters OATP1C1and MCT8are are rich in the capillary endothelial cell membranes of lateral ventricle. Highly expression of OATP1C1and MCT8in adult rats show that they play an important role in the transporting thyroid hormone across the blood-brain-barrier or the choroid plexus-cerebrospinal fluid barrier. With the extension of ischemia time OATP1C1and MCT8gene levels and protein are in different degree of decompensated trend of increasing, although not enough to substitute the need of ischemia rats for thyroid hormone, but indirectly reflect the importance of the thyroid hormone to the central nervous system. |
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