Active vitamin D corrects cerebrovascular dysfunction and aberrant vasopressin expression in the hypertension phenotype of 1α-hydroxylase knockout mice

Active vitamin D corrects cerebrovascular dysfunction and aberrant vasopressin expression in the hypertension phenotype of 1α-hydroxylase knockout mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Active vitamin D corrects cerebrovascular dysfunction and aberrant vasopressin expression in the hypertension phenotype of 1α-hydroxylase knockout mice Wei Zhang, Yingying Hu, Luqing Zhang, Ping Dong, Dongmei Li, Ronghui Du This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4348468/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Under hypertensive conditions, vitamin D has a protective effect on the brain. Our previous research showed that 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] negatively regulates hypertension and central renin–angiotensin system activation partly through a central antioxidative mechanism in 1α-hydroxylase knockout [1α(OH)ase –/– ] mice. To further confirm whether the endogenous 1,25(OH) 2 D 3 deficiency and exogenous 1,25(OH) 2 D 3 supplementation alter cerebrovascular function and vasopressin expression through antioxidation, we provided 1α(OH)ase –/– mice and their wild-type littermates with normal diet; a high-calcium, high-phosphorus rescue diet with N -acetyl- l- cysteine supplementation; or 1,25(OH) 2 D 3 subcutaneous injection. We analysed and compared the changes in arterial blood pressure, brain microvessel reactivity, cerebral blood flow, expression of hypothalamic vasopressin, and brain/blood oxidation and antioxidative indices using caudal artery plethysmography, isolated microvessel pressure myographs, laser Doppler flowmetry, immunohistochemistry, western blot and biochemistry. Results Compared with their wild-type littermates, the hypertension phenotype was present in the 1α(OH)ase –/– mice, hypothalamic paraventricular nucleus and supraoptic nucleus vasopressin expression was significantly upregulated, and the posterior cerebral artery reaction to the vasodilatory effect of acetylcholine and vasoconstrictive effect of the nitric oxide synthase inhibitor L -nitro-arginine was significantly decreased. Brain/blood oxidative stress was increased, but the antioxidative parameters were decreased. These pathologic changes were corrected by 1,25(OH) 2 D 3 or N -acetyl- l- cysteine plus rescue diet. Conclusions our findings indicate that 1,25(OH) 2 D 3 has an inhibitory effect on vasopressin expression and cerebrovascular dysfunction. 1,25(OH) 2 D 3 may be a promising protective intervention to reduce brain impaired induced by oxidative stress in the hypertension phenotype of 1α(OH)ase –/– mice. Cerebral blood flow Vasopressin Oxidative stress Vitamin D 1α-hydroxylase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 BACKGROUND Vitamin D is a necessary nutrient for human growth and development, sustaining life and maintaining health. In the human body, vitamin D can form active 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] after being hydroxylated twice, namely by 25-hydroxylase in the liver and 1α-hydroxylase [1α(OH)ase] in the kidney[ 1 ]. Vitamin D deficiency may increase the risk of hypertension, cardiovascular diseases, and cerebrovascular accident[ 2 – 3 ]. The central nervous system (CNS) has an important regulatory effect on maintaining blood pressure. Therein, the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) have important regulatory effects on the peripheral blood pressure by secreting vasopressin (VP)[ 4 ]. The diverse pathophysiological hypertensive changes may interfere with normal cerebral blood flow (CBF) circulation and normal neuron metabolism, consequently augmenting brain injury[ 3 , 5 ]. Vitamin D involvement in protecting the brain is diverse, and includes decreasing neuron toxicity, resisting oxidative stress, and increasing nerve growth factors[ 6 – 7 ]. Our laboratory had successfully established a mouse model of 1,25(OH) 2 D 3 deficiency through 1α(OH)ase knockout[ 8 ]. Besides hypocalcaemia and hypophosphataemia, the 1α(OH)ase –/– mice also had growth restriction and rickets changes, hypertension, cardiac hypertrophy, ventricular systolic functional injury, and increased systemic renin–angiotensin (RAS) activity[ 9 ]. Our previous findings also revealed that central RAS activity in 1α(OH)ase –/– mice was increased and that 1,25(OH) 2 D 3 could partially correct the hyperirritable RAS in 1α(OH)ase –/– mice brains via the central antioxidative mechanism[ 10 ]. On the other hand, there is cumulative evidence to prove the cerebrovascular dysfunction in hypertensive status and hypertension impairs CBF[ 11 ]. Hypertension alters the structure of cerebral blood vessels that can impair blood flow and disrupts intricate vasoregulatory mechanisms that assure an adequate blood supply to the brain[ 12 ]. Lab experiments reported peripheral vascular and cerebrovascular endothelial dysfunction were found in patients and animal models of hypertension[ 13 – 14 ]. Further, endothelial dysfunction increases the risk of cardiovascular and cerebrovascular diseases. In present study, we hypothesized that, i ) in addition to increased systemic and central RAS, brain VP abnormalities may be implicated in 1,25(OH) 2 D 3 deficiency–induced hypertension phenotype, ii ) cerebrovascular dysfunction may also be involved in 1,25(OH) 2 D 3 deficiency–induced hypertension phenotype, and iii ) Endogenous 1,25(OH) 2 D 3 deficiency–induced oxidative stress is a major factor of altered central VP and cerebrovascular dysfunction. To confirm these hypotheses, we measured the changes in VP expression in the hypothalamus, the classic cardiovascular regulatory centre, cerebrovascular function, hypothalamus and serum oxidative stress, as well as blood pressure, between 10 to 11-week-old 1α(OH)ase –/– and wild-type (WT) mice. We also fed 1α(OH)ase –/– mice a diet supplemented with 1,25(OH) 2 D 3 , or the antioxidant N -acetyl- l -cysteine (NAC) for 4 weeks to observe their rescue effects on increases in their central VP expression, cerebrovascular reactivity, oxidative stress and blood pressure. Our results contribute to illuminate the influence and anti-hypertension mechanism of action of 1,25(OH) 2 D 3 in the mouse brain. 2 MATERIALS AND METHODS 2.1 Animals and treatment We generated and characterised 1α(OH)ase –/– (KO) mice as described by Panda et al [ 8 ]. The KO mice were generated using heterozygous mice and identified by PCR using tail genomic DNA as the template. Their WT littermates were used as controls in all experiments. Twenty-four pairs of age- and gender-matched WT and KO mice (21 days old, n = 24 per group) were randomly divided into three groups. They were weaned onto one of the following dietary regimens for seven weeks: (1) normal diet (Xietong Medicine Bioengineering Co., Ltd., Nanjing, Jiangsu, China) containing 1.0% calcium and 0.67% phosphorus; (2) N -acetyl- l- cysteine (NAC)-supplemented diet: rescue diet (TD96348 Teklad, Madison, WI, USA) containing 2.0% calcium, 1.25% phosphorus, and 20% lactose plus 1 mg ml − 1 NAC in drinking water; (3) 1,25(OH) 2 D 3 -supplemented diet: normal diet plus thrice weekly subcutaneous injections of 1,25(OH) 2 D 3 (1 µg kg − 1 ). 2.2 Blood pressure measurements Systolic and diastolic blood pressure was measured in awake 10 to 11-week-old mice (n = 24 per group) by tail cuff plethysmography using a BP-2000 Blood Pressure Analysis System (Visitech Systems, Apex, NC, USA)[ 10 ]. To avoid the influence of external factors as far as possible, mice received the blood pressure measurement at the same time on three consecutive days after a 7-day training period Ten separate determinations of systolic and diastolic blood pressure were made over a 10 min interval, and the mean arterial pressure (MAP) was calculated; the average value of the 10 MAPs was used for comparisons. 2.3 Cerebrovascular reactivity studies After blood pressure measurements, WT and KO mice ( n = 8 per group) were sacrificed by cervical dislocation, their brains obtained, and placed in Krebs solution (4°C, pH 7.4 ± 0.1, containing 118 mmol l − 1 NaCl, 4.5 mmol l − 1 KCl, 2.5 mmol l − 1 CaCl 2 , 1 mmol l − 1 MgSO 4 , 1 mmol l − 1 KH 2 PO 4 , 25 mmol l − 1 NaHCO 3 , 11 mmol l − 1 glucose). The posterior cerebral artery (PCA) was separated, the arterial ring was attached to the glass sleeve of a pressure myograph (Living Systems Instrumentation, Burlington, VT, USA) and immersed in Krebs solution supplied with 100% oxygen, and the blood vessel was slowly pressurised to 60 mmHg for 1 h at 37°C. Microvessel ring reactivity (precontracted with phenylephrine) was tested against an acetylcholine (ACh) concentration gradient (10 − 11 ~10 − 4 M ACh) using the cumulative concentration method[ 15 ]. We constructed a dose–effect curve, calculated the ACh maximal diastolic pressure ( EA max ) and the − log(EC 50 ) (pD 2 ) using the curve fitting method, and evaluated vascular reactivity to the ACh EA max (shown as the percentage of the altered and baseline diameters of the blood vessel) and pD 2 . The PCA ring was immersed in the nitric oxide synthase (NOS) inhibitor L -nitro-arginine ( L -NNA)/Krebs solution (10 − 5 M) for 35 min, the change in vascular diameter was recorded once every 5 min, and we evaluated the vascular reactivity to the L -NNA EA max . 2.4 Laser Doppler flowmetry Laser Doppler flowmetry measurement of CBF (Transonic Systems Inc. Ithaca, NY, USA) was carried out after blood pressure measurements. WT and KO mice ( n = 8 per group) anesthetized with ketamine (80 mg/kg, intraperitoneally) were fixed in a stereotaxic frame and the bone over the barrel cortex was thinned to translucency using a dental drill. Body temperature was maintained at 37°C using a heating pad. Changes in CBF before, during, and after whisker stimulation (20 seconds at 8 to 10 Hz, electric toothbrush) were recorded, with four to six recordings acquired every 30 to 40 seconds and averaged for each mouse. Cortical CBF change was expressed as percentage increase relative to baseline. 2.5 Immunostaining We measured CBF, and then initially anesthetised them with an intraperitoneal injection of 3.5% chloral hydrate (0.1 ml 10 g − 1 ), followed by 4% paraformaldehyde perfusion. We embedded the mice brains in paraffin and obtained the hypothalamic tissue for continuous coronal sectioning (slice thickness 5 µm). We used three sets of neighbouring sections: one for VP immunohistochemical staining and two for controlled trials: a blank test (one VP immunohistochemical staining antibody was omitted prior to incubation of the tissue sections) and a replacement test (the anti-VP serum was substituted with normal rabbit serum). The sections were rinsed in 0.01 M PBS (pH 7.4) and blocked for 1 h in 5% goat serum before anti-VP rabbit polyclonal antibody (1:5000; Cat#AB1565; Millipore, Billerica, MA, USA) was added and the sections incubated for 12 h at 4°C. Then, the sections were rinsed with PBS, incubated with HRP–IgG (1:200; Cat#MP-7451; Vector, Burlingame, CA, USA) for 1.5 h at room temperature, rinsed with PBS, stained with diaminobenzidine (Elite ABC Kit; Vector), rinsed again with PBS, and the reaction was stopped. The sections were dried and sealed before being observed under light microscopy, photographed and counted under high magnification. P < 0.05 from one-way ANOVA followed by a Newman–Keuls post hoc comparison test indicated a significant difference. 2.6 Western blot Proteins were quantified by Western blot in hypothalamic PVN and SON of the same animals used in the cerebrovascular reactivity studies, VP (Molecular weights 17KD) being measured for all treatments. Western blot was carried out as previously described[ 10 ] using anti-VP rabbit polyclonal antibody (1:8000; Cat#AB1565; Millipore, Billerica, MA, USA). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA). The intensity of the bands was measured using ImageJ version 1.52a (NIH, Bethesda, MD, USA). All the experiments were repeated independently for three times. 2.7 Oxidative/antioxidative parameter assay Mice ( n = 8 per group) were killed by decapitation after received treatment for 2 h. The brains were rapidly dissected on ice, and the brain homogenates were centrifuged and the supernatant was used. Protein content was determined according to the Lowry method[ 16 ]. Serum was separated by centrifugation and stored at − 20°C until analysed. Commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used according to the manufacturer’s instructions to detect malondialdehyde (MDA) levels, total antioxidative capabilities (T-AOC), and total superoxide dismutase (T-SOD) activities in the mouse brain and serum. 2.8 Neuronal cell count According to The Mouse Brain in Stereotaxic Coordinates map[ 17 ], immunohistochemical photomicrographs containing principal part of the PVN (Plates 25–26) and SON (Plates 22–24) were identified using a LAS Image Analysis System (Leica, Wetzlar, Germany). Assessment of the number of VP profile was obtained from the observation of every fourth section of eight mice in the principal region of the SON and PVN in each group. 2.9 Statistical analysis Data are presented as the means ± SEM. Statistical differences were determined by one-way ANOVA followed by a post hoc Dunnett or Newman-Keuls multiple comparison test, or when indicated, by Student’s t -test for comparisons between two groups. All statistical analyses were performed using Prism 5 (GraphPad, San Diego, CA, USA). All tests were two-sided, and P values < 0.05 were considered statistically significant. 3 RESULTS 3.1 1,25(OH) 2 D 3 exerts anti-hypertensive effects in 1α(OH)ase –/– mice The 10 to 11-week-old KO mice had high MAP as compared with their WT littermates, which is in agreement with our previous results[ 10 ]. Both exogenous 1,25(OH) 2 D 3 and the antioxidant NAC plus rescue diet normalised the blood pressure in KO mice, strongly suggesting that the anti-hypertensive effects of 1,25(OH) 2 D 3 are mainly exerted via an antioxidative mechanism (Fig. 1 ). 3.2 1,25(OH) 2 D 3 rescues cerebrovascular dysfunction in 1α(OH)ase –/– mice To determine whether the deficiency of endogenous 1,25(OH) 2 D 3 or the supplementation of exogenous 1,25(OH) 2 D 3 affect cerebrovascular function, the PCA reactivity of 1α(OH)ase –/– mice and their WT littermates with/without 1,25(OH) 2 D 3 or NAC were examined by using on-line videomicroscopy. The PCA diastolic reactivity of KO mice to ACh was significantly decreased ( P < 0.001). Compared with WT mice, the ACh EA max in KO mice decreased by 24.9% (Fig. 2 A and C), but the pD 2 did not differ significantly (WT, 7.3 ± 0.15 vs. KO, 7.8 ± 0.29) (Table 1 ). The PCA EA max of KO mice to L -NNA was also decreased (–20%) as compared with the control group (Fig. 2 B and D); the vascular endothelial NO content of the KO group was clearly lower than that of the WT controls. The exogenous 1,25(OH) 2 D 3 and antioxidant NAC greatly improved the PCA vasoconstrictive and vasodilatory reactions to ACh and L -NNA, respectively. Table 1 Effects of NAC and 1,25(OH) 2 D 3 on cerebrovascular response to ACh and L -NNA WT (ND) WT (NAC) WT (1,25D) KO (ND) KO (NAC) KO (1,25D) ACh (EAmax) 43.0 ± 2.99 39.2 ± 2.52 42.0 ± 3.46 18.1 ± 2.55*** 30.6 ± 3.88 # 36.3 ± 4.10 ## ACh (pD 2 ) 7.3 ± 0.15 7.6 ± 0.15 7.3 ± 0.16 7.8 ± 0.29 7.2 ± 0.26 7.1 ± 0.20 L -NNA (EAmax) 59.0 ± 3.16 57.1 ± 2.13 55.1 ± 2.13 79.6 ± 2.84*** 67.5 ± 2.14 # 62.1 ± 3.04 ## Data are the mean ± SEM from eight mice and are expressed as the maximal agonist response ( EA max ) or potency (pD 2 , −log[EC 50 ]). *** P < 0.001 when compared to normal diet (ND)-treated WT mice by t -test; # P < 0.05, ## P < 0.01, when compared to ND-treated KO mice by t -test. 3.3 1,25(OH) 2 D 3 partially normalizes impaired CBF in 1α(OH)ase –/– mice Hypertension is the prime instigator of alterations in cerebral artery structure and function that can impair blood flow. Here, we observed endogenous 1,25(OH) 2 D 3 deficiency caused the alteration in the evoked CBF in the hypertension phenotype of 1α(OH)ase –/– mice. We confirmed that CBF induced by whisker stimulation in KO mice decreased compared with their WT littermates (12.5%±1.61% versus 23.9%±2.96%, P 0.05). Although not significant, NAC and 1,25(OH) 2 D 3 actually tended to affect the evoked CBF response in 1α(OH)ase –/– mice compared with their WT littermates (Fig. 3 A and B). 3.4 1,25(OH) 2 D 3 inhibits VP expression in 1α(OH)ase –/– mice It is well known that hypothalamic high expression of VP is involved in the pathology of hypertension[ 18 – 20 ]. To determine whether VP is upregulated in 1α(OH)ase –/– mice brains and whether the administration of exogenous 1,25(OH) 2 D 3 or NAC plus the rescue diet normalizes hypertension via downregulation of the central VP level, we used immunohistochemical staining and Western blot to examine the alterations in PVN and SON VP expression levels, and detected VP-immunoreactive neurons (Fig. 4 A-D) and VP protein (Fig. 4 E and F) in the PVN and SON. The Paxinos and Franklin classification standard for mouse PVN subnucleus classifies the PVN into the magnocellular subnuclei and parvocellular subnuclei. The former includes the ventral magnocellular subnucleus (PaV), medial magnocellular subnucleus (PaMM), lateral magnocellular subnucleus (PaLM), and the posterior magnocellular subnucleus (PaPO). VP-immunopositive neurons were distributed mainly in the PaLM (Fig. 4 A) of the mice PVN in a wedge shape. Generally, VP-immunopositive neurons are found mainly in the PaMM and PaLM. VP-immunopositive cells in the PaLM are distributed mainly in the lateral posterior of the PaMM, and are mostly round or ovate, large, intensely stained, and contain brown-yellow cytoplast. VP-immunopositive neurons were not observed in the parvocellular subnuclei of the WT and KO mice. The WT and KO mice PVN contained 40.8 ± 9.41 and 132.6 ± 14.51 VP-immunopositive cells, respectively (Fig. 4 B, P < 0.001). Following the administration of 1,25(OH) 2 D 3 or NAC, the KO mice PVN contained 78.4 ± 13.52 and 91.8 ± 10.47 VP-immunopositive cells, respectively (Fig. 4 B). In both groups, VP-positive neurons were mainly distributed throughout the nucleus in the SON, and were observed as a band-shaped formation in the exterior portion of the optic chiasma, with slightly wider distribution in the medial portion (Fig. 4 C). The cells were large, with large neuron bodies, round nuclei, and cytoplasts filled with brown-yellow immunopositive reactant (Fig. 4 C). Cell counting showed that the SON of the WT and KO mice contained 21.8 ± 3.06 and 41.4 ± 4.09 VP-immunopositive cells, respectively (Fig. 4 D, P < 0.05). Following the administration of 1,25(OH) 2 D 3 or NAC, the KO mice SON contained 27.8 ± 3.88 and 30.0 ± 4.84 VP-immunopositive cells, respectively (Fig. 4 D). Compared to their WT littermates, the PVN and SON of the KO mice initially contained significantly more VP-immunoreactive neurons, which were significantly reduced following the administration of 1,25(OH) 2 D 3 or NAC plus rescue diet (Fig. 3 ). Similar to the immunohistochemical results, western blot’s results show that administration of 1,25(OH) 2 D 3 or NAC plus rescue diet, markedly decreased VP protein levels in PVN of 1α(OH)ase –/– mouse hypothalamus (Fig. 4 E), whereas the decrease in SON caused by 1,25(OH) 2 D 3 or NAC plus rescue diet in KO mice has no significance compared with their WT littermates (Fig. 4 F). 3.5 1,25(OH) 2 D 3 normalises brain and serum oxidative stress in 1α(OH)ase –/– mice To further determine whether hypertension, cerebrovascular dysfunction, and central VP upregulation in 1α(OH)ase –/– mice are associated with central and systemic oxidative stress and whether exogenous 1,25(OH) 2 D 3 inhibits VP overexpression and rescues cerebrovascular function via its antioxidative effect, we evaluated the brain and serum antioxidative capacities in the mice. Compared to the WT mice, the brain and serum MDA levels in KO mice were markedly increased (Fig. 5 A and D), whereas the brain and serum T-AOC (Fig. 5 B and E) and T-SOD activities (Fig. 5 C and F) were decreased significantly in comparison. The administration of 1,25(OH) 2 D 3 or NAC plus rescue diet restored these parameters to normal levels in the KO mice (Fig. 5 ). 4 DISCUSSION There is a negative correlation between blood 1,25(OH) 2 D 3 concentrations and hypertension and cardiovascular diseases[ 21 – 24 ]. Vitamin D supplementation can decrease the blood pressure in a patient with hypertension[ 25 ]. The deficiency of 1,25(OH) 2 D 3 may induce the cellular inflammatory response by activating RAS, and decrease vascular endothelial diastolic function and result in increased blood pressure[ 26 – 27 ]. Additionally, 1,25(OH) 2 D 3 may reduce vascular resistance and decrease blood pressure by regulating smooth muscle cell and endothelial cell functions[ 28 ]. It has been indicated that 1,25(OH) 2 D 3 has inhibitory effects on blood pressure and RAS. Using a 1α(OH)ase KO mice model, we demonstrate for the first time the effect of 1,25(OH) 2 D 3 deficiency on brain VP secretion and cerebrovascular function. The contribution of the CNS to the development and maintenance of high blood pressure is well established. Previously, we reported that endogenous 1,25(OH) 2 D 3 deficiency may increase systemic and central oxidative stress, activating the systemic and central RAS and leading to higher blood pressure[ 10 , 29 ]. Since lowering of blood pressure by the inhibition of the RAS system within systemic and central nervous system was documented by several studies[ 30 – 32 ], it seems that brain angiotensin II may play a key role in the contribution of CNS to hypertension. Angiotensin II has profound effects in the CNS via regulation of VP secretion[ 33 ]. Hypertension in mice with transgenic activation of the brain renin-angiotensin system is VP dependent. VP is required for the hypertension of mouse model induced by the brain RAS[ 34 ]. Injection of an antisense oligonucleotide of angiotensin in both PVN markedly decreased the blood plasma VP in the spontaneous hypertensive rat (SHR), and cerebral ventricular injection of angiotensin also increased PVN secretion of VP[ 35 ]. In this study, hypertension in the KO mice was accompanied by upregulated PVN and SON VP expression. Combined our previous study, we conclude that endogenous 1,25(OH) 2 D 3 deficiency alter the central RAS and neuroendocrine hormone VP in mice. Increased VP is recognized as a driver promoting the development and progression of hypertension in SHR and transgenic mouse[ 34 , 36 ]. VP has a crucial role in regulating blood pressure through V1aR by stimulating vascular contractions, arterial baroreceptor reflex, sympathetic nerve activity, and water re-absorption, and blockade of the VP/V1aR signal results in decreased blood pressure[ 37 ]. Immunohistochemical studies have proven that, in rat hypothalamus, PVN and SON neuron vitamin D–binding proteins and VP coexist to an extent[ 38 ]. In this study, we examined effects of 1,25(OH) 2 D 3 on the expression of VP in PVN and SON. Our results revealed that 1,25(OH) 2 D 3 deficiency up-regulated the expression of VP in PVN and SON, whereas the administration of exogenous 1,25(OH) 2 D 3 and NAC normalized VP expression in both PVN and SON. Our findings suggest 1,25(OH) 2 D 3 downregulated hypothalamic magnocellular neuron VP secretion, subsequently producing an anti-hypertensive effect by regulating central oxidative stress. The CBF and neuron activity communicating mechanism in the brain is termed a neurovascular unit[ 39 ]. Hypertension may cause pathological changes to the cerebral arteriola and arterioles, brain tissue hypoperfusion, and anoxia, damaging the nerve and blood vessel unit coupling activity and worsening brain functional injury. Untreated hypertension, poorly controlled hypertension, and high BP levels are associated with a decline in patients’ parenchymal CBF[ 40 ]. Wiesmann et al. reported that AngII-induced elevated systolic blood pressure results in impaired CBF and a decreased response to blood pressure lowering treatment in a mouse model for Alzheimer's disease[ 41 – 42 ]. As we known, endothelial dysfunction is associated with the oxidative stress in hypertension pathology[ 43 – 46 ]. Amounts of antioxidant scavengers, such as SOD, GPx, catalase and vitamin E, are decreased in patients with hypertension. The antioxidants NAC and tempol, completely normalized cerebrovascular reactivity in aged mice[ 47 ]. 1,25(OH) 2 D 3 is a very effective antioxidant in systemic and central by upregulating the antioxidative defense systems[ 48 – 49 ]. Prophylactic vitamin D 3 supplementation ameliorated neurobehavioral alterations, oxidative stress and neuroinflammation[ 50 ]. Administering 1,25(OH) 2 D 3 to SHR greatly increased the mesenteric resistance arteriola contractility to VP[ 51 ], which shows that under hypertensive conditions, exogenous supplementation of active vitamin D can correct damaged vascular reactivity. Clinical investigation of the MR images of patients with cerebral ischemia revealed that decreased 25-hydroxyl vitamin D in the blood (≤ 25 nmol l − 1 ) is closely related with lacunar infarction, increased white matter signalling, and fine bleeding in the brain, indicating that the deficiency of active vitamin D is involved in brain vascular diseases[ 52 ]. In this experiment, we observed the cumulative vasoconstrictive and vasodilatory effect of ACh and the NOS inhibitor L -NNA, respectively, on the cerebrovascular system in mice, finding significantly decreased brain microvessel reactivity in the mice with 1,25(OH) 2 D 3 deficiency, which was increased after diet correction with 1,25(OH) 2 D 3 and NAC. We also found 1,25(OH) 2 D 3 deficiency attenuates the evoked CBF in the 1α(OH)ase –/– mice compared with their WT littermates. These results indicate that 1,25(OH) 2 D 3 protects cerebrovascular function, to a large extend, through an antioxidative effect. 5 CONCLUSIONS The novel findings here were that 1,25(OH) 2 D 3 deficiency increases hypothalamic VP expression and decreases cerebrovascular reactivity and the evoked of CBF, and that exogenous supplementation of 1,25(OH) 2 D 3 and the antioxidant NAC can correct the above pathological changes. A summary of our previous findings [ 10 ] reported that 1,25(OH) 2 D 3 deficiency increases systemic and central RAS activity in mice and that exogenous 1,25(OH) 2 D 3 can partially correct the activated RAS through central antioxidation. We conclude that 1,25(OH) 2 D 3 regulates the neuroendocrine system and cerebrovascular function through a central antioxidative mechanism with protective effects on the brain, which provides the experimental evidence and theoretical basis for the central mechanism of action of 1,25(OH) 2 D 3 in regulating blood pressure, preventing hypertension, and protecting brain function. Declarations ETHICAL APPROVAL: The Institutional Animal Care and Use Committee of Nanjing Medical University approved the use of animals in this study (NO. 14030117). CONSENT FOR PUBLICATION: Not applicable. AVAILABILITY OF DATA AND MATERIALS: The data used to support the findings of this study are included within the article. COMPETING INTERESTS: The authors declare no conflict of interests. FUNDING : This work was supported by grants from the National Natural Science Foundation of China (81200184 to L.Z.) and (82373884 to R.D.); Fundamental Research Funds for the Central Universities (14380525 to L.Z.); Natural Science Foundation of the Jiangsu Higher Education Institutions (22KJB320016 to W.Z.); Research Talent Training Program of Kangda College of Nanjing Medical University (KD2021KYRC025 to W.Z.); Gathering Talents Plan of Kangda College of Nanjing Medical University (KD2024JXJH007 to W.Z.). AUTHOR' CONTRIBUTIONS: All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conceptualization , L.Z.; Methodology , L.Z., W.Z., and P.D.; Investigation , L.Z. and W.Z.; Formal Analysis , L.Z., W.Z., and Y.H.; Resources , L.Z.; Data Curation , L.Z., W.Z, and D.L.; Writing—Original Draft , L.Z. and W.Z.; Writing—Review& Editing , L.Z.; Visualization , L.Z. and R.D.; Supervision , L.Z.; Project Administration , L.Z.; Funding Acquisition , L.Z, W.Z. and R.D.. ACKNOWLEDGEMENTS: The author wishes to express his gratitude to members of the Bone and Stem Cell Research Center of Nanjing Medical University, Nanjing, China. References Ciobanu AM, Petrescu C, Anghele C, Manea MC, Ciobanu CA, Petrescu DM, et al. Severe Vitamin D Deficiency-A Possible Cause of Resistance to Treatment in Psychiatric Pathology. Med (Kaunas). 2023. 10.3390/medicina59122056 . Al-Oanzi ZH, Alenazy FO, Alhassan HH, Alruwaili Y, Alessa AI, Alfarm NB, et al. The Role of Vitamin D in Reducing the Risk of Metabolic Disturbances That Cause Cardiovascular Diseases. 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Roffe-Vazquez DN, Huerta-Delgado AS, Castillo EC, Villarreal-Calderón JR, Gonzalez-Gil AM, Enriquez C, et al. Correlation of Vitamin D with Inflammatory Cytokines, Atherosclerotic Parameters, and Lifestyle Factors in the Setting of Heart Failure: A 12-Month Follow-Up Study. Int J Mol Sci. 2019. 10.3390/ijms20225811 . Saleem S, Siddiqui A, Iqbal Z. Vitamin D deficiency in patients of type 2 diabetes. Pakistan J Med Health Sci. 2017;11(4):1324–6. Dou D, Yang B, Gan H, Xie D, Lei H, Ye N. Vitamin D supplementation for the improvement of vascular function in patients with chronic kidney disease: a meta-analysis of randomized controlled trials. Int Urol Nephrol. 2019. 10.1007/s11255-019-02088-3 . Zhou C, Lu F, Cao K, Xu D, Goltzman D, Miao D. Calcium-independent and 1,25(OH)2D3-dependent regulation of the renin-angiotensin system in 1alpha-hydroxylase knockout mice. Kidney Int. 2008. 10.1038/ki.2008.101 . Ren CZ, Yang YH, Sun JC, Wu ZT, Zhang RW, Shen D, et al. Exercise Training Improves the Altered Renin-Angiotensin System in the Rostral Ventrolateral Medulla of Hypertensive Rats. Oxid Med Cell Longev. 2016. 10.1155/2016/7413963 . Mirabito Colafella KM, Bovée DM, Danser AHJ. The renin-angiotensin-aldosterone system and its therapeutic targets. Exp Eye Res. 2019. 10.1016/j.exer.2019.05.020 . Ozkayar N, Dede F, Akyel F, Yildirim T, Ateş I, Turhan T, Altun B. Relationship between blood pressure variability and renal activity of the renin-angiotensin system. J Hum Hypertens. 2016. 10.1038/jhh.2015.71 . Iovino M, Lisco G, Giagulli VA, Vanacore A, Pesce A, Guastamacchia E, et al. Angiotensin II-Vasopressin Interactions in The Regulation of Cardiovascular Functions. Evidence for an Impaired Hormonal Sympathetic Reflex in Hypertension and Congestive Heart Failure. Endocr Metab Immune Disord Drug Targets. 2021. 10.2174/1871530321666210319120308 . Littlejohn NK, Siel RB Jr, Ketsawatsomkron P, Pelham CJ, Pearson NA, Hilzendeger AM, et al. Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent. Am J Physiol Regul Integr Comp Physiol. 2013. 10.1152/ajpregu.00082.2013 . Kagiyama S, Tsuchihashi T, Abe I, Matsumura K, Fujishima M. Antisense inhibition of angiotensinogen attenuates vasopressin release in the paraventricular hypothalamic nucleus of spontaneously hypertensive rats. Brain Res. 1999. 10.1016/s0006-8993(99)01375-x . Zhang F, Sun HJ, Xiong XQ, Chen Q, Li YH, Kang YM, et al. Apelin-13 and APJ in paraventricular nucleus contribute to hypertension via sympathetic activation and vasopressin release in spontaneously hypertensive rats. Acta Physiol (Oxf). 2014. 10.1111/apha.12342 . Aoyagi T, Koshimizu TA, Tanoue A. Vasopressin regulation of blood pressure and volume: findings from V1a receptor-deficient mice. Kidney Int. 2009. 10.1038/ki.2009.319 . Jirikowski GF, Kaunzner UW, Dief Ael E, Caldwell JD. 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Angiotensin II, hypertension and angiotensin II receptor antagonism: Roles in the behavioural and brain pathology of a mouse model of Alzheimer's disease. J Cereb Blood Flow Metab. 2017. 10.1177/0271678X16667364 . Huang YJ, Nan GX. .Oxidative stress-induced angiogenesis. J Clin Neurosci. 2019. 10.1016/j.jocn.2019.02.019 . Ugusman A, Kumar J, Aminuddin A. Endothelial function and dysfunction: Impact of sodium-glucose cotransporter 2 inhibitors. Pharmacol Ther. 2021. 10.1016/j.pharmthera.2021.107832 . Patil PD, Melo AC, Westwood BM, Tallant EA, Gallagher PE. A Polyphenol-Rich Extract from Muscadine Grapes Prevents Hypertension-Induced Diastolic Dysfunction and Oxidative Stress. Antioxid (Basel). 2022. 10.3390/antiox11102026 . Sebastian A, Cordain L, Frassetto L, Banerjee T, Morris RC. Postulating the major environmental condition resulting in the expression of essential hypertension and its associated cardiovascular diseases: Dietary imprudence in daily selection of foods in respect of their potassium and sodium content resulting in oxidative stress-induced dysfunction of the vascular endothelium, vascular smooth muscle, and perivascular tissues. Med Hypotheses. 2018. 10.1016/j.mehy.2018.08.001 . Nicolakakis N, Aboulkassim T, Ongali B, Lecrux C, Fernandes P, Rosa-Neto P, et al. Complete rescue of cerebrovascular function in aged Alzheimer's disease transgenic mice by antioxidants and pioglitazone, a peroxisome proliferator-activated receptor gamma agonist. J Neurosci. 2008. 10.1523/JNEUROSCI.3348-08.2008 . Uberti F, Morsanuto V, Bardelli C, Molinari C. Protective effects of 1α,25-Dihydroxyvitamin D3 on cultured neural cells exposed to catalytic iron. Physiol Rep. 2016. 10.14814/phy2.12769 . Kasatkina LA, Tarasenko AS, Krupko OO, Kuchmerovska TM, Lisakovska OO, Trikash IO. Vitamin D deficiency induces the excitation/inhibition brain imbalance and the proinflammatory shift. Int J Biochem Cell Biol. 2020. 10.1016/j.biocel.2019.105665 . Yamini P, Ray RS, Chopra K. Vitamin D3 attenuates cognitive deficits and neuroinflammatory responses in ICV-STZ induced sporadic Alzheimer's disease. Inflammopharmacology. 2018. 10.1007/s10787-017-0372-x . Bukoski RD, Xue H. On the vascular inotropic action of 1,25-(OH)2 vitamin D3. Am J Hypertens. 1993. 10.1093/ajh/6.5.388 . Chung PW, Park KY, Kim JM, Shin DW, Park MS, Chung YJ, et al. 25-hydroxyvitamin D status is associated with chronic cerebral small vessel disease. Stroke. 2015. 10.1161/STROKEAHA.114.007706 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4348468","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299055307,"identity":"cf0178d0-98fb-47bf-9962-79e32928edf5","order_by":0,"name":"Wei Zhang","email":"","orcid":"","institution":"Kangda College, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":299055309,"identity":"1d89bfd8-99fa-4715-a28a-c6b9cf0e58b9","order_by":1,"name":"Yingying Hu","email":"","orcid":"","institution":"Kangda College, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Hu","suffix":""},{"id":299055312,"identity":"4e88fbe3-0018-4692-a2c7-6f5a6c6dc792","order_by":2,"name":"Luqing Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYBACPgYGNhAtx8BwgAHKJgDYoMqMSdeS2ADjEtbCnv7swc8dtenbGc8YMHwoO8zAP7uBgBaeB+mGvWeO5+5sOGPAOOPcYQaJOwcIaJFIOCbB23Ysd8OBMwbMvG2HGQwkEghpSWyT/Nt2LN0ApOUvcVqS2aR522oSwFoYidLC84xNWrbtgOGGA8cKDvacS+eRuEFACz8wxCTfttXJG9w4vPHBjzJrOf4ZBLQwMIAVHGZgkDgAjkweQuphWuqA9jUQoXgUjIJRMApGJAAAP6pEOBZVgnwAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing University","correspondingAuthor":true,"prefix":"","firstName":"Luqing","middleName":"","lastName":"Zhang","suffix":""},{"id":299055315,"identity":"1132d87e-6751-4cb2-8739-24a9394a01da","order_by":3,"name":"Ping Dong","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Dong","suffix":""},{"id":299055319,"identity":"16d80a40-8dff-4436-9605-b988e359b5a5","order_by":4,"name":"Dongmei Li","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Li","suffix":""},{"id":299055322,"identity":"f402b4d3-40e9-4f3c-a47b-d0c0463e5743","order_by":5,"name":"Ronghui Du","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Ronghui","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2024-04-30 10:51:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4348468/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4348468/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55992541,"identity":"f49e11af-d296-4c2a-b6d2-b535f30660ad","added_by":"auto","created_at":"2024-05-07 09:41:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC and 1,25(OH)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e on the MAP in 1α(OH)ase\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e–/–\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter weaning, WT and KO mice received normal diet (ND), rescue diet plus NAC in drinking water (NAC), or normal diet plus thrice weekly subcutaneous injections of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e (1,25D). The systolic and diastolic blood pressure of the mice were measured and used to calculate their MAP. Values are the mean ± SEM (\u003cem\u003en\u003c/em\u003e = 24 mice per group). **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 when compared to ND-treated WT mice; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 when compared to ND-treated KO mice.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/6bd8c0019637428cf2a35c44.png"},{"id":55993201,"identity":"20d97324-fb9c-4f1f-9f04-46eb610558bc","added_by":"auto","created_at":"2024-05-07 09:49:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC and 1,25(OH)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e on cerebrovascular response to ACh and NOS inhibition with \u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e-NNA in 1α(OH)ase\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e–/–\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice from each group were treated as described in Fig. 1. (A and C) ACh-induced vasodilation and (B and D) \u003csub\u003eL\u003c/sub\u003e-NNA–induced vasoconstriction were measured in each group. Each value is the mean ± SEM (\u003cem\u003en\u003c/em\u003e = 8 mice per group). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 when ND-treated KO mice were compared with ND-treated WT mice; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 when NAC- or 1,25D-treated KO mice were compared with ND-treated KO mice.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/bca80fde5627b05e7b3acd03.png"},{"id":55992542,"identity":"829ef9ed-5132-4ca2-b931-a4f39c6cc8f4","added_by":"auto","created_at":"2024-05-07 09:41:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC and 1,25(OH)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e on CBF in 1α(OH)ase\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e–/–\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice from each group were treated as described in Fig. 1. (A and B) CBF induced by whisker stimulation as measured by laser Doppler flowmetry. Each value is the mean ± SEM (\u003cem\u003en\u003c/em\u003e = 8 mice per group). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 when ND-treated KO mice were compared with ND-treated WT mice.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/64de4d85f3afe93a0658676c.png"},{"id":55992545,"identity":"c5dd8a61-0016-4c2b-8b37-842a700689e8","added_by":"auto","created_at":"2024-05-07 09:41:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1887847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC and 1,25(OH)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e on hypothalamic VP expression in 1α(OH)ase\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e–/–\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice from each group were treated as described in Fig. 1. Representative micrographs of WT and KO mouse brain sections stained immunohistochemically for VP showing VP-immunoreactive neurons in (A) the PVN and (C) the SON. The VP-immunoreactive neurons in the (B) PVN and (D) SON were counted by computer-assisted analysis. (E and F) Representative western blots of hypothalamus extracts to determine VP protein levels. VP in the (E) PVN and (F) SON protein relative to actin protein levels assessed by densitometric analysis and expressed as a percentage of the levels in WT mice fed a normal diet. Each value is the mean ± SEM (\u003cem\u003en\u003c/em\u003e = 8 mice per group). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 when compared with ND, NAC, or 1,25D-treated WT mice; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 when compared with ND-treated KO mice. Bar = 100 μm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/eed6078813e224b0695d539f.png"},{"id":55992544,"identity":"a70ac43c-5953-40a2-9812-68abba6b1efc","added_by":"auto","created_at":"2024-05-07 09:41:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194682,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of NAC and 1,25(OH)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e on brain and serum oxidative stress in 1α(OH)ase\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e–/–\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice from each group were treated as described in Fig. 1. Brain/serum (A and D) MDA, (B and E) T-AOC, and (C and F) T-SOD levels were determined. Each value is the mean ± SEM (\u003cem\u003en\u003c/em\u003e = 8 mice per group). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 when compared with ND-treated WT mice; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 when compared with ND-treated KO mice.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/a8e49a8faa08f2f10f450837.png"},{"id":56234855,"identity":"3b5027ff-d710-439a-a375-a5723c4eba2e","added_by":"auto","created_at":"2024-05-10 08:28:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2440668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4348468/v1/792c28b2-cd2d-4845-a73d-74f2725bda6d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Active vitamin D corrects cerebrovascular dysfunction and aberrant vasopressin expression in the hypertension phenotype of 1α-hydroxylase knockout mice","fulltext":[{"header":"1 BACKGROUND","content":"\u003cp\u003eVitamin D is a necessary nutrient for human growth and development, sustaining life and maintaining health. In the human body, vitamin D can form active 1,25-dihydroxyvitamin D\u003csub\u003e3\u003c/sub\u003e [1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e] after being hydroxylated twice, namely by 25-hydroxylase in the liver and 1α-hydroxylase [1α(OH)ase] in the kidney[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Vitamin D deficiency may increase the risk of hypertension, cardiovascular diseases, and cerebrovascular accident[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe central nervous system (CNS) has an important regulatory effect on maintaining blood pressure. Therein, the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) have important regulatory effects on the peripheral blood pressure by secreting vasopressin (VP)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The diverse pathophysiological hypertensive changes may interfere with normal cerebral blood flow (CBF) circulation and normal neuron metabolism, consequently augmenting brain injury[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Vitamin D involvement in protecting the brain is diverse, and includes decreasing neuron toxicity, resisting oxidative stress, and increasing nerve growth factors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur laboratory had successfully established a mouse model of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency through 1α(OH)ase knockout[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Besides hypocalcaemia and hypophosphataemia, the 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice also had growth restriction and rickets changes, hypertension, cardiac hypertrophy, ventricular systolic functional injury, and increased systemic renin\u0026ndash;angiotensin (RAS) activity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our previous findings also revealed that central RAS activity in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice was increased and that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e could partially correct the hyperirritable RAS in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice brains via the central antioxidative mechanism[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the other hand, there is cumulative evidence to prove the cerebrovascular dysfunction in hypertensive status and hypertension impairs CBF[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hypertension alters the structure of cerebral blood vessels that can impair blood flow and disrupts intricate vasoregulatory mechanisms that assure an adequate blood supply to the brain[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Lab experiments reported peripheral vascular and cerebrovascular endothelial dysfunction were found in patients and animal models of hypertension[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Further, endothelial dysfunction increases the risk of cardiovascular and cerebrovascular diseases.\u003c/p\u003e \u003cp\u003eIn present study, we hypothesized that, \u003cb\u003ei\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e in addition to increased systemic and central RAS, brain VP abnormalities may be implicated in 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency\u0026ndash;induced hypertension phenotype, \u003cb\u003eii\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e cerebrovascular dysfunction may also be involved in 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency\u0026ndash;induced hypertension phenotype, and \u003cb\u003eiii\u003c/b\u003e\u003cem\u003e)\u003c/em\u003e Endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency\u0026ndash;induced oxidative stress is a major factor of altered central VP and cerebrovascular dysfunction.\u003c/p\u003e \u003cp\u003eTo confirm these hypotheses, we measured the changes in VP expression in the hypothalamus, the classic cardiovascular regulatory centre, cerebrovascular function, hypothalamus and serum oxidative stress, as well as blood pressure, between 10 to 11-week-old 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e and wild-type (WT) mice. We also fed 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice a diet supplemented with 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e, or the antioxidant \u003cem\u003eN\u003c/em\u003e-acetyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-cysteine (NAC) for 4 weeks to observe their rescue effects on increases in their central VP expression, cerebrovascular reactivity, oxidative stress and blood pressure. Our results contribute to illuminate the influence and anti-hypertension mechanism of action of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e in the mouse brain.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and treatment\u003c/h2\u003e \u003cp\u003eWe generated and characterised 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e (KO) mice as described by Panda et al [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The KO mice were generated using heterozygous mice and identified by PCR using tail genomic DNA as the template. Their WT littermates were used as controls in all experiments. Twenty-four pairs of age- and gender-matched WT and KO mice (21 days old, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;24 per group) were randomly divided into three groups. They were weaned onto one of the following dietary regimens for seven weeks: (1) normal diet (Xietong Medicine Bioengineering Co., Ltd., Nanjing, Jiangsu, China) containing 1.0% calcium and 0.67% phosphorus; (2) \u003cem\u003eN\u003c/em\u003e-acetyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el-\u003c/span\u003ecysteine (NAC)-supplemented diet: rescue diet (TD96348 Teklad, Madison, WI, USA) containing 2.0% calcium, 1.25% phosphorus, and 20% lactose plus 1 mg ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NAC in drinking water; (3) 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e-supplemented diet: normal diet plus thrice weekly subcutaneous injections of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e (1 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Blood pressure measurements\u003c/h2\u003e \u003cp\u003eSystolic and diastolic blood pressure was measured in awake 10 to 11-week-old mice (n\u0026thinsp;=\u0026thinsp;24 per group) by tail cuff plethysmography using a BP-2000 Blood Pressure Analysis System (Visitech Systems, Apex, NC, USA)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To avoid the influence of external factors as far as possible, mice received the blood pressure measurement at the same time on three consecutive days after a 7-day training period Ten separate determinations of systolic and diastolic blood pressure were made over a 10 min interval, and the mean arterial pressure (MAP) was calculated; the average value of the 10 MAPs was used for comparisons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cerebrovascular reactivity studies\u003c/h2\u003e \u003cp\u003eAfter blood pressure measurements, WT and KO mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 per group) were sacrificed by cervical dislocation, their brains obtained, and placed in Krebs solution (4\u0026deg;C, pH 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, containing 118 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, 4.5 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KCl, 2.5 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e, 1 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaHCO\u003csub\u003e3\u003c/sub\u003e, 11 mmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose). The posterior cerebral artery (PCA) was separated, the arterial ring was attached to the glass sleeve of a pressure myograph (Living Systems Instrumentation, Burlington, VT, USA) and immersed in Krebs solution supplied with 100% oxygen, and the blood vessel was slowly pressurised to 60 mmHg for 1 h at 37\u0026deg;C. Microvessel ring reactivity (precontracted with phenylephrine) was tested against an acetylcholine (ACh) concentration gradient (10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e~10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e M ACh) using the cumulative concentration method[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We constructed a dose\u0026ndash;effect curve, calculated the ACh maximal diastolic pressure (\u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) and the \u0026minus;\u0026thinsp;log(EC\u003csub\u003e50\u003c/sub\u003e) (pD\u003csub\u003e2\u003c/sub\u003e) using the curve fitting method, and evaluated vascular reactivity to the ACh \u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e (shown as the percentage of the altered and baseline diameters of the blood vessel) and pD\u003csub\u003e2\u003c/sub\u003e. The PCA ring was immersed in the nitric oxide synthase (NOS) inhibitor \u003csub\u003eL\u003c/sub\u003e-nitro-arginine (\u003csub\u003eL\u003c/sub\u003e-NNA)/Krebs solution (10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M) for 35 min, the change in vascular diameter was recorded once every 5 min, and we evaluated the vascular reactivity to the \u003csub\u003eL\u003c/sub\u003e-NNA \u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Laser Doppler flowmetry\u003c/h2\u003e \u003cp\u003eLaser Doppler flowmetry measurement of CBF (Transonic Systems Inc. Ithaca, NY, USA) was carried out after blood pressure measurements. WT and KO mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 per group) anesthetized with ketamine (80 mg/kg, intraperitoneally) were fixed in a stereotaxic frame and the bone over the barrel cortex was thinned to translucency using a dental drill. Body temperature was maintained at 37\u0026deg;C using a heating pad. Changes in CBF before, during, and after whisker stimulation (20 seconds at 8 to 10 Hz, electric toothbrush) were recorded, with four to six recordings acquired every 30 to 40 seconds and averaged for each mouse. Cortical CBF change was expressed as percentage increase relative to baseline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Immunostaining\u003c/h2\u003e \u003cp\u003eWe measured CBF, and then initially anesthetised them with an intraperitoneal injection of 3.5% chloral hydrate (0.1 ml 10 g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), followed by 4% paraformaldehyde perfusion. We embedded the mice brains in paraffin and obtained the hypothalamic tissue for continuous coronal sectioning (slice thickness 5 \u0026micro;m). We used three sets of neighbouring sections: one for VP immunohistochemical staining and two for controlled trials: a blank test (one VP immunohistochemical staining antibody was omitted prior to incubation of the tissue sections) and a replacement test (the anti-VP serum was substituted with normal rabbit serum).\u003c/p\u003e \u003cp\u003eThe sections were rinsed in 0.01 M PBS (pH 7.4) and blocked for 1 h in 5% goat serum before anti-VP rabbit polyclonal antibody (1:5000; Cat#AB1565; Millipore, Billerica, MA, USA) was added and the sections incubated for 12 h at 4\u0026deg;C. Then, the sections were rinsed with PBS, incubated with HRP\u0026ndash;IgG (1:200; Cat#MP-7451; Vector, Burlingame, CA, USA) for 1.5 h at room temperature, rinsed with PBS, stained with diaminobenzidine (Elite ABC Kit; Vector), rinsed again with PBS, and the reaction was stopped. The sections were dried and sealed before being observed under light microscopy, photographed and counted under high magnification. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 from one-way ANOVA followed by a Newman\u0026ndash;Keuls post hoc comparison test indicated a significant difference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot\u003c/h2\u003e \u003cp\u003eProteins were quantified by Western blot in hypothalamic PVN and SON of the same animals used in the cerebrovascular reactivity studies, VP (Molecular weights 17KD) being measured for all treatments. Western blot was carried out as previously described[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] using anti-VP rabbit polyclonal antibody (1:8000; Cat#AB1565; Millipore, Billerica, MA, USA). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA). The intensity of the bands was measured using ImageJ version 1.52a (NIH, Bethesda, MD, USA). All the experiments were repeated independently for three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Oxidative/antioxidative parameter assay\u003c/h2\u003e \u003cp\u003eMice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 per group) were killed by decapitation after received treatment for 2 h. The brains were rapidly dissected on ice, and the brain homogenates were centrifuged and the supernatant was used. Protein content was determined according to the Lowry method[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Serum was separated by centrifugation and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysed. Commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used according to the manufacturer\u0026rsquo;s instructions to detect malondialdehyde (MDA) levels, total antioxidative capabilities (T-AOC), and total superoxide dismutase (T-SOD) activities in the mouse brain and serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Neuronal cell count\u003c/h2\u003e \u003cp\u003eAccording to \u003cem\u003eThe Mouse Brain in Stereotaxic Coordinates\u003c/em\u003e map[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], immunohistochemical photomicrographs containing principal part of the PVN (Plates 25\u0026ndash;26) and SON (Plates 22\u0026ndash;24) were identified using a LAS Image Analysis System (Leica, Wetzlar, Germany). Assessment of the number of VP profile was obtained from the observation of every fourth section of eight mice in the principal region of the SON and PVN in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical differences were determined by one-way ANOVA followed by a post hoc Dunnett or Newman-Keuls multiple comparison test, or when indicated, by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test for comparisons between two groups. All statistical analyses were performed using Prism 5 (GraphPad, San Diego, CA, USA). All tests were two-sided, and \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e exerts anti-hypertensive effects in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eThe 10 to 11-week-old KO mice had high MAP as compared with their WT littermates, which is in agreement with our previous results[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Both exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and the antioxidant NAC plus rescue diet normalised the blood pressure in KO mice, strongly suggesting that the anti-hypertensive effects of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e are mainly exerted via an antioxidative mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e rescues cerebrovascular dysfunction in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eTo determine whether the deficiency of endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or the supplementation of exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e affect cerebrovascular function, the PCA reactivity of 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice and their WT littermates with/without 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC were examined by using on-line videomicroscopy. The PCA diastolic reactivity of KO mice to ACh was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared with WT mice, the ACh \u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e in KO mice decreased by 24.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and C), but the pD\u003csub\u003e2\u003c/sub\u003e did not differ significantly (WT, 7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 vs. KO, 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The PCA \u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of KO mice to \u003csub\u003eL\u003c/sub\u003e-NNA was also decreased (\u0026ndash;20%) as compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and D); the vascular endothelial NO content of the KO group was clearly lower than that of the WT controls. The exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and antioxidant NAC greatly improved the PCA vasoconstrictive and vasodilatory reactions to ACh and \u003csub\u003eL\u003c/sub\u003e-NNA, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of NAC and 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e on cerebrovascular response to ACh and \u003csub\u003eL\u003c/sub\u003e-NNA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT (ND)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT (NAC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT (1,25D)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKO (ND)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eKO (NAC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eKO (1,25D)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACh (EAmax)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e43.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e39.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e36.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACh (pD\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csub\u003eL\u003c/sub\u003e-NNA (EAmax)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e59.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e57.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e55.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e79.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e62.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eData are the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from eight mice and are expressed as the maximal agonist response (\u003cem\u003eEA\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e) or potency (pD\u003csub\u003e2\u003c/sub\u003e, \u0026minus;log[EC\u003csub\u003e50\u003c/sub\u003e]). ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 when compared to normal diet (ND)-treated WT mice by \u003cem\u003et\u003c/em\u003e-test; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, when compared to ND-treated KO mice by \u003cem\u003et\u003c/em\u003e-test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e partially normalizes impaired CBF in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eHypertension is the prime instigator of alterations in cerebral artery structure and function that can impair blood flow. Here, we observed endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency caused the alteration in the evoked CBF in the hypertension phenotype of 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice. We confirmed that CBF induced by whisker stimulation in KO mice decreased compared with their WT littermates (12.5%\u0026plusmn;1.61% versus 23.9%\u0026plusmn;2.96%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, neither NAC nor 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e restored the increase in CBF induced by whisker stimulation of KO mice (18.08%\u0026plusmn;1.34%, or 16.88\u0026thinsp;+\u0026thinsp;1.25 versus 12.5%\u0026plusmn;1.61%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Although not significant, NAC and 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e actually tended to affect the evoked CBF response in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice compared with their WT littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e inhibits VP expression in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eIt is well known that hypothalamic high expression of VP is involved in the pathology of hypertension[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To determine whether VP is upregulated in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice brains and whether the administration of exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC plus the rescue diet normalizes hypertension via downregulation of the central VP level, we used immunohistochemical staining and Western blot to examine the alterations in PVN and SON VP expression levels, and detected VP-immunoreactive neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D) and VP protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F) in the PVN and SON.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Paxinos and Franklin classification standard for mouse PVN subnucleus classifies the PVN into the magnocellular subnuclei and parvocellular subnuclei. The former includes the ventral magnocellular subnucleus (PaV), medial magnocellular subnucleus (PaMM), lateral magnocellular subnucleus (PaLM), and the posterior magnocellular subnucleus (PaPO). VP-immunopositive neurons were distributed mainly in the PaLM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) of the mice PVN in a wedge shape. Generally, VP-immunopositive neurons are found mainly in the PaMM and PaLM. VP-immunopositive cells in the PaLM are distributed mainly in the lateral posterior of the PaMM, and are mostly round or ovate, large, intensely stained, and contain brown-yellow cytoplast. VP-immunopositive neurons were not observed in the parvocellular subnuclei of the WT and KO mice.\u003c/p\u003e \u003cp\u003eThe WT and KO mice PVN contained 40.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.41 and 132.6\u0026thinsp;\u0026plusmn;\u0026thinsp;14.51 VP-immunopositive cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Following the administration of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC, the KO mice PVN contained 78.4\u0026thinsp;\u0026plusmn;\u0026thinsp;13.52 and 91.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.47 VP-immunopositive cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn both groups, VP-positive neurons were mainly distributed throughout the nucleus in the SON, and were observed as a band-shaped formation in the exterior portion of the optic chiasma, with slightly wider distribution in the medial portion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The cells were large, with large neuron bodies, round nuclei, and cytoplasts filled with brown-yellow immunopositive reactant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Cell counting showed that the SON of the WT and KO mice contained 21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06 and 41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.09 VP-immunopositive cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following the administration of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC, the KO mice SON contained 27.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 and 30.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.84 VP-immunopositive cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eCompared to their WT littermates, the PVN and SON of the KO mice initially contained significantly more VP-immunoreactive neurons, which were significantly reduced following the administration of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC plus rescue diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilar to the immunohistochemical results, western blot\u0026rsquo;s results show that administration of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC plus rescue diet, markedly decreased VP protein levels in PVN of 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mouse hypothalamus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), whereas the decrease in SON caused by 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC plus rescue diet in KO mice has no significance compared with their WT littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e normalises brain and serum oxidative stress in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eTo further determine whether hypertension, cerebrovascular dysfunction, and central VP upregulation in 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice are associated with central and systemic oxidative stress and whether exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e inhibits VP overexpression and rescues cerebrovascular function via its antioxidative effect, we evaluated the brain and serum antioxidative capacities in the mice. Compared to the WT mice, the brain and serum MDA levels in KO mice were markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and D), whereas the brain and serum T-AOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and E) and T-SOD activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and F) were decreased significantly in comparison. The administration of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or NAC plus rescue diet restored these parameters to normal levels in the KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eThere is a negative correlation between blood 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e concentrations and hypertension and cardiovascular diseases[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Vitamin D supplementation can decrease the blood pressure in a patient with hypertension[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The deficiency of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e may induce the cellular inflammatory response by activating RAS, and decrease vascular endothelial diastolic function and result in increased blood pressure[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e may reduce vascular resistance and decrease blood pressure by regulating smooth muscle cell and endothelial cell functions[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It has been indicated that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e has inhibitory effects on blood pressure and RAS. Using a 1α(OH)ase KO mice model, we demonstrate for the first time the effect of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency on brain VP secretion and cerebrovascular function.\u003c/p\u003e \u003cp\u003eThe contribution of the CNS to the development and maintenance of high blood pressure is well established. Previously, we reported that endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency may increase systemic and central oxidative stress, activating the systemic and central RAS and leading to higher blood pressure[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Since lowering of blood pressure by the inhibition of the RAS system within systemic and central nervous system was documented by several studies[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], it seems that brain angiotensin II may play a key role in the contribution of CNS to hypertension. Angiotensin II has profound effects in the CNS via regulation of VP secretion[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Hypertension in mice with transgenic activation of the brain renin-angiotensin system is VP dependent. VP is required for the hypertension of mouse model induced by the brain RAS[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Injection of an antisense oligonucleotide of angiotensin in both PVN markedly decreased the blood plasma VP in the spontaneous hypertensive rat (SHR), and cerebral ventricular injection of angiotensin also increased PVN secretion of VP[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, hypertension in the KO mice was accompanied by upregulated PVN and SON VP expression. Combined our previous study, we conclude that endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency alter the central RAS and neuroendocrine hormone VP in mice.\u003c/p\u003e \u003cp\u003eIncreased VP is recognized as a driver promoting the development and progression of hypertension in SHR and transgenic mouse[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. VP has a crucial role in regulating blood pressure through V1aR by stimulating vascular contractions, arterial baroreceptor reflex, sympathetic nerve activity, and water re-absorption, and blockade of the VP/V1aR signal results in decreased blood pressure[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Immunohistochemical studies have proven that, in rat hypothalamus, PVN and SON neuron vitamin D\u0026ndash;binding proteins and VP coexist to an extent[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, we examined effects of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e on the expression of VP in PVN and SON. Our results revealed that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency up-regulated the expression of VP in PVN and SON, whereas the administration of exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and NAC normalized VP expression in both PVN and SON. Our findings suggest 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e downregulated hypothalamic magnocellular neuron VP secretion, subsequently producing an anti-hypertensive effect by regulating central oxidative stress.\u003c/p\u003e \u003cp\u003eThe CBF and neuron activity communicating mechanism in the brain is termed a neurovascular unit[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Hypertension may cause pathological changes to the cerebral arteriola and arterioles, brain tissue hypoperfusion, and anoxia, damaging the nerve and blood vessel unit coupling activity and worsening brain functional injury. Untreated hypertension, poorly controlled hypertension, and high BP levels are associated with a decline in patients\u0026rsquo; parenchymal CBF[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Wiesmann et al. reported that AngII-induced elevated systolic blood pressure results in impaired CBF and a decreased response to blood pressure lowering treatment in a mouse model for Alzheimer's disease[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As we known, endothelial dysfunction is associated with the oxidative stress in hypertension pathology[\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Amounts of antioxidant scavengers, such as SOD, GPx, catalase and vitamin E, are decreased in patients with hypertension. The antioxidants NAC and tempol, completely normalized cerebrovascular reactivity in aged mice[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e is a very effective antioxidant in systemic and central by upregulating the antioxidative defense systems[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Prophylactic vitamin D\u003csub\u003e3\u003c/sub\u003e supplementation ameliorated neurobehavioral alterations, oxidative stress and neuroinflammation[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Administering 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e to SHR greatly increased the mesenteric resistance arteriola contractility to VP[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which shows that under hypertensive conditions, exogenous supplementation of active vitamin D can correct damaged vascular reactivity. Clinical investigation of the MR images of patients with cerebral ischemia revealed that decreased 25-hydroxyl vitamin D in the blood (\u0026le;\u0026thinsp;25 nmol l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) is closely related with lacunar infarction, increased white matter signalling, and fine bleeding in the brain, indicating that the deficiency of active vitamin D is involved in brain vascular diseases[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this experiment, we observed the cumulative vasoconstrictive and vasodilatory effect of ACh and the NOS inhibitor \u003csub\u003eL\u003c/sub\u003e-NNA, respectively, on the cerebrovascular system in mice, finding significantly decreased brain microvessel reactivity in the mice with 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency, which was increased after diet correction with 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and NAC. We also found 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency attenuates the evoked CBF in the 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice compared with their WT littermates. These results indicate that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e protects cerebrovascular function, to a large extend, through an antioxidative effect.\u003c/p\u003e"},{"header":"5 CONCLUSIONS","content":"\u003cp\u003eThe novel findings here were that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency increases hypothalamic VP expression and decreases cerebrovascular reactivity and the evoked of CBF, and that exogenous supplementation of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e and the antioxidant NAC can correct the above pathological changes. A summary of our previous findings [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] reported that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency increases systemic and central RAS activity in mice and that exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e can partially correct the activated RAS through central antioxidation. We conclude that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e regulates the neuroendocrine system and cerebrovascular function through a central antioxidative mechanism with protective effects on the brain, which provides the experimental evidence and theoretical basis for the central mechanism of action of 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e in regulating blood pressure, preventing hypertension, and protecting brain function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eETHICAL APPROVAL:\u0026nbsp;\u003c/strong\u003eThe Institutional Animal Care and Use Committee of Nanjing Medical University approved the use of animals in this study (NO. 14030117).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS:\u0026nbsp;\u003c/strong\u003eThe data used to support the findings of this study are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS:\u003c/strong\u003e The authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the National Natural Science Foundation of China (81200184 to L.Z.) and (82373884 to R.D.); Fundamental Research Funds for the Central Universities (14380525 to L.Z.); Natural Science Foundation of the Jiangsu Higher Education Institutions (22KJB320016 to W.Z.); Research Talent Training Program of Kangda College of Nanjing Medical University (KD2021KYRC025 to W.Z.); Gathering Talents Plan of Kangda College of Nanjing Medical University (KD2024JXJH007 to W.Z.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026apos; CONTRIBUTIONS:\u0026nbsp;\u003c/strong\u003eAll authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.\u003cem\u003eConceptualization\u003c/em\u003e, L.Z.; \u003cem\u003eMethodology\u003c/em\u003e, L.Z., W.Z., and P.D.; \u003cem\u003eInvestigation\u003c/em\u003e, L.Z. and W.Z.; \u003cem\u003eFormal Analysis\u003c/em\u003e, L.Z., W.Z., and Y.H.; \u003cem\u003eResources\u003c/em\u003e, L.Z.; \u003cem\u003eData Curation\u003c/em\u003e, L.Z., W.Z, and D.L.; \u003cem\u003eWriting\u0026mdash;Original Draft\u003c/em\u003e, L.Z. and W.Z.; \u003cem\u003eWriting\u0026mdash;Review\u0026amp; Editing\u003c/em\u003e, L.Z.; \u003cem\u003eVisualization\u003c/em\u003e, L.Z. and R.D.; \u003cem\u003eSupervision\u003c/em\u003e, L.Z.; \u003cem\u003eProject Administration\u003c/em\u003e, L.Z.; \u003cem\u003eFunding Acquisition\u003c/em\u003e, L.Z, W.Z. and R.D..\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS:\u003c/strong\u003e The author wishes to express his gratitude to members of the Bone and Stem Cell Research Center of Nanjing Medical University, Nanjing, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCiobanu AM, Petrescu C, Anghele C, Manea MC, Ciobanu CA, Petrescu DM, et al. 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Am J Hypertens. 1993. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ajh/6.5.388\u003c/span\u003e\u003cspan address=\"10.1093/ajh/6.5.388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChung PW, Park KY, Kim JM, Shin DW, Park MS, Chung YJ, et al. 25-hydroxyvitamin D status is associated with chronic cerebral small vessel disease. Stroke. 2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/STROKEAHA.114.007706\u003c/span\u003e\u003cspan address=\"10.1161/STROKEAHA.114.007706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cerebral blood flow, Vasopressin, Oxidative stress, Vitamin D, 1α-hydroxylase","lastPublishedDoi":"10.21203/rs.3.rs-4348468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4348468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e Under hypertensive conditions, vitamin D has a protective effect on the brain. Our previous research showed that 1,25-dihydroxyvitamin D\u003csub\u003e3\u003c/sub\u003e [1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e] negatively regulates hypertension and central renin\u0026ndash;angiotensin system activation partly through a central antioxidative mechanism in 1α-hydroxylase knockout [1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e] mice. To further confirm whether the endogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e deficiency and exogenous 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e supplementation alter cerebrovascular function and vasopressin expression through antioxidation, we provided 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice and their wild-type littermates with normal diet; a high-calcium, high-phosphorus rescue diet with \u003cem\u003eN\u003c/em\u003e-acetyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el-\u003c/span\u003ecysteine supplementation; or 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e subcutaneous injection. We analysed and compared the changes in arterial blood pressure, brain microvessel reactivity, cerebral blood flow, expression of hypothalamic vasopressin, and brain/blood oxidation and antioxidative indices using caudal artery plethysmography, isolated microvessel pressure myographs, laser Doppler flowmetry, immunohistochemistry, western blot and biochemistry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e Compared with their wild-type littermates, the hypertension phenotype was present in the 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice, hypothalamic paraventricular nucleus and supraoptic nucleus vasopressin expression was significantly upregulated, and the posterior cerebral artery reaction to the vasodilatory effect of acetylcholine and vasoconstrictive effect of the nitric oxide synthase inhibitor \u003csub\u003eL\u003c/sub\u003e-nitro-arginine was significantly decreased. Brain/blood oxidative stress was increased, but the antioxidative parameters were decreased. These pathologic changes were corrected by 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e or \u003cem\u003eN\u003c/em\u003e-acetyl-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el-\u003c/span\u003ecysteine plus rescue diet.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e our findings indicate that 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e has an inhibitory effect on vasopressin expression and cerebrovascular dysfunction. 1,25(OH)\u003csub\u003e2\u003c/sub\u003eD\u003csub\u003e3\u003c/sub\u003e may be a promising protective intervention to reduce brain impaired induced by oxidative stress in the hypertension phenotype of 1α(OH)ase\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice.\u003c/p\u003e","manuscriptTitle":"Active vitamin D corrects cerebrovascular dysfunction and aberrant vasopressin expression in the hypertension phenotype of 1α-hydroxylase knockout mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 09:41:12","doi":"10.21203/rs.3.rs-4348468/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"df10ebd9-dc28-4f58-bf5a-78349fc3ef26","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-10T08:20:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 09:41:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4348468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4348468","identity":"rs-4348468","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}