Efficacy of Shaobei injection in the treatment of grade II-III hemorrhoids and the effect on fibulin protein expression: A study protocol of a randomized controlled trial.

BACKGROUND: Hemorrhoids are a common and seriously disruptive condition that seriously affects people's lives in terms of treatment. Injection therapy is an effective minimally invasive scheme for the treatment of grade II-III hemorrhoids, but its clinical application is limited by the adverse reactions caused by injection drugs. Some clinical studies have confirmed the efficacy and safety of Shaobei injection as a traditional Chinese medicine extract. However, there is no standard randomized controlled study to verify its efficacy and explore its potential mechanism. METHODS: This is a prospective, randomized, single blind, parallel controlled trial to study the efficacy of Shaobei injection in the treatment of grade II-III hemorrhoids and its effect on the expression of fibulin-3 and fibulin-5 in fibulin protein family. The patients will be randomly divided into a treatment group and control group. The treatment group will be treated with Shaobei injection, and the control group will be treated with rubber band ligation. The observation indexes include: visual analysis scale, postoperative hospital stay, total use of painkillers, fibulin-3 and fibulin-5, hemorrhoids recurrence, and adverse events. Finally, the data will be statistically analyzed by SPASS 18.0 software. DISCUSSION: This study will compare the efficacy of Shaobei injection with the rubber band ligation method in the treatment of grade II-III haemorrhoids and investigate its effect on the expression of fibulin-3 and fibulin-5 in the fibulin protein family. The results of this study will provide a basis for the clinical use of Paeoniflora injection as an alternative to traditional sclerosing agent in the treatment of grade II-III haemorrhoids.Trial registration: OSF Registration number:DOI 10.17605/OSF.IO/MKVDB.

Effect of Berberine Isolated from Barberry Species by Centrifugal Partition Chromatography on Memory and the Expression of Parvalbumin in the Mouse Hippocampus Proper.

Neurodegenerative diseases associated with memory disturbances are important health issues occurring due to a prolonged life span. This article presents the results of a study targeting the emergence of a drug candidate with antiamnesic properties. The effect of berberine (BBR), an isoquinoline alkaloid isolated from the overground parts of Berberis sibirica Pall., on memory and expression of parvalbumin in the mouse hippocampus proper were determined. High-purity BBR was isolated by centrifugal partition chromatography from a methanolic extract from B. sibirica by using a methyl-tert-butyl ether and water (1:1 v/v) solvent system with 10 mmol/L of triethylamine and hydrochloric acid. In an in vivo study, we assessed the influence of the chronic administration of BBR on different stages of memory-related responses in mice. Our results indicated that the chronic administration of BBR in a higher dose (5 mg/kg) improves long-term memory acquisition in mice, as determined in the passive avoidance test. The hippocampal CA1-CA3 fields showed an increased number of parvalbumin-immunoreactive neurons (PV-IR) and nerve fibers as compared to the control. No significant changes in the dentate gyrus were observed between the groups. The HPLC-ESI-QTOF-MS/MS analysis of the biological material revealed the content of BBR as 363.4 ± 15.0 ng (4.11% of RSD) per brain, 15.06 ± 0.89 ng (5.91% of RSD) per hippocampus, and 54.45 ± 1.40 (4.05% of RSD) ng in 100 µL plasma. The study showed that BBR could be a factor influencing the expression of PV in hippocampal neurons. We speculate that BBR may modulate the level of Ca2+ in neurons and thus potentially act as a neuroprotective factor against neuronal damages.

Inhibition of lung cancer by vitamin D depends on downregulation of histidine-rich calcium-binding protein.

Introduction: Intrinsic vitamin D affects the proliferation, apoptosis, invasion, metastasis, and tumorigenesis of lung cancer by regulating tumor signaling pathways. Histidine-rich calcium-binding protein (HRC) maintains Ca2+ homeostasis, which plays crucial roles in the occurrence and development of cancer. Objectives: Our study aims to investigate the ability of vitamin D in the regulation of HRC and the role of HRC playing in lung cancer. Methods: We investigated the effects of vitamin D on lung cancer and the underlying mechanisms, by measuring HRC and vitamin D receptor (VDR) expression in lung cancer, paracancer, and normal tissues from patients using immunohistochemistry, western blotting, and real time RT-PCR. We transfected H460 lung cancer cells (supplemented or not with vitamin D) with PX458-HRC and pcDNA3.1-HRC plasmids and injected mice with lung cancer cells harboring pcDNA3.1-vector or pcDNA3.1-HRC plasmids. Results: Vitamin D inhibited HRC expression and H460 cell migration and proliferation, and promoted apoptosis compared with controls. The expression of HRC and VDR was significantly upregulated and downregulated, respectively, in lung cancer versus paracancer or normal tissues. Cell proliferation and migration were reduced, apoptotic cells were more and tumors were smaller in mice treated with vitamin D/cholecalciferol cholesterol emulsion (CCE) than in vitamin D/CCE+HRC+/+ mice. Conclusion: Vitamin D inhibited lung cancer tumor growth, migration, and proliferation by downregulating HRC.

Melatonin alleviates angiotensin-II-induced cardiac hypertrophy via activating MICU1 pathway.

Mitochondrial calcium uptake 1 (MICU1) is a pivotal molecule in maintaining mitochondrial homeostasis under stress conditions. However, it is unclear whether MICU1 attenuates mitochondrial stress in angiotensin II (Ang-II)-induced cardiac hypertrophy or if it has a role in the function of melatonin. Here, small-interfering RNAs against MICU1 or adenovirus-based plasmids encoding MICU1 were delivered into left ventricles of mice or incubated with neonatal murine ventricular myocytes (NMVMs) for 48 h. MICU1 expression was depressed in hypertrophic myocardia and MICU1 knockdown aggravated Ang-II-induced cardiac hypertrophy in vivo and in vitro. In contrast, MICU1 upregulation decreased cardiomyocyte susceptibility to hypertrophic stress. Ang-II administration, particularly in NMVMs with MICU1 knockdown, led to significantly increased reactive oxygen species (ROS) overload, altered mitochondrial morphology, and suppressed mitochondrial function, all of which were reversed by MICU1 supplementation. Moreover, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α)/MICU1 expression in hypertrophic myocardia increased with melatonin. Melatonin ameliorated excessive ROS generation, promoted mitochondrial function, and attenuated cardiac hypertrophy in control but not MICU1 knockdown NMVMs or mice. Collectively, our results demonstrate that MICU1 attenuates Ang-II-induced cardiac hypertrophy by inhibiting mitochondria-derived oxidative stress. MICU1 activation may be the mechanism underlying melatonin-induced protection against myocardial hypertrophy.

Should vitamin K be supplemented instead of antagonised in patients with idiopathic pulmonary fibrosis?

INTRODUCTION: There is an ongoing need for additional interventions in idiopathic pulmonary fibrosis (IPF) as antifibrotic drugs currently available only inhibit and do not stall disease progression. Vitamin K is a co-factor for the activation of coagulation factors. However, it is also required to activate proteins with functions outside of the coagulation cascade, such as matrix Gla protein (MGP), a defender against soft tissue calcification. Vitamin K antagonists are anticoagulants that are, for unknown reasons, associated with increased mortality in IPF. Areas covered: We advance the hypothesis that modulation of vitamin K-dependent MGP activation in IPF patients by either vitamin K antagonism or administration may result in acceleration and deceleration of fibrosis progression, respectively. Furthermore, shortfall in vitamin K could be suspected in IPF based on the high prevalence of certain co-morbidities, such as vascular calcification and lung cancer. Expert commentary: We hypothesize that vitamin K status is reduced in IPF patients. This, in combination with studies suggesting that vitamin K may play a role in lung fibrosis pathogenesis, would provide a rationale for conducting a clinical trial assessing the potential mitigating effects of vitamin K administration on progression of lung fibrosis, prevention of co-morbidities and mortality in IPF.

Plasma Desphospho-Uncarboxylated Matrix Gla Protein as a Marker of Kidney Damage and Cardiovascular Risk in Advanced Stage of Chronic Kidney Disease.

BACKGROUND/AIMS: Desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) is formed as a result of vitamin K insufficiency. The aim of this study was to investigate the association between plasma dp-ucMGP, kidney function and cardiovascular risk factors before and after 9-months substitution of vitamin K2 in non-dialysis patients with chronic kidney disease (CKD) stage 4 and 5. METHODS: 38 CKD patients were supplemented for 270±12 days with 90 µg vitamin K2 and 10 µg cholecalciferol or 10 µg cholecalciferol alone. At baseline and at follow-up circulating calcium, phosphate, lipids, hemoglobin, albumin and total protein, dp-ucMGP, osteoprotegerin, fetuin A, osteocalcin and fibroblast grown factor 23 (FGF-23) were assessed. Proteinuria was assessed in the first morning void. RESULTS: Baseline plasma dp-ucMGP was 1018.6±498.3 pmol/l and was significantly higher in patients at stage 5 CKD (1388.3 ±505.4 pmol/l) than at stage 4 (885.1±419.7 pmol/l), p=0.04. Vitamin K2 supplementation resulted in a decrease of dp-ucMGP level by 10.7%. Plasma dp-ucMGP was positively associated with proteinuria, serum creatinine, PTH and FGF-23; and inversely associated with glomerular filtration rate, serum hemoglobin and albumin. CONCLUSIONS: High dp-ucMGP level, reflecting a poor vitamin K status seems to be associated with kidney damage and may be also a marker of cardiovascular risk in CKD patients. Supplementation with vitamin K2 may improve the carboxylation status of MGP.

Supplementation of apelin increase plasma levels of nesfatin-1 in normal and DOCA-salt hypertensive rats.

AIMS: We aimed to observe the effects of apelin supplementation on the plasma levels of nesfatin-1 in DOCA-salt hypertensive and normal rats. METHODS: For this purpose, 28 young Wistar albino male rats were divided into four groups; Control (C), Control + Apelin (C+A), Hypertension (HT) and Hypertension + Apelin (HT+A). Hypertension was induced by injection of DOCA-salt (25 mg/kg, s.c.) twice weekly, 4 weeks, whereas intraperitoneal apelin was administered (200 µg.kg-1) for 17 days. Plasma nesfatin-1 and apelin levels were measured with ELISA. Systolic blood pressure was monitored using a tail cuff system. The relationships between plasma nesfatin levels and blood pressure were assessed. RESULTS: Plasma nesfatin-1 levels was found lower in control animals compared to C+A, HT and HT+A groups (p = 0.002, p = 0.026 and p = 0.011, respectively). Systolic blood pressures were similar in the C and C+A groups, but systolic blood pressures of the HT and HT+A groups was found significantly higher than the C and C+A groups. CONCLUSIONS: In conclusion, apelin administration induced an increment of nesfatin-1 in normal rats and plasma levels of nesfatin-1 increase in DOCA-salt hypertension rats. But apelin addition in hypertension did not cause an extra increase in nesfatin-1 levels. This is the first report to investigate the effect of apelin administration on plasma nesfatin levels of normal and hypertensive rats (Fig. 2, Ref. 44).

Pyrano-isochromanones as IL-6 inhibitors: synthesis, in vitro and in vivo antiarthritic activity.

Bergenin (1), a unique fused C-glycoside isolated from Bergenia species, possesses interesting anti-inflammatory and antipain activities. To study SAR of this scaffold, first-generation derivatives were synthesized and evaluated for inhibition of lymphocyte proliferation and production of pro-inflammatory cytokines. The C-7 substituted derivatives showed inhibition of IL-6 as well as TNF-α production. Bergenin and its most potent IL-6 inhibitor derivatives 4e and 4f were then investigated in a panel of in vitro and in vivo inflammation/arthritis models. These compounds significantly decreased the expression of NF-kB and IKK-ß in THP-1 cells. In in vivo study in BALB/c mice, a dose-dependent inhibition of SRBC-induced cytokines, reduction in humoral/cell-mediated immunity, and antibody titer was observed. The CIA study in DBA/1J mice indicated that compounds led to reduction in swelling of paws, cytokine levels, and anticollagen IgG1/IgG2a levels. The significant in vivo immunosuppressive efficacy of pyrano-isochromanones demonstrates the promise of this scaffold for development of next-generation antiarthritic drugs.

Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Glial cell activity is maintained during prolonged inflammatory challenge in rats.

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Circulating matrix γ-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome.

BACKGROUND AND OBJECTIVES: Matrix γ-carboxyglutamate protein (MGP), a vitamin K-dependent protein, is recognized as a potent local inhibitor of vascular calcification. Studying patients with Keutel syndrome (KS), a rare autosomal recessive disorder resulting from MGP mutations, provides an opportunity to investigate the functions of MGP. The purpose of this study was (i) to investigate the phenotype and the underlying MGP mutation of a newly identified KS patient, and (ii) to investigate MGP species and the effect of vitamin K supplements in KS patients. METHODS: The phenotype of a newly identified KS patient was characterized with specific attention to signs of vascular calcification. Genetic analysis of the MGP gene was performed. Circulating MGP species were quantified and the effect of vitamin K supplements on MGP carboxylation was studied. Finally, we performed immunohistochemical staining of tissues of the first KS patient originally described focusing on MGP species. RESULTS: We describe a novel homozygous MGP mutation (c.61+1G>A) in a newly identified KS patient. No signs of arterial calcification were found, in contrast to findings in MGP knockout mice. This patient is the first in whom circulating MGP species have been characterized, showing a high level of phosphorylated MGP and a low level of carboxylated MGP. Contrary to expectations, vitamin K supplements did not improve the circulating carboxylated mgp levels. phosphorylated mgp was also found to be present in the first ks patient originally described. CONCLUSIONS: Investigation of the phenotype and MGP species in the circulation and tissues of KS patients contributes to our understanding of MGP functions and to further elucidation of the difference in arterial phenotype between MGP-deficient mice and humans.

The cardiac force-frequency relationship and frequency-dependent acceleration of relaxation are impaired in lipopolysaccharide-treated rats: is the phospholamban-SERCA axis a therapeutic target?

Sepsis-induced myocardial dysfunction has traditionally been thought of as principally affecting systolic heart function. One of the primary reasons for this concept is that systolic dysfunction is relatively easy to conceptualize, visualize, and measure. With the advent of preload-independent measurements for diastolic function, both measurement and conceptual difficulties are being resolved, and a new realm of evidence is beginning to emerge regarding the aberrations that are found during cardiac relaxation in sepsis. A recent article in Critical Care brings this issue into sharper focus.

Insulin-dependent rescue from cardiogenic shock is not mediated by phospholamban phosphorylation.

INTRODUCTION: We used immunoblots to determine whether inotropic and lusitropic effects of high-dose insulin (HDI) in cardiogenic shock, induced by a beta-blocker (BB) or a calcium channel blocker (CCB), are mediated by phosphorylation of phospholamban (PLB). PLB is a membrane protein that regulates calcium uptake into the sarcoplasmic reticulum (SR) by inhibition of the cardiac calcium pump (SERCA2a). Phosphorylation of PLB relieves SERCA inhibition, thus enhancing diastolic relaxation and preload. METHODS: Our Institutional Animal Care and Use Committee approved this research. Swine myocardia from six groups were flash frozen immediately upon death or sacrifice. Groups 1-6 received: (1) no medications, (2) HDI and glucose only, (3) toxic propranolol infusions and saline resuscitation, (4) toxic propranolol infusions and HDI resuscitation, (5) toxic verapamil infusions and saline resuscitation, and (6) toxic verapamil infusions and HDI resuscitation. Groups 3-6 were resuscitated for 4 h. Tissue samples from all six groups were analyzed by quantitative immunoblots, using antibodies to both unphosphorylated PLB (uPLB) and phosphorylated PLB (pPLB), to determine the total PLB content and the fraction of PLB phosphorylated. RESULTS: There were no differences in either pPLB or total PLB in cardiac tissue among any of the six groups. However, infusion of a pig with the beta-adrenergic agonist, isoproterenol, produced enhanced PLB phosphorylation. CONCLUSION: The mechanism by which HDI produces its inotropic and lusitropic effects in CCB- and BB-induced cardiovascular toxicity, resulting in resuscitation, is not due to changes in phosphorylation of PLB or a change in the total PLB in the SR.

Cardioprotective effect of salvianolic acid B on large myocardial infarction mediated by reversing upregulation of leptin, endothelin pathways, and abnormal expression of SERCA2a, phospholamban in rats.

AIM: To study the cardioprotective effect of salvianolic acid B (Sal B) on cardiac dysfunction. We hypothesized that hyperleptinemia may correlate with abnormal expression of sarco/endoplasmic reticulum ATPase 2a (SERCA2a), phospholamban (PLB) and endothelin-reactive oxygen species (ET-ROS) pathways in rats with large myocardial infarction (MI). METHODS: Large MI was produced by coronary artery ligation for 4 weeks in rats. The rats were divided into four groups: sham, MI, MI+l-Sal B (50 mg/(kg d)), p.o. for 4 weeks), and MI+h-Sal B (100 mg/(kg d)), p.o. for 4 weeks). RESULTS: In MI rats, hemodynamic and echocardiographic abnormalities, cardiac remodeling, and histological changes with features of cardiac failure were associated with hyperleptinemia accompanied by oxidative stress and upregulated OB-Rb, ET pathway mRNA expression and downregulated SERCA2a and PLB mRNA and protein expressions in the myocardium. CONCLUSIONS: The studies demonstrated that an activated leptin pathway correlated with abnormal expression of SERCA2a, PLB and an activated ET-ROS system in the affected myocardium. Sal B exerts beneficial actions on cardiac function in rats with large MI, mainly suppressing upregulation of leptin and ET pathways and oxidative stress, and recovering the normal expressions of SERCA2a and PLB in myocardium.

Involvement of GABA(A) receptors in the neuroprotective effect of theanine on focal cerebral ischemia in mice.

We investigated the involvement of gamma-aminobutyric acid(A) (GABA(A)) receptors in the neuroprotective effect of gamma-glutamylethylamide (theanine), a component of Japanese green tea, following a 4-h middle cerebral artery (MCA) occlusion in mice. Theanine (1 mg/kg) reduced the size of the cerebral infarct and alterations of NeuN, GFAP, and Iba 1 expression levels at 24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by bicuculline (GABA(A)-receptor antagonist, 10 mg/kg) but not 3-mercaptopropionic acid (glutamate decarboxylase inhibitor). These results suggest that the neuroprotective effect of theanine is mediated, at least in part, by GABA(A) receptors.

Nonoperative management of hyperparathyroidism: present and future.

Parathyroidectomy provides effective treatment for primary and secondary hyperparathyroidism with a predictable response of symptoms related to hypercalcemia and elevated parathyroid hormone. Calcium and vitamin D supplementation has reduced the need for parathyroidectomy in dialysis patients with secondary hyperparathyroidism. However, surgery continues to be the only effective treatment of primary hyperparathyroidism. Potential nonoperative treatments for hyperparathyroidism have included the use of estrogen replacement, bisphosphonates, and a new class of drugs known as calcimimetics. Hormone replacement therapy with estrogen has been reported to improve cortical bone density in postmenopausal women with asymptomatic or mildly symptomatic primary hyperparathyroidism. Calcimimetic agents are a new class of drugs that increase the sensitivity of the calcium receptor to ionized calcium. Initial studies have shown that calcimimetics can acutely lower parathyroid hormone levels in patients with primary and secondary hyperparathyroidism. These drugs are currently being evaluated in phase II clinical trials. Ultimately, these medical modalities will need to be compared to parathyroidectomy in randomized controlled clinical trials.

Phytoestrogens decrease brain calcium-binding proteins but do not alter hypothalamic androgen metabolizing enzymes in adult male rats.

Phytoestrogen [plant estrogenic-like molecule(s)] research has grown rapidly in recent years due to their potential health benefits. However, little is known about phytoestrogen's effects on the CNS. Androgen metabolizing enzymes are known to regulate neuroendocrine functions and reproductive behaviors, while calcium-binding proteins are associated with protecting against neurodegenerative diseases. Therefore, we examined aromatase and 5alpha-reductase enzyme activities in the medial basal hypothalamic and preoptic area (mbh-poa) and characterized mbh-poa and amygdala (amy) calbindin and calretinin levels (via Western analysis) from animals fed a phytoestrogen-free (P-free) vs. a phytoestrogen-containing diet [(P-600); that had 600 microg/g of phytoestrogens]. After approximately 5 weeks on the diets, the male rats were killed at 105 days. P-600 plasma phytoestrogen levels were 78-fold higher than the P-free values and the mbh-poa phytoestrogen content was 8-fold higher than the P-free group, demonstrating the passage of phytoestrogens into brain. In general, brain aromatase or 5alpha-reductase activity levels were not significantly altered by the experimental diets. However, independent of brain site (i.e., mbh-poa or amy) the abundance of calbindin from male P-600 rats was significantly lower than P-free animals. Conversely, for calretinin there were no significant alterations in the mbh-poa tissue site, while in the amy a similar pattern of expression was seen to that of the calbindin results. These data suggest that consumption of phytoestrogens via a soy diet for a relatively short interval can significantly: (1) elevate plasma and brain phytoestrogens levels and (2) decrease brain calcium-binding proteins without altering brain androgen metabolizing enzymes.

Effects of dexamethasone and linoleic acid on hepatic secretion of biliary lipids and anionic polypeptide factor: In vivo and in vitro studies.

Synthetic glucocorticoids, such as dexamethasone, and diets enriched with unsaturated fatty acids have been shown to stimulate hepatic bile salt synthesis. This fact led us to investigate the effects of dexamethasone and linoleic acid supplementation on bile secretion. Cholesterol (Ch) and phospholipid secretions are bile acid dependent. Ch and phospholipid in bile are also highly bound to a small apoprotein, the anionic polypeptide factor (APF). In bile, APF may play a physiological role in stabilizing cholesterol:phospholipid vesicles and might also be important in the regulatory process of bile lipid secretion. In order to study the factors influencing bile secretion, the biliary secretion rates of bile lipids and APF were experimentally modulated in perfused rat liver (PRL) and HepG2 cells. As expected, dexamethasone induced an increase in the biliary secretion rate of bile salts (BS) in the two models (PRL: 34 up to 67 nmol/l/min/g liver; HepG2 cells: 234% vs. 100% in controls). The bile secretion rates for phospholipids (PRL: from 5 down to 1.5 nmol/l/min/g liver; HepG2 cells: 93 vs. 100% in controls) and APF (PRL: from 0.34 down to 0.12 microg/l/min/g liver; cells: 86 vs. 100% in controls) rapidly decreased independently from those of BS. The data from experimental cell models supplemented with linoleic acid indicated a correlation between the BS and APF levels (APF: 71 and 63%; BS: 161 and 197% vs. 100% in controls). The phospholipid level was regulated independently from that of APF and BS and increased (106 and 111% vs. 100% in controls), while Ch remained nevertheless unchanged. Our data showed that dexamethasone induced changes in bile and that linoleic acid clearly impaired the regulation exerted by the dexamethasone on bile lipids.

Expression of calcium-binding protein regucalcin mRNA in the cloned human hepatoma cells (HepG2): stimulation by insulin.

The expression of hepatic Ca(2+)-binding protein regucalcin in the cloned human hepatoma cells (HepG2) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in HepG2 cells, although the mRNA was markedly expressed in normal rat liver. Moreover, regucalcin protein in HepG2 cells was detected by Western blot analysis using a polyclonal rabbit anti-regucalcin antibody. Regucalcin mRNA expression in HepG2 cells was clearly stimulated by the culture with insulin (10(-8) M) of the effective concentration. Regucalcin protein in HepG2 cells was also increased by the treatment of insulin (10(-8) M). The present results demonstrate that regucalcin is expressed in the transformed HepG2 cells, and that the expression is stimulated by insulin.

Ultrastructural alterations and retention of the C-propeptide of type II collagen in human chondrocytes exposed in vitro to brefeldin A.

Normal human chondrocytes grown in vitro were exposed to 10 micrograms/ml Brefeldin A (BFA) for 24 h, 1 microgram/ml for 4 h, or 0.1 microgram/ml for 4 h and evaluated for ultrastructural alterations. BFA in the amount of 0.1 microgram/ml resulted in vacuolization, disappearance of the Golgi, and moderate increases in rough endoplasmic reticulum (rER) vesicles. After 1 microgram/ml BFA exposure large interconnected cisternae were identified. BFA treatment of 10 micrograms/ml was associated with large dilated ER cisternae which contained material of variable electron densities. Immunocytochemical localization showed markedly increased type II procollagen intracellular retention in BFA-treated cells. High dose BFA-treated cells showed ultrastructural similarities to those seen in the skeletal dysplasia hypochondrogenesis. Results presented here show that in vitro culture of normal human chondrocytes results in retention of the C-propeptide of type II collagen and marked alterations in cytoplasmic ultrastructure.