Beta mRNA Research Articles

Abstract 3849: Triptolide reduces activity of RNA polymerase II

Abstract Triptolide (TPL) possesses potent anticancer and anti-inflammatory effects. Our previous study demonstrated that radiation-induced pulmonary fibrosis in mice can be significantly reduced by TPL treatment (0.25 mg/kg/every 3 days for 1-3 months via intravenous injection). Treatment was associated with the reduction of a panel of inflammation molecules, as evidenced by a membrane array of 62 inflammatory molecules. We found that TPL has a high level of biointeraction with RNA polymerase II (Pol II). Pol II is a major enzyme complex responsible for the production of mRNA and could explain improved tumor response and reduced normal tissue toxicity. The mRNA of interleukin-1 beta (IL-1α), tumor necrosis factor-alpha (TNFα), IL-6, and Cox-2 were used as downstream products of Pol II. Alpha-amanitin (a specific inhibitor of Pol II) and actinomycin D (a pan Pol inhibitor) were used as positive controls. The results showed that: 1) Pol II was suppressed as early as 1 hour after TPL was added to cells; 2) TPL (effective at ng/ml levels) is more potent than α-amanitin and actinomycin D (effective at µg/ml levels) at reducing housekeeping genes L32 and S14; 3) the suppression of Pol II by TPL was dose dependent; and 4) TPL inhibited radiation-induced mRNA of IL-1α and TNFα at 3 hours after irradiation and IL-6 and Cox-2 at 8 hours after irradiation. It is unknown whether these early effects of TPL on Pol II impact either tumor response or normal tissue complications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3849. doi:1538-7445.AM2012-3849

Acute inflammation with induction of anaphylatoxin C5a and terminal complement complex C5b-9 associated with multiple intra-articular injections of hylan G-F 20: a case report

The purpose of this case report was to investigate local immune mechanisms present during an acute inflammatory flare initiated by viscosupplementation with hylan G-F 20 in a patient with osteoarthritis (OA) and past meniscectomy. A patient with a history of bilateral OA and partial left knee meniscectomy, who had received three injections of hylan G-F 20, was diagnosed with an acute flare reaction in the left knee. Her chart was evaluated for clinical, radiological, and laboratory findings and for clinical follow-up. Histopathological synovial examination and real-time polymerase chain reaction (RT-PCR) for genes with major roles in local inflammation and enzyme-linked immunosorbent assays (ELISAs) for markers of complement activation and cytokines were performed. To study the impact of the inflammatory and immune features we compared the case patient with groups of three representative OA and three rheumatoid arthritis (RA) patients. The patient exhibited evidence of highly increased acute phase reactant C-reactive protein (CRP) in the blood. The pathological examination of the synovial membrane identified abundant fibrinous exudate with numerous particles of hyaluronan surrounded by a dense infiltrate of neutrophils and eosinophils. The synovium had moderate hypertrophy and sclerosis as well as an inflammatory infiltrate predominantly composed of T lymphocytes and macrophages with scattered perivascular eosinophils and neutrophils. Immunoperoxidase staining identified numerous deposits of C5b-9 in the fibrinous exudates and the synovial membrane of the patient. Similar findings were observed in the RA patients, whereas deposits were rare in OA synovial samples. In addition, both anaphylatoxin C5a and the terminal complement complex C5b-9 were present at high levels, comparable to those in RA patients. The levels of mRNA for interleukin-1 beta (IL-1β), IL-6, and the neutrophil marker myeloperoxidase (MPO) were markedly increased compared to those in the RA and OA patients. This present study is indicative of a pseudo-septic acute inflammatory reaction in response to local accumulation of hylan G-F 20 with the activation of complement and local invasion of pro-inflammatory cells.

Transgenerational toxicity of Zearalenone in pigs

Zearalenone (ZEN) is a mycotoxin that can be a contaminant of food and feed commodities. ZEN acts as a xenoestrogen and is considered an endocrine disruptor. Since estrogens influence oogenesis during fetal growth, the effect of ZEN on oocytes was investigated in the F1-generation. Pregnant and lactating pigs were exposed to feed naturally contaminated with ZEN (200, 500 and 1000μg/kg feed). Ovaries of F1-animals were examined for follicle development, expression of estrogen converting enzymes and estrogen receptors, and oocyte quality. In F1-newborns, ZEN did not affect follicle dynamics, but follicle integrity decreased with increasing ZEN concentrations. Expression of estrogen receptor beta mRNA increased following ZEN exposure, whereas expression of genes coding for estrogen converting enzymes remained unchanged. In F1-prepubertal gilts, follicular atresia and oocyte maturation with subsequent embryo development remained unchanged. In conclusion, ZEN reduced the quantity of healthy follicles, which may lead to premature oocyte depletion in adulthood.

Prognostic Significance of High Expression of ER-beta in Surgically Treated ER-Positive Breast Cancer Following Endocrine Therapy

PurposeThis study evaluated estrogen receptor (ER)-beta mRNA and ER-beta protein expression and its prognostic implications in hormone receptor-positive breast cancer.MethodsParaffin sections from 139 hormone receptor-positive breast cancer cases were prepared. The expression of ER-beta mRNA and protein were analyzed by branched-chain assay and immunohistochemistry (IHC), respectively.ResultsThe Allred score of ER-beta IHC was correlated with smaller tumor size (p=0.043), the Allred score of ER-alpha IHC (p<0.001), and the Allred score of progesterone receptor (PR) IHC (p=0.022) but not with the HER2 IHC score. ER-beta mRNA level was correlated with PR mRNA levels (p<0.001) but not with the Allred score of ER-beta IHC, ER-alpha IHC, and PR IHC, nor with the HER2 IHC score and ER-alpha mRNA level. In survival analysis, high expression of ER-beta mRNA was associated with worse disease-free survival along with poor differentiation, lymph node metastasis and absence of PR protein expression in univariate analysis (p=0.040, p=0.002, p=0.018, and p=0.007, respectively) and multivariate analysis (p=0.044, p=0.002, p=0.035, and p=0.007, respectively).ConclusionHigh expression of ER-beta mRNA is an independent predictor of disease recurrence in hormone-receptor-positive breast cancer.

Ginsenoside Rg&lt;sub&gt;3&lt;/sub&gt; Attenuates Microglia Activation Following Systemic Lipopolysaccharide Treatment in Mice

Neuroinflammation, characterized by activation of microglia and expression of major inflammatory mediators, contributes to neuronal damage in addition to acute and chronic central nervous system (CNS) disease progression. The present study investigated the immune modulatory effects of ginsenoside Rg3, a principle active ingredient in Panax ginseng, on pro-inflammatory cytokines and microglia activation in brain tissue induced by systemic lipopolysaccharide (LPS) treatment in C57BL/6 mice. Systemic LPS treatment induces immediate microglia activation in the brain. Based on this information, ginsenoside Rg3 was treated orally with 10, 20, and 30 mg/kg 1 h prior to the LPS (3 mg/kg, intraperitoneally (i.p.)) injection. Ginsenoside Rg3 at 20 and 30 mg/kg oral doses significantly attenuated up-regulation of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6 mRNA in brain tissue at 4 h after LPS injection. Morphological activation of microglia and Iba1 protein expression by systemic LPS injection were reduced with ginsenoside Rg3 (30 mg/kg) treatment. In addition, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in brain tissue were also attenuated with oral treatment of ginsenoside Rg3 at 30 mg/kg. These results indicate that ginsenoside Rg3 plays a modulatory role in neuroinflammation. This study shows that ginsenoside Rg3 attenuates microglia activation using an in vivo animal model.

Bee venom inhibits 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced mouse skin inflammation

Bee venom (BV) has been used to treat diseases such as arthritis, rheumatism and pain in many countries. Although BV has recently been used as an additive of functional cosmetics, there is no evidence of the effects of BV on skin inflammation or disease. Here, we show that BV patch attachment or intra-peritoneal injection inhibited in vivo acute inflammation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) using the mouse ear model of inflammation. When we examined the TNF-alpha and IL-1 beta mRNA expression in BV treated ear samples by real time PCR, even though BV treatment had no significant effects, it inhibited TNF-alpha and IL-1 beta mRNA expression when compared to samples treated with TPA alone. BV also inhibited edema (based on ear weight) and inhibited neutrophil infiltration (based on myeloperoxidase (MPO) activity measured as a neutrophil marker). Taken together, these results showed that BV exerted an anti-inflammatory effect in vivo and was beneficial in an animal model of skin inflammation. These results suggest that BV might be beneficial as a topical agent for the treatment of skin inflammation.

HSP90 Chaperone – An Important Player in CML: Study At RNA and Protein Level

Abstract 4878Chronic myeloid leukemia (CML) is the myeloproliferative disease characterised by presence of BCR-ABL tyrosine kinase, which has been proved to play the basic role in CML ontogenesis. BCR-ABL as well as other kinases active during disease progression (e.g. Src kinase) belong to HSP90 client proteins. It is known, that HSP90 protein is overexpressed in cancer cells and leukemias and is related to therapy resistance in CML cells (Gorre et.al. Blood 2002;100:3041-3044).HSP90 exists in two isoforms: alpha - inducible and beta – constitutively expressed. Taherian et.al. (Biochemistry and Cell Biology, 2008, 86:(1) 37–45) demonstrated that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different substrate specificity under stress conditions. Those results reveal both functional similarities and key functional differences between the individual members of this protein family.In our previous study we found high protein expression of total HSP90 in leukocytes of CML patients in advanced disease states and in patients with poor responses (Zackova et.al. EHA 2011). The HSP90 seems to be an indicator of disease deterioration in patients with chronic myeloid leukemia.In the current study, we extended our field of interest on HSP90 isoforms alpha and beta on mRNA level. We aimed to find out possible correlation between protein and mRNA expression levels, as well as between mRNA levels and response to the therapy. We wondered to know whether the monitoring of total HSP90 or its isoforms in CML can early predict the disease deterioration.We tested HSP90 alpha and HSP90 beta mRNA expression levels in patients with various response of CML by real-time RT-PCR method. The mRNA profiles showed high similarity with the data obtained from previous western blot analyses. The analyses showed that high HSP90 levels are associated with poor response to therapy and in advanced disease phases. These levels are probably represented by HSP90-beta isoform (constitutively expressed), which is expressed in higher levels comparing to HSP90 alpha in all samples tested. Expression of HSP90 alpha isoform is much lower while its mRNA expression level highly increases only in blast crisis.Studying both isoformes separately could distinguish various mechanisms in disease progression.The results of this and previous studies suggest HSP90 (on protein and mRNA levels) is an important molecule for studying of prognosis in CML patients. Thus HSP90 appears to be a candidate for novel marker of CML progression. Disclosures:No relevant conflicts of interest to declare.

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain as well as wild-type RVFV strains, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level. IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs), which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2+/−) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2−/−) and wild-type (mCxcr2+/+) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis.

Changes of thyroid hormone levels and related gene expression in zebrafish on early life stage exposure to triadimefon

In this study, zebrafish was exposed to triadimefon. Thyroid hormones levels and the expression of related genes in the hypothalamic–pituitary–thyroid (HPT) axis, including thyroid-stimulating hormone ( TSH-beta), deiodinases ( dio1 and dio2) and the thyroid hormone receptor ( thraa and thrb) were evaluated. After triadimefon exposure, increased T4 can be explained by increased thyroid-stimulating hormone ( TSH-beta). The conversion of T4 to T3 (deiodinase type I- dio1) was decreased, which reduced the T3 level. Thyroid hormone receptor beta ( thrb) mRNA levels were significantly down-regulated, possibly as a response to the decreased T3 levels. The overall results indicated that triadimefon exposure could alter gene expression in the HPT axis and that mechanisms of disruption of thyroid status by triadimefon could occur at several steps in the synthesis, regulation, and action of thyroid hormones.

Investigation of inflammasome during excitation of IgG-HepG2 cells for evaluation of allergenic ingredients

To investigate the alteration of inflammasome and receptor during IgG promoter transfected to HepG2 cells. By assay of Elisa to evaluate the secretion of IL-1 beta, IL-8, TNF-alpha and MCP-1 after puerarine and LPS administration, and by assay of real time PCR to evaluate the expression of mRNA of IL-1 beta, IL-8,TNF-alpha and MCP-1, as well as the receptors of TLR2, 4 and NOD2, MyD88. IgG promoter did not active innate immunity and enhance the expression and secretion of inflammasome in HepG2. Puerarine did not active the inflammasome either. LPS activated the innate immunity and increased the secretion of IL-8, TNF-alpha and MCP-1. IgG-HepG2 cells could be used specifically as the model of allergy type II for ingredients screening. It is suggested that puerarine was suite for the activator for this type of allergy as positive control.

Reproductive Experience Alters Prolactin Receptor Expression in Mammary and Hepatic Tissues in Female Rats1

Recent studies have reported that reproductive experience in female rats alters prolactin (PRL) receptor gene expression in the brain as well as neural sensitivity to PRL. Given PRL's actions in nonneural tissues, that is, mammary tissue and liver, it was asked whether reproductive experience may also alter prolactin receptor (Prlr) gene expression in these tissues. Groups of age-matched female rats were generated with varying reproductive histories. Separate groups of primiparous (first lactation) and multiparous (second lactation) had mammary tissue and liver samples collected on Day 3 or 10 of lactation. A fifth group raised one litter to weaning and then resumed estrous cyclicity. This group and a final group of age-matched, virgin controls were killed on diestrus. Tissue was processed by quantitative PCR for expression rates of the long and short forms of Prlr mRNA as well as casein beta mRNA (mammary tissue only). Western blots were performed to quantify receptor protein content. Multiple lactations as well as lactation itself resulted in alterations in Prlr expression. Prlr gene expression in mammary tissue was increased in primiparous mothers compared with that in multiparous dams, whereas in the liver, Prlr expression was reduced during an initial lactation. In contrast, PRLR protein levels declined during lactation in mammary, but not hepatic, tissues. Overall, the results demonstrate that the prolactin receptor system is altered in nonneural tissues as a result of the female's reproductive history. The findings are discussed in the context of milk and bile production and PRL's possible role in breast cancer.

Abstract 2330: Role of RET finger protein in the integrin beta1 expression in cancer cells and its significance in cancer patients

Abstract Integrins play central roles in the biology by regulating cell adhesion to the extracellular matrix and cell migration, growth, differentiation and apoptosis. Although contribution of integrins on cancer development has been well studied, the mechanism of the regulation of integrins expression remains elusive. Here we report that RET finger protein (RFP) regulates integrin beta1 expression in cancer cells. RNAi-mediated depletion of RFP resulted in decrease of integrin beta1 expression and impaired cell migration/invasion. RNA immunoprecipitation assay revealed that RFP interacts with integrin beta1 mRNA, and RFP knockdown caused destabilization of the mRNA. Furthermore, we examined the expression of RFP in human colon cancer and endometrial cancer, and assessed its clinical significance. High levels of RFP expression correlated with poor clinical outcome in colon cancer and endometrial cancer patients. These findings suggest that RFP regulates the cancer cell migration/invasion through the upregulation of integrin beta1 expression by stabilization of its mRNA, and expression of RFP can be a prognostic marker for an unfavorable clinical outcome in patients with colon cancer and endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2011-2330

ET-1 induced Elevation of intracellular calcium in clonal neuronal and embryonic kidney cells involves endogenous endothelin-A receptors linked to phospholipase C through Gαq/11

Endothelin-1 (ET-1) is a pain mediator, elevated in skin after injury, which potentiates noxious thermal and mechanical stimuli (hyperalgesia) through the activation of ETA (and, perhaps, ETB) receptors on pain fibers. Part of the mechanism underlying this effect has recently been shown to involve potentiation of neuronal TRPV1 by PKCɛ. However, the early steps of this pathway, which are recapitulated in HEK 293 cells co-expressing TRPV1 and ETA receptors, remain unexplored. To clarify these steps, we investigated the pharmacological profile and signaling properties of native endothelin receptors in immortalized cell lines including HEK 293 and ND7 model sensory neurons. Previously we showed that in ND7/104, a dorsal root ganglia-derived cell line, ET-1 elicits a rise in intracellular calcium ([Ca2+]in) which is blocked by BQ-123, an ETA receptor antagonist, but not by BQ-788, an ETB receptor antagonist, suggesting that ETA receptors mediate this effect. Here we extend these findings to HEK 293T cells. Examination of the expression of ETA and ETB receptors by RT-PCR and [125I]-ET-1 binding experiments confirms the slight predominance of ETA receptor binding sites and messenger RNA in both ND7/104 and HEK 293T cells. In addition, selective agonists of the ETB receptor (sarafotoxin 6c, BQ-3020 or IRL-1620) do not induce a transient increase in [Ca2+]in. Furthermore, reduction of ETB mRNA levels by siRNA does not abrogate calcium mobilization by ET-1 in HEK 293T cells, corroborating the lack of an ETB receptor role in this response. However, in HEK 293 cells with low endogenous ETA mRNA levels, ET-1 does not induce a transient increase in [Ca2+]in. Observation of the [Ca2+]in elevation in ND7/104 and HEK 293T cells in the absence of extracellular calcium suggests that ET-1 elicits a release of calcium from intracellular stores, and pretreatment of the cells with pertussis toxin or a selective inhibitor of phospholipase C (PLC) point to a mechanism involving Gαq/11 coupling.These results are consistent with the hypothesis that a certain threshold of ETA receptor expression is necessary to drive a transient [Ca2+]in increase in these cells and that this process involves release of calcium from intracellular stores following Gαq/11 activation.

Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: A drill-hole defect model

To establish a drill-hole defect model in osteoporotic mouse femur by comparing temporal cortical bone healing pattern between OVX-induced osteoporotic bone and sham-operated bone. 3-month-old female C57BL/6 mice were randomly divided into an ovariectomy group (OVX) and a sham-operated group (Sham). At 6 weeks post-surgery, 7 mice from each group were sacrificed to examine the distal femur and femoral shaft by both micro-CT and mechanical testing for confirming established osteoporosis induced by OVX. In the remaining mice, a cortical bone defect 0.8mm in diameter was created on the mid-diaphysis of the right femur. The local repair process at days 0, 3, 7, 10, 14 and 21 after creation of the drill-hole was in vivo monitored by high-resolution micro-CT scanning. At each time point, each animal was scanned four times and was removed from the scanner between scans to determine reproducibility. Mice were sacrificed at each time point (n=12 at days 0, 3, 7, 10 and 14; n=20 at day 21). Before sacrifice, sera were collected to examine expression of bone formation marker P1NP (procollagen type I N-terminal propeptide) and bone resorption marker CTX (C-terminal telopeptide of type I collagen). After sacrifice, callus samples were collected and subjected to the following analyses: micro-CT-based angiography; histological examination; immunohistochemical staining to determine estrogen receptor expression; quantitative real-time PCR analysis of collagen type I, collagen type II, collagen type X, osteocalcin, tartrate-resistant acid phosphatase, estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) gene expression; and three-point mechanical testing. At 6 weeks post-surgery, OVX mice had significantly lower bone mass, impaired bone micro architecture and compromised mechanical properties compared to the Sham mice. In vivo micro-CT analysis revealed that the bone volume fraction in the defect region was significantly lower in the OVX group from day 10 to day 21 post-injury as compared to the Sham group, and was significantly lower in the intra-medulla region in the OVX group from day 7 to day 14 as compared to the Sham group, consistent with the histological data. Analysis of bone biochemical markers indicated that circulating P1NP levels normalized by baseline in the OVX mice were significantly lower than in the Sham mice from day 7 to day 10, and that temporal expression of circulating CTX levels normalized by baseline was also lower in the OVX mice as compared to the Sham mice. These results were consistent with quantitative real-time PCR analysis. ER alpha mRNA expression was significantly lower in the OVX mice, whereas ER beta mRNA expression was significantly higher in the OVX mice as compared to the Sham mice at all time points examined, consistent with immunohistochemical staining. The restoration of femoral mechanical property, determined based on ultimate load and energy-to-failure, was significantly lower in the OVX mice than in the Sham mice. In addition, in vivo micro-CT scanning for quantifying new bone formation in the defect site was highly reproducible in this model. The bone healing of the drill-hole defect was impaired in mice with OVX-induced osteoporosis. The present study provides a model to investigate the functional role of specific gene in osteoporotic bone healing and may facilitate development of novel therapeutic strategies for promoting osteoporotic bone healing.

An herbal fruit, Amomum xanthoides, ameliorates thioacetamide-induced hepatic fibrosis in rat via antioxidative system

Amomum xanthoides is a well-known traditional herbal medicine mainly for diverse digestive system disorders in Asia for a long time. In the present study, we investigate the effects and action mechanism of methanol fraction of Amomum xanthoides (MFAX) on thioacetamide (TAA)-induced liver fibrosis in rat model. TAA (200mg/kg, ip on twice a week for 14 weeks) treated rats were orally administered with MFAX (25, 50 or 100mg/kg) once a day from the 7th week until 14th week. Significantly elevated serum bilirubin, liver tissue hydroxyproline and malondialdehyde (MDA) in liver fibrosis were ameliorated by MFAX treatment. Further, MFAX treatment attenuated the reactive oxygen species (ROS) levels and restored glutathione (GSH) content and glutathione-peroxidase (GPx) activity. Histopathological data showed that MFAX treatment inhibited collagen accumulation and activation of hepatocyte stellate cells (HSCs) in the liver tissue. Compared to the TAA group, activation of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), platelet-derived growth factor beta (PDGF-β) mRNAs and the level of pro-fibrotic cytokines PDGF-β and connective tissue growth factor (CTGF) in the liver tissue were attenuated in MFAX treated groups. The above evidences collectively indicate that MFAX is a potential herb which can be used as an anti-hepatofibrotic remedy.

Expression of Human Glucocorticoid Receptor in T Lymphocytes in Acute-on-Chronic Hepatitis B Liver Failure

Glucocorticoid receptor (GR) participates in the pathogenesis of liver inflammation. However, the potential role of GR in acute-on-chronic hepatitis B liver failure (ACHBLF) is still obscure. This present study was aimed to determine peripheral GR expression in ACHBLF patients. Forty patients with ACHBLF, 20 patients with chronic hepatitis B (CHB) and 16 healthy controls were included in this retrospectively study. Flow cytometry was used to determine the peripheral expression of GR+T lymphocytes. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed for assessing relative mRNA levels of GR alpha and beta isoforms in peripheral blood mononuclear cells. Serum cortisol level was evaluated using radioimmunoassay. The serum cortisol level and the percentage of GR+T lymphocytes in ACHBLF patients were significantly decreased compared with CHB patients and healthy controls. However, there were no significant differences in mean fluorescence intensity (MFI) of GR+T lymphocytes within three groups. The relative GR alpha mRNA expression in ACHBLF patients was significant decreased compared with healthy controls. However, the relative GR beta mRNA expression in ACHBLF patients was significantly increased compared with CHB patients and healthy controls. In ACHBLF patients, the percentage of GR+T lymphocyte was significantly positively associated with relative GR alpha expression, prothrombin activity, and HBV DNA level, but negatively correlated with serum cortisol level and MELD score. Furthermore, the percentage of GR+T lymphocytes was also obviously elevated in survivors than non-survivors. It was strongly suggested that GR play an important role in the pathogenesis of ACHBLF.

Elevated estrogen receptor expression in hypothalamic preoptic area decreased by electroacupuncture in ovariectomized rats

In the present study, effects of electroacupuncture (EA) on estrogen receptor alpha (ERα) and beta (ERβ) mRNA and protein expression in the hypothalamus of ovariectomized (OVX) rats were detected by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot analysis. Gonadotropin-releasing hormone (GnRH) release and GnRH mRNA level in hypothalamic preoptic area (POA) were evaluated by push–pull perfusion and qRT-PCR. Our results showed that elevated mRNA and protein expression of ERα and ERβ in hypothalamus were restrained following EA treatment in OVX rats. EA treatment also inhibited GnRH release and GnRH mRNA levels in OVX rats. These results provide novel evidence that EA treatment down regulates the expression of hypothalamic estrogen receptors (ERs), thus restores the response of GnRH neurons to estrogen depression, and partially resets the negative feedback of estrogen to hypothalamus–pituitary–ovary axis (HPOA) in OVX rats, which may be a critical mechanism for EA on female reproductive disorders.

血液透析および持続携行式腹膜透析患者の末梢血単核球におけるTNF-α, IL-1βおよびIL-6遺伝子発現

Inflammatory cytokines, including TNF-alpha, IL-1 beta and IL-6, have been reported to be responsible for acute or chronic complications and biocompatibility issues in dialysis patients have focused on the role of these cytokines. Expression of mRNA for TNF-alpha, interleukin (IL)-1 beta and IL-6 in peripheral blood mononuclear cells (PBMC) and their plasma concentrations was examined in 10 hemodialysis and 10 continuous ambulatory peritoneal dialysis (CAPD) patients. Plasma TNF-alpha and IL-1 beta levels in hemodialysis, CAPD and in every healthy subject were below the detection limits. Plasma IL-6 concentration was increased significantly in hemodialysis and CAPD patients when compared to normal controls (p < 0.01). Expression of mRNA for TNF-alpha, IL-1 beta and IL-6 in PBMCs was increased significantly (p < 0.01) in hemodialysis and CAPD patients compared to normal subjects. These mRNA expressions were lower in CAPD patients than in hemodialysis patients (p < 0.05). Significant correlation was observed between the plasma IL-6 concentration and PBMC IL-6 mRNA expression in patients on hemodialysis or CAPD (p < 0.01). IL-6 mRNA expression in PBMCs did not correlate with the duration of hemodialysis or CAPD, blood urea nitrogen (BUN) or serum creatinine levels. These findings suggest that mRNA expression indicates stimulation by dialysis procedures in patients, and CAPD is tolerated better than hemodialysis by patients with end-stage renal failure.

N‐methyl‐4‐isoleucine cyclosporine attenuates CCl4‐induced liver fibrosis in rats by interacting with cyclophilin B and D

N-methyl-4-isoleucine cyclosporine (NIM811), a new analogue of cyclosporine A, can inhibit collagen deposition in vitro and reduce liver necrosis in a bile-duct-ligation animal model. However, whether NIM811 effects on CCl(4) -induced rat liver fibrosis, and the related mechanism has not been determined. A liver fibrosis model was induced in Wistar rats using CCl(4) for 6 weeks. Meanwhile, two different doses of NIM811 (low-dose 10 mg/kg and high-dose 20 mg/kg) were given to the CCl(4) -treated rats. Liver fibrosis was then evaluated according to histopathological scoring and liver hydroxyproline content. Serum alanine aminotransferase, aspartate aminotransferase and albumin levels, expression of matrix metalloproteinase-13, tissue inhibitor of metalloproteinase-1, α-smooth muscle actin and cyclophilin B and D in liver tissue were determined. Cyclophilin B and D were also studied in an hepatic stellate cell line. Hydroxyproline content was decreased in both NIM811 groups compared with the model (P < 0.05). Liver necrosis and fibrosis were also attenuated in the NIM811 groups. NIM811 suppressed the expression of tissue inhibitor of metalloproteinase-1, transforming growth factor beta mRNA and α-smooth muscle actin protein in liver tissue. Expression of cyclophilin B in the fibrosis model was increased compared with the normal group (P < 0.05), and was decreased significantly in the low-dose NIM811 treatment group (P < 0.05), which indicated that cyclophilin B might have a profibrotic effect. In vitro studies revealed that cyclophilin B and/or D knockout were associated with collagen inhibition. NIM811 attenuates liver fibrosis in a CCl(4)-induced rat liver fibrosis model, which may be related to binding with cyclophilin B and D.