Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 μg/mL produced a 5.2-fold increase in IL-8 mRNA by 8 h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.     

血清铜蓝蛋白联合透明质酸检测在肝纤维化患者诊断中的应用

目的探讨血清铜蓝蛋白(CP)联合透明质酸(HA)检测在肝纤维化患者诊断中的应用前景。方法选取2015年1月至2018年12月本院收治的疑似肝纤维化患者226例为研究对象,对患者的CP、HA水平进行联合检测,根据各项指标检测水平对患者做出诊断,并与金标准诊断方法肝穿刺活检结果进行对比。对CP、HA单独检测和联合检测的诊断结果进行统计对比,并对各组检查方法的灵敏度、特异度、假阳性率、假阴性率、准确度等进行统计对比,比较不同检查方法在不同肝纤维化分级患者中的诊断正确率。结果 226例患者中金标准诊断201例(88.94%)为肝纤维化,CP单独检测检出154例(68.14%),HA单独检测检出143例(63.27%),CP+HA联合检测检出195例(86.28%)。经Kappa一致性检验分析CP+HA联合检测与金标准结果有高度一致性,CP、HA单独检测与金标准结果为中等一致性。CP+HA联合检测的灵敏度、特异度、准确度均高于CP、HA单独检测,方法间比较差异有统计学意义(χ~2=11.602、55.959、78.047,P=0.003、0.039、0.002)。在不同分级肝纤维化患者中,CP+HA联合检测在Ⅰ级、Ⅱ级纤维化检出率高于CP、HA单独检测,方法间比较差异有统计学意义(χ~2=9.432、8.324,P=0.007、0.010)。结论 CP联合HA检测在肝纤维化患者诊断中应用情况良好,有较高的诊断正确率,且对症状程度较轻的肝纤维化患者有较高的检出率,可作为肝纤维化诊断检查的主要手段加以推广应用。     

不同二步酶灌注法分离大鼠肝星状细胞的比较研究

目的:在现有二步酶灌注法分离大鼠肝星状细胞(hepatic stellate cells,HSC)的基础上,探索更加高效的分离HSC方法。方法:分别采用链酶蛋白酶+胶原酶循环灌注、链酶蛋白酶非循环灌注+胶原酶循环灌注以及胶原酶单独循环灌注法分离大鼠HSC,比较三种方法的细胞获得率、活性和纯度差异。应用0.4%台盼蓝染色判断活性,结蛋白(desmin)、波形蛋白(vimentin)细胞免疫荧光方法鉴定纯度。结果:链酶蛋白酶非循环灌注+胶原酶循环灌注法细胞获得率高于另两种方法,细胞活力高于链酶蛋白酶循环灌注+胶原酶循环灌注法,三组得到的细胞纯度均高于90%且无显著差异。结论:在三种二步酶灌注方法中,链酶蛋白酶非循环灌注+胶原酶循环灌注法能显著提高HSC获得率,且对细胞活力影响小,不降低细胞纯度,是一种高效的分离方法,有利于HSC相关肝脏疾病的生物学研究。     

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.     

Cardiorenal fibrosis and dysfunction in aging: Imbalance in mediators and regulators of collagen

Cardiorenal fibrosis is a biological process that increases with age and contributes to dysfunction of the heart and kidney. While numerous circulating and tissue hormones, cytokines and enzymes have been identified in the development of cardiorenal fibrosis, several reports have suggested that the anti-fibrotic natriuretic peptide system (NPS), pro-fibrotic renin–angiotensin–aldosterone system (RAAS), transforming growth factor-beta 1 (TGF-β1), matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are fundamental regulators and mediators of this process. However, the simultaneous assessment of these components in the development of age-mediated cardiorenal fibrotic remodeling is not completely understood. Thus, we assessed cardiorenal structure and function, the circulating NPS and RAAS and the cardiorenal tissue gene expression of collagen (Col) I, Col III, TGF-β1, MMP-9 and TIMP-1 in 2 and 20 month old Fischer rats. Our studies determined that aging was characterized by an increase in cardiorenal fibrosis that was accompanied with cardiorenal dysfunction. These alterations were associated with lower circulating atrial and C-type natriuretic peptides and higher angiotensin II and aldosterone levels in the aged rats. Moreover, we observed a decrease in Col I and III and an increase in TIMP- mRNA expressions in the aged heart and kidney, while TGF-β1 expression increased and MMP-9 decreased only in the aged kidney. We conclude that the age-mediated alterations in these fibrotic regulator and mediator profiles favors collagen accumulation due to an imbalance between the NPS and RAAS as well as a decline in the degradative pathway, thus suggesting a therapeutic opportunity to target these components.     

Relation of exaggerated cytokine responses of CF airway epithelial cells to PAO1 adherence

In many model systems, cystic fibrosis (CF) phenotype airway epithelial cells in culture respond to P. aeruginosa with greater interleukin (IL)-8 and IL-6 secretion than matched controls. In order to test whether this excess inflammatory response results from the reported increased adherence of P. aeruginosa to the CF cells, we compared the inflammatory response of matched pairs of CF and non CF airway epithelial cell lines to the binding of GFP-PAO1, a strain of pseudomonas labeled with green fluorescent protein. There was no clear relation between GFP-PAO1 binding and cytokine production in response to PAO1. Treatment with exogenous aGM1 resulted in greater GFP-PAO1 binding to the normal phenotype compared to CF phenotype cells, but cytokine production remained greater from the CF cell lines. When cells were treated with neuraminidase, PAO1 adherence was equalized between CF and nonCF phenotype cell lines, but IL-8 production in response to inflammatory stimuli was still greater in CF phenotype cells. The polarized cell lines 16HBEo-Sense (normal phenotype) and Antisense (CF phenotype) cells were used to test the effect of disrupting tight junctions, which allows access of PAO1 to basolateral binding sites in both cell lines. IL-8 production increased from CF, but not normal, cells. These data indicate that increased bacterial binding to CF phenotype cells cannot by itself account for excess cytokine production in CF airway epithelial cells, encourage investigation of alternative hypotheses, and signal caution for therapeutic strategies proposed for CF that include disruption of tight junctions in the face of pseudomonas infection.     

Regeneration capacity of oceanic-montane liverworts: implications for community distribution and conservation

Abstract

Scotland’s mountains are home to a rare and unique liverwort community, ‘the oceanic-montane liverwort-rich heath’, but its component species are absent from regions where they could potentially thrive. Many biological characteristics of these species are unknown, making it impossible to explain the reasons for their rarity; however, they have not been observed to produce sporophytes within Britain. We use ex situ cultivation of whole liverworts and fragments, and in situ cultivation of fragments, to assess the growth rate and the potential for vegetative reproduction of several species. Most of the species grew from both fragments and as whole plants, indicating that the rarity of the liverwort heath is not due to poor powers of regeneration. We propose that growth rate and the potential to regenerate from fragments are important factors structuring the liverwort heath community, at least locally. Furthermore, this study demonstrates that there is potential for ex situ conservation of rare liverwort species, in situ enhancement of existing populations, and creation of new ones.     

Dual effect of chemokine CCL7/MCP-3 in the development of renal tubulointerstitial fibrosis

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis – TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0–8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0–3 days) decrease of inflammatory cell infiltration followed (3–8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10–14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.     

Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.