Infusion of Trx-1-Overexpressing hucMSC Prolongs the Survival of Acutely Irradiated NOD/SCID Mice by Decreasing Excessive Inflammatory Injury

A protective reagent for ARI should have the ability to repair injured tissue caused by radiation and prevent continuous damage from secondary risk factors. Trx-1 was explored as a candidate therapy for ARI, as it scavenges reactive oxygen species, regulates cell growth and differentiation, participates in immune reactions, and inhibits apoptosis by acting inside and/or outside cells. Trx-1 can also decrease excessive inflammation in ARI by regulating the creation of inflamed media, by inhibiting the activation of complement, and by reducing the chemotaxis, adhesion, and migration of inflammatory cells. As effectively and stably expressing exogenous genes in the long term and regulating immune inflammation and tissue repair, MSC are a good choice for Trx-1 gene therapy. In this study, Trx-1-overexpressing hucMSC-Trx-1 were obtained by adenoviral vector-mediated infection. We first measured the redox capacity of hucMSC-Trx-1 with an antioxidant capacity (T-AOC) assay, a hydrogen peroxide (H₂O₂) content determination assay in vivo, a H₂O₂-induced oxidation hemolysis assay, and a lipid peroxidation assay in vitro. Then, we measured survival time, the protection of the hematopoietic system, and the regulation of inflammation in important organs in three treatment groups of NOD/SCID mice (treated with hucMSC-Trx-1, with hucMSC, and with saline) that were exposed to 4.5 Gy ⁶⁰Co-γ-ray radiation. The hucMSC-Trx-1 group achieved superior antioxidation results, protecting bone marrow hematopoietic stem cells (Lin⁻CD117⁺: hucMSC-Trx-1 vs. hucMSC, P<0.05; hucMSC-Trx-1 vs. NS, P<0.01), promoting the formation of red blood cells and hemoglobin (hucMSC-Trx-1 vs. hucMSC or NS, P<0.05), reducing inflammation and damage in important organs (Bone marrow and lung: hucMSC-Trx-1 vs. NS, P<0.01; hucMSC-Trx-1 vs. hucMSC, P<0.05. Liver and intestine: hucMSC-Trx-1 vs. NS, P<0.05; hucMSC-Trx-1 vs. hucMSC, P<0.05), and prolonging survival (hucMSC-Trx-1 vs. hucMSC or NS, P<0.01). Therefore, hucMSC-Trx-1 combines the merits of gene and cell therapy as a multifunctional radioprotector for ARI.     

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis.METHODS: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3^rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot.RESULTS: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05).CONCLUSION: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.     

Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (G_αgust) and α-transducin (G_αtran) G protein subunits. This study tested whether G_αtran and G_αgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, G_αgust and G_αtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, G_αgust and G_αtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of G_αtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of G_αgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included G_αtran / 5HT-IR and G_αtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as G_αgust /5-HT-IR or G_αgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in G_αtran-IR cells in the duodenal crypts and a significant increase of G_αgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of G_αtran-G_αgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of G_αtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of G_αgust / G_αtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.     

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C _max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC_0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), V_d increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C _max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.     

Regional Arterial Infusion with Lipoxin A4 Attenuates Experimental Severe Acute Pancreatitis

Objective

Investigate the therapeutic effect of regional arterial infusion (RAI) with Aspirin-Triggered Lipoxin A₄ (ATL) in experimental severe acute pancreatitis (SAP) in rats.

Materials and Methods

SAP was induced by injection of 5% sodium taurocholate into the pancreatic duct. Rats with SAP were treated with ATL (the ATL group) or physiological saline (the SAP group) infused via the left gastric artery 30 min after injection of sodium taurocholate. The sham group was subjected to the same surgical procedure, though without induction of SAP. Serum levels of amylase, phospholipase A2 (PLA2), interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured at 12 and 24 h after induction of SAP. Ascitic fluid, the pancreatic index (wet weight ratio) and myeloperoxidase (MPO) levels in the pancreas were determined and histopathological findings were evaluated. The expression of intercellular adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), NF-κB p65, and heme oxygenase-1 (HO-1) in the pancreas were estimated by immunofluorescence and western blot, respectively.

Results

ATL rats had lower serum levels of TNF-α, IL-1β, and IL-6 (P<0.01), PLA₂ (P<0.05), and amylase levels (P<0.05) studied as compared with the SAP group. The pancreatic index in the ATL group decreased only at 24 h as compared with the SAP group (P<0.05). The histopathological findings and MPO levels in the pancreas significantly decreased in the ATL group as compared to the SAP group (P<0.05 and P<0.01, respectively). Immunofluorescence and western blot showed that ATL attenuated the expression of NF-κB p65, ICAM-1 and PECAM-1 in the pancreas, and increased the expression of HO-1 in SAP animals.

Conclusions

We demonstrated that RAI with ATL attenuated the severity of experimental SAP, maybe achieved by improving the expression of HO-1, and down-regulating the NF-κB signaling pathway, with decreased expression of ICAM-1 and PECAM-1 and reduced generation of pro-inflammatory cytokines.     

Suppression of TGF-β1/Smad Signaling Pathway by Sesamin Contributes to the Attenuation of Myocardial Fibrosis in Spontaneously Hypertensive Rats

This study investigated the effect of sesamin on myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the possible mechanisms involved. Twenty-eight male SHRs were randomly allocated to SHR group, Ses160 group (sesamin 160 mg/kg), Ses80 group (sesamin 80 mg/kg) and Cap30 group (captopril 30 mg/kg). Seven male WKY rats were used as control. Sesamin and captopril were administered intragastrically for 12 weeks. Captopril significantly reduced systolic blood pressure and angiotensin II (Ang II) levels in SHRs, accompanied by a marked attenuation of left ventricular hypertrophy (LVH) and collagen deposition (P <0.05 or P <0.01). Though sesamin had no significant influence on Ang II levels, and the hypotensive effect was also significantly inferior to that of captopril (P <0.05 or P <0.01), however, the improvement of LVH and collagen deposition was similar to that in captopril group. Sesamin markedly reduced transforming growth factor-β₁ (TGF-β₁) content in cardiac tissues, with Smad3 phosphorylation decreased and Smad7 protein expression increased notably (P <0.05 or P <0.01). Protein expression of type I collagen and type III collagen, target genes of Smad3, was down-regulated markedly by sesamin (P <0.05 or P <0.01). In addition, sesamin significantly increased total antioxidant capacity and superoxide dismutase protein in cardiac tissues (P <0.05 or P <0.01), while the expression of NADPH oxidase subunit p47phox and malondialdehyde content were reduced markedly (P <0.05 or P <0.01). In vitro studies also demonstrated that sesamin was able to suppress Ang II induced phosphorylation of Smad3 and secretion of TGF-β₁ and type I and type III collagen in cultured rat cardiac fibroblasts. These data suggest that sesamin is capable of attenuating hypertensive myocardial fibrosis through, at least partly, suppression of TGF-β₁/Smad signaling pathway.     

The influence of environmental temperature on appetite-related hormonal responses

Background

Acute exercise in the heat has been shown to reduce appetite. However, the influence of exercise in the cold on appetite regulation remains unclear. The aim of this study was to compare exercise-induced appetite regulation under three different environmental temperatures.

Methods

Eleven male participants completed three experimental trials on the following separate days: exercise in the heat (36°C), exercise at neutral temperature (24°C), and exercise in the cold (12°C). The exercise trials consisted of pedaling exercises for 30 min at 65% of maximal oxygen uptake (VO_2max). Blood samples were collected repeatedly to determine plasma ghrelin, peptide YY (PYY) and other hormonal concentrations. Subjective feelings of hunger and tympanic temperature were also monitored.

Results

Tympanic temperature was significantly higher in the 36°C trial than that of the other two trials (P < 0.05). The subjective feelings of hunger in the 36°C and 24°C trials were significantly lower than those in the 12°C trial (P < 0.05). Plasma ghrelin concentration decreased significantly with exercise in all conditions (P < 0.05), and the responses were not significantly different among the three conditions. Plasma PYY concentration increased significantly after the exercise in the 24°C trial only (P < 0.05), with no significant difference among the three trials.

Conclusions

These results suggest that exposure to hot or cold temperatures during exercise did not affect exercise-induced plasma ghrelin and PYY responses. However, the exercise-induced reduction of subjective hunger was significantly attenuated in a cold environment.     

Personality and Dietary Intake – Findings in the Helsinki Birth Cohort Study

Background

Personality traits are associated with health outcomes including non-communicable diseases. This could be partly explained by lifestyle related factors including diet. The personality traits neuroticism, extraversion, openness, agreeableness, and conscientiousness are linked with resilience, meaning adaptability in challenging situations. Resilient people usually comply with favorable health behaviors.

Objective

Our objective was to explore the associations between food and nutrient intake, personality traits and resilience.

Design

A validated semi-quantitative food frequency questionnaire was used to measure diet and the NEO-personality inventory to assess personality in 1681 subjects. Linear regression analysis was used to explore diet-personality associations and cluster analysis to define resilient and non-resilient personality profiles.

Results

Adjusting for age, education and energy intake, and applying Bonferroni corrections, openness in men was associated with higher vegetable (14.9 g/d for 1 SD increase in the personality score, P_Bonf <0.01) and lower confectionery and chocolate (−2.8 g/d, P_Bonf <0.01) intakes. In women, neuroticism was associated with lower fish (−4.9 g/d, P_Bonf <0.001) and vegetable (−18.9 g/d, P_Bonf <0.01) and higher soft drink (19.9 g/d, P_Bonf <0.001) intakes. Extraversion, in women, associated with higher meat (5.9 g/d, P_Bonf <0.05) and vegetable (24.8 g/d, P_Bonf<0.001) intakes, openness with higher vegetable (23.4 g/d, P_Bonf <0.001) and fruit (29.5 g/d, P_Bonf <0.01) intakes. Agreeableness was associated with a lower soft drink (−16.2 g/d, P_Bonf <0.01) and conscientiousness with a higher fruit (32.9 g/d, P_Bonf<0.01) intake in women. Comparing resilient and non-resilient subjects, we found resilience in women to be associated with higher intakes of vegetables (52.0 g/d, P<0.001), fruits (58.3 g/d, P<0.01), fish (8.6 g/d, P<0.01) and dietary fiber (1.6 g/d, P<0.01).

Conclusion

Personality traits are associated with dietary intake and especially subjects with resilient personality profiles had healthier dietary intakes. These associations were stronger in women than in men.     

Resistance Training Improves Hemodynamic Function,Collagen Deposition and Inflammatory Profiles: Experimental Model of Heart Failure

The role of resistance training on collagen deposition, the inflammatory profile and muscle weakness in heart failure remains unclear. Therefore, this study evaluated the influence of a resistance training program on hemodynamic function, maximum strength gain, collagen deposition and inflammatory profile in chronic heart failure rats. Thirty-two male Wistar rats submitted to myocardial infarction by coronary artery ligation or sham surgery were assigned into four groups: sedentary sham (S-Sham, n = 8); trained sham (T-Sham, n = 8); sedentary chronic heart failure (S-CHF, n = 8) and trained chronic heart failure (T-CHF, n = 8). The maximum strength capacity was evaluated by the one maximum repetition test. Trained groups were submitted to an 8-week resistance training program (4 days/week, 4 sets of 10–12 repetitions/session, at 65% to 75% of one maximum repetition). After 8 weeks of the resistance training program, the T-CHF group showed lower left ventricular end diastolic pressure (P<0.001), higher left ventricular systolic pressure (P<0.05), higher systolic blood pressure (P<0.05), an improvement in the maximal positive derivative of ventricular pressure (P<0.05) and maximal negative derivative of ventricular pressure (P<0.05) when compared to the S-CHF group; no differences were observed when compared to Sham groups. In addition, resistance training was able to reduce myocardial hypertrophy (P<0.05), left ventricular total collagen volume fraction (P<0.01), IL-6 (P<0.05), and TNF-α/IL-10 ratio (P<0.05), as well as increasing IL-10 (P<0.05) in chronic heart failure rats when compared to the S-CHF group. Eight weeks of resistance training promotes an improvement of cardiac function, strength gain, collagen deposition and inflammatory profile in chronic heart failure rats.     

The natural organosulfur compound dipropyltetrasulfide prevents HOCl-induced systemic sclerosis in the mouse

Introduction

The aim of this study was to test the naturally occurring organosulfur compound dipropyltetrasulfide (DPTTS), found in plants, which has antibiotic and anticancer properties, as a treatment for HOCl-induced systemic sclerosis in the mouse.

Methods

The prooxidative, antiproliferative, and cytotoxic effects of DPTTS were evaluated ex vivo on fibroblasts from normal and HOCl mice. In vivo, the antifibrotic and immunomodulating properties of DPTTS were evaluated in the skin and lungs of HOCl mice.

Results

H₂O₂ production was higher in fibroblasts derived from HOCl mice than in normal fibroblasts (P < 0.05). DPTTS did not increase H₂O₂ production in normal fibroblasts, but DPTTS dose-dependently increased H₂O₂ production in HOCl fibroblasts (P < 0.001 with 40 μM DPTTS). Because H₂O₂ reached a lethal threshold in cells from HOCl mice, the antiproliferative, cytotoxic, and proapoptotic effects of DPTTS were significantly higher in HOCl fibroblasts than for normal fibroblasts. In vivo, DPTTS decreased dermal thickness (P < 0.001), collagen content in skin (P < 0.01) and lungs (P < 0.05), αSMA (P < 0.01) and pSMAD2/3 (P < 0.01) expression in skin, formation of advanced oxidation protein products and anti-DNA topoisomerase-1 antibodies in serum (P < 0.05) versus untreated HOCl mice. Moreover, in HOCl mice, DPTTS reduced splenic B-cell counts (P < 0.01), the proliferative rates of B-splenocytes stimulated by lipopolysaccharide (P < 0.05), and T-splenocytes stimulated by anti-CD3/CD28 mAb (P < 0.001). Ex vivo, it also reduced the production of IL-4 and IL-13 by activated T cells (P < 0.05 in both cases).

Conclusions

The natural organosulfur compound DPTTS prevents skin and lung fibrosis in the mouse through the selective killing of diseased fibroblasts and its immunomodulating properties. DPTTS may be a potential treatment for systemic sclerosis.     

1,25-dihydroxyvitamin D3 Protects against Macrophage-Induced Activation of NFκB and MAPK Signalling and Chemokine Release in Human Adipocytes

Increased accumulation of macrophages in adipose tissue in obesity is linked to low-grade chronic inflammation, and associated with features of metabolic syndrome. Vitamin D₃ may have immunoregulatory effects and reduce adipose tissue inflammation, although the molecular mechanisms remain to be established. This study investigated the effects of vitamin D₃ on macrophage-elicited inflammatory responses in cultured human adipocytes, particularly the signalling pathways involved. Macrophage-conditioned (MC) medium (25% with adipocyte maintenance media) markedly inhibited protein expression of the nuclear factor-κB (NFκB) subunit inhibitor κBα (IκBα) (71%, P<0.001) and increased NFκB p65 (1.5-fold, P = 0.026) compared with controls. Treatment with 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) abolished macrophage-induced activation of NFκB signalling by increasing IκBα expression (2.7-fold, P = 0.005) and reducing NFκB p65 phosphorylation (68%; P<0.001). The mitogen-activated protein kinase (MAPK) signalling was activated by MC medium, which was also blunted by 1,25(OH)₂D₃ with a downregulation of phosphorylated p38 MAPK (32%, P = 0.005) and phosphorylated Erk1/2 (49%, P = 0.001). Furthermore, MC medium (12.5% or 25%) dose-dependently upregulated secretion of key proinflammatory chemokines/cytokines (22-368-fold; all P<0.001) and this was significantly decreased by 1,25(OH)₂D₃: IL-8 (61% and 31%, P<0.001), MCP-1 (37%, P<0.001 and 36%, P = 0.002), RANTES (78% and 62%, P<0.001) and IL-6 (29%, P<0.001 and 34%, P = 0.019). Monocyte migration-elicited by adipocytes treated with 1,25(OH)₂D₃ was also reduced (up to 25%, P<0.001). In conclusion, vitamin D₃ could be anti-inflammatory in adipose tissue, decreasing macrophage-induced release of chemokines and cytokines by adipocytes and the chemotaxis of monocytes. Our data suggests these effects are mediated by inhibition of the NFκB and MAPK signalling pathways.     

Ovariectomy differential influence on some hemostatic markers of mice and rats

Rodent ovariectomy is an experimental method to eliminate the main source of sexual steroids. This work explored for the first time the ovariectomy temporal changes induced in the hemostatic coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB) along with uterine weight on adult female CD1 mice and Wistar rats. Uterine weight (Uw) was assessed before ovariectomy (control), and 1, 3, 5, 7, 9, 16, and 21 days after surgery. PT, aPTT, TT and FIB were estimated the same days, using reported standard techniques. Ovariectomy decreased Uw, since day 1; and from day 10 to 21 reached the lowest values for both species. After day 1, mice hemostatic parameters changed (PT +10%, P<0.05; aPTT +53%, P<0.05; TT −24%, P<0.05; FIB +67%, P<0.05). Rats showed significant changes only in TT and FIB (TT −13%, P<0.001; FIB +65%, P<0.001). Neither mice PT, aPTT and TT, recovered control values after 21 days. In the rats from day 5 to 16 aPTT diminished (18–23%, P<0.05) recovering to control values on day 21, TT after 9 days and PT on day 16. In both species, FIB returned to its control values after 9 days. Ovariectomy differentially altered the PT hemostatic parameter of mice and rats indicating a non-equivalence among both species behaviour for experimental studies of blood coagulation.     

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.     

Suppression of Experimental Choroidal Neovascularization by Curcumin in Mice

Purpose

To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.

Methods

C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.

Results

Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).

Conclusion

Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.     

Mitochondrial Regulation of NADPH Oxidase in Hindlimb Unweighting Rat Cerebral Arteries

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Relation of Leptin,Ghrelin and Inflammatory Cytokines with Body Mass Index in Pulmonary Tuberculosis Patients with and without Type 2 Diabetes Mellitus

Background

Pulmonary tuberculosis (TB) patients often suffer from anorexia and poor nutrition, causing weight loss. The peptide hormones leptin and its counterpart ghrelin, acting in the regulation of food intake and fat utilization, play an important role in nutritional balance. This study aimed to investigate the association of blood concentrations of leptin, ghrelin and inflammatory cytokines with body mass index (BMI) in TB patients with and without type 2 diabetes mellitus (T2DM).

Methods

BMI, biochemical parameters and plasma levels of leptin, ghrelin and inflammatory cytokines were measured before the start of treatment in 27 incident TB patients with T2DM, 21 TB patients and 23 healthy subjects enrolled in this study.

Results

The levels of leptin were significantly higher in TB patients (35.2±19.1 ng/ml) than TB+T2DM (12.6±6.1 ng/ml) and control (16.1±11.1 ng/ml) groups. The level of ghrelin was significantly lower in TB (119.9±46.1 pg/ml) and non-significantly lower in TB+T2DM (127.7±38.6 pg/ml) groups than control (191.6±86.5 pg/ml) group. The levels of TNF-α were higher, while IFN-γ and IL-6 levels were lower in patients than in the control group. Leptin showed a negative correlation with BMI in TB (r=-0.622, p<0.05) and TB+T2DM (r= -0.654, p<0.05) groups, but a positive correlation with BMI in the control group (r=0.521, p<0.05). Contrary ghrelin showed a positive correlation with BMI in TB (r=0.695, p<0.05) and TB+T2DM (r= 0.199, p>0.05) groups, but negative correlation with BMI in the control (r=-0.693, p<0.05) group. Inflammatory cytokines were poorly correlated with BMI in this study. Only IFN-γ showed a significant negative correlation with BMI in the control group (r=-0.545, p<0.05).

Conclusions

This study may suggest that possible abnormalities in ghrelin and leptin regulation (high levels of leptin and low levels of ghrelin) may be associated with low BMI and may account for the poor nutrition associated with TB and TB+T2DM.     

H-Index Is Important for Postural Control for People with Impaired Foot Sole Sensation

Introduction

People with Peripheral Neuropathy (PN), especially those with impaired sensory inputs through the small-afferent fiber (type II afferent fibers) reflex loop (SAF), might depend more on the large-afferent fiber (type I afferent fibers) reflex loop (LAF) for postural control.

Purpose

To examine whether the function of the LAF reflex loop, reflected by the H-reflex and ankle joint proprioception, influences postural control when the SAF reflex loop is impaired, as indicated by reduced foot sole cutaneous sensation.

Methods

Thirteen participants (8 women, 5 men) diagnosed with PN and 12 age-matched controls (7 women, 5 men) completed the testing protocol. Measures of interest included the H-index, active (AAP) and passive (PAP) ankle proprioception, plantar pressure sensitivity (PPS), average sway velocity (V_AVG) and area (A₉₅) during 30 seconds eyes-closed standing, 6-minute walk distance (6MWD) and timed up-and-go duration (TUG).

Results

Statistically significant group-dependent regression was observed between V_AVG and H-index. Compared to the control group, the PN group demonstrated reduced PPS (2.0 ± 1.9 vs. 4.2 ± 1.2, P < .05) and H-index (63.6 ± 10.9 vs. 76.4 ± 16.0, P < .05), greater V_AVG (3.5 ± 2.1 vs. 1.6 ± 0.6cm/s, P < .05) and A₉₅ (10.0 ± 10.1 vs. 2.5 ± 1.5cm², P < .05), shorter 6MWD (442.2 ± 93.0 vs. 525.3 ± 68.2m, P < .05), and longer TUG (9.4 ± 1.6 vs. 6.5 ± 1.3s, P < .05). Within the PN group, but not the control group, the H-index was correlated with V_AVG (r = -.56, P < .05). Moreover, within the PN group only, PAP scores were correlated with 6MWD (r = -.68, P < .05) and TUG (r = -.59, P < .05) performance. No other statistically significant group difference, correlation or group-dependent regression was observed.

Conclusion

V_AVG, 6MWD, and TUG correlated with LAF reflex loop function observed among those with impaired functioning of the SAF reflex loop. This observation suggests that the LAF reflex loop may be critical to the control of balance in those individuals suffering from small-fiber PN.     

CT Perfusion with Acetazolamide Challenge in C6 Gliomas and Angiogenesis

Background

This study was performed to investigate the correlation between CT perfusion with acetazolamide challenge and angiogenesis in C6 gliomas.

Methods

Thirty-two male Sprague-Dawley rats were evaluated. The rats were divided randomly to four groups: eight rats with orthotopically implanted C6 gliomas at 10-days old (Group A), eight rats with gliomas at 14-days old (Group B), eight rats with gliomas at 18-days old (Group C), eight rats with orthotopically injected normal saline served as controls. CT perfusion was performed before and after administration of acetazolamide. Changes in perfusion parameters due to acetazolamide administration were calculated and analyzed.

Results

Elevated carbon dioxide partial pressure and decreased pH were found in all 32 rats post acetazolamide challenge (P<0.01). Cerebral blood flow_{pre-challenge} was increased in group C (95.0±2.5 ml/100g/min), as compared to group B (80.1±11.3 ml/100g/min) and group A (63.1±2.1 ml/100g/min). Cerebral blood flow percentage changes were detected with a reduction in group C (54.2±4.8%) as compared to controls (111.3±22.2%). Cerebral blood volume _{pre-challenge} was increased in group C (50.8±1.7ml/100g), as compared to group B (45.7±1.9 ml/100g) and group A (38.2±0.8 ml/100g). Cerebral blood volume percentage changes were decreased in group C (23.5±4.6%) as compared to controls (113.5±30.4%). Angiogenesis ratio = [(CD105-MVD) / (FVIII-MVD)] ×100%. Positive correlations were observed between CD105-microvessel density, angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow_{pre-challenge}, cerebral blood volume _{pre-challenge}. Negative correlations were observed between CD105-microvessel density and cerebral blood flow percentage changes (P<0.01, correlation coefficient r=-0.788), cerebral blood volume percentage changes (P<0.01, r=-0.703). Negative correlations were observed between angiogenesis ratio, vascular endothelial growth factor, proliferation marker and cerebral blood flow percentage changes, cerebral blood volume percentage changes.

Conclusion

Our findings suggest that CT perfusion with challenge can provide new insight into non-invasive assessment of rat C6 glioma angiogenesis.