Experimental Biology and Medicine

Clara cell protein (CC16) is a well-known anti-inflammatory protein secreted by the epithelial Clara cells of the airways. It is involved in the development of airway inflammatory diseases such as chronic obstructive pulmonary disease and asthma. Previous studies suggest that CC16 gene transfer suppresses expression of interleukin (IL)-8 in bronchial epithelial cells. However, its role in the function of these cells during inflammation is not well understood. In this study, we evaluated the effect of CC16 on the expression of matrix metalloproteinase (MMP)-9 in lipopolysaccharide (LPS)-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. We generated recombinant rat CC16 protein (rCC16) which was bioactive in inhibiting the activity of phospholipase A₂. rCC16 inhibited LPS-induced MMP-9 expression at both mRNA and protein levels in a concentration-dependent (0–2 µg/mL) manner, as demonstrated by real time RT-PCR, ELISA, and zymography assays. Gene transcription and DNA binding studies demonstrated that rCC16 suppressed LPS-induced NF-κB activation and its binding of gene promoters as identified by luciferase reporter and gel mobility shift assays, respectively. Western blotting and immunofluorescence staining analyses further revealed that rCC16 concentration dependently inhibited the effects of LPS on nuclear increase and cytosol reduction of NF-κB, on the phosphorylation and reduction of NF-κB inhibitory IκBα, and on p38 MAPK-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. These data indicate that inhibition of LPS-mediated NF-κB activation by rCC16 involves both translocation- and phosphorylation-dependent signaling pathways. When the tracheal epithelial cells were pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, cellular uptake of rCC16 and its inhibition of LPS-induced NF-κB nuclear translocation and also MMP-9 production were significantly abolished. Taken together, our data suggest that clathrin-mediated uptake of rCC16 suppresses LPS-mediated inflammatory MMP-9 production through inactivation of NF-κB and p38 MAPK pathways in tracheal epithelial cells.

Recent research has shown that the occurrence of gender disparity in liver cancer associated with sex differences in MyD88-dependent IL-6 production, but the role of this signaling pathway in sex differences of non-alcoholic steatohepatitis (NASH) remains unknown. To investigate the effects of sex hormone-specific intervention on pathology and progression of NASH, and on the inflammatory TLR-MyD88-IL-6 signaling pathway NASH was modeled in C57/BL6 mice by feeding a methionine and choline-deficient (MCD) diet for 4 weeks. Male mice were subjected to sex hormone-related interventions such as orchidectomy, and orchidectomy combined with administration of either testosterone propionate or estradiol benzoate. Next, the degree of non-alcoholic fatty liver disease activity score (NAS), serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the expression level of MyD88 and IL-6, were compared between these groups. Males developed more serious inflammatory problems and had a higher NAS than the females. Sex-specific intervention in male mice by orchidectomy reduced NAS, ALT, and AST, and the expression level of MyD88 and IL-6. But administration of exogenous androgen had no influence on either NAS or the expression of ALT, AST, MyD88, and IL-6. On the other hand, exogenous estrogen could alleviate the pathological damage caused by NASH, as well as reduce NAS, ALT and AST, and the expression of MyD88 and IL-6. The result show different sex hormone-related interventions affected the severity of NASH, possibly by modulating the level of sex hormones and regulating the TLR-MyD88-IL-6 signaling pathway.

The most important etiologic agent in the pathogenesis of cervical cancers (CCs) is human papillomavirus (HPV), while the mechanisms underlying are still not well known. Glucose-6-phosphate dehydrogenase (G6PD) is reported to elevate in various tumor cells. However, no available references elucidated the correlation between the levels of G6PD and HPV-infected CC until now. In the present study, we explored the possible role of G6PD in the pathology of CC induced by HPV infection. Totally 48 patients with HPV + CC and another 63 healthy women enrolled in the clinical were employed in the present study. Overall, prevalence of cervical infection with high-risk-HPV (HR-HPV) type examined was HPV-16, followed by HPV-18. The expressions of G6PD in CC samples were also detected by immunohistochemistry (IHC), qRT-PCR, and Western blot. Regression analysis showed elevated G6PD level was positively correlated with the CC development in 30–40 aged patients with HR-HPV-16/18 infection. The HPV16 + Siha, HPV18 + Hela, and HPV-C33A cell lines were employed and transfected with G6PD deficient vectors developed in vitro. MTT and flow cytometry were also employed to determine the survival and apoptosis of CC cells after G6PD expressional inhibition. Our data revealed that G6PD down-regulation induced poor proliferation and more apoptosis of HPV18 + Hela cells, when compared with that of HPV16 + Siha and HPV-C33A cells. These findings suggest that G6PD expressions in the HR-HPV + human CC tissues and cell lines play an important role in tumor growth and proliferation.

The physiology of vascular cells depends on stimulating mechanical forces caused by pulsatile flow. Thus, mechano-transduction processes and responses of primary human endothelial cells (ECs) and smooth muscle cells (SMCs) have been studied to reveal cell-type specific differences which may contribute to vascular tissue integrity. Here, we investigate the dynamic reorientation response of ECs and SMCs cultured on elastic membranes over a range of stretch frequencies from 0.01 to 1 Hz. ECs and SMCs show different cell shape adaptation responses (reorientation) dependent on the frequency. ECs reveal a specific threshold frequency (0.01 Hz) below which no responses is detectable while the threshold frequency for SMCs could not be determined and is speculated to be above 1 Hz. Interestingly, the reorganization of the actin cytoskeleton and focal adhesions system, as well as changes in the focal adhesion area, can be observed for both cell types and is dependent on the frequency. RhoA and Rac1 activities are increased for ECs but not for SMCs upon application of a uniaxial cyclic tensile strain. Analysis of membrane protrusions revealed that the spatial protrusion activity of ECs and SMCs is independent of the application of a uniaxial cyclic tensile strain of 1 Hz while the total number of protrusions is increased for ECs only. Our study indicates differences in the reorientation response and the reaction times of the two cell types in dependence of the stretching frequency, with matching data for actin cytoskeleton, focal adhesion realignment, RhoA/Rac1 activities, and membrane protrusion activity. These are promising results which may allow cell-type specific activation of vascular cells by frequency-selective mechanical stretching. This specific activation of different vascular cell types might be helpful in improving strategies in regenerative medicine.

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.

The study examined the putative role of ovarian hormones in shaping of rat peripheral T-cell compartment during post-reproductive period. In 20-month-old rats ovariectomized (Ox) at the very end of reproductive period, thymic output, cellularity and composition of major TCRαβ + peripheral blood lymphocyte and splenocyte subsets were analyzed. Ovariectomy led to the enlargement of CD8 + peripheral blood lymphocyte and splenocyte subpopulations. This reflected: (i) a more efficient thymic generation of CD8 + cells as indicated by increased number of CD4+CD8 + double positive and the most mature CD4-CD8+TCRαβ^high thymocytes and CD8 + recent thymic emigrants (RTEs) in peripheral blood, but not in the spleen of Ox rats, and (ii) the expansion of CD8 + memory/activated peripheral blood lymphocytes and splenocytes. The latter was consistent with a greater frequency of proliferating cells among freshly isolated memory/activated CD8 + peripheral blood lymphocytes and splenocytes and increased proliferative response of CD8 + splenocytes to stimulation with plate-bound anti-CD3 antibody. The former could be related to the rise in splenic IL-7 and IL-15 mRNA expression. Although ovariectomy affected the overall number of CD4 + T cells in none of the examined compartments, it increased CD4+FoxP3 + peripheral blood lymphocyte and splenocyte counts by enhancing their generation in periphery. Collectively, the results suggest that ovariectomy-induced long-lasting disturbances in ovarian hormone levels (mirrored in diminished progesterone serum level in 20-month-old rats) affects both thymic CD8 + cell generation and peripheral homeostasis and leads to the expansion of CD4+FoxP3 + cells in the periphery, thereby enhancing autoreactive cell control on account of immune system efficacy to combat infections and tumors.

This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg⁻¹, which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn’s method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD₅₀) of acidolysis product in mice was >9.09 g kg⁻¹ body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient.

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [³H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.

The continuous changes in pulmonary hemodynamic properties and right ventricular (RV) function in pulmonary arterial hypertension (PAH) have not been fully characterized in large animal model of PAH induced by a carotid artery–jugular vein shunt. A minipig model of PAH was induced by a surgical anastomosis between the left common carotid artery and the left jugular vein. The model was validated by catheter examination and pathologic analyses, and the hemodynamic features and right-ventricle functional characteristics of the model were continuously observed by Doppler echocardiography. Of the 45 minipigs who received the surgery, 27 survived and were validated as models of PAH, reflected by mean pulmonary artery pressure ≥25 mmHg, and typical pathologic changes of pulmonary arterial remodeling and RV fibrosis. Non-invasive indices of pulmonary hemodynamics (pulmonary artery accelerating time and its ratio to RV ventricular ejection time) were temporarily increased, then reduced later, similar to changes in tricuspid annular displacement. The Tei index of the RV was elevated, indicating a progressive impairment in RV function. Surgical anastomosis between carotid artery and jugular vein in a minipig is effective to establish PAH, and non-invasive hemodynamic and right-ventricle functional indices measured by Doppler echocardiography may be used as early indicators of PAH.

The release of mediators by mast cells triggers allergic symptoms involving various physiological systems and, in the most severe cases, the development of anaphylactic shock compromising mainly the nervous and cardiovascular systems. We aimed to establish variables to objectively study the anaphylactic response (AR) after an oral challenge in an allergy model. Brown Norway rats were immunized by intraperitoneal injection of ovalbumin with alum and toxin from Bordetella pertussis. Specific immunoglobulin (Ig) E antibodies were developed in immunized animals. Forty days after immunization, the rats were orally challenged with the allergen, and motor activity, body temperature and serum mast cell protease concentration were determined. The anaphylaxis induced a reduction in body temperature and a decrease in the number of animal movements, which was inversely correlated with serum mast cell protease release. In summary, motor activity is a reliable tool for assessing AR and also an unbiased method for screening new anti-allergic drugs.

A proposed intervention for newborn infants in countries with suspected vitamin A (VA) deficiency is to administer 50,000 IU retinyl palmitate at birth to reduce mortality risk. However, no studies have investigated birth weight effects. In this study, low birth weight (LBW; <1 kg, n = 18) and healthy birth weight (HBW) piglets (>1.5 kg, n = 18) from VA-depleted sows were dosed with 25,000 or 50,000 IU retinyl palmitate (26.2 or 52.4 µmol retinol equivalents) at birth to compare VA reserves. Blood was collected at varying times (n = 3–5/time/dose), and piglets were killed at 12 or 24 h for blood, liver, kidneys, spleen, lungs, adrenal gland, and intestinal contents. HBW piglets had significantly higher birth, death, and organ weights than LBW (P < 0.0001 for all). HBW and LBW piglets, which received VA, had higher liver and kidney VA concentrations (0.18 ± 0.09, 0.24 ± 0.10 µmol/g liver and 13.4 ± 4.1, 14.2 ± 4.5 nmol/g kidney, respectively) than controls (n = 10) (0.051 ± 0.01 µmol/g liver and 1.01 ± 0.43 nmol/g kidney) (P = 0.0061 and < 0.0001, respectively). Total liver (9.75 ± 5.16 µmol) and kidney retinol (204 ± 79.1 nmol) were higher in HBW than LBW piglets (P < 0.0001). Extrahepatic tissues, except lung, had higher VA concentration than controls (P < 0.0001). Serum retinol and ester concentrations were higher in treated than control piglets (P = 0.0028, P < 0.0001, respectively), and significantly changed during the times sampled (P = 0.022, P = 0.011, respectively). Peak serum retinyl ester concentrations, which occurred at 3 h, were higher in piglets that received 50,000 IU (4.2 ± 4.4 µmol/L) than 25,000 IU (2.7 ± 2.3 µmol/L) (P = 0.031). Regardless of dose amount, HBW piglets stored more supplemental VA than LBW piglets when administered at birth.