Effects of nalmefene hydrochloride on TLR4 signaling pathway in rats with lung ischemia-reperfusion injury.

OBJECTIVE: To investigate the effect of nalmefene hydrochloride on TLR4 signaling pathway in rats with lung ischemia-reperfusion injury. MATERIALS AND METHODS: Altogether 64 pure inbred male SD rats were divided into groups A, B, C, and D according to the principle of body weight similarity, with 24 rats in each group. Four groups of rats were respectively twisted on the left testis to establish unilateral testicular torsion rats. Group A was the control group, treated with normal saline, group B was the nalmefene hydrochloride high-dose group, treated with 20 µg/kg of nalmefene hydrochloride, group C was the nalmefene hydrochloride low-dose group, treated with 10 µg/kg of nalmefene hydrochloride, and group D was the sham operation group. Lung tissue was collected 60 h later. Western blotting was used to detect the expression levels of HMGB1, TLR4, CD14, and NF-κB protein, qPCR was used to detect the mRNA expression level, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors IL-17, IL-6, and ICAM-1. RESULTS: The expression levels of HMGB1, TLR4, CD14, NF-κB protein, mRNA, IL-17, IL-6, and ICAM-1 in group A were significantly higher than those in groups B, C, and D (p<0.05), while were significantly lower in group D than in groups B and C (p<0.05), and were significantly lower in group B than in group C (p<0.05). CONCLUSIONS: Nalmefene hydrochloride can effectively inhibit the signal pathway of TLR4, and can effectively reduce the injury caused by lung ischemia-reperfusion. The large dose is closely related to the good effect, which is worthy of promotion.

Expression of ERCC1 and BRCA1 predict the clinical outcome of non-small cell lung cancer in patients receiving platinum-based chemotherapy.

We examined mRNA expression levels of ERCC1, BRCA1, RRM1, and human ß-tubulin-III (TUBB3) in non-small-cell lung carcinoma (NSCLC) patients and investigated the association between expression of these genes and the clinical outcome of NSCLC treatment. A total of 366 patients who underwent surgery for NSCLC were included in this study. All patients received third-generation platinum-based chemotherapy as first-line treatment. The relative cDNA quantification for ERCC1, RRM1, BRCA1, and TUBB3 was determined using a fluorescence-based, real-time detection method. We found that low expression of ERCC1 and BRCA1 was associated with a good response to platinum-based chemotherapy, with an odds ratio [95% confidence interval (CI)] of 2.09 (1.33-3.27) and 2.92 (1.85-4.62), respectively. Multivariate Cox regression analysis indicated that patients with low expression of ERCC1 and BRCA1 attained a longer overall survival time than those with high expression, with a hazard ratio (95%CI) of 0.42 (0.23-0.77) and 0.39 (0.21-0.71), respectively. However, RMM1 and TUBB2 expressions were not correlated with clinical outcome of NSCLC. In conclusion, we found that low expression of ERCC1 and BRCA1 can be useful for selecting NSCLC patients who would benefit from chemotherapy and warrants further investigation in prospective studies.

Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells.

The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents.

[Inhibitory effects of co-cinobufotalin oral liquor on hepatitis B in vitro].

Co-Cinobufotalin Oral Liquor (CCOL) was studied for its ability to inhibit hepatitis B virus DNA replication, HBsAg and HBeAg expression in a HBV-transfected cell line (2.2.15 cell). The result showed that ID50 (the drug concentration that inhibits HBsAg or HBeAg secretion by 50%) was 0.08 mg/ml and 0.07 mg/ml on HBsAg and HBeAg respectively. CD50 (the drug concentration that reduces cell growth by 50%) was 2.5 mg/ml. TI (therapeutic index) was 31.3 and 35.7 respectively. The present data suggest that CCOL could exert a potent antiviral activity against HBV in vitro. Southern blot showed that CCOL inhibited HBV-DNA repication in a dose-dependent manner.

Mitogenesis and retinal pigment epithelial cell antigen expression in the rat after krypton laser photocoagulation.

PURPOSE: Polypeptide growth factors, such as the acidic and basic fibroblast growth factors (aFGF and bFGF), may be important in the pathogenesis of subretinal neovascularization. The authors studied the relationship of aFGF and bFGF expression to retinal pigment epithelial (RPE) cell and choriocapillary endothelial cell proliferation in krypton-laser-treated regions of the retina, RPE, and choroid of a model of subretinal neovascularization in the pigmented rat they developed. METHODS: Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals up to 80 days after treatment, the retinas, RPE, and choroid of these animals were examined by [3H]-thymidine autoradiography and electron microscopic immunocytochemistry, using antibodies to aFGF, bFGF, cytoplasmic retinaldehyde-binding protein, opsin, and various basement membrane macromolecules. RESULTS: Nuclear radiolabeling became evident in these layers when the label was injected as late as 75 days after photocoagulation, but the number of labeled nuclei was greatest when isotope was injected 2-5 days after laser treatment. Although there were labeled nuclei in the retina, RPE, and choroid, the frequency of labeling as a fraction of the total number of nuclei appeared to be greatest in the RPE and choriocapillaris. Non-laser-damaged RPE cells were immunocytochemically strongly positive for cytoplasmic retinaldehyde-binding protein, but were negative for aFGF and bFGF. After laser treatment, many RPE cells lost their cytoplasmic retinaldehyde-binding protein positivity but stained strongly for aFGF and bFGF within intracellular structures of variable shape and homogeneous appearance. Although these structures had an appearance suggestive of basement membrane, they did not stain with antibodies to collagens type IV or V, to laminin, or to heparan sulfate proteoglycan core protein. They also did not stain with an antibody to the N-terminal peptide of opsin. Choriocapillary endothelial cells were unreactive with antibodies to aFGF, bFGF, or cytoplasmic retinaldehyde-binding protein either before or after laser treatment. FGF-positive RPE cells persisted for the 80-day duration of the experiment. CONCLUSIONS: Because the presence of FGF-positive RPE cells coincides temporally with increased nuclear thymidine labeling in the RPE and choriocapillaris, aFGF and/or bFGF may be at least partly responsible for initiating the process of cell proliferation and subretinal neovascularization in these animals.

Ultrastructural immunocytochemistry of subretinal neovascular membranes in age-related macular degeneration.

BACKGROUND: The authors studied various cellular and extracellular matrix components of subretinal neovascular membranes (SRNVM) from patients with age-related macular degeneration (ARMD). METHODS: Electron microscopic immunocytochemistry was used on the subfoveal neovascular membranes surgically removed from three patients with disciform lesions due to ARMD. FINDINGS: The SRNVMs always contained large "feeder" vessels along with many new capillaries in different stages of maturation. Capillaries were sparse and embedded in an abundant stroma. The majority of the nonvascular cells were either retinal pigment epithelial (RPE) cells or fibroblast-like cells. The RPE cells formed single or multiple layers on one side of the membranes. The stroma was composed mainly of collagen types I and IV and fibronectin, with small amounts of collagen types III, V, and VI. The absence of Bruch's membrane suggests that a splitting may occur between the RPE cells and Bruch's membrane with the new vessels growing into this cleft. A thickened layer of collagen type IV was often present under the RPE cells. The basement membranes of the newly formed capillaries were morphologically ill-defined, and contained substantial amounts of collagen type IV and fibronectin, but, unlike the basement membranes of normal capillaries, they lacked laminin or heparan sulfate proteoglycan. CONCLUSION: These results on the ultrastructural components of the SRNVMs may be useful in clarifying the nature of the disciform process in ARMD.

Sorbinil does not prevent galactose-induced glomerular capillary basement membrane thickening in the rat.

We investigated the role of the polyol pathway in the pathogenesis of glomerular basement membrane thickening in galactosaemic rats, an animal model that develops basement membrane lesions comparable to those of human diabetic subjects. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for nine months developed significant glomerular basement membrane thickening by comparison with rats on a control test diet (p = 0.008). However, addition of an aldose reductase inhibitor, sorbinil (250 mg/kg diet), to the galactose diet did not prevent the increase in glomerular basement membrane thickness. Furthermore, by using a quantitative electron microscopic immunogold technique, we examined biochemical alterations in the composition of glomerular basement membranes in this animal model. The labelling density (comparable to relative concentration) of collagen type IV in thickened glomerular basement membranes of galactosaemic animals was significantly increased by comparison to those of control rats (p = 0.015). However, there was no significant difference in labelling densities of laminin and heparan sulfate proteoglycan core protein of these animals. Thus, our results indicate that an increase in glomerular basement membrane thickness accompanied by an increase in the labelling density of collagen type IV occurs in the galactosaemic rats, but this thickening is not prevented by sorbinil at the dose used in this experiment. Our study raises the strong possibility that glomerular basement membrane thickening in galactosaemic rats may not be due to excessive polyol pathway activity.

Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials.

To investigate the effects of extracellular matrix components on cellular function, we cultured several types of ocular cells on substrates composed of extracellular matrix materials that were layered on culture dishes either as dried films or as gels. We measured cellular proliferation on these substrates and on a series of gels composed of varying proportions of rat tail tendon type I collagen and Matrigel, a commercially available extract of a basement membrane-producing murine tumor. In addition, we studied the biosynthesis of collagens and of proteoglycans by these cultured cells using [3H]-L-proline and [35S]-sulfate. The proliferative abilities of the various types of ocular cells on the dried film substrates, on uncoated plastic culture vessels, and on pure type I collagen gel, were similar. However, proliferation of ocular cells cultured on gels composed of greater than or equal to 90% Matrigel was markedly reduced. There was little or no inhibition of growth of two types of non-ocular cells: rat C6 astrocytoma cells, and human dermal fibroblasts. Histologic studies showed that the ocular cells tested often formed long strands and capillary-like tubes, and tended to "burrow" beneath the surface of substrates containing high percentages of Matrigel. Fibroblasts infrequently formed tubes, and exhibited the burrowing property also on gels containing primarily type I collagen, while C6 cells showed neither of these behaviors on any of the matrices tested. The elution pattern of newly synthesized [3H]-labeled and [35S]-labeled macromolecules produced by all of the cultured cell types, and detected by Sepharose CL-4B chromatography in the medium and in the cell layer plus matrix fractions did not vary following culture on the different substrates. Approximately twofold more of the newly synthesized collagens and proteoglycans were deposited in the cell layer plus matrix, and proportionately less appeared in the medium, when cells were cultured on type I collagen gels and on Matrigel than on the dried film substrates. These experiments demonstrate the influence of the extracellular matrix on several aspects of cell behavior, and provide further evidence that modification of the composition of the extracellular matrix may be an important determinant of normal or pathological cell function.

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening--prevention by an aldose reductase inhibitor.

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.

Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch's membrane.

We have used the electron microscopic immunogold technique to localize precisely various extracellular matrix components in human retinal vessels and Bruch's membrane. Collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein were localized in basement membranes of retinal capillaries. In addition to the capillary basement membrane components, collagen types I and III and fibronectin were found in the basement membranes of retinal arterioles and venules. The basement membranes of choriocapillaries and retinal pigment epithelial cells in Bruch's membrane also showed a similar distribution of the retinal capillary basement membrane components. Both the inner and outer collagenous layers of Bruch's membrane contained collagen types I and III along with fibronectin, whereas collagen type VI was mostly limited to the central elastic lamina. The precise localization and distribution of various extracellular matrix components in human retinal vessels and Bruch's membrane may be important for understanding their normal function as well as their alteration in disease.

[A mathematical simulation of Chinese population dynamics].

PIP: Using mathematical and computer simulation methods, the authors suggest that it is possible to reflect realistically demographic factors such as changes in Chinese birth rates or age ratios and to provide estimates such as the number of children who will enter school in a given year.^ieng