BRL Cells Research Articles

RNA interference against stromal interacting molecule-1 (STIM1) ameliorates ethanol-induced hepatotoxicity

Previously we have demonstrated that stromal interacting molecule-1 (STIM1) was involved in ethanol induced liver injury. However, the exact pathogenic mechanism of STIM1 in alcoholic liver disease (ALD) is still unknown. We constructed plasmid vectors encoding short-hairpin RNA against STIM1 to investigate its role in ALD in the rat liver cell line BRL and in Sprague-Dawley rats. The results showed that STIM1 targeted sh-RNA (Sh-STIM1) significantly ameliorated ethanol-induced BRL cells injury and liver injury in rats with 20 weeks-induced alcoholic liver disease. Inhibition of STIM1 also reduced intracellular calcium ion concentration, reactive oxygen species (ROS) production, lipid peroxidation, NF-kappa B activation and TNF-α production under ethanol exposure. STIM1 may play an important role in the pathogenesis of alcoholic liver disease. Silencing STIM1 may be effective in preventing alcoholic liver disease.

An approach for cell viability online detection based on the characteristics of lensfree cell diffraction fingerprint

To overcome the drawbacks such as low automation and high cost, an approach for cell viability online detection is proposed, based on the extracted lensfree cell diffraction fingerprint characteristics. The cell fingerprints are acquired by a constructed large field-of-view (FOV) diffraction imaging platform without any lenses. The approach realizes distinguishing live and dead cells online and calculating cell viability index based on the number of live cells. With theoretical analysis and simulation, diffraction fingerprints of cells with different morphology are simulated and two characteristics are discovered to be able to reflect cell viability status effectively. Two parameters, fringe intensity contrast (FIC) and fringe dispersion (FD), are defined to quantify these two characteristics. They are verified to be reliable to identify live cells. In a cytotoxicity assay of different methyl mercury concentration on BRL cells, the proposed approach is used to detect cell viability. MTT method is also employed and the results of correlational analysis and Bland-Altman analysis prove the validity of the proposed approach. By comparison, it can be revealed that the proposed approach has some advantages over other present techniques. Therefore it may be widely used as a cell viability measurement method in drug screening, nutritional investigation and cell toxicology studies.

Angiotensin‑(1‑7) prevents lipopolysaccharide‑induced hepatocellular inflammatory response by inhibiting the p38MAPK/AP‑1 signaling pathway.

The pathological mechanism of lipopolysaccharide (LPS)‑induced liver injury involves a number of inflammatory mediators and cytokines. Angiotensin (Ang)‑(1‑7), a ligand for the proto‑oncogene Mas (Mas) receptor, antagonizes the actions of Ang II in the renin angiotensin system and exerts an anti‑inflammatory effect on LPS‑induced macrophages. The present study investigated the potential role of Ang‑(1‑7) in the regulation of inflammatory responses in LPS‑induced hepatocytes using the rat liver BRL cell line. The results of the present study demonstrated that the inflammatory mediator, tumor necrosis factor (TNF)‑α, its upstream transcriptional regulatory factor activator protein (AP)‑1 and p38 mitogen activated protein kinase (MAPK) which were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting were upregulated in LPS‑induced hepatic cells in a time‑dependent manner, peaking 12h following LPS stimulation. By contrast, treatment with Ang‑(1‑7) significantly attenuated the expression of TNF‑α, AP‑1 and p38MAPK in a concentration‑dependent manner. The anti‑inflammatory effect of Ang‑(1‑7) was reversed by the Mas receptor selective antagonist, A779, in BRL cells. Furthermore, the p38MAPK inhibitor, SB 203580, abolished the protective effects of Ang‑(1‑7), suggesting the involvement of the p38MAPK pathway in the anti‑inflammatory activity of Ang‑(1‑7). The results of the present study indicated that Ang‑(1‑7) may serve an anti‑inflammatory role in LPS‑induced hepatocyte injury via the regulation of the p38MAPK/AP‑1 signaling pathway.

Real-Time Visualization of Cysteine Metabolism in Living Cells with Ratiometric Fluorescence Probes.

Sulfite from cysteine metabolism in living cells plays a crucial role in improving the water solubility of metabolic xenobiotics for their easier excretion in urine or bile. However, an imbalance of sulfite in vivo would lead to oxidative stress or age-related diseases, and an effective strategy for real-time imaging of cysteine metabolism in living cells is still lacking due to its low metabolite concentration and rapid reaction kinetics. Herein, a cyanine moiety based ratiometric fluorescence probe was developed for highly selective and sensitive detection of sulfite in aqueous solution and living cells. The free probe exhibited an orange emission color, and the fluorescence color would gradually change to blue once sulfite anions selectively reacted with the unsaturated carbon double bonds in the probe molecule. This ratiometric fluorescence manner endowed the probe excellent sensitivity with a detection limit of 0.78 nM, which was then explored to image the kinetic process of sulfite release in hepatic BRL cells after incubating with an excess amount of cysteine. This strategy opens new opportunities for revealing thiol-containing species metabolism and even quantitatively tracking their distributions in live cells or organelles.

The Favored Mechanism for Coping with Acute Cold Stress: Upregulation of miR-210 in Rats

Background/Aims: The main aim of this study was to determine the mechanisms by which rno-miR-210-3p affects changes in gene expression, metabolism, apoptosis and proliferation of cells under acute cold stress (ACS) conditions. Methods: The treatment group (n=6, weight 340±20 g) was exposed to ACS (temperature 4±0.5°C, relative humidity 45±0.5%) and the control group (n=6, weight 340±20 g) to normal temperature (NT) (temperature 24±0.5°C, relative humidity 45±0.5%). Rat liver samples were collected for qRT-PCR and western blot analyses to detect relative expression of rno-miR-210-3p, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2. For cell experiments, 100 pmol/dish rno-miR-210-3p mimic and 150 pmol/dish rno-miR-210-3p inhibitor were used. Mitochondrial glucose flux and glycolysis were measured using the XFe24 Extracellular Flux Analyzer. Cells were collected for apoptosis analysis 24 h after transfection and proliferation was quantified using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China), according to the manufacturerʹs instructions. Results: In the rat experiment, expression of rno-miR-210-3p under ACS was increased sharply while ISCU, E2F3, RAD52, and PSMB6 levels declined, along with protein expression of ISCU and PSMB6. In cell experiments, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes were downregulated while ISCU and PSMB6 protein expression decreased with upregulation of rno-miR-210-3p. Conversely, in response to decreased rno-miR-210-3p expression, ISCU, E2F3, RAD52, PSMB6 and GPD2 genes were upregulated, in addition to ISCU and PSMB6 proteins. Upregulation of miR-210 inhibited cell proliferation and induced cell death whereas its downregulation promoted cell proliferation. Upregulation or downregulation of miR-210 promoted glycolysis and mitochondrial respiration of BRL cells. However, downregulation of miR-210 caused acid production in cells. Conclusion: Expression of rno-miR-210-3p is significantly increased under ACS. Upregulation of rno-miR-210-3p inhibits the expression of ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes, promotes glycolysis of liver and enhances the mitochondrial respiratory capacity of cells, but may also cause cell death. Our findings collectively indicate that regulation of rno-miR-210-3p is a preferential mechanism of choice used by the body to cope with ACS.

Akebia saponin D alleviates hepatic steatosis through BNip3 induced mitophagy

Akebia Saponin D (ASD) is the most abundant constituent of the rhizome of Dipsacus asper Wall. The prior studies have shown that ASD alleviates hepatic steatosis targeted at the modulation of autophagy and exerts hepatoprotective effects through mitochondria. However, it is still unclear which signal transduction pathway that ASD increase autophagy and protect the mitochondria. The purpose of this paper was to explore the mechanisms through which ASD alleviates hepatic steatosis. ASD significantly reduced lipid accumulation in BRL cells. Furthermore, ASD significantly increased the mitophagy acting as increase the colocalization between mitochondria and punctate EGFP-LC3. ASD treatment increased the expression of BNip3, phospho-AMPK, prevented oleic acid (OA) induced LC3-II and phospho-mTOR expression. These effects were similar to the effects cotreatment with rapamycin. ASD treatment could not attenuate the expression of BNip3 blocked by chloroquine (CQ) or siRNA-mediated knockdown of BNip3. These results suggest that Akebia saponin D alleviates hepatic steatosis targeted at BNip3 mediated mitophagy. Activation of BNip3 via ASD may offer a new strategy for treating NAFLD.

Genistein Ameliorates Fat Accumulation Through AMPK Activation in Fatty Acid-Induced BRL Cells.

Genistein is able to ameliorate fat accumulation through regulation of fatty acid metabolism mediated by AMPK activation in vitro.

Hydroxyapatite nanoparticle-induced mitochondrial energy metabolism impairment in liver cells: in vitro and in vivo studies.

Hydroxyapatite nanoparticles (HAP-NPs) have been extensively developed as drug carriers, bone implants, coating materials, etc. in the human body. However, research focusing on the potential side effects of HAP-NPs on the mitochondria-associated energy metabolism in liver cells is lacking. In this study, HAP-NPs with a long diameter of 80 nm and a short diameter of 20 nm were evaluated for their ability to induce mitochondrial energy metabolism dysfunction in vitro and in vivo. In the in vitro system, the buffalo rat hepatocyte (BRL) cell line was directly exposed to the HAP-NPs. The results of these experiments showed that the HAP-NPs induced inhibition of mitochondrial dehydrogenase activity, which was accompanied by a decrease in the mitochondrial membrane potential (MMP). In addition, HAP-NPs elevated the hepatic levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased the levels of GSH and SOD. These data indicated that HAP-NPs induced a lowered rate of electron transfer in the mitochondrial respiratory chain, accompanied by a decrease in the activity of the mitochondrial respiratory chain complexes I, II and III. Furthermore, HAP-NPs induced a decline in the enzymatic expression in the Krebs cycle. We also investigated the role of Kupffer cells (KCs, rat-derived) in the effects induced by the HAP-NPs. The supernatant from the HAP-NP-treated KCs was used to stimulate the BRL cells. We observed that the HAP-NPs had the ability to induce KC activation. The activation of KCs then led to the release of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen species (ROS), and induced the inhibition of mitochondrial respiratory chain complexes I, II and III in the BRL cells. In the in vivo study, the TEM examination revealed mitochondrial swelling and vacuolar degeneration in the HAP-NP-treated hepatocytes. In addition, the amount of succinate (Suc), an intermediate in the mitochondrial Krebs cycle, also declined in the 1 H NMR spectroscopic measurements. Copyright © 2017 John Wiley & Sons, Ltd.

Clerosterol from vinegar-baked radix bupleuri modifies drug transport.

Vinegar-baked Radix Bupleuri (VBRB) is reportedly used to treat liver cancer when combined with traditional chemotherapy and data show that this combination may modify drug transport. We isolated clerosterol from VBRB and studied its effect on drug transporters in normal or transporter-overexpressing cells. Transporter activity was assayed using cellular substrate concentration and transporter expression with Western blot and RT-qPCR. Clerosterol decreased cisplatin uptake in BRL cells mainly through increasing Mrp2 gene expression. Clerosterol also decreased the uptake of colchicine in HEK 293 cells by increasing both Pgp and Mrp1 activity; in detail, it could increase Pgp protein but had marginal effects on Mrp1 protein and gene expression. Further study showed clerosterol increased OCT2 activity in HEK293-Pgp cells by increasing OCT2 protein and mRNA. Clerosterol could suppress Pgp overexpression but not by regulating protein and gene expression. And clerosterol had marginal effects on Mrp2 and Mrp1 activity in Mrp2- and Mrp1-overexpressing HEK293 cells. Thus, Clerosterol may be an active constituent of VBRB and may work against cancer multidrug resistance by inhibiting Pgp activity.

Protective role of licochalcone B against ethanol-induced hepatotoxicity through regulation of Erk signaling.

Objective(s):Oxidative stress has been established as a key cause of alcohol-induced hepatotoxicity. Licochalcone B, an extract of licorice root, has shown antioxidative properties. This study was to investigate the effects and mechanisms of licochalcone B in ethanol-induced hepatic injury in an in vitro study.Materials and Methods:An in vitro model of Ethanol-induced cytotoxicity in BRL cells was used in this study. Cell injury was assessed using WST-1 assay and lactate dehydrogenase, alanine transaminase, and aspartate aminotransferase release assay. Cell apoptosis were quantified by flow cytometric analysis. The intracellular oxidative level was evaluated by reactive oxidative species, malondialdehyde and glutathione detection. Furthermore, the expression level of Erk, p-Erk, Nrf-2 were assessed using Western blot.Results:Treatment with ethanol induced marked cell injury and cell apoptosis in BRL cells. Licochalcone B significantly attenuated ethanol-induced cell injury, and inhibited cell apoptosis. Furthermore, licochalcone B significantly inhibited ethanol-induced intracellular oxidative level, upregulated the expression of p-Erk, and promoted nuclear localization of Nrf2. Additionally, this hepatoprotective role was significantly abolished by inhibition of Erk signaling. However, no apparent effects of Erk inhibition were observed on ethanol-induced hepatotoxicity.Conclusion:This study demonstrates that licochalcone B protects hepatocyte from alcohol-induced cell injury, and this hepatoprotective role might be attributable to apoptosis reduction, inhibition of oxidative stress, and upregulation of Erk–Nrf2. Therefore, licochalcone B might possess potential as a novel therapeutic drug candidate for alcohol-related liver disorders.

Schisandra polysaccharide increased glucose consumption by up-regulating the expression of GLUT-4

In our previous study, a polysaccharide was extracted from Schisandra Chinensis (Trucz.) Baill and found with anti-diabetic effects. The aim of this study was to investigate the anti-diabetic effects of the low weight molecular polysaccharide (SCPP11) purified from crude Schisandra polysaccharide and illustrate the underlying mechanism in buffalo rat liver cells. The insulin resistance model of BRL cells was established by incubating with insulin solution for 24h. The effects of SCPP11 on regulating related protein and mRNA expression in an insulin and AMPK signal pathway were investigated by western blot and RT-PCR analysis. SCPP11 showed no cytotoxicity to BRL cells and could improve the glucose consumption in BRL cells. SCPP11 increased the protein expression of Akt, p-AMPK and GLUT-4 in BRL cells. Moreover, SCPP11 could enhance the mRNA expression levels of IRS-1, PI3K, Akt, GLUT-4, AMPKα and PPAR-γ in BRL cells at the same time. In conclusion, SCPP11 possessed effects in improving glucose consumption by up-regulating the expression of GLUT-4 which might occur via insulin and AMPK signal pathway and could be a potential functional food to prevent and mitigate the insulin resistance condition.

Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2.

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2.

P53-dependent apoptosis contributes to di-(2-ethylhexyl) phthalate-induced hepatotoxicity

Di-(2-ethylhexyl) phthalate (DEHP) is used extensively in many personal care and consumer products, resulting in widespread non-occupational human exposure through multiple routes and media. DEHP has various deleterious effects including hepatotoxicity. p53 protein is a central sensor in cell apoptosis. In order to clarify the roles of p53 in DEHP-induced hepatotoxicity, Sprague–Dawley (SD) rats were dosed daily with DEHP by gavage for 30 days; BRL cells (rat liver cell line) were treated with DEHP for 24 h after pretreatment with NAC or small interfering RNA (siRNA). Results indicated that after exposure to DEHP, hepatic histological changes such as hepatocyte edema, vacuolation and hepatic sinusoidal dilation, and increased apoptosis index were observed. In the liver, DEHP induced oxidative stress and DNA damage, which activated p53 in vivo and in vitro. Pretreatment with NAC significantly reduced ROS level and p53 expression in BRL cells. The suppressed Mdm2 also contributed to p53 accumulation. Activated p53 mediated hepatocyte apoptosis via the intrinsic mitochondrial pathway, inhibiting anti-apoptotic Bcl-2 and Bcl-xL and inducing pro-apoptotic Bax, cytochrome c and caspases. In p53-silenced BRL cells, hepatocyte apoptosis mediated by p53 was attenuated. PCNA protein level was upregulated after p53 gene silencing. However, the Fas/FasL apoptotic pathway did not exhibit activated signs in DEHP-caused hepatotoxicity. Taken together, DEHP-caused oxidative stress and Mdm2 downregulation contribute to p53 activation. The p53-dependent apoptotic pathway plays critical and indispensable roles in DEHP-induced hepatotoxicity, while the Fas/FasL pathway does not involve in this molecular event.

Elevated cyclin A associated kinase activity promotes sensitivity of metastatic human cancer cells to DNA antimetabolite drug.

Drug resistance is a major obstacle in successful systemic therapy of metastatic cancer. We analyzed the involvement of cell cycle regulatory proteins in eliciting response to N (phosphonoacetyl)-L-aspartate (PALA), an inhibitor of de novo pyrimidine synthesis, in two metastatic variants of human cancer cell line MDA-MB-435 isolated from lung (L-2) and brain (Br-1) in nude mouse, respectively. L-2 and Br-l cells markedly differed in their sensitivity to PALA. While both cell types displayed an initial S phase delay/arrest, Br-l cells proliferated but most L-2 cells underwent apoptosis. There was distinct elevation in cyclin A, and phosphorylated Rb proteins concomitant with decreased expression of bcl-2 protein in the PALA treated L-2 cells undergoing apoptosis. Markedly elevated cyclin A associated and cdk2 kinase activities together with increased E2F1-DNA binding were detected in these L-2 cells. Induced ectopic cyclin A expression sensitized Br-l cells to PALA by activating an apoptotic pathway. Our findings demonstrate that elevated expression of cyclin A and associated kinase can activate an apoptotic pathway in cells exposed to DNA antimetabolites. Abrogation of this pathway can lead to resistance against these drugs in metastatic variants of human carcinoma cells.

Blockade of store-operated calcium entry alleviates ethanol-induced hepatotoxicity via inhibiting apoptosis

Extracellular Ca2+ influx has been suggested to play a role in ethanol-induced hepatocyte apoptosis and necrosis. Previous studies indicated that store-operated Ca2+ entry (SOCE) was involved in liver injury induced by ethanol in HepG2 cells. However, the mechanisms underlying liver injury caused by SOCE remain unclear. We aimed to investigate the effects and mechanism of SOCE inhibition on liver injury induced by ethanol in BRL cells and Sprague–Dawley rats. Our data demonstrated that ethanol (0–400mM) dose-dependently increased hepatocyte injury and 100mM ethanol significantly upregulated the mRNA and protein expression of SOC for at least 72h in BRL cells. Blockade of SOCE by pharmacological inhibitors and sh-RNA knockdown of STIM1 and Orai1 attenuated intracellular Ca2+ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and inhibited ethanol-induced apoptosis. STIM1 and Orai1 expression was greater in ethanol-treated than control rats, and the SOCE inhibitor corosolic acid ameliorated the histopathological findings and alanine transaminase and aspartate transaminase activity as well as decreased cytochrome C release and inhibited alcohol-induced cell apoptosis. These findings suggest that SOCE blockade could alleviate alcohol-induced hepatotoxicity via inhibiting apoptosis. SOCE might be a useful therapeutic target in alcoholic liver diseases.

Chemical and biological insights into uranium-induced apoptosis of rat hepatic cell line.

Uranium release into the environment is a threat to human health, and the mechanisms of cytotoxicity caused by uranium are not well-understood. To improve our understanding in this respect, we herein evaluated the effects of uranium exposure on normal rat hepatic BRL cells. As revealed by scanning electron microscopy and transmission electron microscope analysis, uranyl nitrate was found to be transformed into uranyl phosphate particles in the medium and taken up by BRL cells in an endocytotic uptake manner, which presumably initiates apoptosis of the cell, although soluble uranyl ion may also be toxic. The apoptosis of BRL cells upon uranium exposure was also confirmed by both the acridine orange and ethidium bromide double staining assay and the Annexin V/propidium iodide double staining assay. Further studies revealed that uranium induced the loss of mitochondrial membrane potential in a dose-dependent manner. Moreover, the uranium-induced apoptosis was found to be associated with the activation of caspase-3, caspase-8 and caspase-9, indicating both a mitochondria-dependent signaling pathway and a death receptor pathway by a crosstalk. This study provides new chemical and biological insights into the mechanism of uranium toxicity toward hepatic cells, which will help seek approaches for biological remediation of uranium.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

Vinegar-baked Radix Bupleuri modulates the cell membrane constituents and inhibits the P-gp activity in rat hepatocytes.

BackgroundVinegar-baked Radix Bupleuri (VBRB) enhances the effects of other drugs on the liver by increasing drug distribution to the liver, but the mechanism of action remains unclear. The present study was designed to determine the effects of VBRB on the membrane permeability, constituents, and P-glycoprotein (P-gp) activity of hepatocyte BRL cells, in order to interpret the liver targeting enhancing effects of VBRB.MethodsThe membrane permeability and P-gp expression were analyzed by flow cytometry. The membrane constituents were determined by an automatic biochemistry analyzer and thin-layer chromatography.ResultsThe results showed that, compared with the control, VBRB enhanced the membrane permeability by 41-67% (P < 0.05), which occurred in the absence of any cytotoxicity. VBRB had marginal effects on the cholesterol content, but significantly affected the total protein contents and the lipid constituents of the cell membrane in a dose- and time-dependent manner. VBRB inhibited P-gp expression in the cell membrane by 59-86% (P < 0.01).ConclusionVBRB affects the constituents of BRL cells and increases its permeability, which may help explain its liver-targeting effects.

Hydrodynamics-based transfection of rat interleukin-10 gene attenuates porcine serum-induced liver fibrosis in rats by inhibiting the activation of hepatic stellate cells

Liver fibrosis is the common pathological outcome for the majority of chronic liver diseases. Interleukin-10 (IL-10) is a cytokine that downregulates proinflammatory responses and has a modulatory effect on liver fibrogenesis. However, little is known regarding the effect of rat interleukin-10 (rIL-10) gene by hydrodynamics-based transfection (HBT) on liver fibrosis in rats. The aim of this study was to investigate the effect of the rIL-10 gene by HBT on the progression of liver fibrosis induced by porcine serum (PS) in rats and explore its possible mechanism. Plasmid-expressing rIL-10 was transferred into rats by HBT and immunohistochemistry and RT-PCR were used to detect the major organ expressing rIL-10. Liver fibrosis was induced in rats by intraperitoneal administration of PS for 8 weeks. Plasmid pcDNA3-rIL-10 solution was administered weekly by HBT starting at the 5th week. Liver function and hepatic histology were examined. The possible molecular mechanisms of rIL-10 gene therapy were assessed in liver tissue and hepatic stellate cells (HSCs) co-cultured with BRL cells (a hepatocyte line) in vitro. The results showed rIL-10 expression occurred mainly in the liver following rIL-10 gene transfer by HBT. Maintaining a stable expression of rIL-10 in serum was assessed by repeated administration. The rIL-10 gene treatment attenuated liver inflammation and fibrosis in PS-induced fibrotic rats, reduced the deposition of collagen and the expression of α-smooth muscle actin (α-SMA) in fibrotic rats. The in vitro experiment showed that the expression of a-SMA and procollagen type I in HSCs co-cultured with the BRL-transfected rIL-10 gene were significantly decreased. These findings indicate that rIL-10 gene therapy by HBT attenuates PS-induced liver fibrosis in rats and that its mechanism is associated with rIL-10 inhibiting the activation of HSCs and promoting the degeneration of collagen.

Utilization of Chum Salmon (Oncorhynchus keta) Skin Gelatin Hydrolysates to Attenuate Hydrogen Peroxide-Induced Oxidative Injury in Rat Hepatocyte BRL Cell Model

Six chum salmon skin gelatin hydrolysates with degree of hydrolysis of 4.7–13.5% were generated by Alcalase and papain and showed scavenging activities to 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals. Potential protection of these hydrolysates against H2O2-induced oxidative injury in rat hepatocyte line (BRL cells) was assessed in vitro, based on cell viability, leakage of lactate dehydrogenase, thiobarbituric acid reactive substances (TBARS), glutathione content, glutathione reductase, and catalase of the cells. The hydrolysates exhibited protection on the hepatocytes, reflected by an enhanced cell viability (about 6.2–87.9%), glutathione reductase (about 28.2–85.9%) or catalase (about 43.3–116.9%) activity, and decreased lactate dehydrogenase leakage (about 2.7–34.7%) or TBARS content (about 4.1–39.3%). On the contrary, cellular glutathione content gave an unnoticeable difference among the investigated groups. These hydrolysates also showed protection against DNA damage in the cells. Cytoprotection of these hydrolysates possessed a dosage-dependence manner. More importantly, there was a high correlation (0.7 < r < 0.9) between DPPH or superoxide anion radical scavenging activity of these hydrolysates and three cellular antioxidant indices (glutathione content, glutathione reductase, and catalase activity).