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Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
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Humanos , Proteínas Bacterianas , Toxina Diftérica/genética , Escherichia coli/genética , Chaperonas Moleculares/genética , Proteínas Recombinantes/genéticaRESUMEN
Objective To study the effects of extracted active components of Chaenomeles Speciosa (EACCS) on non-alcoholic fatty liver disease (NAFLD) in mice; To discuss the possible molecular mechanism. Methods Forty male KM mice were randomized into four groups, namely normal group, model group, low-dose (50 mg/kg) EACCS group and high-dose (100 mg/kg) EACCS group. Except that the normal group was daily given routine diet, the other groups were given high-fat–high-fructose diet (HFFD). The mice were put to death 4 weeks later. Body weight, liver weight and serum TG were measured. HE and oil red O staining were used to observe liver tissue morphology. RT-PCR and Western blot were used to detect the expression of lipid metabolism related genes. Results Compared with the normal group, the liver size, liver index (P<0.01) and epididymal fat index (P<0.05) increased significantly;The ALT and GLU in serum increased (P<0.05), TG increased (P<0.05), and pathological findings showed significant steatosis; RT-PCR and Western blot showed that the expression levels of SIRT1 and FoxO1 mRNA decreased and the level of SERBP-1c increased in the model group. Compared with the model group, the hepatic lipid accumulation of EACCS groups was obviously improved, and the serum ALT, GLU, and TG levels significantly decreased, the expression levels of hepatic SIRT1 and FoxO1 mRNA increased. Conclusion EACCS has protective effects on NAFLD mice induced by HFFD, and its mechanism may be related to the activation of SIRT1-FoxO1 signaling pathway in the liver tissues.
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Objective To study the drug dosage and time dependency characteristics of high-fructose-high-fat-feeding plus dexamethason for inducing the mouse acute fatty liver model and to optimize the condition of drug induced fatty liver model.Methods Male KM mice were divided into the normal control group and high-fructose-high-fat-feeding plus peritoneal injection of dexamethason group.The mice were killed at 3 different time points.The mouse body mass and liver mass were detected.The liver index was calculated.The serum and liver tissue homogenate TG and serum glucose(GLU) levels were detected.The liver tissue pathological change was observed by HE staining.Total RNA reverse expression related gene was extracted from the liver tissue.The total protein was extracted from the liver tissue and the related protein expression was detected by Western Blot.Results Compared with the control group,blood and liver homogenate TG after 7 d in the dexamethason model group was increased,the liver index was increased,the pathological section displayed that the fatty liver was formed.RT-PCR showed that lipid metabolism related gene expression had obvious change.Western Blot showed that SIRT1 was significantly decreased.But with the dexamethason dosage decrease and time extending,the fatty liver related indexes were decreased,lipid metabolic gene PPAR,FOXO3 and FXR were gradually increased,while LXR was gradually decreased and protein SIRT1 was gradually increased.Conclusion High-fructose-high-fatfeeding plus peritoneal injection of dexamethason could establish the mouse acute fatty liver model,moreover the model maintenance has dependency on dexamethason dosage and medication time,which has a guidance significance for the drug interventional experiment.
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Aim To study the effects of total saponins of Panax japonicus(TSPJ) on liver inflammation of natural aging rats.Methods The experimental rats were allocated into seven groups (twelve rats in each group): three months group, nine months group, fifteen months group, twenty-four months group, and TSPJ low-, mid-and high-dose groups(10, 30, 60 mg·kg-1).When the rats were eighteen months old, the TSPJ low-, mid-and high-dose groups of rats were given lavage treatments with TSPJ 10,30, 60 mg·kg-1 respectively until twenty-four months.During lavage, we stopped a day every week for six consecutive months.HE staining was used to observe the pathological morphological changes.Western blot was utilized to test IL-1β and TNF-α protein expressions.RT-PCR method was adopted to test IL-1β, IL-6, IL-12, IL-17α, TNF-α, IFN-γ mRNA expressions.Results HE staining observation showed that as the rats grew older the hepatic cord and sinusoid were arranged in more severe disorder, and the fat vacuole and inflammatory cells were increased significantly.While every dose group of TSPJ could improve these pathological changes distinctly.The IL-1β, TNF-α protein and IL-1β, IL-6, IL-12, IL-17α, TNF-α, IFN-γ mRNA expressions were increased gradually as the rats grew older, and every dose group of TSPJ could reduce their expressions to some extent.Conclusion TSPJ could protect the aging rat liver to some extent by inhibiting the liver inflammation.
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Aim To investigate SIRT1 activity and an-ti-inflammatory effects of Chikusetsu oleanane saponin ( COS) on the RAW264. 7 macrophage cells induced by lipopolysaccharide ( LPS ) . Methods The nitric oxide ( NO) release was detected with Griess method. Western blot was used to detect the expression of tumor necrosis factor-α( TNF-α) , interleukin 1β( IL-1β) . Nuclear factor-κB ( NF-κB) and silent information ad-justment factor 1 ( SIRT1 ) were tested by immunofluo-rescence. Results 1 mg · L-1 LPS co-cultured with COS at 25~300 mg·L-1 had no significant effect on the growth of RAW264. 7 cells. Compared with the LPS group, COS effectively inhibited the NO release and suppressed the expression of TNF-α and IL-1β, and also inhibited the translocation of NF-κB and up-regulation of SIRT1 . Conclusion COS has protective effects on RAW264. 7 cells stimulated by LPS, which may be related to up-regulating the expression of SIRT1 and promoting the deacetylation of NF-κB, thereby in-hibiting the translocation of NF-κB and reducing the expression of TNF-α and IL-1β.
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Objective To explore the protective effects of polysaccharide from Panax japonicus on free fatty acid in different parts of hepatic cell injury. Methods Polysaccharide of Panax japonicus was prepared through different concentrations of ethanol precipitation and was named as 30%polysaccharide component (pc), 60% pc and 90% pc. Palmitic acid (PA) was used to induce a cellular model of steatosis in HepG2 cells in order to screen the intervention viability of polysaccharide of Panax japonicus. MTT method was used to detect cell viability, and Oil Red O staining was used to demonstrate steatosis. Total RNA was extracted to detect the expression level of the relevant genes. Results MTT results showed that the 30% pc significantly increased cell viability compared with the model group;Oil Red O staining showed that the number of intracellular lipid droplets was significantly reduced in the 30% pc compared with the model group;RT-PCR results showed that expressions of the endoplasmic reticulum stress-related gene glucoese-regulated protein, CCAAT enhancer binding protein homologous protein and TNF-αwere significantly lower in the 30% pc compared with the model group, and there was no significant difference compared with normal control group. Conclusion The 30%ethanol precipitation fraction of polysaccharide from Panax japonicus significantly reduced PA-induced steatosis in HepG2 cells. Its mechanism was possibly realized through intervention in endoplasmic reticulum stress-related response.
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Objective To study the adjuvant immunoactivity of polysaccharides from Panax japonicus by alcohol of different concentrations;To discuss its part with the strongest adjuvant immunoactivity. Methods Polysaccharides from Panax japonicus was sunk with alcohol of different concentrations, and 30%alcohol compound, 60%and 90%alcohol polysaccharide were obtained. Different segments of polysaccharide and OVA protein were injected to mice once a week for three times for immunity. Five days after the last immunity, the mice were executed to collect blood, and the antibody titer was determined. The three parts of alcohol compound were scanned by infrared spectrum to determine the type of polysaccharide preliminarily. Results Compared with the control group, the antibody titer of different segments of polysaccharide obviously increased, especially the polysaccharide sunk by 60%alcohol. Infrared spectrum analysis showed that polysaccharides from Panax japonicus contained pyranose ring structure. Conclusion Polysaccharides from Panax japonicus has significant adjuvant immunoactivity, and polysaccharide sunk by 60%alcohol has the strongest adjuvant effects.
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Objective To discuss the protection mechanism of total saponins from Panax japonicus on acute hepatic injury induced by carbon tetrachloride. Methods HepG2 cells were used to establish CCl4-induced liver cell injury model in vitro experiments. Mouse model of acute liver injury was caused by 1%CCl4 oil on Balb/c. Mice were randomly divided into normal group, model group and medicine group. Mice in the medicine group were given a gavage with 20 mL/kg total saponins from Panax japonicus, while mice in the other two groups were given a gavage with the same amount of stroke-physiological saline solution. MTT method was used to detect the activity of hepatic cells. The pathological changes of mouse liver were examined by HE staining. RT-PCR was used to detect changes in the expression of transforming growth TGF-β, TNF-α, and TLR4 mRNA. Results Compared with normal group, less hepatic cells survived in model group (P<0.01);compared with model group, more hepatic cells survived in medicine group (P<0.01). HE staining showed that damages in liver tissues of medicine group significantly improved than those in model group. RT-PCR results showed that the levels of TGF-β, TNF-α, and expression of TLR4 mRNA increased more significantly than those in model group;the expression of TGF-β, TNF-α, and TLR4 mRNA in medicine group decreased more significantly than those in model group, with statistical significance (P<0.01). Conclusion The total saponins from Panax japonicus has a protective effect on liver injury induced by carbon tetrachloride.
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Prostate cancer is the most prevalent malignancies in elderly men. At present, gene therapy is the most po-tential treatment for late-stage prostate cancer. However, virus vector or nonvirus vector are less safety, weaker targeting function and higher immunogenic. Ultrasound microcapsular contrast agent as a drug transporter is a new type of disease tar-geted treatment strategies. It has provided a high-targeted, an effective and a safety gene therapy for prostate cancer, and brings a new hope for late-stage prostate cancer patients. This article aims to summarize the recent progress in using the mi-crobubble technology in prostate cancer.
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Objective To research the protective effects of totalPanax Japonicus extract on learning memory, antioxidation, and anti-apoptosis of aging mice,and explore the mechanism. Methods Male mice were randomly divided into normal group, model group, Vitamin E (VE) group, Panax Japonicus extract low and high dose group. Except for the normal group, the other groups were injected withD-gal on the back of the neck subcutaneously to establish aging model. Normal group and model group were given a gavage with saline and each treatment group was given a gavage with totalPanax Japonicus extract and VE once a day for 7 weeks after the aging model established. All mice were be measured their learning and memory ability in the eighth week. After the test, the morphological changes of CA1 neurons were observed by HE stain. SOD, GSH-Px, MDA levels in brain tissue were measured by biochemical method, the expressions of Bcl-2 and Bax mRNA were evaluated by RT-PCR.Results Mice inPanax Japonicus extract low and high dose group could spend less time in searching for the platform, improve the learning and memory ability. TotalPanax Japonicus extract increased the activity of SOD and GSH-Px, while decreased the content of MDA. In addition, it could increase the expression level of Bcl-2 mRNA and reduce the expression level of Bax mRNA as well.Conclusion TotalPanax Japonicus extract has anti-aging effect.