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Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties. In this study, we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32 (WB800-KR32) using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli (ETEC) K88 in weaned piglets. Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet. The feed of the control group (CON) was infused with normal sterilized saline; meanwhile, the ETEC, ETEC+WB800, and ETEC+WB800-KR32 groups were orally administered normal sterilized saline, 5×1010 CFU (CFU: colony forming units) WB800, and 5×1010 CFU WB800-KR32, respectively, on Days 1‒14 and all infused with ETEC K88 1×1010 CFU on Days 15‒17. The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance, improved the mucosal activity of antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)) and decreased the content of malondialdehyde (MDA). More importantly, WB800-KR32 downregulated genes involved in antioxidant defense (GPx and SOD1). Interestingly, WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum. WB800-KR32 markedly changed the richness estimators (Ace and Chao) of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces. The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway, providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.
Assuntos
Animais , Suínos , Escherichia coli Enterotoxigênica , Proteína 1 Associada a ECH Semelhante a Kelch , Bacillus subtilis , Fator 2 Relacionado a NF-E2 , Antioxidantes , Estresse OxidativoRESUMO
Objective To compare the hepatotoxicity of matrine (MT) and oxymatrine (OMT) and explore the severity and characteristics of their toxicity, and to preliminarily elucidate their toxic mechanisms. Methods Liver cell line LO-2 cells were treated with acetaminophen (APAP), matrine and oxymatrine for 24 h, and the IC values, the contents of enzymes in the liver cells, the pathological changes, the contents of malondialdehyde (MDA) and glutathione (GSH) and the cell apoptosis rate were detected. In addition, adult zebrafish were treated with APAP, matrine and oxymatrine for 96 h, and the LC50 values, the pathological morphology of the liver cells, the contents of MDA and GSH and the apoptosis rate were detected. Meanwhile, the expression of oxidative stress-related gene, zgc: 136383, and the apoptosis-related genes, EIF4EBP3 and zgc: 123120, was also detected. Results Matrine and oxymatrine had toxic effects on liver cells in vitro. The IC50 value of matrine was 5. 3 mmol/L, and that of oxymatrine was ＞ 19 mmol/L. The contents of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in the liver cells treated with matrine or oxymatrine were increased, and the cells appeared swollen, with an increase in the MDA level and a decrease in the GSH level. The cell apoptosis rate was also increased (P ＜ 0. 05). Furthermore, matrine and oxymatrine had toxic effects on the zebrafish. The LC50 value of matrine was 0. 41 mmol/L, and that of oxymatrine was ＞3. 8 mmol/L. The hepatocytes of zebrafish treated with matrine and oxymatrine appeared vacuolization in a mild to moderate degree, with an increase of the MDA content and a decrease of the GSH content. The cell apoptosis rate was increased (P ＜0. 05 for all). Expression of the oxidative stress-related gene zgc: 136383 (P ＜ 0. 05) and the apoptosis-resistant gene EIF4EBP3 (P ＜ 0. 05) was down-regulated by matrine, but that of the apoptosis-promoting gene zgc: 123120 was up-regulated (P ＜ 0. 05). Conclusions Results of the experiments using liver cells in vitro are consistent with those using the in vivo zebrafish model. Matrine (MT) and oxymatrine (OMT) both have hepatotoxicity, with similar toxic characteristics, and the toxicity of matrine is greater than oxymatrine. The mechanism of their hepatotoxicity is related with oxidative stress and cell apoptosis. Matrine reduces lipid transportation and activates oxidative stress reactions through down-regulation of gene zgc: 136383. In addition, matrine induces apoptosis in the liver cells via up-regulation of the apoptosis-promoting gene zgc: 123120 and down-regulation of the apoptosis-resistant gene EIF4EBP3.
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OBJECTIVE To compare the liver toxicity of matrine and oxymatrine ,and to explore their toxic mechanism. METHODS Thirty ICR mice were randomly divided into normal control ,matrine 200 mg · kg-1 and oxymatrine 200 mg · kg-1 groups,10 mice per group. After single ig administration of corresponding drugs or water, animal mortality was calculated at the 15th day. The content of glutamic-pyruvic transami?nase(GPT),glutamic-oxalacetic transamin(GOT),alkaline phosphatase(ALP)and lactate dehydroge?nase (LDH) in serum were detected. Histopathological changes of the liver were examined by HE stain. The content of superoxide dismutase(SOD)and glutathione(GSH)in liver homogenates were detected by ELISA. Hepatocyte apoptosis was detected by Tunel stain. RESULTS The mortality rate of mice in two groups was 80% and 0,respectively. GPT,GOT and ALP contents of dead mice in matrine group were significantly higher than that in normal control group(P0.05). The content of SOD and GSH of dead mice in matrine group was lower than that in control group(P<0.05,P<0.01). The content of GSH in oxymatrine group was also decreased(P<0.05). The apoptosis rate of liver cells in dead mice in matrine group was increased(P<0.05). CONCLUSION A large dose of matrine and oxymatrine can produce liver toxicity. At an equal dosage,the liver toxicity of matrine is significantly higher than that of oxymatrine. The toxic mechanism is related to oxidative stress and apoptosis.
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Objective To prove the advantages of telemetry by comparing with the traditional methods in safety pharmacology.Methods To monitor continuously the heart rate, respiratory rate, blood pressure and ECG of Beagle dogs by traditional and telemetry methods respectively, analyze and compare the changes between anesthetized and conscious dogs before and after feeding.Results Maintenance of anesthesia changed significantly the heart rate, respiratory rate, blood pressure and QT interval in the ECG of animals.The changes of physiological indicators in 24 h is not obvious in conscious animals, and showed a certain biorhythm.Compared with the conscious animals, the anesthetized dogs’ heart rate was significantly higher, blood pressure increased significantly, QRS and QTcf interval prolonged significantly, respiratory frequency decreased, heart rate increased significantly after feeding, and QTcf interval extended very significantly.Conclusions Traditional methods in safety pharmacology affect animal physiological indicators such as heart rate, blood pressure, respiratory rate and QT interval, which affect the objectivity of drug evaluation.Using conscious animals by telemetry can reduce these errors, however, the interference from outside should be eliminated.
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Objective Beagle dogs are commonly used animal for drug safety evaluation .As the necessary parameters , blood biochemical indicators are detected in acute or chronic toxicity tests .This study aims at assessing the influence of different preservation conditions and different preservation time on blood biochemical indicators to ensure the reliability of test results of long-term toxicity assessment .Methods Six Beagle dogs (3 males and 3 females) were used in this study .After collection and preparation of serum samples , biochemical indicators were detected after preservation in refrigerator at 2-8℃for 1, 2, 5, 8, and 12 hours;after preservation in ice transportation boxes at 2-10℃for 2, 5, and 8 hours;and after preservation in refrigerator at -20℃ for 1, 3, and 5 days.The biochemical indicators included alanine aminotransferase ( ALT ) , aspartate aminotransferase ( AST ) and alkaline phosphatase ( ALP ) , total protein ( TP ) , albumin (propagated), urea, creatinine (CREA), glucose (GLU), total cholesterol (TCHO), total bilirubin (TBIL), creatine kinase ( CK ) , gamma pancreatic acyl transferase ( GGT ) , calcium ( CA ) , lactate dehydrogenase ( LDH ) , phosphorus ( P) , high-density lipoprotein cholesterol ( HDL-C) , low density lipoprotein cholesterol ( LDL-C) , triglyceride ( TG) , sodium ( Na+) , potassium ( K+) and chloride ( Cl -) .Results Compared with the results of samples preserved for 1 hour, the LDL-C result of that preserved in refrigerator at -20℃for 5 days was significantly increased (P0.05 ) , and the coefficient of variation of LDH was 41%.Conclusions According to the test results of blood biochemical indicators in the Beagle dogs detected after different preservation conditions and different preservation time in this study , detection test should be done within 1 hour, if not, detection should be done within 12 hours for the samples preserved at 2~8℃, or within 3 days for the sample preserved at -20℃.For transportation of serum samples , the serum samples should be placed in the ice box at 2~10℃, and detection test should be done within 8 hours .
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Objective To investigate the kinetics of absorbing timosaponin by AB-8 resin.Methods Static absorption experiment,dynamic absorption experiment and series dynamic absorption experiment were carried out,and then the content of timosaponin was detected by spectrophotometry.Finally,the static absorption curve and the dynamic absorption curve were figured out.Results The result of static absorption experiment showed that the parameters of Lagergren equation are Kad=0.0186,r=0.9986,and the parameters of Dumwals-Wagner are K=0.0081,r=0.9958.In dynamic absorption experiment,the leaking of timosaponin is found and the penetrating point is at 600mL(0.1g/mL).Conclusion The mechanism of AB-8 resin absorbing timosaponin is characterized as liquid membrane diffusion at the early phase and intragranular diffusion at the later phase.Leaking phenomena in dynamic absorption experiment can be solved by three tubes in series.Therefore,this method can be used for the research of Chinese herbal medicine.
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AIM: To investigate the effect of Gengnianle Granule on hypothalamic-pituitary-ovarian(HPO) axis in climacteric rats.METHODS: 12-15 months SD female rats were randomized into climacteric group and young control group was selected additionally.After being administrated for thirty days,the level of E_2、P、Te、FSH、LH in serum were examined by the radio-immunity method,the expression of GnRH in hypothalame,the expression of ER in hypothalame and pituitary appendage were examined by the immunohistochemical method. RESULTS: Compared with climacteric group,Gengnianle Granule could increase the level of E_2、P in serum(P