ABSTRACT
Objective To prepare the survival motor neuron(SMN)polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy(SMA).Methods A prokaryotic expressional plasmid named pET-28? (+)/SMN was constructed and SMN-His fusion protein was induced.The fusion protein was used to immunize New Zealadd rabbits to prepare SMN polyclonal antibody.A eukaryotic expressional plasmid named pcDNA3.1/myc-HisB-SMN was constructed and used to transfect CHO cells.Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ, 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis.Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients.Results Correct pET-28a(+)/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a(+)/SMN.Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein.SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus.The results of Western-blotting were as follows:the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase(GAPDH)band density (SMN/GAPDH)is 0.619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type Ⅲ and Ⅱ were 0.347 and 0.340 respectively, which were lower than that of normal controls.However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0.079, which was quite lower than that of normal controls.Conclusions The rabbit anti-human full-length SMN polyclonal antibody is of high specificity and sensitivity, which makes the basis for the research of SMN function and SMA pathogenesis.There may be a correlation between the SMN level in skeletal muscle and the severity of disease.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the possible protection provided by oral quercetin pretreatment against hepatic ischemia-reperfusion injury in rats.</p><p><b>METHODS</b>The quercetin (0.13 mmol/kg) was orally administrated in 50 min prior to hepatic ischemia-reperfusion injury. Ascorbic acid was also similarly administered. The hepatic content of quercetin was assayed by high performance liquid chromatography (HPLC). Plasma glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) activities and malondialdehyde (MDA) concentration were measured as markers of hepatic ischemia-reperfusion injury. Meanwhile, hepatic content of glutathione (GSH), activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and xanthine oxidase (XO), total antioxidant capacity (TAOC), contents of reactive oxygen species (ROS) and MDA, DNA fragmentation were also determined.</p><p><b>RESULTS</b>Hepatic content of quercetin after intragastric administration of quercetin was increased significantly. The increases in plasma GPT, GOT activities and MDA concentration after hepatic ischemia-reperfusion injury were reduced significantly by pretreatment with quercetin. Hepatic content of GSH and activities of SOD, GSH-Px and TAOC were restored remarkably while the ROS and MDA contents were significantly diminished by quercetin pretreatment after ischemia-reperfusion injury. However, quercetin pretreatment did not reduce significantly hepatic XO activity and DNA fragmentation. Ascorbic acid pretreatment had also protective effects against hepatic ischemia-reperfusion injury by restoring hepatic content of GSH, TAOC and diminishing ROS and MDA formation and DNA fragmentation.</p><p><b>CONCLUSION</b>It is indicated that quercetin can protect the liver against ischemia-reperfusion injury after oral pretreatment and the underlying mechanism is associated with improved hepatic antioxidant capacity.</p>
Subject(s)
Animals , Male , Rats , Administration, Oral , Antioxidants , Pharmacokinetics , Therapeutic Uses , Ascorbic Acid , Pharmacokinetics , Therapeutic Uses , Biological Availability , Biomarkers , Blood , DNA Fragmentation , Glutathione Peroxidase , Metabolism , Liver , Malondialdehyde , Blood , Quercetin , Pharmacokinetics , Therapeutic Uses , Rats, Wistar , Reactive Oxygen Species , Metabolism , Reperfusion Injury , Blood , Metabolism , Superoxide Dismutase , Metabolism , Transaminases , Blood , Xanthine Oxidase , MetabolismABSTRACT
<p><b>AIM</b>To compare the TAOC of quercetin, rutin, vitamin C, vitamin E in vitro and examine the effect of quercetin on TAOC of rat plasma after intragastric administration.</p><p><b>METHODS</b>Fe3+ reducing ability assay, UV spectrum analysis and HPLC analysis were used to measure TAOC of plasma and the contents of quercetin and rutin after intragastric administration.</p><p><b>RESULTS</b>The TAOC of quercetin was stronger than that of rutin and roughly equal to vitamin C and vitamin E in vitro. After intragastric administration of quercetin (40 mg/kg bw), the TAOC and content of quercetin in rat plasma increased significantly. Vitamin C also increased plasma TAOC significantly, but rutin and vitamin E didn't after intragastric administration. However, there was no remarkable absorption peak of quercetin on HPLC chromatograms and on the other hand, the peak areas of two unknown peaks near quercetin peak were increased after intragastric administration of quercetin.</p><p><b>CONCLUSION</b>The antioxidant capacity of quercetin was stronger than rutin and comparable to vitamin C both in vitro and in vivo. After absorption, quercetin is metabolized to its derivatives.</p>