ABSTRACT
@#Objective To study the effects of Dengzhan Shengmai (DZSM) capsules on the autophagy in brains after cerebral Ischemia-reperfusion in rats. Methods 50 Sprague-Dawley rats were divided into sham group (n=10), 3-methyladenine (3-MA) group (n=10), 3-MA-control group (n=10), DZSM group (n=10) and DZSM-control group (n=10). The middle cerebral arteries were occluded for 1 hour and re-perfused in all the rats except the sham group. The 3-MA and 3-MA-control groups were injected 3-MA or normal saline (NS) into the right lateral ventricle 1 hour before operation. The DZSM and DZSM-control groups accepted DZSM or NS by gavage daily for 3 days since 4 hours after reperfusion. The rats were assessed with Longa's score 3 days after operation, and the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in the brain were detected with immunofluorescent labeling and Western blotting; and the level of reactive oxygen species (ROS) were detected with chemiluminescence in the DZSM and DZSM-control groups. Results The expression of LC3 and Beclin1 increased in the ipsilateral ischemic hemisphere, especially in the cortex and striatum surrounding the infarct core (P< 0.001, compared with the sham group). The expression of LC3 and Beclin1 decreased in the 3-MA group compared with the 3-MA-control group (P<0.001), with the decrease of Longa's score (P<0.001). The expression of LC3-II and Beclin1 decreased in the DZSM group compared with the DZSM-control group (P<0.001), with the decrease of Longa's score (P<0.001) and level of ROS (P<0.001). Conclusion Inhibition of autophagy after focal cerebral ischemia-reperfusion plays a role in neuroprotection, which may be a way of DZSM to work.
ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.