In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U·mg-1. Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
ObjectiveTo investigate the anti-apoptotic effect of ginsenoside Rg1 in neonatal rats with hypoxia ischemia brain damage (HIBD), and to explore the possible signaling pathway involved in anti-apoptosis. MethodsForty-eight 10-day-old Sprague Dawley (SD) rats (weighing 17-21 g, male or female) were randomly allocated into 4 groups (12 rats in each group): sham-operation group (sham group), HIBD group (HI group), HIBD+ginsenoside Rg1 group (HI+Rg1 group), and HIBD+ginsenoside Rg1+U0126 group (HI+Rg1+U0126 group). SD rats in HI group, HI+Rg1 group, and HI+Rg1+U0126 group underwent ligation of the right common carotid artery (CCA) and hypoxic ventilation (8%O2+92%N2) for 2.5 hours to prepare the HIBD model, and rats in sham group underwent only separation of the right CCA. SD rats in HI+Rg1+U0126 group received intraventricular injection of 5 μL phosphate buffer saline (PBS) containing U0126 (25 μg/kg) at 1 hour before HIBD, and rats in the other three groups received intraventricular injection of PBS at the same time. The rats in HI+Rg1 group and HI+Rg1+U0126 group received intraperitoneal injection of 0.1 mL normal saline (NS) containing Rg1 (40 mg/kg) at immediate after HIBD, while rats in HI group and sham group received intraperitoneal injection of 0.1 mL NS at immediate after HIBD. At 4 and 24 hours after HIBD, the right hemisphere and hippocampus were collected to detect the protein expression and distribution of extracellular signal-related protein kinase 1/2 (Erk1/2), phospho-Erk1/2 (p-Erk1/2), hypoxia inducible factor 1α (HIF-1α), and cleaved Caspase-3 (CC3) by Western blot and immunohistochemistry staining. TUNEL staining was used to evaluate neural apoptosis in situ. ResultsWestern blot results showed that there were expressions of Erk1/2, p-ERK1/2, HIF-1α, and CC3 proteins at 4 and 24 hours after HIBD in each group. The expressions of HIF-1α and CC3 protein at 4 and 24 hours, and expression of p-Erk1/2 protein at 4 hours were significantly increased in HI group when compared with sham group (P < 0.05). When compared with HI group, the expressions of p-Erk1/2 and HIF-1α protein in HI+Rg1 group were significantly increased (P < 0.05), while the expression of CC3 protein was significantly decreased at 4 and 24 hours (P < 0.05). When compared with HI+Rg1 group, the expressions of p-Erk1/2 and HIF-1α proteins in HI+Rg1+U0126 group were significantly decreased (P < 0.05), while expression of CC3 protein was significantly increased at 4 and 24 hours (P < 0.05). There was no significant difference in Erk1/2 protein expression between groups at different time points (P > 0.05). Immunohistochemistry staining displayed that HIF-1α and CC3 proteins mainly distributed in the nucleus and cytoplasma, while Erk1/2 and p-Erk1/2 proteins mainly distributed in the cytoplasma. The expression levels of protein by immunohistochemistry results were similar to the results by Western blot. TUNEL staining showed that the apoptotic neurons were characterized by yellow or brown particle in the nucleus. The apoptotic index (AI) of neurons at 4 and 24 hours was significantly increased in HI group when compared with sham group (P < 0.05), and the AI of neurons was significantly decreased in HI+Rg1 group when compared with HI group and HI+Rg1+U0126 group at 24 hours (P < 0.05). ConclusionRg1 could enhance HIBD induced HIF-1α expression and inhibit activation of Caspase-3 by Erk1/2 signaling pathway, and play an anti-apoptotic role in neonatal rats with HIBD.