Ferroptosis is a non-apoptotic regulated cell death caused by iron accumulation and subsequent lipid peroxidation. Currently, the therapeutic role of ferroptosis on cancer is gaining increasing interest. Baicalin an active component in

Objective:To evaluate the effect of flurbiprofen postconditioning on the permeability of blood brain barrier in a rat model of focal cerebral ischemia-reperfusion (I/R) injury.Methods:Eighty healthy male Wistar rats, aged 8-9 weeks, weighing 280-320 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group Sham), focal cerebral I/R group (group I/R), lipo-microballoons group (group V) and flurbiprofen 10 mg/kg group (group F). Focal cerebral I/R model was established by left middle cerebral artery occlusion for 2 h followed by 24-h reperfusion in anesthetized rats.Flurbiprofen 10 mg/kg (group F), the equal volume of lipo-microballoons (group V) or the equal volume of normal saline (group Sham and group I/R) was injected via the tail vein at the onset of reperfusion.The rats were sacrificed at 24 h of reperfusion, brains were immediately removed, and cerebral tissues were obtained for measurement of brain water content, Evans blue content, expression of matrix metalloproteinase-9 (MMP-9) in ischemic penumbra (by immuno-histochemistry), and expression of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and inducible nitric oxide synthase (iNOS) in ischemic penumbra (by Western blot). Results:Compared with Sham group, brain water content and Evans blue content in brain tissues were significantly increased, and the expression of MMP-9, p-p38 MAPK and iNOS in ischemic penumbra was up-regulated in I/R, V and F groups ( P<0.05). Compared with group I/R, brain water content and Evans blue content in brain tissues were significantly decreased, and the expression of MMP-9, p-p38 MAPK and iNOS in ischemic penumbra was down-regulated in group F ( P<0.05), and no significant change was found in the above parameters in group V ( P>0.05). Conclusion:Flurbiprofen postconditioning can decrease the permeability of blood brain barrier during focal cerebral I/R in rats, and the mechanism may be related to inhibiting the activation of p38 MAPK/iNOS signaling pathway and down-regulating the expression of MMP-9.

OBJECTIVETo characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.

METHODSThe full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.

RESULTSThe recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.

CONCLUSIONThe pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; immunology ; Calmodulin ; immunology ; Clonorchiasis ; immunology ; Clonorchis sinensis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Immunoglobulin G ; blood ; Inflammation ; Liver Cirrhosis ; parasitology ; Male ; Mice ; Rats ; Recombinant Proteins ; immunology

Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1:51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis. Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis-associated hepatic fibrosis.

Objective To characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis. Methods The full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein. Results The recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1:51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis. Conclusion The pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis-associated hepatic fibrosis.

The aim of this study is to investigate the effect and mechanism of angiotensin (Ang) II on E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression in rat brain microvascular endothelial cells (BMEC) and evaluate the effect of compound EXP-2528, a novel Ang Ⅱ type 1 (AT1) receptor antagonist. The experiment was performed in cultured BMEC of rat. The mRNA and protein expression of E-selectin and VCAM-1 in BMEC was analyzed by RT-PCR and Western blotting, respectively. The results showed that the mRNA and protein expression of E-selectin and VCAM-1 in BMEC were significantly upregulated by 4 h or 18 h exposure to 1×10-7 mol·L-1 Ang Ⅱ. These effects were abolished by pretreatment with the selective AT1 receptor antagonists losartan and compound EXP-2528, but not with the AT2 selective antagonist PD123319. Combining losartan with PD123319 also significantly inhibited Ang Ⅱ-induced E-selectin and VCAM-1 expression in BMEC, but there was no significant difference compared with losartan group. These findings indicated that Ang Ⅱ upregulated E-selectin and VCAM-1 in BMEC by activating AT1 receptor and then involved in the development of cerebrovascular disease.

This study is to investigate the effect of hydroxyethylpuerarin on the expression of tumor necrosis factor-alpha (TNF-α) and activity of nuclear factor kappa B (NF-κB) after middle cerebral artery occlusion (MCAO) in rats. Rats were subjected to cerebral ischemia-reperfusion injury induced by MCAO. Hydroxyethylpuerarin (10, 20, 40 mg·kg-1, iv) was administered just 30 min before occlusion and immediately after reperfusion. After a 24 h reperfusion following 2 h of MCAO, the number of viable neurons in hippocampal CA1 region was counted by hematoxylin and eosin (HE) staining. TNF-α protein and its mRNA expression were examined with radioimmunoassay (RIA) and reverse transcriptase-polymerase chain reaction (RT-PCR) respectively. NF-κB activity was observed by electrophoretic mobility shift assay (EMSA), and inhibition of NF-κB α (IκBα) protein expression was evaluated by Western blotting analysis. Animals treated with hydroxyethylpuerarin had a significant increase in neuronal survival in comparison with vehicle-treated group. Hydroxyethylpuerarin significantly reduced the protein and mRNA expression of TNF-α following 2 h of ischemia with 24 h of reperfusion. NF-κB DNA binding activity and the degradation of IκBα in the cytoplasm also decreased by hydroxyethylpuerarin treatment. The protective effects of hydroxyethylpuerarin against ischemia-reperfusion injury may be mediated by decreasing the expression of TNF-α and the activity of NF-κB in rats.

In order to determine whether H1 histone proteins are associated with innate immune antimicrobial response in goldfish, we extracted the total RNA from the hemocytes of goldfish (Carassius auratus), designed 3 pairs of primers based on the previous antimicrobial peptide sequences from fish and performed RT-PCR. Among 3 obtained-PCR products, we identified a novel histone H1 coding sequence of 576 bp which belongs to the histone H1 family and is 78% homologous with the amino acid sequence of histone H1 from Salmon salar that had been found with an important role in salmon defenses against infectious pathogens. The H1 histone of goldfish contained 3 predicting cleavage sites that divided the protein into 4 parts. We successfully cloned and expressed the whole CDs (No ATG) of H1 histone and its N-terminal part (2-38aa) in Pichia pPIC9K expression system. The products of H1 histone and its N-terminal deriving peptide (AEVAPAASAPPAKAPKKKSAAKAKKAGPAVGDLIVKA) show antimicrobial activity. The results suggested that the H1 histone fragment reported in this paper is a novel antimicrobial peptide found in goldfish. H1 histone plays an important role in innate immune responses of goldfish.
Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; biosynthesis ; pharmacology ; Cloning, Molecular ; Goldfish ; genetics ; metabolism ; Histones ; biosynthesis ; genetics ; pharmacology ; Molecular Sequence Data ; Peptide Fragments ; biosynthesis ; genetics

Aim To observe the damages induced by hydrogen peroxide in cultured bovine cerebral microvascular endothelial cells (BCMEC) and evaluate the protective effects of hydroxyethylpuerarin on hydrogen peroxide-injured BCMEC. Methods BCMEC were cultured and transferred into modified Eagle medium (MEM). The viability of cells was detected by MTT assay. Cell injury was determined by lactate dehydrogenase (LDH) activity in the extracellular medium. Flow cytometry was employed to observe the occurrence of apoptosis. Morphologic changes of cells were visualized under phase contrast and electron occurrence of apoptosis. Hydroxyethylpuerarin was shown to increase the survival rate and decrease the activity of LDH of BCMEC damaged by hydrogen peroxide. Hydroxyethylpuerarin was also found to protect BCMEC against apoptosis induced by hydrogen peroxide. Conclusion Hydrogen peroxide induces BCMEC injury either by apoptosis or through necrosis. Hydroxyethylpuerarin protects BCMEC against hydrogen peroxide-induced injury in a concentration-dependent manner. Its antioxidant effects might be involved as the mechanism protection.

Aim To investigate the effect of low molecular N-trimethyl chloride chitosan(LMTC) on the growth of bovine vascular smooth muscle cells(BVSMCs) in vitro. Methods The antiproliferation activities of LMTC were evaluated in BVSMCs by means of crystal violet staining and MTT assay. The morphological changes of LMTC in BVSMCs were observed under transmission electron microscope. Cell survival ratio influenced by LMTC was assessed by flow cytometer. [WTHZ]Results After BVSMCs were treated by LMTC for 72 h,the growth of the cells was inhibited in vitro and was dependent on concentration; there were some changes of apoptosis by transmission electron microscope and flow cytometer. Conclusion LMTC can inhibit the proliferation of BVSMCs and induce their apoptosis.