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Aim To study the effect of Astragalus polysaccharide (APS) on the apoptosis of cardiomyocytes induced by lipopolysaccharide (LPS) in mice and to explore its mechanism.Methods Kunming mice were treated with APS for 14 days,and intraperitoneal injection of LPS (10 mg · kg-1) was performed to establish cardiomyocyte apoptosis model in vivo,and H9c2 cells were pre-administered with APS.After 30 min,LPS (1 mg · L-1) was incubated for 24 hours to establish a model of cardiomyocyte apoptosis.Cardiac ejection fraction (EF) and left ventricular shortening score (FS) were measured using echocardiography in mice.TUNEL was carried to measure myocardial cell apoptosis.Enzyme-linked immunosorbent assay (ELISA) was employed to measure serum levels of IL-1β,TNF-α.Western blot was used to detect the expression of JNK,NF-κB signaling pathway and Bcl-2 family and caspase-3 protein in vivo and in vitro.Results LPS could significantly inhibit the left ventricular systolic function in mice and promote myocardial cell apoptosis.The levels of IL-1β,TNF-α in serum,JNK,p-JNK,Bax,caspase-3 and the concentration of NF-κB in nucleus were all increased,while the level of Bcl-2 and the concentration of NF-κB,IκB-α in cytoplasm were reduced.APS could significantly inhibit left ventricular systolic weakening in LPS-induced mice and cut down the apoptosis of cardiomyocytes.The secretion of IL-1β,TNF-α decreased to varying degrees.The protein levels of p-JNK,Bax,caspase-3 in myocardium and NF-κB in nucleus were down-regulated,but those of Bcl-2 and NF-κB,IκB-α in cytoplasm were up-regulated,and JNK had no significant change in vivo and in vitro.Conclusion APS ameliorates the LPS-induced apoptosis of myocardial cells by inhibiting NF-κB and JNK signaling pathway in mice.
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<p><b>OBJECTIVE</b>To observe the effect of naringin of Drynaria Rhizome, a Chinese medical component of Zhuanggu Jianxi Recipe (ZJR) containing serum on caveolin-p38MAPK signal factors (such as caveolin-1, p-p38, p-ATF-2, IL-1β, and TNF-α) in IL-1β induced rabbit degenerated chondrocytes, and further to explore its mechanism for protecting articular cartilages.</p><p><b>METHODS</b>Naringin of Drynaria Rhizome was obtained and analyzed by HPLC-TOF/MS. Four weeks old New Zealand rabbits were killed and their bilateral knee joints were isolated aseptically. CDs were isolated and then cultured in vitro. The second generation of CDs were used for later experiment. The effect of naringin on CDs proliferation was detected by MTT assay. The effect of naringin on the expression of IL-1β-induced collagen II in CDs was detected by immunohistochemical method. The effect of naringin on caveolin-1, p-p38, and p-ATF-2 protein in IL-1β-induced CDs was detected by Western blot. The effect of naringin on mRNA expression of IL-1β and TNF-α in IL-1β-induced CDs was detected by RT-PCR.</p><p><b>RESULTS</b>The appearance time of naringin in flow graphs of naringin standard solution and ZJR containing serum was 23.5 min, and the molecular weight ranged between 581.0 and 581.5 m/z. Naringin could promote the proliferation of CDs, and inhibit the effect of IL-1β on collagen II in CDs. Compared with the model group, naringin could reduce the expression of caveolin-1, p-p38, p-ATF-2, IL-1β, and TNF-α in IL-1β induced CDs (P < 0.05), which was approximate to the level of the normal group.</p><p><b>CONCLUSIONS</b>Naringin could not only promote the proliferation of CDs, but also protect IL-1β-induced CDs. Its mechanism might be associated with decreasing the expression of caveolin-1, p-p38, and p-ATF-2 proteins, inhibiting caveolin-p38MAPK signal pathway, and further reducing mRNA expression of IL-1β and TNF-α in the downstream of caveolin-p38MAPK signal pathway.</p>
Subject(s)
Animals , Rabbits , Blotting, Western , Cartilage, Articular , Caveolins , Chondrocytes , Metabolism , Drugs, Chinese Herbal , Pharmacology , Flavanones , Pharmacology , Interleukin-1beta , Metabolism , Polypodiaceae , Rhizome , Signal Transduction , Tumor Necrosis Factor-alpha , Metabolism , p38 Mitogen-Activated Protein Kinases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 β (IL-1 β)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.</p><p><b>METHODS</b>Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 β stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 β, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction.</p><p><b>RESULTS</b>IL-1 β stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-α, MMP-3 and MMP-13. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.</p><p><b>CONCLUSION</b>ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.</p>
Subject(s)
Animals , Male , Rabbits , Base Sequence , Blotting, Western , Caveolins , Metabolism , Chondrocytes , Metabolism , DNA Primers , Drugs, Chinese Herbal , Pharmacology , Gene Expression Profiling , Interleukin-1beta , Physiology , MAP Kinase Signaling System , RNA, Messenger , Genetics , p38 Mitogen-Activated Protein Kinases , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the effects of kappa-opioid receptor stimulation on high glucose induced myocardial hypertrophy of neonatal rats.</p><p><b>METHODS</b>Using cultured myocardial cells as a model, the protein content was assayed with Lowrys method. The cardiomyocytes volumes were measured by computer photograph analysis system. The level of p-ERK44/42 was determined by Western blot.</p><p><b>RESULTS</b>Compared with the control, U50488H significantly inhibited the protein content and volumes of cultured hypertrophic myocardial cells induced by high glucose. Meanwhile the role of ERK was important.</p><p><b>CONCLUSION</b>The stimulation of kappa-opioid can inhibit myocardial hypertrophy induced by high glucose, which is possibly via attenuating p-ERK.</p>
Subject(s)
Animals , Female , Male , Rats , 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer , Pharmacology , Animals, Newborn , Cardiomegaly , Metabolism , Cells, Cultured , Glucose , MAP Kinase Signaling System , Myocytes, Cardiac , Rats, Sprague-Dawley , Receptors, Opioid, kappa , MetabolismABSTRACT
OBJECTIVE@#To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia.@*METHODS@#Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei.@*CONCLUSION@#HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.
Subject(s)
Animals , Female , Male , Rats , Asphyxia/metabolism , Disease Models, Animal , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Ischemia, Brain/metabolism , Immunohistochemistry , Kidney/pathology , Liver/pathology , Lung/pathology , Myocardium/pathology , Nitrogen/poisoning , Random Allocation , Rats, Sprague-Dawley , Time FactorsABSTRACT
<p><b>OBJECTIVE</b>To evaluate the therapeutic effect of a new recombinant immunotoxin mMIP-1alpha-DT390 on experimental autoimmune encephalomyelitis (EAE).</p><p><b>METHODS</b>EAE was induced in the low-sensitive strain C57BL/6 mice with intraperitoneal injection of myelin basic protein (MBP) to simulate the human disease multiple sclerosis, followed by intramuscular injection of cationic liposome carrying the plasmid DNA SRalpha-mMIP-1alpha-DT390 in the leg muscle to elicit resistance to EAE development. The mice were then examined daily for clinical signs of EAE by an observer blind to the treatment protocol. For immunohistochemistry the mice were anesthetized and perfused with sterile PBS and paraformaldehyde, and the cerebrum, cerebellum, medulla and spinal cord were removed for preparation of serial sections. The mononuclear cells (MNCs) from the EAE mouse spleens were prepared for three-color flow cytometry analysis of the surface markers with appropriate antibodies following the BD Pharmingen cytokine staining protocol.</p><p><b>RESULTS</b>EAE model was successfully established by active MBP immunization in C57BL/6 mice. Administration of the immunotoxin mMIP-1alpha-DT390 significantly delayed the disease onset and lowered the mean clinical score for EAE as compared with the control mice. Immunohistochemistry demonstrated much less CCR5(+) infiltrating cells in the central nervous system in mMIP-1alpha-DT390-treated mice than in the control. The treatment also eliminated reactive T cells in the periphery blood without affecting the number of B cells.</p><p><b>CONCLUSION</b>The immunotoxin mMIP-1alpha-DT390 can attenuate the disease activity of EAE in mice, suggesting its potential use in the treatment of other autoimmune disorders.</p>
Subject(s)
Animals , Female , Mice , Antigens, CD19 , B-Lymphocytes , Cell Biology , Metabolism , CD3 Complex , Chemokine CCL3 , Genetics , Metabolism , Diphtheria Toxin , Genetics , Metabolism , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental , Drug Therapy , Flow Cytometry , Immunoglobulin Fragments , Genetics , Metabolism , Immunohistochemistry , Immunologic Factors , Therapeutic Uses , Immunotoxins , Therapeutic Uses , Meninges , Chemistry , Pathology , Mice, Inbred C57BL , Multiple Sclerosis , Drug Therapy , NIH 3T3 Cells , Receptors, CCR5 , Recombinant Fusion Proteins , Genetics , Metabolism , Therapeutic Uses , T-Lymphocytes , Cell Biology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To understand the role of mitochondria associated signaling pathway in the apoptosis of human vascular endothelial cell induced by homocysteine (Hcy).</p><p><b>METHODS</b>The mRNA and protein expression levels of the up-stream signaling molecules of caspase 3, Bcl 2, caspase 9, and cytosolic cytochrome-c, were investigated. The in vitro cultured human umbilical vein endothelial cells with homocysteine at different concentrations were incubated for 24 h. The expressions of Bcl 2 and caspase 9 at mRNA and protein levels were analyzed by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Cytochrome-c in cytoplasm was also detected by Western blot.</p><p><b>RESULTS</b>The expression levels of three signaling molecules were all down-regulated by homocysteine at both mRNA and protein levels in a dose-dependent manner.</p><p><b>CONCLUSION</b>Homocysteine could affect the formation of apoptosome through repressing the expression of Bcl 2 gene and release of cytochrome-c from mitochondria. Decreasing of apoptosome could disturb the activation of caspase 9. The results also indicate that the mitochondria pathway is not the major signaling pathway involved in Hcy-induced apoptosis.</p>
Subject(s)
Humans , Apoptosis , Blotting, Western , Caspase 3 , Genetics , Metabolism , Caspase 9 , Genetics , Metabolism , Cells, Cultured , Cytochromes c , Metabolism , Dose-Response Relationship, Drug , Endothelial Cells , Cell Biology , Metabolism , Flow Cytometry , Homocystine , Pharmacology , Proto-Oncogene Proteins c-bcl-2 , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To study the genetic polymorphisms of the mitochondrial DNA (mtDNA) control region in Chengdu Han population.</p><p><b>METHODS</b>Sequence polymorphisms of the mtDNA control region, hypervariable regions I and II from 100 unrelated Chinese Hans were determined by PCR and direct sequencing.</p><p><b>RESULTS</b>Sequences of 404 nucleotides for hypervariable region I and 379 nucleotides for region II were obtained. Ninety-two and fifty variable sites were revealed in region I and region II respectively as compared to the reference sequence, and a total of 97 different genetic patterns from both the regions I and II were determined. The probability of identity was estimated at 1.84% for region I, 1.94% for region II, and 1.18% for both the regions.</p><p><b>CONCLUSION</b>These results suggest that sequence polymorphism of mtDNA control region would be very useful in forensic practice as a marker for individual identification.</p>