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AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pan-creatic cancer cell line Panc-1.METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent.The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry.Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c ( Cyt-c) and Bcl-2, were analyzed by Western blot.The activity of caspase-3 was measured by colorimetric method.RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids.The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group.The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group.The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group.CON-CLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and en-hancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.
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[ ABSTRACT] AIM:To investigate the effects of Chaihu Shugan powder ( CSP) on lipid metabolism and the pro-teins involved in adenosine 5’-monophosphate-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) pathway in the liver tissues of the rats with non-alcoholic fatty liver disease (NAFLD).METHODS: Sprague-Dawley rats were randomly di-vided into normal control ( NC) group, with HFD ( HFD) group and CSP group.The NAFLD models were established by feeding with HFD for 16 weeks in the rats.The rats in CSP group were intragastrically administered with CSP extracts (9.6 g· kg-1 · d-1 ) , and blood and liver samples were collected 16 weeks later.Serum and liver levels of total cholesterol ( TC) and triglyceride ( TG) , and serum levels of alanine aminotransferase ( ALT) and aspartate aminotransferase ( AST) were measured using an automatic biochemical analyzer.The histological changes of liver tissues were observed with HE staining, while the lipid deposition was observed with Oil Red O staining.The ultrastructural changes of the liver tissues were observed under transmission electron microscope.Moreover, the protein levels of AMPK, phosphorylated AMPK (pAMPK), SIRT1 and uncoupling protein 2 (UCP2) in the liver were detected by Western blot.RESULTS:The results of HE staining, Oil Red O staining and electron microscopy demonstrated that NAFLD rat model was successfully estab-lished.Compared with NC group, the serum and liver levels of TC and TG, and serum level of AST in model group were markedly elevated ( P<0.01) .Moreover, the protein levels of pAMPK and SIRT1 in HFD group were markedly reduced (P<0.01), whereas UCP2 level was elevated (P<0.01).Furthermore, liver levels of TC and TG, and serum level of AST in GSP group were markedly reduced as compared with HFD group ( P<0.05 ) .The protein levels of pAMPK and SIRT1 were elevated ( P<0.05 ) , whereas the UCP2 level was reduced as compared with HFD group ( P<0.01 ) .The protein level of AMPK between the 3 groups had no significant difference.CONCLUSION: CSP attenuates hepatic lipid disorder and hepatic lipid deposition in NAFLD rats induced by feeding with HFD for 16 weeks, which is associated with the activation of AMPK/SIRT1 pathway.
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[ ABSTRACT] AIM:To explore the role of Huoxue Jiangzhi Recipe in preventing and treating fatty liver in mice and its underlying mechanisms.METHODS:Healthy Kunming mice were fed with high-fat diet and treated intragastrically with different doses of Huoxue Jiangzhi Recipe ( compound of ginseng, panax notoginseng and rhizoma gastrodiae, named as GST) for 2 weeks.The levels of blood lipids and triglyceride ( TG) in hepatic tissues were measured.Meanwhile, liver in-dex and hepatic pathology were observed.The optimized dosage of Huoxue Jiangzhi Recipe was determined by the experi-ments.The mice were divided into normal control group ( NC group, fed with normal diet) and model group ( fed with high-fat diet) .The model mice were subdivided into 3 subgroups 12 weeks later:HF group ( fed continuously with high-fat di-et) , ND group ( fed with normal diet) , GSL group ( fed with normal diet and treated intragastrically with GSL) .The mice in NC, HF and ND groups were given distilled water by gastric perfusion.Two weeks later, all mice were killed, and blood was collected for measuring serum total cholesterol (TC),TG,high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol ( LDL-C) contents, hepatic TC, TG, malondialdehyde ( MDA ) levels and superoxide dismutase ( SOD) activity were detected.Moreover, liver index and hepatic pathology were also observed.The mRNA expression of peroxisome proliferator-activated receptor alpha (PPARα) and cytochrome-P450 2E1 (CYP2E1) in the liver was examined by RT-PCR.RESULTS:GST significantly decreased serum lipid, hepatic lipid and MDA levels and elevated SOD activi- ty.Furthermore, GST markedly reduced liver index, improved hepatic adipose infiltration, increased PPARαmRNA ex-pression and inhibited CYP2E1 mRNA expression.CONCLUSION:GST is effective in the treatment of fatty liver in mice by up-regulating PPARα, thus reducing serum and hepatic TG levels, down-regulating CYP2E1 and inhibiting lipid peroxi-dation.
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AIM:To investigate the effects of thalidomide ( THD) on the activation of connective tissue growth factor ( CTGF) gene promoter induced by transforming growth factor β1 ( TGF-β1 ) in human embryonic lung fibroblasts ( HELF) .METHODS:DNA sequence of CTGF gene promoter was cloned into luciferase reporter gene vector to construct the recombinant eukaryotic expression vector pGL 3-CTGFP, and the recombinant vector was transfected into HELF cell line.The effects of TGF-β1 and THD on the activation of CTGF gene promoter were detected by dual-luciferase analysis . RESULTS:TGF-β1 increased the reporter gene activity dose-dependently (P<0.05), with a plateau at 5 μg/L being 2.16 folds as high as the control .TGF-β1-induced increase in the reporter gene activity was also time-dependent ( P<0.05).After exposure to TGF-β1(5 μg/L), the level of luciferase activity reached its peak at 12 h and was 2.52 folds as high as the control .THD significantly inhibited TGF-β1-induced increase in the reporter gene activity in a dose-dependent manner , but its basal activity was not changed .CONCLUSION: TGF-β1 stimulates the transcriptional activity of CTGF gene promoter in HELF cells in a dose-and time-dependent manner , while THD may inhibit the effects dose-dependently .
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AIM: To investigate the role of fibrocystin/polycystin (FPC) in autosomal recessive polycystic kidney disease (ARPKD) development by means of screening the protein interaction using yeast two-hybrid approach. METHODS: The constructed pGBKT7-FPC was used as the bait to screen the pre-transformed human fetal kidney cDNA expression library by yeast two-hybrid assay to obtain the host cell protein which interacted with C-terminal region of FPC. The sequence transformation screening experiment was applied to confirm the protein interactions in yeast. RESULTS: After yeast mating and co-transformation screening analysis, Klotho (KL) was selected from the host cells and the interaction between KL and FPC was further confirmed. CONCLUSION: C-terminal region of FPC can interact with KL, which probably provide the approach for further studying the role and biochemistry mechanism of FPC protein in ARPKD.
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Objective To investigate the role of NF-KB binding element in regulation of NOD2. Methods Promoter region of NOD2 containing the NF-κB binding site was amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-N3 which had been cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD2 gene promoter. The constructed plasmids were transiently transferred into cell line HeLa by LipofectAMINETM2000 and the GFP expression was ob- served by the inversion fluorescence microscope. The NF-κB binding site in the constructed vector pEGFP- N3-NOD2wt was deleted by the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEG- FP-N3-NOD2 was transiently transferred into cell line HeLa by LipofectAMINETM2000, and the GFP expres- sion was observed by the inversion fluorescence microscope. Results The constructed pEGFP-N3-NOD2wt plasmids and mpEGFP-N3-NOD2 were the same as the design confirmed by restriction digestion and se- quence analysis. The results of the cell transient transfection indicated that different strength of GEP ex- pressed by recombinant plasmids in HeLa cells could be observed. The GFP expression of constructed mpEGFP-N3-NOD2 was lower than that of pEGFP-N3-NOD2wt. Conclusion The GFP expression vector driven by human NOD2 gene promoter which contains the NF-κB binding site, and the site deleted plasmid were successfully constructed. The GFP expression of recombinant plasmid mpEGFP-N3-NOD2, deletion of the NF-KB binding site, was obviously weaken in HeLa. The results indicate that NF-KB binding element may play a positive role in regulation of NOD2 gene, which establishes favourable bases for further study on the mechanism of NOD2 gene expression and regulation.
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Objective To explore the clinical value of laparoscopic supracervical hysterectomy (LSH). Methods From July 2006 to September 2007,78 cases of LSH and 59 cases of abdominal supracervical hysterectomy (ASH) were performed in our hospital. The clinical data of the patients,including intraoperative blood loss,operation time,recovery time of bowel movement,postoperative time of out-of-bed activity and postoperative hospital stay,were analyzed and compared between the two groups.Results No significant difference was found on the mean intraoperative blood loss between the LSH group and ASH group [(65.1?25.5) ml vs (72.9?23.6) ml,t=-1.830,P=0.069]. Whereas,the LSH group had significantly longer operation time and earlier recovery of the gastrointestinal function [(80.3?29.6) min vs (62.4?13.1) min,t=4.332,P=0.000;and (26.5?8.5) h vs (30.9?7.0) h,t=-3.232,P=0.001]. Furthermore,the LSH patients had out-of-bed activity and were discharged from hospital significantly earlier than the ASH group [(32.8?6.7) h vs (40.4?9.7)h,t=-5.421,P=0.000;and (7.1?0.6) d vs (7.9?0.5) d,t=-8.291,P=0.000]. No major complication occurred in both the groups. Conclusions LSH shows great advantages over ASH. As long as surgeons are skilled in laparoscopic operation,LSH can be an ideal procedure for hysterectomy.
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α-MSH is an endogenous neuropetide that is effective of all categories of expreimental inflammation. The mechanisms underlying the anti-inflammatory influence of α-MSH are reviewed in the article. α-MSH suppresses inflammation in the CNS and periphery by downregulating the activation of NF-κB, then inhibiting production of proinflammatory cytokines, nitric oxide, chemokines and adhension molecules, and increasing synthesis of anti-inflammatory cytokines. α-MSH is useful in the treatment of many pathological situations in humans.
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AIM: To study the relationship between prostaglandins and acute pulpitis. METHODS: Rat traumatic pulpitis model was established by pulp exposure. The kinetic pathological changes in dental pulpal tissues and changes of PGE 2?6-Keto-PGF 1? and TXB 2 concentration in dental pulp were observed. RESULTS: After pulpal trauma, the dental pulp showed inflammatory changes and the concentrations of PGE 2?6-Keto-PGF 1? and TXB 2 were increased, which peaked at 6 hour post-trauma. CONCLUSION: Prostaglandins play a significant role in the pathogenesis of pulpitis.
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Over the past ten years studies in animals and human have supported the links between inflammation and tumors. The inflammatory cells and agents found in tumors play an important role in the pathogenesis of malignant disease facilitating tumor growth, invasion and metastasis. This article reviewed the molecular mechanism of inflammatory action in promoting tumors and suggested that inflammatory cytokine network is more likely to contribute to immunosuppression than to mount an effective host anti-tumor response. [
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AIM:To explore the role of reactive oxygen species(ROS)and energy metabolism dysfunction in hepatocyte adipose degeneration induced by alcohol and calf serum(CS).METHODS:The growing L02 cells were treated with different concentrations of alcohol.To screen the proper concentration of alcohol,the proliferation of cells was measured by MTT.Lipid droplets in the cells were observed through oil red staining.Triglyceride(TG)content was detected with analyzed kit.The level of ROS and changes of mitochondrial membrane potential(??m)in cells were tested by flow cytometry.Reversed-phase high performance liquid chromatography(RP-HPLC)was applied to assay the cellular ATP content.RESULTS:Lipid droplets were observed under light microscope in the cells treated with 2% alcohol and 50% CS(A+CS)for 36 h.Compared to control group,the cellular TG and ROS levels in model group markedly increased while ??m and ATP content in cells significantly decreased(P
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Nucleotide-binding oligomerization domain (NOD) proteins are members of a growing family of cytosolic factors related to the apoptosis regulator Apaf-1 and a class of plant disease resistance proteins. NOD proteins have been implicated in the induction of NF-?B activity and in the activation of caspases. Biochemical evidence has unraveled the role of NOD1 and NOD2 as intracellular sensors of bacterial peptidoglycan. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CⅡTA in humans is associated with inflammatory disease or increased susceptibility to bacterial infections. NOD proteins may be involved in the recognition of microorganisms and regulation of inflammatory responses. [
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This experiment was carried out in 65 New Zealand rabbits. The resultsrevealed that the febrile response and increaed PGE_2 level in cerebrospipel fluid (CSF) were significantly reduced when the calcium-channel-blocking agent (verapamil) wasintravenously injected prior to ET. But, verapamil had no marked effect on the increasedcAMP level in csf during ET-induced fever. It was suggested that most likely this anti-pyretic action was due to the effect on biosynthesis of PGE in hypothalamus, while,cAMP might not be involved in the mechanism of this antipyretic action.
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0.05) after non-fever limit (non-FL) dose and FL dose endotoxin (ET) was intravenously injected into rabbits. The increase of PGE_2 in CSF was not limited during the occurrence of ET FL. 2. Intracerebroventricular injection (ICV) of different dose of PGE_2 into rabbits induced dose-dependent fever, but there was no more rise in body temperature when the febrile response had reached a certain height. This is termed "PGE FL". 3. The concentration of cyclic adenosine-3′, 5′-monophosphate (cAMP)in CSF paralleled the fluctuation of temperature (r=0.9906, P
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AIM: To investigate association between children interleukin 1?(-511) gene polymorphisms and susceptibility to febrile seizures. METHODS: Children were devided into two groups: those with febrile seizures (FS) and healthy control, FS group was also devided into family history group and non-family history group. Single nucleotide polymorphisms of IL-1?-511 gene in promoters were detected by PCR-RFLP among these groups. RESULTS: Genotypes for IL-1? promoter -511 (T-T, T-C, C-C) in total febrile seizures and healthy controls were not significantly differen. Genotype IL-1?-511(T-T) in family history patients is 46.2% ,which is much higher than those in non-family history group (10.3%) and healthy control group (5.7%). The genotype proportions for IL-1? promoter-511 in family history group were markedly different from those in non-family history patient group and control group (P
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AIM: To investigate the protective effect of Siduqing, a Chinese medicine, on LPS-induced myocardium injury in mice and its mechanisms. METHODS: Mice were divided into 4 group: control, LPS, Siduqing treatment and Siduqing group, and administered intragastrically with Siduqing decoction or distilled water (0 2 mL/10 g) twice a day for 3 days, two hours after Chinese herbal medicine treatment on day 3, LPS (30 mg/kg) or normal saline was injected intraperitoneally. The serum creatine kinase (CK) and myocardial superoxide dismutase (SOD) activities were determined, and myocardial tumor necrosis factor ? (TNF?) and malondialdehyde (MDA) contents were also detected. In addition, the histological changes and ultrastructure of heart were examined. RESULTS: Histological examination showed edema in myocardium and architectural disarray at 12, 24 h after LPS injection, mitochondrial swelling, condensation and margination of chromatin, irregular nuclear envelope and loss of contractile filaments at 24 h post LPS administration, while Siduqing treatment attenuated the above pathological changes of myocardium. CK activity in serum and myocardial TNF? content were higher in LPS group than control and Siduqing treatment group. Myocardial SOD activity in siduqing treatment group was higher than that in LPS group, but there was no difference in myocardial MDA content between control, LPS and Siduqing treatment group. CONCLUSION: These data suggest that Siduqing protects myocardium against LPS- induced injury via inhibiting myocardial TNF? production.
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AIM: To explore the anti-LPS mechanisms of ?-melanocyte-stimulating hormone (?-MSH), the effects of ?-MSH on the expression of SOCS-3 mRNA and the production of NO in murine peritoneal macrophages induced by LPS were investigated. METHODS: BALB/c mouse peritoneal macrophages were cultured in vitro and induced by LPS, ?-MSH and LPS with ?-MSH, respectively. The expression of SOCS-3 mRNA was detected using reverse transcription polymerase chain reaction (RT-PCR). NO produced in macrophages was tested with Griess reagent. RESULTS: The level of NO and the expression of SOCS-3 mRNA were significantly increased in macrophages stimulated with LPS.?-MSH markedly decreased the expression of SOCS-3 mRNA and almost completely inhibited the production of NO induced by LPS. CONCLUSION: These results suggest that the negative regulative circuits operated by SOCS are activated during the inflammation induced by LPS, but SOCS might not be involved in the anti-LPS mechanism of ?-MSH.
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AIM:To observe protective effect of L-glutamine on myocardial cell injury induced by endotoxin. METHODS:22 male SD rats,weighting(250?30)g,were randomly divided into three groups:control group( n= 7),endotoxin group( n= 7),glutamine/endotoxin group( n= 8). Heart were isolated from rats and perfused on Langendorff apparatus with Krebs- Henseleit(K-H)buffer(Saturation 95%O 2+5%CO 2)at a constant pressure(8.33 kPa)and temperature(37℃). The activity of superoxide dismutase (SOD)and malondialdehyde (MDA)content of efflux from coronary artery, monophasic action potential(MAP)and contractile force were measured at certain timepoints (0 min,20 min,50 min,80 min). RESULTS: Monophasic action potential and contractile force were markedly improved in Gln/ET group. The activity of SOD and MDA level in Gln/ET group restored closely to that in control group. CONCLUSION:L-gluta mine protects myocardial cells from injure induced by endotoxin.
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AIM and METHODS: To examine the relationship of glutathione S-transferase M1( GSTM 1) and T1( GSTT 1) with the occurrence of lung cancer, The case-control study was conducted among 161 lung cancer and 165 healthy controls. The genetic polymorphisms of GSTM1 and GSTT1 were detected with the method of multiplex polymerase chain reaction. Logistic regression analyses were used to assess the interaction of between different genotypes as well as between null genotypes and smoking. RESULTS: The frequences of GSTM 1 and GSTT 1 null genotypes had no obvious difference between lung cancer and healthy controls. In non-smoking subjects, the frequence of GSTM 1 null genotype was significantly different between lung cancer and healthy controls. Furthermore, GSTM 1 null genotype was significantly overrepresented in adenocarcinoma patients aged 60 or over, compared with controls.The results from interaction analyses showed although smoking and GSTM 1 deletion were associated with an increased risk of lung cancer, GSTM1 and GSTT 1 null genotypes combined with smoking did not have interaction effect on the risk of lung cancer. The risk for adnocarcinoma in the individuals at the age of 60 or over and in nonsmokers without GSTM1 gene but with GSTT1 functional genotype decreased by 48.5% and 45.3%, respectively. CONCLUSION: Our results suggest that GSTM1 deletion is an important host risk for lung cancer, and imply that GSTT1 functional genotype protects the old (aged 60 or over) and nonsmokers who are lack of GSTM1 gene from the risk of adenocarcinoma.
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?-MSH is an endogenous neuropetide that is effective of all categories of expreimental inflammation. The mechanisms underlying the anti-inflammatory influence of ?-MSH are reviewed in the article. ?-MSH suppresses inflammation in the CNS and periphery by downregulating the activation of NF-?B, then inhibiting production of proinflammatory cytokines, nitric oxide, chemokines and adhension molecules, and increasing synthesis of anti-inflammatory cytokines. ?-MSH is useful in the treatment of many pathological situations in humans.