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1.
Shanghai Kou Qiang Yi Xue ; 29(3): 250-256, 2020 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-33043340

RESUMO

PURPOSE: To compare the mechanical properties of 3D-printed titanium meshes and pre-shaped titanium meshes, and to evaluate the effects of 3D-printed titanium meshes on cell proliferation and differentiation. METHODS: 3D- printed titanium meshes were produced and prepared with laser printing machine. The mechanical properties were analyzed by static tension and compression load test. Bone marrow mesenchymal stem cells (BMSCs) were extracted from 4-week-old male SD rats. BMSCs were co-cultured with 3D-printed titanium meshes of different apertures. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Alkaline phosphatase (ALP) activity assay was used to test ALP activity. The expression of related osteogenic genes was tested by real-time PCR. The adhesion and growth of BMSCs were investigated by scanning electron microscopy (SEM) and living / dead cell staining. SPSS 22.0 software package was used for statistical analysis of the results. RESULTS: The results of 3D-printing Ti-meshes tension and compression loading experiment were excellent. The 3D-printing Ti-meshes showed no inhibitory effects on cell proliferation, survival and adhesion, but had a positive effect on osteogenesis of BMSCs. CONCLUSIONS: The mechanical properties of 3D-printed Ti-meshes are excellent. The 3D-printed Ti-meshes have good biocompatibility.


Assuntos
Implantes Dentários , Titânio , Animais , Masculino , Impressão Tridimensional , Ratos , Ratos Sprague-Dawley , Telas Cirúrgicas
2.
PLoS Pathog ; 16(9): e1008891, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956401

RESUMO

The transitions between developmental stages are critical points in the Plasmodium life cycle. The development of Plasmodium in the livers of their mammalian hosts bridges malaria transmission and the onset of clinical symptoms elicited by red blood cell infection. The egress of Plasmodium parasites from the liver must be a carefully orchestrated process to ensure a successful switch to the blood stage of infection. Cysteine protease activity is known to be required for liver-stage Plasmodium egress, but the crucial cysteine protease(s) remained unidentified. Here, we characterize a member of the papain-like cysteine protease family, Plasmodium berghei serine repeat antigen 4 (PbSERA4), that is required for efficient initiation of blood-stage infection. Through the generation PbSERA4-specific antisera and the creation of transgenic parasites expressing fluorescently tagged protein, we show that PbSERA4 is expressed and proteolytically processed in the liver and blood stages of infection. Targeted disruption of PbSERA4 results in viable and virulent blood-stage parasites. However, upon transmission from mosquitoes to mice, Pbsera4(-) parasites displayed a reduced capacity to initiate a new round of asexual blood-stage replication. Our results from cultured cells indicate that this defect results from an inability of the PbSERA4-deficient parasites to egress efficiently from infected cells at the culmination of liver-stage development. Protection against infection with wildtype P. berghei could be generated in animals in which Pbsera4(-) parasites failed to establish infection. Our findings confirm that liver-stage merozoite release is an active process and demonstrate that this parasite-encoded cysteine protease contributes to parasite escape from the liver.


Assuntos
Cisteína Proteases/metabolismo , Fígado/parasitologia , Malária/enzimologia , Plasmodium berghei/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Cisteína Proteases/genética , Fígado/metabolismo , Malária/genética , Camundongos , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Ratos , Ratos Sprague-Dawley
3.
Life Sci ; 259: 118382, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32898532

RESUMO

AIM: Vancomycin (VCM) is a glycopeptide antibiotic widely used to treat serious infections caused by methicillin-resistant Staphylococcus aureus and has been associated with some severe side effects such as hepatotoxicity and nephrotoxicity. However, the underlying mechanism of VCM-induced hepatotoxicity is not yet fully understood. Therefore, the current study was designed to evaluate the protective effects of zingerone (Zin) against VCM-induced hepatotoxicity in rats. MATERIALS AND METHODS: VCM was intraperitoneally administered at a dose of 200 mg/kg body weight (b.w.) for 7 days alone and in combination with the orally administered Zin (25 and 50 mg/kg b.w). KEY FINDINGS: Zin treatment significantly improved VCM-induced hepatic lipid peroxidation, glutathione depletion, reduced antioxidant enzyme (superoxide dismutase, catalase and glutathione peroxidase) activities and liver function markers (aspartate aminotransferase, alkaline phosphatase and alanine aminotransferase). Histopathological integrity and immunohistochemical expression of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the VCM-induced liver tissue were ameliorated after Zin administration. In addition, Zin reversed the changes in levels and/or activities of inflammatory and apoptotic parameters such as nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), p53, cysteine aspartate specific protease-3 (caspase-3), cysteine aspartate specific protease-8 (caspase-8), cytochrome c, Bcl-2 associated X protein (Bax) and B-cell lymphoma-2 (Bcl-2) in the VCM-induced hepatotoxicity. SIGNIFICANCE: Collectively, these results reveal probable ameliorative role of Zin against VCM-induced hepatotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Guaiacol/análogos & derivados , Inflamação/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Vancomicina/toxicidade , Animais , Western Blotting , Ciclo-Oxigenase 2/metabolismo , Guaiacol/uso terapêutico , Interleucina-1beta/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
4.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(7): 958-964, 2020 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895155

RESUMO

OBJECTIVE: To investigate the protective effect of melatonin against myocardial ischemia reperfusion (IR) injury in isolated rat hearts and explore the underlying mechanisms. METHODS: The isolated hearts from 40 male SD rats were randomly divided into 4 groups (n=10): the control group, where the hearts were perfused with KH solution for 175 min; IR group, where the hearts were subjected to global ischemia for 45 min followed by reperfusion for 120 min; IR+melatonin (Mel+IR) group, where melatonin (5 µmol/L) was administered to the hearts 1 min before ischemia and during the first 5 min of reperfusion, followed by 115 min of reperfusion; and IR+2, 3-butanedione monoxime (IR+BDM) group, where the hearts were treated with BDM (20 mmol/L) in the same manner as melatonin treatment. Myocardial injury in the isolated hearts was assessed based on myocardial injury area, caspase-3 activity, and expressions of cytochrome C and cleaved caspase-3 proteins. Cardiac contracture was assessed using HE staining and by detecting lactate dehydrogenase (LDH) activity and the content of cardiac troponin I (cTnI) in the coronary outflow, measurement of left ventricular end-diastolic pressure (LVEDP) and electron microscopy. The content of ATP in the cardiac tissue was also determined. RESULTS: Compared with those in the control group, the isolated hearts in IR group showed significantly larger myocardial injury area and higher caspase-3 activity and the protein expressions of cytochrome C and cleaved caspase-3 with significantly increased LDH activity and cTnI content in the coronary outflow and elevated LVEDP at the end of reperfusion; HE staining showed obvious fractures of the myocardial fibers and the content of ATP was significantly decreased in the cardiac tissue; electron microscopy revealed the development of contraction bands. In the isolated hearts with IR, treatment with Mel or BDM significantly reduced the myocardial injury area, caspase-3 activity, and protein expressions of cytochrome C and cleaved caspase-3, obviously inhibited LDH activity, lowered the content of cTnI and LVEDP, reduced myocardial fiber fracture, and increased ATP content in the cardiac tissue. Both Mel and BDM inhibited the formation of contraction bands in the isolated hearts with IR injury. CONCLUSIONS: Mel can alleviate myocardial IR injury in isolated rat hearts by inhibiting cardiac contracture, the mechanism of which may involve the upregulation of ATP in the cardiac myocytes to lessen the tear of membrane and reduce cell content leakage.


Assuntos
Contratura , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Masculino , Melatonina , Miocárdio , Miócitos Cardíacos , Ratos , Ratos Sprague-Dawley
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(7): 1018-1022, 2020 Jul 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895167

RESUMO

OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 µg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). CONCLUSIONS: Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.


Assuntos
Edema Encefálico , Lesões Encefálicas Traumáticas , Sistema de Sinalização das MAP Quinases , Animais , Barreira Hematoencefálica , Metaloproteinase 9 da Matriz , Ratos , Ratos Sprague-Dawley
6.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(8): 1148-1154, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895174

RESUMO

OBJECTIVE: To study the inhibitory effect of Biejiajian pills (BJJ) agaisnt diethylnitrosamine (DEN)-induced hepatocarcinogenesis and explore the relation between this effect and the inflammasome signaling pathway. METHODS: Sixty-five male SD rats were randomly divided into control group, DEN model group, and 3 BJJ treatment groups at low, medium and high dose (with daily dose of 0.55, 1.1 and 2.2 g/kg, respectively, for 12 consecutive weeks starting from the 5th week after modeling). The pathological changes of the liver tissue were observed with HE and Masson staining, and serum levels of alanine transaminase (ALT), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) of the rats were detected using ELISA. Oxidation stress in the liver tissue was assessed with ELISA, and Western blotting and ELISA were used to detect the molecular expressions of inflammasome-related pathway. RESULTS: BJJ significantly inhibited tumor growth in the liver of the rats. HE and Masson staining showed that BJJ treatment obviously ameliorated liver fibrosis and reduced cancer cell and inflammatory cell infiltration in the liver. BJJ significantly reduced elevations of serum ALT, AST, ALP and TBIL levels, increased the contents of superoxide dismutase, catalase and glutathione peroxidase in the liver and suppressed malondialdehyde in Den-treated rats. BJJ also dose-dependently decreased the expressions of NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, pro-IL-1ß, pro-IL-18, IL-1ß and IL-18 in the liver of Den-treated rats. CONCLUSIONS: BJJ treatment can dose-dependently inhibit DEN-induced hepatocarcinogenesis by enhancing antioxidant capacity and down-regulating inflammatory-related pathways in rats.


Assuntos
Neoplasias Hepáticas , Animais , Aspartato Aminotransferases , Dietilnitrosamina , Fígado , Masculino , Ratos , Ratos Sprague-Dawley
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(8): 1081-1089, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895186

RESUMO

OBJECTIVE: To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice. METHODS: The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria (Escherichia coli and Staphylococcus aureus) or specific monoclonal antibody against type Ⅱ collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse. RESULTS: The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type Ⅱ collagen triple helix structure peptide antibody exhibited strong reactivity. CONCLUSIONS: Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type Ⅱ collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.


Assuntos
Cartilagem Articular , Inflamação , Animais , Artrite Experimental , Autoanticorpos , Citrulina , Humanos , Camundongos , Ratos , Ratos Sprague-Dawley
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(8): 1207-1212, 2020 Aug 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895191

RESUMO

OBJECTIVE: To compare the effects of different materials for partial sciatic nerve ligation on glial cell activation in the spinal cord in a rat model of chronic constriction injury (CCI). METHODS: SD rats were randomly divided into the sham group (n=15), silk suture CCI group (n=15) and chromic catgut CCI group (n=14). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of the rats were detected at 3, 7, 11 and 15 days after the operation. The changes in the sciatic nerve, the activation of spinal cord glial cells and the expression of inflammatory factors were observed using Western blotting and RT-PCR. RESULTS: At 3 to 15 days after the surgery, MWT and TWL of the rats were significantly lower in silk suture group and chromic catgut group than in the control group (P < 0.05), and was significantly lower in chromic catgut group than in the silk suture group (P < 0.05) at 3 days after the surgery. The results of sciatic nerve myelin staining showed that the sciatic nerve was damaged and demyelinated in both the ligation groups. The expressions of CD11b, GFAP, IL-1ß and TNF-α in the two ligation groups were similar and all significantly higher than those in the control group (P < 0.05). IL-6 mRNA level was significantly higher in chromic catgut group than in the silk suture group (P < 0.05). CONCLUSIONS: The CCI models established by partial sciatic nerve ligation with silk suture and chromic catgut all show glial activation, and the inflammatory response is stronger in chromic catgut group.


Assuntos
Nervo Isquiático , Animais , Constrição , Neuroglia , Ratos , Ratos Sprague-Dawley , Medula Espinal
9.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(6): 850-855, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895208

RESUMO

OBJECTIVE: To investigate the effects of Shoutai pills (a traditional Chinese medicinal preparation) on immune functions and oxidative stress in pregnant rats exposed to di(2-ethylhexyl) phthalate (DEHP). METHODS: Thirty-six mature female SD rats were randomly divided into 3 groups (n=12). After pregnancy was confirmed, the rats were given 10 mL/kg corn oil +10 mL/kg saline (control group), 500 mg/kg DEHP+10 mL/kg saline (model group), and 500 mg/kg DEHP+10 mL/kg Shoutai pills (treatment group). At 19 days of gestation, the rats were sacrificed and the fetal rats were weighed and the numbers of live and stillborn fetal rats were recorded. Serum levels of interleukin-6 (IL-6), interleukin-2 (IL-2), tumor necrosis factor-ɑ (TNF-ɑ), estradiol (E2) and progesterone (P) levels were detected. The appearance, color and quality of the placenta in each group were recorded, and the placental tissues were examined pathologically. The total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH- Px), catalase (CAT), reactive oxygen species (ROS) and malondialdehyde (MDA) in the placental tissues were measured. RESULTS: Compared with the control group, the rats with DEHP exposure showed slow weight gain in the middle and late gestation period and significantly lower fetal weight (P < 0.05) with lowered serum levels of IL-2, IL-6 and TNF-ɑ, increased estradiol level (P < 0.05), decreased placental T-AOC, GSH-Px, SOD and CAT levels, and increased ROS and MDA levels (P < 0.01). Compared with the model group, the rats treated with Shoutai pills had significantly increased weight gain in mid and late pregnancy and greater fetal weight (P < 0.05) with significantly increased serum IL-2 and IL-6 levels, decreased estradiol level (P < 0.05), slightly increased TNF-ɑ expression (P> 0.05), increased placenta T-AOC, GSH- Px and CAT levels, decreased MDA level (P < 0.05), and slightly increased SOD and decreased ROS levels (P>0.05). No significant difference was found in progesterone levels among the groups (P>0.05). HE staining showed that the trophoblast in the placental tissue sponge in the model group was loose and irregular with numerous vacuoles. In the treatment group, the structure of the placenta remained intact with clearly visible labyrinth zone, sponge trophoblast and giant cell trophoblast, and the cell distribution in each layer was better than that in the model group. CONCLUSIONS: Shoutai pills can regulate the immune function of DEHP-exposed pregnant rats possibly by antagonizing the estrogenlike effect of DEHP and regulating serum immune factors; Shoutai pills can also reduce placental tissue damage and improve pregnancy outcome by correcting DEHP-induced imbalance of oxidative stress in the placental tissues.


Assuntos
Estresse Oxidativo , Animais , Dietilexilftalato , Feminino , Ácidos Ftálicos , Gravidez , Ratos , Ratos Sprague-Dawley
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(6): 899-906, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895211

RESUMO

OBJECTIVE: To investigate the mechanism by which Shexiang Tongxin dripping pills (STDP) improves coronary microcirculation disorder (CMD) and cardiac dysfunction in a porcine model of myocardial ischemia-reperfusion injury. METHODS: Fourteen minipigs were randomly selected for interventional balloon occlusion of the middle left anterior descending branch to induce CMD, and another 7 pigs received sham operation. The pig models of CMD were randomized equally into the model group and STDP-treated group. All the animals were fed with common feed for 8 weeks, and in STDP-treated group, the pigs were given STDP at the daily dose of 3 mg/kg (mixed with feed) for 8 weeks. Before and at the 8th week after the operation, the pigs underwent coronary angiography and echocardiography to determine the vessel lumen diameter and TIMI frame count (CTFC). The pathologies of the myocardium and the microvessels were examined with HE staining at the 8th week. Western blotting was used to detect the expression of silencing information regulator (Sirt1), peroxidase proliferator-activated receptor-γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor α (PPARα), extracellular signal-regulated kinase1/2 (ERKI/2), Toll-like receptor 4 (TLR4), and uncoupling protein 2 (UCP2) in myocardial tissue. RESULTS: Before and at the 8th week after the operation, the diameter of the anterior descending vessel in the 3 groups did not differ significantly (P > 0.05). At the 8th week, the number of CTFC frames in the model group increased significantly compared with that in the sham-operated group, but was obviously lowered by treatment with STDP (P < 0.05). Myocardial ischemia-reperfusion injury significantly increased the interventricular septal thickness at end-diastole, left ventricular end-diastole dimension, end-diastole volume, interventricular septal thickness at end-systole and left ventricular mass at 8 weeks after the modeling (P < 0.05), but such changes were significantly alleviated by treatment with STDP (P < 0.05). STDP treatment markedly alleviated myocardial microvascular congestion, thrombosis and peripheral inflammatory cell infiltration induced by myocardial ischemia-reperfusion, but atrophy of the myocardial muscle fiber remained distinct. STDP obviously suppressed the down-regulation of Sirt1, PGC-1α, and PPARα and the up-regulation of ERK1/ 2, TLR4, and UCP2 in the myocardial tissues induced by myocardial ischemia-reperfusion injury. CONCLUSIONS: STDP has anti-inflammatory effects and regulates energy metabolism in the myocardium through modulating Sirt1, PGC-1α, PPARα, ERKI/2, TLR4, and UCP2 to improve CMD and cardiac dysfunction after myocardial ischemia-reperfusion.


Assuntos
Medicamentos de Ervas Chinesas , Traumatismo por Reperfusão Miocárdica , Animais , Microcirculação , Miocárdio , Ratos , Ratos Sprague-Dawley , Suínos
11.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(6): 806-813, 2020 Jun 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895213

RESUMO

OBJECTIVE: To prepare the recombinant peptide MVF-HER3 I composed of the 183-227aa peptide segment of human epidermal growth factor receptor 3 (HER3 I) and the measles virus protein 288-302 peptide segment (MVF), and prepare polyclonal antibodies (PcAb) against this recombinant peptide. METHODS: The MVF-HER3 I gene was synthesized chemically and subcloned into pET21b or pET32a plasmid containing Thioredoxin (Trx) tag gene. The recombinant plasmids were identified by endonuclease digestion. MVF-HER3 I was expressed in E.coli BL21(DE3) cells under an optimal bacterial expression condition. The fusion protein Trx-MVF-HER3 I was purified using nickel ion affinity chromatography, and the purified protein was digested by enterokinase to remove Trx tag. The digested mixture underwent further nickel ion affinity chromatography to obtain purified MVF-HER3 I. The purified MVF-HER3 I was used to immunize SD rats subcutaneously for preparing anti-MVF-HER3 I PcAb. The titer of PcAb was determined using ELISA. The bindings of anti-MVF-HER3 I PcAb to MVF-HER3 I, native HER3 and MCF7 cells were analyzed using immunoblotting, immunoprecipitation and laser confocal microscopy. The growth inhibition effect of the antibodies on MCF7 cells cultured in the absence or presence of NRG was assessed using sulforhodamine B. RESULTS: The recombinant peptide gene could not be expressed alone, but could be efficiently expressed after fusion with Trx gene under optimized conditions. The fusion peptide MVF-HER3 I was successfully prepared from Trx-MVF-HER3 I. The anti-MVF-HER3 I PcAb, with a titer reaching 1: 512 000, specifically bound to MVF-HER3 I, recognized native HER3 and bound to the membrane of MCF7 cells. The obtained PcAb could dose-dependently inhibit the growth of MCF7 cells irrespective of the presence or absence of NRG. CONCLUSIONS: We successfully obtained the recombinant peptide MVF-HER3 I and prepared its PcAb, which can facilitate further functional analysis of HER3 signaling pathway.


Assuntos
Receptor ErbB-3/imunologia , Animais , Anticorpos , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Humanos , Plasmídeos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão
12.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 640-646, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897197

RESUMO

OBJECTIVE: To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-ß1 (TGF-ß1)-stimulated collagen I production in rat biliary fibroblasts. METHODS: Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-ß1 (10 ng/mL), Smad3 siRNA+TGF-ß1, CTGF siRNA+TGF-ß1, metformin (10 mmol/L)+ TGF-ß1, or Compound C (10 µmol/L)+metformin+TGF-ß1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting. RESULTS: TGF-ß1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-ß1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-ß1-induced collagen I secretion (P < 0.01). Activation of AMPK by metformin significantly reduced TGF-ß1-induced collagen I production by suppressing Smad3-driven CTGF expression (P < 0.01), and the application of Compound C reversed such changes in the fibroblasts (P < 0.01). CONCLUSIONS: Metformin inhibits TGF-ß1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.


Assuntos
Fibroblastos , Animais , Células Cultivadas , Colágeno , Feminino , Masculino , Metformina , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Smad3 , Fator de Crescimento Transformador beta1
13.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 609-615, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897201

RESUMO

OBJECTIVE: To investigate the potential neural pathway connecting the nucleus accumbens (NAc) and the rostral ventrolateral medulla (RVLM), and whether the pathway participates in the regulation of cardiovascular function in a model rat of anorexia nervosa (AN). METHODS: Rat models of AN were established by allowing voluntary activity in a running wheel with restricted feeding, with the rats having free access to normal chow without exercise as the control group. FluoroGold (FG) retrograde tracing method and multi-channel simultaneous recording technique were used to explore the possible pathway between the NAc and the RVLM. RESULTS: The rats in AN group exhibited significantly reduced systolic blood pressure (SBP), mean arterial pressure (MAP) and heart rate (HR) with significantly increased discharge frequency of RVLM neurons in comparison with the control rats. After the injection of FG into the RVLM, retrograde labeled neurons were observed in the NAc of the rats in both the normal control and AN groups. In both groups, SBP and HR were significantly decreased in response to 400 µA electrical stimulation of the NAc accompanied by an obvious increase in the discharge frequency of the RVLM neurons; the diastolic blood pressure (DBP) and MAP were significantly lower in AN model rats than in the normal rats in response to the stimulation. CONCLUSIONS: We successfully established a rat model of AN via hyperactivity and restricted feeding and confirm the presence of a neural pathway connecting the NAc and the RVLM. This pathway might participate in the regulation of cardiovascular function in AN model rats.


Assuntos
Anorexia Nervosa , Núcleo Accumbens , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Bulbo , Vias Neurais , Ratos , Ratos Sprague-Dawley
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 632-639, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897203

RESUMO

OBJECTIVE: To investigate the role of endoplasmic reticulum (ER)-stress of Kupffer cells (KCs) and KCs-derived tumor necrosis factor-α (TNF-α) in medicating apoptosis of hepatic stellate cell (HSC). METHODS: Sixty male SD rats were randomized into control group, model group, ER- stress group, depletion group and KCs block group (n=15). The 4 groups of rats were given intraperitoneal injections (twice a week for 8 weeks) of normal saline (2 mg/kg); 40% CCl4 solution (in peanut oil, 2 mg/kg); 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg); and 40% CCl4 solution (2 mg/kg) and tunicamycin (1 mg/kg) followed by clodronate liposomes (50 mg/kg), respectively. After the treatments, samples of the liver tissue and serum were collected from the rats from the 4 groups to isolate KC cells, which were co-cultured with LX2 cells. In the depletion group, the rats were injected with anti-rat TNFR mAb (0.35 mg/kg) via the portal vein before isolating the KCs. Liver function examination, Eirius red staining, ELISA, immuno- histochemical staining, and RT-PCR were performed to assess the liver function, liver fibrosis, KC phenotypes, expression of the in fl ammatory factors, and the number of active HSC was detected. The isolated KCs were treated with tunicamycin before co-culture with LX2 cells, and ELISA, RT-PCR and Western blot were performed to examine KC phenotypes, in fl ammatory factors, LX2 cell apoptosis and TNFR/caspase8 pathway activity. RESULTS: Compared with the rats in the control group, the rats in the model group had significantly increased ALT and AST levels, Sirius red staining-positive area, and Desmin-positive cells (activated HSCs) (P < 0.05) with significantly lowered number of CD16-positive KCs (M1), and TNF-α protein and mRNA expression (P < 0.05). Compared with those in the model group, the rats in ER-stress group showed significantly decreased ALT and AST levels, Sirius red staining positivity and Desmin-positive cells (P < 0.05) and increased number of CD16-positive KCs and TNF-α expressions (P < 0.05). In the depletion group, compared with the ER-stress group, the rats had significantly increased ALT and AST levels of, Sirius red staining positivity and Desmin-positive cells (P < 0.05) and reduced CD16- positive KCs and TNF-αexpressions (P < 0.05). In the cell co-culture experiment, the model group showed significantly reduced TUNEL-positive LX2 cells, CD16-positive cells, and expressions of TNFR1, cleaved caspase- 8 and cleaved caspase- 3 in the KCs (P < 0.05) with increased Desmin-positive LX2 cells (P < 0.05). Compared with the model group, the ER- stress group exhibited significantly increased TUNEL-positive LX2 cells, CD16-positive cells and expressions of TNFR, cleaved caspase-8 and cleaved caspase-3 in the KCs (P < 0.05) and decreased Desmin-positive LX2 cells (P < 0.05). In the depletion group, blocking TNFR resulted in significantly decreased expressions of cleaved caspase-8 and cleaved caspase-3 compared with those in ER- stress group (P < 0.05) although there was no significant changed in TNFR expression. CONCLUSIONS: ER stress of KCs promotes the transformation of KCs towards M1 phenotype and increases the expression of TNF-α, which triggers the apoptosis of HSCs through the TNFR/caspase-8 pathway.


Assuntos
Estresse do Retículo Endoplasmático , Células Estreladas do Fígado , Macrófagos do Fígado , Animais , Apoptose , Caspase 8 , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa
15.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 708-712, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897204

RESUMO

OBJECTIVE: To observe the effect of traditional Chinese medicine for intervention of phlegm and blood stasis in regulating TGF-ß1/Smad3 signaling and relieving nephropathy in diabetic rats. METHODS: SD rats were divided into blank group (NC), diabetic model group (MC group), intervention of phlegm and blood stasis (RPDBS) group, phlegm-removing (RP) group and blood-removing (DBS) group. Diabetic models were established in all the rats except for those in the blank group. After 4 weeks of feeding, the rats in RPDBS group, RP group and DBS group were given corresponding drug intervention for 8 weeks. HE staining was used to observe the changes in renal histopathology. Western blotting and real-time fluorescence quantitative PCR were used to detect the expression levels of transforming growth factor-ß1 (TGF-ß1) and Smad3. RESULTS: The structure and arrangement of the glomeruli and renal tubules improved significantly in the treatment groups in comparison with those in the MC group. The expression levels of TGF-ß1, Smad3 and p-Smad3 were significantly downregulated at both the protein and mRNA levels in the treatment groups (P < 0.05), and the down-regulation was more obvious in RPDBS group than in RP group and DBS group (P < 0.05). CONCLUSIONS: Intervention of phlegm and blood stasis may inhibit the activation of TGF-ß1/Smad3 signaling pathway and delay diabetic nephropathy and fibrosis to protect the renal function in diabetic rats.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Animais , Rim , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Smad3 , Fator de Crescimento Transformador beta1
16.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 647-653, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897210

RESUMO

OBJECTIVE: To investigate the role of cholinergic anti-inflammatory pathway (CAP) in neuro-regulation of inflammatory and immune response in the early stage of sepsis. METHODS: Sixty-four SD rats were randomly divided into control Group (n=8) with normal feeding without any treatment; sham operation group (n=8) with laparotomy but without cecal ligation and puncture (CLP), followed by intraperitoneal injection 50 mg/kg piperacillin 3 times a day for 3 consecutive days; and sepsis group (n=48) with CLP-induced sepsis. The rat models of sepsis were randomized into model groups (n=16) with intraperitoneal injection of piperacillin (50 mg/kg) and normal saline (1 mL/100 g) for 3 times a day for 3 days; GTS-21 group (n=16) with additional intraperitoneal injection of 4 mg/kg GTS-21 (once a day for 3 days); and methyllycaconitine (MLA) group (n=16) with intraperitoneal injection of MLA (4.8 mg/kg) in addition to piperacillin (once a day for 3 days). Murine Sepsis Score (MSS) of the rats and short-range HRV analysis were recorded. Three days later, the rats were sacrificed and serum levels of TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 were measured with ELISA. The percentages of CD4+CD25+ Treg and TH17 lymphocytes and their ratios were measured using flow cytometry. RESULTS: Compared with the control rats, the septic rats had significantly increased MSS scores and lowered HRV indexes (SDNN, RMSSD, HF, SD1, and SD2; P < 0.05); treatment with GTS-21 significantly decreased while MLA increased MSS scores (P < 0.05), but neither of them obviously affected HRV of the rats. Serum levels TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 and the percentages of CD4+CD25+ Treg and TH17-positive lymphocytes were significantly higher and Treg/TH17 ratio was significantly lower in the septic rats compared with those in the control group (P < 0.05); treatment with GTS-21 significantly decreased the levels of serum levels of TNF-α, IL-1α, IL-6, HMGB1, and sCD14 and TH17 lymphocyte percentage (P < 0.05), whereas MLA treatment significantly increased serum levels of TNF-α, IL-1α, IL-10, IL-6, HMGB1, and sCD14 and the percentages of CD4+ CD25+ Treg and TH17-positive lymphocytes and decreased Treg/TH17 ratio in the septic rats (P < 0.05). CONCLUSIONS: CAP plays negative regulatory role in early inflammatory and immune response to sepsis, and some of the HRV indicators can well reflect the regulatory effect of CAP on inflammation and immunity in the septic rats.


Assuntos
Neuroimunomodulação , Sepse , Animais , Modelos Animais de Doenças , Camundongos , Ratos , Ratos Sprague-Dawley , Linfócitos T Reguladores
17.
Zhen Ci Yan Jiu ; 45(8): 603-10, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32869568

RESUMO

OBJECTIVE: To investigate the effect of electroacupuncture (EA) on the p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription 3 (STAT3) pathway in hippocampus and frontal cortex of diabetic rats with cognitive impairment (CI), as well as the mechanism of EA in protection against CI in diabetic rats. METHODS: Thirty SD rats were divided into normal, model and EA groups (n=10 rats/group). The diabetic model was established by i.p.injection of Streptozotocin solution(25 mg/kg), followed by high-fat diet raising for 1 month, and the CI rats was confirmed by Morris water maze tasks. The rats in the EA group were given acupuncture at "Zusanli" (ST36) "Neiting" (ST44) and "Yishu" (EX-B3) 20 min/d, among which ST36 and ST44 were treated with EA. The treatment was conducted 6 times a week for 4 weeks. The fasting blood glucose (FBG) contents were assayed by glucometer before and after treatment. The rats' learning-memory ability was detected by Morris water maze tasks. The expression levels of IL-6、IL-1ß、TNF-α、p38 MAPK、p-p38 MAPK、STAT3 and p-STAT3 in hippocampus and frontal cortex were detected by Western blot and quantitative real-time PCR, separately. The mean fluorescence intensity of p38 MAPK and STAT3 was observed by immunofluorescence histochemistry. RESULTS: After modeling, FBG and the escape latency of Morris water maze tasks were significantly increased in the model group compared with the normal group (P<0.001, P<0.01). Following EA treatment, the increased FBG and average escape latency were markedly reversed in the EA group relevant to the model group (P<0.05). Compared with the normal group, the proteins and mRNAs expression of IL-6, IL-1ß, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3 in hippocampus and frontal cortex were significantly increased in the model group (P<0.001), as well as the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex (P<0.001). Following EA intervention, the proteins and mRNAs expression of IL-6, IL-1ß, TNF-α, p38 MAPK, p-p38 MAPK, STAT3 and p-STAT3, and the mean fluorescence intensity of p38 MAPK and STAT3 in hippocampus and frontal cortex were down-regulated(P<0.001, P<0.05). CONCLUSION: EA can inhibit the over production of pro-inflammatory cytokines in diabetic rats with CI, possibly by regulating the expression of p38 MAPK and STAT3 pathway.


Assuntos
Disfunção Cognitiva , Diabetes Mellitus Experimental , Eletroacupuntura , Animais , Citocinas , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3 , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno
18.
Zhen Ci Yan Jiu ; 45(8): 617-22, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32869570

RESUMO

OBJECTIVE: To compare the effect of electroacupuncture (EA) of acupoint group for "reinforcing the kidney and regulating Governor Vessel" and acopoint group for "reinforcing the kidney and lung and regulating Governor Vessel" on lear-ning-memory ability and expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) proteins in the hippocampus and prefrontal cortex (PFC) in Alzheimer's disease (AD) rats, so as to explore the efficacy of the two acupoint groups and mechanisms underlying improvement of AD. METHODS: Forty male SD rats were randomly divided into control, sham operation, model, "Baihui" + "Shenshu" (GV20+BL23, for "reinforcing the kidney and regulating Governor Vessel") EA and GV20+BL23+ "Feishu" (BL13, GV20+BL23+BL13, for "reinforcing the kidney and lung and regulating Governor Vessel") EA groups (n=8 rats in each group). The AD model was established by bilateral injection of amyloid ß peptide (Aß25-35,10 µL) into bilateral hippocampus, and rats of the sham operation group received injection of normal saline. After successful establishment of the model,EA (2 Hz, 2 mA) was applied to these acupoints for 15 min, once daily for 10 days. Then, the learning-memory ability was assessed by using Morris water maze tests, and the expression levels of TNF-α and IL-1ß proteins in the PFC and hippocampus tissues were detected by using Western blot. RESULTS: Following modeling, the average escape latency of place navigation test were significantly increased (P<0.05) and the platform crossing times of spatial probe test was significantly decreased in the model group than in the control and sham operation groups (P<0.05). The expression levels of IL-1ß and TNF-α proteins in the PFC and hippocampus were apparently up-regulated in the model group than in the control and the sham operation groups (P<0.000 1, P<0.001, P<0.01). After the intervention, the increase of the average escape latency and expression of IL-1ß and TNF-α in the PFC and hippocampus, and the decrease of space exploration test were revised in both GV20+BL23 EA and GV20+BL23+BL13 EA groups (P<0.05,P<0.01). No significant differences were found between the GV20+BL23 and GV20+BL23+BL13 EA groups in the above mentioned indexes (P>0.05). CONCLUSION: EA of both GV20+BL23 and GV20+BL23+BL13 acupoint can improve learning-memory ability of AD rats, which is associated with their effects in down-regulating the expression of IL-1ß and TNF-α in the PFC and hippocampus to reduce inflammatory reaction. There were no significant differences between the two acupoint groups in the therapeutic effects.


Assuntos
Pontos de Acupuntura , Doença de Alzheimer , Eletroacupuntura , Peptídeos beta-Amiloides , Animais , Hipocampo , Interleucina-1beta , Masculino , Córtex Pré-Frontal , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa
19.
Zhen Ci Yan Jiu ; 45(8): 623-7, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32869571

RESUMO

OBJECTIVE: To observe the effect of wrist-ankle acupuncture (WA) stimulation at "R4"- "R5" - "R6" on the expression of glutamate (Glu) and phosphorylated protein NMDAR1(p-NMDAR1) of the spinal dorsal horn in spared nerve injury (SNI) rats, so as to explore its mechanism underlying improvement of SNI. METHODS: A total of 36 SD rats were randomly divi-ded into sham operation, model and WA groups, with 12 rats in each group. The SNI procedure comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact. Rats of the WA group were treated by acupuncture at "R4"-"R5"-"R6" points from the 5th day to the 14th day after modeling. The mechanical pain thresholds were measured before and 5, 10 and 14 d after SNI, respectively. The cold allodynia was dectected by Acetone solution dropped onto the lateral plantar surface of the paw. Glu content and p-NMDAR1 expression of spinal dorsal horn were detected by 1H-MRS, ELISA and immunohistochemistry Methods. RESULTS: Compared with the sham operation group, the mechanical pain threshold of the model group was significantly decreased (P<0.01), the duration of cold stimulation foot contraction was increased (P<0.01), and the Glu content and p-NMDAR1 expression in the spinal dorsal horn were significantly increased (P<0.05, P<0.01). After WA intervention, the mechanical pain threshold was significantly increased (P<0.01), the duration of cold stimulation was significantly shortened (P<0.01), and Glu content and p-NMDAR1 protein expression of spinal dorsal horn were decreased significantly (P<0.05, P<0.01) in the WA group compared with the model group. CONCLUSION: WA can reduce pain sensitivity in rats with neuropathic pain, possibly by inhibiting the expression of Glu and p-NMDAR1 in the spinal dorsal horn.


Assuntos
Terapia por Acupuntura , Neuralgia , Animais , Ácido Glutâmico , Extremidade Inferior , N-Metilaspartato , Ratos , Ratos Sprague-Dawley , Medula Espinal , Corno Dorsal da Medula Espinal , Extremidade Superior
20.
Zhen Ci Yan Jiu ; 45(8): 633-9, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32869573

RESUMO

OBJECTIVE: To observe the effect of moxibustion on serum inflammatory factors, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor Kappa B (NF-κB) in colon tissue of rats with diarrhea-predominant irritable bowel syndrome (IBS-D),so as to explore the mechanism of moxibustion in the treatment of IBS-D. METHODS: Healthy male SD rats were randomly divided into blank group, model group and moxibustion group, with 8 rats in each group. The IBS-D model was established by chronic restraint combined with gavage of Senna-leaf solution. The rats of the moxibustion group was treated with moxibustion at "Tianshu"(ST25)and "Shangjuxu"(ST37)for 30 min, once a day for 7 d. After the intervention, the rate of loose stools and the minimum threshold volume of abdominal withdrawal reflex (AWR) induced by colorectal distension were observed. The contents of serum inflammatory factors IL-1ß, TNF-α and IL-6 were detected by ELISA. The pathological changes of rat colon were observed by H.E. staining. The average optical density of TLR4 and NF-κB (p65) in colon tissue was detected by immunohistochemistry. The relative expressions of TLR4, MyD88 and NF-κB (p65) mRNAs in colon tissues were detected by real-time fluorescence quantitative PCR, and the expression levels of TLR4, MyD88 and NF-κB (p65) proteins in colon tissues were detected by Western blot. RESULTS: There was a mild degree of inflammation in colon in rats of the model group. Compared with the blank group, the minimum volume threshold of AWR decreased significantly (P< 0.01), and the rate of loose stools, the contents of IL-1ß, IL-6 and TNF-α in serum as well as the expressions of TLR4, MyD88, NF-κB (p65) mRNAs and protein in colon tissues were significantly increased in the model group (P<0.01). Compared with the model group, the inflammation was alleviated in colonic mucosa, the minimum volume threshold of AWR increased significantly (P<0.01), and rate of loose stools decreased significantly (P<0.01) in the moxibustion group, the contents of IL-1ß, TNF-α and IL-6 in serum and the expression levels of TLR4, MyD88, NF-κB (p65) mRNAs and proteins in colon tissue decreased significantly in the moxibustion group (P<0.01). CONCLUSION: Moxibustion ST25 and ST37 can improve diarrhea symptom and visceral hypersensitivity in IBS-D rats, which may be related with its effects in inhibiting TLR4/MyD88/NF-κB signaling pathway and reducing the expression of inflammatory factors.


Assuntos
Síndrome do Intestino Irritável , Moxibustão , Animais , Diarreia , Masculino , Fator 88 de Diferenciação Mieloide , NF-kappa B , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Receptor 4 Toll-Like
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