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Objective:To explore the protective effect of Gegen Qinliantang on the intestinal mucosal epithelial barrier function of ulcerative colitis （UC） mice， and to explore its mechanism of action in the treatment of ulcerative colitis via matrix metallopeptidase-9 （MMP-9）/p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway. Method:The 48 female C57BL/6 mice were randomly divided into normal group， model group， sulfasalazine group （0.3 g·kg<sup>-1</sup>） and Gegen Qinliantang high， medium and low dose groups （2.84，1.42，0.71 g·kg<sup>-1</sup>）. The UC murine model was established by 3% dextran sulfate sodium （DSS）. Gegen Qinliantang and sulfasalazine were intragastrically administered on the 8<sup>th</sup> day after the model was established for 7 days， and the normal group was treated with the same amount of normal saline. Colon tissues were collected after the last administration， and the pathological changes of colon tissues were detected by hematoxylin-eosin （HE） staining. The expression of tight junction （TJ） proteins such as Occludin and zonula occludens-1（ZO-1） in colon tissues was detected by immunohistochemistry （IHC）， and the expression levels of tumor necrosis factor-alpha （TNF-<italic>α</italic>）， interleukin-1<italic>β</italic> （IL-1<italic>β</italic>）， and MMP-9 mRNA in colon tissues were detected by Real-time polymerase chain reaction （Real-time PCR）. The expression of phosphorylated p38 MAPK （p-p38 MAPK）， p38 MAPK and MMP-9 protein in colon tissues was detected by Western blot. Result:Compared with normal group， the body weight of mice decreased （<italic>P</italic><0.01） and disease activity index （DAI） score increased significantly （<italic>P</italic><0.01） in model group， the colon tissues of the model group were damaged more obviously， the expression of occludin and ZO-1 proteins in model group was significantly reduced （<italic>P</italic><0.01）， and the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA in model group were significantly increased （<italic>P</italic><0.01）， the expression of p-p38 MAPK and MMP-9 in model group was significantly increased （<italic>P</italic><0.01）. Compared with model group， the body mass and DAI score of the sulfasalazine group and Gegen Qinliantang group were significantly improved （<italic>P</italic><0.05，<italic>P</italic><0.01）， the colonic tissues damage were significantly improved， and the expression of Occludin and ZO-1 protein was significantly increased （<italic>P</italic><0.05，<italic>P</italic><0.01）， the relative expression levels of TNF-<italic>α</italic>， IL-1<italic>β</italic>， and MMP-9 mRNA were significantly decreased （<italic>P</italic><0.01）， and the expression of p-p38 MAPK and MMP-9 was significantly decreased （<italic>P</italic><0.01）. The changes in the middle dose group were the most obvious among the various dose groups of Gegen Qinliantang. Conclusion:Gegen Qinliantang repairs the intestinal mucosal barrier function by inhibiting the expressions of MMP-9 and inflammatory cytokines such as TNF-<italic>α</italic> and IL-1<italic>β</italic>， blocking the activation of the p38 MAPK signaling pathway， and increasing the expressions of tight junction protein.
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BACKGROUND: A high fracture of the mandibular condyle is often accompanied by cartilage damage. At the same time, the muscle attached to the condyle is avulsed and becomes a free bone mass. How to speed up the concurrent healing of cartilage during bone healing has always been a clinical difficulty and challenge. OBJECTIVE: To investigate the effect of parathyroid hormone on the healing of condylar cartilage in rabbits with high condylar fracture after free reduction. METHODS: An experimental model of free reduction and fixation of condylar fracture was established in 48 New Zealand big-eared rabbits, which were randomly divided into experimental group and control group (n=20 per group). The experimental group was injected with parathyroid hormone (20 μg/kg) subcutaneously every other day, and the control group was injected with 1 mL of normal saline. The animals were sacrificed at 1, 2, 3, and 4 weeks postoperatively. The mandible condyle was histologically observed. Immunohistochemistry staining and PCR were used to detect the expression of Sox9 and matrix metalloproteinase 13 in the condylar cartilage. The study protocol was approved by the Animal Experimental Ethics Committee of Guizhou Medical University (approval No. 1700456). RESULTS AND CONCLUSION: The results of safranine O-fast green staining and hematoxylin-eosin staining indicated that there were more chondrocytes and cartilage matrix deposition in the experimental group than the control group. In the immunohistochemistry, the average absorbance of Sox9 in the experimental group was significantly higher than that in the control group within 1-3 postoperative weeks (P < 0.05). The average absorbance of matrix metalloproteinase 13 in the experimental group was lower than that in the control group within 1-3 postoperative weeks (P < 0.05). The expression of Sox9 mRNA in the experimental group was significantly higher than that in the control group within 1-3 postoperative weeks, P < 0.05). The expression of matrix metalloproteinase 13 mRNA in the experimental group was significantly lower than that in the control group within 1-3 postoperative weeks (P < 0.05). These findings indicate that intermittent subcutaneous injection of parathyroid hormone can up-regulate the expression of Sox9, inhibit the expression of matrix metalloproteinase 13, promote the transformation of mesenchymal stem cells to cartilage, and accelerate the repair of cartilage damage.
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Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.
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Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.
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PURPOSE: The fourth state of matter, plasma is known as an ionized gas with electrons, radicals and ions. The use of non-thermal plasma (NTP) in cancer research became possible because of the progresses in plasma medicine. Previous studies on the potential NTP-mediated cancer therapy have mainly concentrated on cancer cell apoptosis. In the present study, we compared the inhibitory effect of NTP on cell migration and invasion in the oral squamous cancer cell lines. MATERIALS AND METHODS: We used oral squamous cancer cell lines (SCC1483, MSKQLL1) and different gases (N₂, He, and Ar). To investigate the mechanism of plasma treatment, using different gases (N₂, He, and Ar) which induces anti-migration and anti-invasion properties, we performed wound healing assay, invasion assay and gelatin zymography. RESULTS: The results showed that NTP inhibits cancer cell migration and invasion of oral squamous cancer cell. In addition, focal adhesion kinase expression and matrix metalloproteinase-2/9 activity were also inhibited. CONCLUSION: The suppression of cancer cell invasion by NTP varied depending on the type of gas. Comparison of the three gases revealed that N₂ NTP inhibited cell migration and invasion most potently via decreased expression of focal adhesion kinase and matrix metalloproteinase activity.
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Apoptose , Linhagem Celular , Movimento Celular , Células Epiteliais , Proteína-Tirosina Quinases de Adesão Focal , Gases , Gelatina , Íons , Neoplasias de Células Escamosas , Paxilina , Plasma , Gases em Plasma , CicatrizaçãoRESUMO
Recently, several studies showed that gastrointestinal tract may be associated with pathophysiology of Parkinson's disease (PD). Intestine tight junction protein zonula occluden-1 (ZO-1) is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 (MMP-9). In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-1 expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvastatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that pass across the intestinal barrier.
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Animais , Camundongos , Modelos Animais de Doenças , Inibidores de Hidroximetilglutaril-CoA Redutases , Farmacologia , Doença de Parkinson , Metabolismo , Sinvastatina , Farmacologia , Proteína da Zônula de Oclusão-1RESUMO
Objective To investigate the correlation of matrix metallopeptidase-9 (MMP-9 )and tissue inhibitor of metalloprotei-nase-1(TIMP-1)with hemorrheology in male patients with diabetic cerebral infarction.Methods 56 patients with type 2 diabetic cerebral infarction(group A),49 cases with non-diabetic cerebral infarction(group B),and 40 cases of community healthy men (group C)were enrolled in the study.The serum levels of MMP-9 and TIMP-1 and hemorrheology indexes were detected.Results The levels of MMP-9 and MMP-9/TIMP-1 in group A were significantly higher than those in group B and C(P<0.05),however, TIMP-1 level in group A was significantly lower than that of group C(P<0.05).The levels of MMP-9 and MMP-9/TIMP-1 in group B existed significant increasing,compared with group C(P<0.05).The levels of high shear viscosity,plasma viscosity,and platelet aggregation index in group A were significantly higher than those in group B and C(P<0.05).While the levels of low shear viscosity,plasma specific viscosity,and platelet aggregation index showed significant differences between group B and C(P<0.05). MMP-9 positively correlated with high shear viscosity,plasma specific viscosity,and platelet aggregation index(P<0.05).TIMP-1 negatively correlated with high shear viscosity and plasma ratio viscosity(P<0.05).And MMP-9/TIMP-1 showed significant cor-relation with high shear viscosity,low shear viscosity,plasma specific viscosity,and platelet aggregation index(P<0.05).Conclusion In patients with diabetic cerebral infarction,MMP-9,TIMP-1 and their ratio show some correlation with hemorrheology indexes. Detecting these indexes would be helpful for the diagnosis of diabetic cerebral infarction.
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Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.
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Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/isolamento & purificação , Azotobacter/enzimologia , Azotobacter/isolamento & purificação , Fermentação , Metaloexopeptidases/análise , Metaloexopeptidases/isolamento & purificação , Peptídeo Hidrolases/análise , Serina/análise , Ativação Enzimática , Métodos , Padrões de Referência , MétodosRESUMO
Objective To explore the preventive and therapeutic role of silencing type Ⅰ rat platelet-binding protein motifs depolymerization protein-like metalloproteinase 2(ADAMTS2)by siRNA on experimental liver fibrosis in vitro.By studying the mechanism of siRNA silencing of ADAMTS2,we also aim to evaluate the feasibility of ADAMTS2 as a target for anti-liver fibrosis therapy.Methods Three pairs of siRNAs targeting ADAMTS2 mRNA 2237,2597 and 690 targets were designed and synthesized by utilizing RNA design software.The most effective siRNA was chosen to transfect HSC-T6 cell line to test the tendency of hepatic stellate cell(HSC)activation and ex pression of ADAMTS2,COL1α1,COL(I),α-SMA,TGF-β1,MMP-2 and TIMP-3.These were quantified using real time-PCR,Western blotting,and MTT assays.Results Of the same dosage and time of injection,siRNA 2237 inhibited ADAMTS2 gene expression significantly more than other siRNAs.siRNA-ADAMTS2 2237 markedly inhibited ADAMTS2 gene and protein expression of HSCT6 with more than 80％ efficiency.Conversely,siRNA-ADAMTS2 2237 markedly reduced the gene and protein expressions of COL(I),α-SMA and TGF-β1 on HSC-T6 and inhibited the proliferation of HSC.Conclusions siRNA-ADAMTS2 2237 could effectively knockdown the gene and protein expression of ADAMTS2 in HSC-T6 cell lines.Silencing ADAMTS2 by siRNA significantly inhibited the activation,proliferation of HSC and the gene and protein expressions of COL(I),α SMA,and TGF-β1,and it may have a potential anti-fibrotic effect.ADAMTS2 might be an efficient target for anti-fibrotic therapy.
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The aim of this study was to determine whether single nucleotide polymorphisms (SNPs) of matrix metallopeptidase 2 (MMP2) are associated with obesity. MMP2 is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. To identify the relationship between polymorphisms of MMP2 and overweight/obese, we genotyped 5 SNPs (rs17242319, rs1053605, rs243849, rs2287074, and rs10775332) of the coding region of MMP2 using the Golden Gate assay on an Illumina BeadStation 500 GX. One hundred and forty two overweight/ obese (BMI > or =23) and 145 normal (BMI 18 to <23) subjects were analyzed. SNPStats, Haploview, HapAnalyzer, SNPAnalyzer, and Helixtree programs were used for the analysis of genetic data. A linkage disequilibrium (LD) block was discovered among the 5 SNPs selected, including rs17242319, rs1053605, rs243849, and rs2287074. Of the 5 polymorphisms, 2 synonymous SNPs [rs17242319 (Gly226Gly) and rs10775332 (Phe602Phe)] were found significant associations with overweight/obese. Recently, rs1132896 replaced rs17242319 as a new number (SNP database, BUILD 129). In haplotype analysis using Haploview, a haplotype (haplotype: CCCA) containing a meaningful polymorphism (rs17242319) was found to be significantly different. The results suggest that MMP2 may be associated with overweight/obese in Korean population.