Effect of ANKRD49 on migration of human lung adenocarcinoma cells and its mechanism / 中国生物制品学杂志

@#Objective To study the effect of ankyrin repeat domain 49(ANKRD49)on the migration of human lung adenocarcinoma cell line NCI-H1299 and its mechanism.Methods NCI-H1299 cells were infected with lentivirus vector carrying ANKRD49 gene and shRNA targeting ANKRD49 to construct the cell models stably overexpressing and knocking down ANKRD49. Meanwhile,the control cell models infected with empty lentivirus vector and lentivirus vector with scramble sequences were constructed respectively. The expression levels of ANKRD49 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot. The effect of ANKRD49 on cell migration was measured by scratch test. The mRNA and protein levels of matrix metalloproteinase(MMP)-2/9 and tissue inhibitor of metalloproteinase(TIMP)-1/2 were detected by real-time fluorescence quantitative PCR and Western blot. The protein expression levels of p65,p-p65,IκBα and p-IκBα were detected by Western blot.Results The levels of ANKRD49 mRNA and protein in the ANKRD49 overexpression group were significantly higher than those in the control group(t = 70. 02 and 45. 68,respectively,each P < 0. 001). Compared with the control group,the migration ability of cells in the ANKRD49 overexpression group significantly increased at 24 h and 48 h(t = 5. 343 and 3. 282,P = 0. 005 9 and 0. 030 4,respectively);The mRNA transcription levels and protein expression levels of MMP-2 and MMP-9 significantly increased(t = 9. 304 and 6. 193,P =0. 000 7 and 0. 003 5,respectively),while the mRNA and protein expression of TIMP-1 and TIMP-2 decreased significantly(t = 3. 858 and 3. 517,P = 0. 018 2 and 0. 024 5,respectively),and the values of MMP-2/TIMP-1 and MMP-9/TIMP-2 significantly increased(t = 17. 7 and 9. 682,P < 0. 001 and < 0. 01,respectively);The expression of p-p65 and pIκBα significantly increased,the total protein levels of p65 and IκBα showed no obvious change,and the values of p-p65/p65 and p-IκBα/IκBα significantly increased(t = 3. 962 and 5. 370,P = 0. 016 7 and 0. 005 8,respectively). However,knocking down of ANKRD49 presented the opposite results.Conclusion ANKRD49 promotes the migration of NCI-H1299cells by enhan-cing the expression of MMP-2/9,the values of MMP-9/TIMP-1 and MMP-2/TIMP-2 via activating NF-κB/p65 signa-ling pathway.

Therapeutic Efficacy of Jingangteng Capsules Combined with Guizhi Fuling Capsules in the Treatment of Chronic Pelvic Inflammation and Their Effect on Serum Granulocyte-Macrophage Colony-Stimulating Factor and Matrix Metalloproteinase 2 Levels / 广州中医药大学学报

Objective To investigate the efficacy of Jingangteng Capsules combined with Guizhi Fuling Capsules(GFC)for the treatment of patients with chronic pelvic inflammation of damp-heat and stasis obstruction type and to observe their effects on serum granulocyte-macrophage colony-stimulating factor(GM-CSF)and matrix metalloproteinase 2(MMP-2)levels.Methods Ninety patients with chronic pelvic inflammation of damp-heat and stasis obstruction type were randomly divided into the combined group and the GFC group,with 45 patients in each group.Patients in the GFC group were treated with Guizhi Fuling Capsules,while patients in the combined group were given Jingangteng Capsules together with GFC.The treatment period lasted for 2 weeks and then one-month follow-up was conducted.The changes of traditional Chinese medicine(TCM)scores,serum GM-CSF and MMP-2 levels in the two groups were observed before and after treatment.And the clinical efficacy,time for the relief of symptoms,recurrence of disease and occurrence of adverse reactions in the two groups were compared.Results(1)After 2 weeks of treatment,the total effective rate of the combined group was 93.33%(42/45),and that of the GFC group was 66.67%(30/45).The intergroup comparison showed that the therapeutic effect of the combined group was significantly superior to that of the GFC group when comparing the two groups(P＜0.01).(2)After treatment,the scores of TCM symptoms of lower abdominal pain,lumbosacral pain,leukorrhagia,profuse menstruation,dysmenorrhea,and fatigue in both groups were significantly lower than those before treatment(P＜0.05),and the reduction of TCM syndrome scores in the combined group was significantly superior to that in the GFC group(P＜0.05).(3)The time for leucorrhea recovering normal and the time for the relief of lower abdominal distension and abdominal pain in the combined group were significantly shorter than those in the GFC group after treatment(P＜0.01).(4)After treatment,the serum serological indicators of GM-CSF and MMP-2 levels in the two groups were significantly decreased compared with those before treatment(P＜0.05),and the reduction of serum GM-CSF and MMP-2 levels in the combined group was significantly superior to that in the GFC group(P＜0.05 or P＜0.01).(5)The recurrence rate and the incidence rate of adverse reactions in the combined group were 11.11%(5/45)and 13.33%(6/45),respectively,and were significantly lower than those in the GFC group[all being 35.56%(16/45)],the differences being all statistically significant(P＜0.05 or P＜0.01).Conclusion Jingangteng Capsules combined with Guizhi Fuling Capsules can significantly enhance the clinical efficacy of the patients with chronic pelvic inflammatory of damp-heat and stasis obstruction type,effectively shorten the time for the relief of symptoms,and decrease the serum GM-CSF and MMP-2 levels.

Prediction and validation of potential targets for the glucagon-like peptide-1 receptor agonist in the treatment of Alzheimer's disease / 中国组织工程研究

BACKGROUND:In the process of exploring the mechanism of Alzheimer's disease,the important role of bioinformatics for common target screening has been revealed,enabling the use of its screening results as a basis for exploring the therapeutic effects of drugs on the disease. OBJECTIVE:To predict the targets of liraglutide,a glucagon-like peptide-1 receptor agonist,in the treatment of Alzheimer's disease by bioinformatics and molecular biology. METHODS:DisGeNET database and SEA database were used to obtain the common genes of Alzheimer's disease and liraglutide.GO/KEGG enrichment analysis of common targets was conducted using DAVID online database.Protein-protein interaction networks were constructed using STRING database.The optimal dosage of liraglutide was determined using cell counting kit-8 assay.Expression of key proteins was analyzed using immunofluorescence and immunoblotting techniques.The mouse hippocampal neuron HT22 cell line was used for ex vivo experiments,and the cells were randomly divided into three groups:HT22 group,HT22+Aβ group,and HT22+Aβ+Lir group.No special treatment was done in the HT22 group,while Aβ1-42 was used to intervene in the HT22 cell line for 24 hours to construct an Aβ injury cell model in the HT22+Aβ group.In additional to modeling,liraglutide was added to the HT22+Aβ+Lir group for 12 hours. RESULTS AND CONCLUSION:A total of 3 333 genes associated with Alzheimer's disease were screened from DisGeNET database.Then 147 potential targets of liraglutide were obtained from SEA database.Finally,64 common targets of Alzheimer's disease and Liraglutide were determined using R packets.GO/KEGG analysis of common targets using DAVID online database suggested that common targets were mainly enriched in the following biological processes:neuroactive ligand-receptor interaction,renin-angiotensin system,bladder cancer,endopeptidase activity,peptide receptor activity,G protein-coupled peptide receptor activity,and transport vesicles.The obtained 64 common target proteins were imported into SRTING online database for protein-protein interaction network construction,and the top three genes,matrix metalloproteinases 2,9 and interleukin 1β,were obtained.The activity of cultured cells was detected by the cell counting kit-8 kit.Liraglutide at 100 nmol/L was the optimal concentration for antagonizing Aβ1-42.In the western blot and immunofluorescence assays,the expression of matrix metalloproteinases 2,9 and interleukin 1β was significantly increased in the HT22+Aβ group compared with the HT22 group(P＜0.05)but significantly decreased in the HT22+Aβ+Lir group compared with the HT22+Aβ group(P＜0.05).To conclude,the above bioinformatics data and secondary validation of differential genes in the GEO database suggest that both matrix metalloproteinases 2,9 and interleukin 1β could be potential targets of liraglutide in the treatment of Alzheimer's disease.

The relationship between MMP-2 rs243865, MMP-9 rs398242 and CXCL-12 rs1801157 gene polymorphisms with Japanese encephalitis disease and disease outcome in North Indian population

Background & objectives: Japanese encephalitis virus (JEV) is one of the most important causes of acute and uncontrolled inflammatory disease in Asia. Matrix metalloproteinases (MMPs) and chemokines play a detrimental role in the host response to JE disease, aetiology, and disease outcome. Evidently, MMPs are widely circulated in the brain and regulate various process including microglial activation, inflammation, blood-brain barrier disruption as well as affects central nervous system (CNS). The present study was to assess the association of single nucleotide polymorphisms of MMP-2, MMP-9 and chemokine (CXCL-12/SDF1-3’) in the north Indian population. Methods: We performed case-control study comprising of 125 patients and 125 healthy controls in north Indian population. Genomic DNA was extracted from whole blood and gene polymorphism have been determined by PCR-RFLP method. Results: MMP-2, MMP-9 and CXCL-12 gene was not significantly associated with JE disease, but homozygous (T/T) genotype of MMP-2 was statically associated with disease outcome (p=0.05, OR=0.110). A/G and G/G genotype of CXCL-12 was significantly associated with severity of disease. (p=0.032, OR=5.500, p=0.037, OR= 9.167). The serum level of MMP-2 was observed significantly increased in JE patients with homozygous (T/T) genotype whereas increased MMP-9 level was associated with heterozygous genotype. Interpretation & conclusion: MMP-2, MMP-9 and CXCL-12 gene polymorphism were not associated with JE susceptibility, but MMP-2 may be contributed to disease protection. CXCL-12 was associated with disease severity. In our concern this is the first report from northern India.

On-demand integrated nano-engager converting cold tumors to hot via increased DNA damage and dual immune checkpoint inhibition

Cancer immunotherapy has become a promising strategy. However, the effectiveness of immunotherapy is restricted in "cold tumors" characterized with insufficient T cells intratumoral infiltration and failed T cells priming. Herein, an on-demand integrated nano-engager (JOT-Lip) was developed to convert cold tumors to hot via "increased DNA damage and dual immune checkpoint inhibition" strategy. JOT-Lip was engineered by co-loading oxaliplatin (Oxa) and JQ1 into liposomes with T-cell immunoglobulin mucin-3 antibodies (Tim-3 mAb) coupled on the liposomal surface by metalloproteinase-2 (MMP-2)-sensitive linker. JQ1 inhibited DNA repair to increase DNA damage and immunogenic cell death (ICD) of Oxa, thus promoting T cells intratumoral infiltration. In addition, JQ1 inhibited PD-1/PD-L1 pathway, achieving dual immune checkpoint inhibition combining with Tim-3 mAb, thus effectively promoting T cells priming. It is demonstrated that JOT-Lip not only increased DNA damage and promoted the release of damage-associated molecular patterns (DAMPs), but also enhanced T cells intratumoral infiltration and promoted T cell priming, which successfully converted cold tumors to hot and showed significant anti-tumor and anti-metastasis effects. Collectively, our study provides a rational design of an effective combination regimen and an ideal co-delivery system to convert cold tumors to hot, which holds great potential in clinical cancer chemoimmunotherapy.

Sequential delivery of PD-1/PD-L1 blockade peptide and IDO inhibitor for immunosuppressive microenvironment remodeling via an MMP-2 responsive dual-targeting liposome

Intelligent responsive drug delivery system opens up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of tumor cascade-targeted responsive liposome (NLG919@Lip-pep1) is developed by conjugating polypeptide inhibitor of PD-1 signal pathway (AUNP-12), which is also a targeted peptide that conjugated with liposome carrier through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGD). This targeted liposome is prepared through a mature preparation process, and indoleamine-2,3-dioxygenase (IDO) inhibitor NLG919 was encapsulated into it. Moreover, mediated by the enhanced permeability and retention effect (EPR effect) and AUNP-12, NLG919@Lip-pep1 first targets the cells that highly express PD-L1 in tumor tissues. At the same time, the over-expressed MMP-2 in the tumor site triggers the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of T cells. The exposure of secondary targeting module II VRGDC-NLG919@Lip mediated tumor cells targeting, and further relieved the immunosuppressive microenvironment. Overall, this study offers a potentially appealing paradigm of a high efficiency, low toxicity, and simple intelligent responsive drug delivery system for targeted drug delivery in breast cancer, which can effectively rescue and activate the body's anti-tumor immune response and furthermore achieve effective treatment of metastatic breast cancer.

Protective mechanism of RNA binding protein LIN28 on diabetic nephropathy / 局解手术学杂志

Objective To explore the protective mechanism of RNA binding protein LIN28 on diabetic nephropathy(DN).Methods LIN28 was overexpressed or knocked down by adenovirus in HEK 293T cells.The gene and functional signaling pathway significantly changed after overexpression of LIN28 were obtained through RNA Seq transcriptome sequencing and bioinformatics analysis.HEK 293T cells were divided into the Control group,the Treatment group,the Adv-LIN28-OE+D-ribose group and the Adv-LIN28-NC+D-ribose group in in vitro LIN28 overexpression studies.And HEK 293T cells were divided into the Control group,the Treatment group,the Adv-shRNA LIN28+D-ribose group and the Adv-shRNA NT+D-ribose group in in vitro LIN28 knockdown studies.ELISA was used to detect the levels of human advanced glycation end products(AGEs)of cells supernatants in the above groups.Western blot was used to detect the expression levels of RAGE,NF-κB and MMP-2 of cells in the above groups.Results The results of RNA-Seq transcriptome sequencing,GO analysis and KEGG analysis showed that overexpression of LIN28 resulted in a significant down-regulation of AGE-RAGE signaling pathway in HEK 293T cells(P<0.05).The results of ELISA showed that compared with the Control group,the cell in the Treatment group produced a large amount of AGEs(P<0.05);compared with the Treatment group,the AGEs level of cells in the Adv-LIN28-OE+D-ribose group was significantly decreased(P<0.05),while the AGEs level of cells in the Adv-shRNA LIN28+D-ribose group was increased(P>0.05).The results of Western blot showed that compared with the Control group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Treatment group were significantly increased(P<0.05);compared with the Treatment group,the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-LIN28-OE+D-ribose group were significantly decreased(P<0.05),while the expression levels of RAGE,NF-κB and MMP-2 of cells in the Adv-shRNA LIN28+D-ribose group were significantly increased(P>0.05).Conclusion Overexpression of LIN28 by adenovirus at the cellular level in vitro can lead to significant differential expression of thousands of genes.In particular,it can inhibit the diabetes and complications-related AGE-RAGE signaling pathway,which is critical in the progression of DN disease,and play a role in protecting DN.

New insights about myofibrillar myopathies: the role of metalloproteinases 2 and 9 in the pathogenesis

Backgroung: There are few reports suggesting that gene expression and activation of various matrix metalloproteinases (MMPs) are deregulated. MMP-2 and MMP-9 represent the two MMPs, which degrade type IV collagen, the component of basement membrane. Methods: We analysed the involvement of gelatinases, MMP-2 and MMP-9, in the pathogenesis of myofibrillar myopathy (MFM). Muscle specimens from 23 patients well diagnosed with MFM, were immunostained by MMP-2 and MMP-9. We analysed qualitatively the immunoexpression in three compartments: subsarcolemmal (SSC), intracytoplasmic (ICC) and perinuclear (PNC).Results: 95,7% and 100% samples showed MMP-2 and MMP-9 upregulation ICC, respectively. PNC showed MMP-2 (82,6%) and MMP-9 (8,7%) regulation (p<0.001). SSC and ICC did not present statistical significance. There was no correlation between mutated gene and immunohistochemical pattern distribution.Conclusion: Our results suggest that MMP-2 and/or MMP-9 could participate in the pathomechanism of MFM, causing damage of sarcomere and deposition of protein aggregates.

Introdução: Existem poucos relatos sugerindo que a expressão gênica e a ativação de várias metaloproteinases de matriz (MMPs) estão desreguladas. MMP-2 e MMP-9 representam as duas MMPs, que degradam o colágeno tipo IV, o componente da membrana basal.Método: Analisamos o envolvimento das gelatinases, MMP-2 e MMP-9, na patogênese da miopatia miofibrilar (MFM). Amostras de músculos de 23 pacientes bem diagnosticados com MFM foram imunocoradas por MMP-2 e MMP-9. Analisamos qualitativamente a imunoexpressão em três compartimentos: subsarcolemal (SSC), intracitoplasmático (ICC) e perinuclear (PNC).Resultados: 95,7% e 100% das amostras apresentaram ICC de regulação positiva de MMP-2 e MMP-9, respectivamente. PNC mostrou regulação MMP-2 (82,6%) e MMP-9 (8,7%) (p <0,001). SSC e ICC não apresentaram significância estatística. Não houve correlação entre o gene mutado e a distribuição do padrão imunohistoquímico.Conclusão: Nossos resultados sugerem que MMP-2 e / ou MMP-9 podem participar do patomecanismo da MFM, causando dano ao sarcômero e deposição de agregados proteicos.

Prevention of liver fibrosis in mice by ethanol extract of Euonymus alatus and its preliminary mechanism / 中华消化杂志

Objective:To study the role of ethanol extract of Euonymus alatus stems (EAT) and ethanol extract of Euonymus alatus wings (EAW) in anti-hepatic fibrosis induced by carbon tetrachloride in mice, and to explore its preliminary mechanism. Methods:Sixty C57BL/6 mice were selected and randomly divided into healthy control group, carbon tetrachloride model (CTM) group, EAW low dose (EAW-L) group, EAW high dose (EAW-H) group, EAT low dose (EAT-L) group and EAT high dose (EAT-H) group, with 10 mice in each group. Three days before modeling, the mice of EAT-L, EAT-H, EAW-L and EAW-H group were gavaged with EAT or EAW at 2.0 or 8.0 g/kg, respectively, and the mice of healthy control group and CTM group were gavaged with equal volume of pure water, once a day till the 30th day after modeling (total 33 times). Five percent carbon tetrachloride olive oil solution was intraperitoneally injected at 8 mL/kg to establish liver fibrosis model in CTM, EAT-L, EAT-H, EAW-L and EAW-H groups. The mice in the healthy control group were intraperitoneally injected with equal volume of 0.9% sodium chloride solution, twice per week for 30 days, and a total of 9 times of injection. The liver index, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and interleukin-6 (IL-6) were detected. Hematoxylin-eosin and Masson staining were used to observe the pathological changes of mouse liver tissue and calculate the collagen volume fraction. The liver inflammatory response and fibrosis degree were evaluated by histological activity index (HAI) and Ishak system score. The level of α-smooth muscle actin(α-SMA)in liver tissue was both detected by immunohistochemistry and Western blotting. The expression of matrix metalloproteinase 2 (MMP2) and extracellular signal-regulated kinase (ERK) 1/2 at protein and mRNA level was detected by Western blotting and fluorescent quantitative polymerase chain reaction. Analysis of variance, Tukey test and Dunn test were used for statistical analysis.Results:The hepatic indexes of EAW-L, EAW-H and EAT-H groups were lower than that of CTM group(0.06±0.01, 0.05±0.01 and 0.05±0.01 vs. 0.07±0.01), and the differences were statistically significant ( q=5.12, 7.70, 7.11; all P<0.01). The serum ALT and AST levels of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than those of CTM group((601.76±141.38), (283.35±42.32), (734.74±116.06) and (391.60±34.33) U/L vs.(982.45±96.04) U/L, (509.49±152.29), (345.41±67.39), (282.30±65.72) and(243.23±45.20) U/L vs.(766.01±114.49) U/L), and the differences were statistically significant ( qALT =9.88, 20.81, 7.65, 17.58, qAST =5.11, 12.52, 14.92, 15.56; all P<0.001). The serum TBil levels of EAW-H and EAT-H groups were lower than that of CTM group((6.81±0.49) and (7.08±1.78) μmol/L vs.(12.68±3.28) μmol/L), and the differences were statistically significant( q=6.31, 6.01; both P<0.01). The serum IL-6 levels of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that of CTM group((29.26±5.42), (24.28±4.75), (9.05±1.74) and (8.01±1.24) ng/L vs.(53.21±10.05) ng/L); the serum IL-6 level of EAT-L group was lower than that of EAW-L group; the serum IL-6 level of EAT-H group was lower than that of EAW-H group, and the differences were statistically significant( q=12.20, 14.73, 22.48, 22.11, 10.28, 7.96; all P <0.001). The collagen volume fractions of EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that of CTM group (6.15±1.09, 2.91±0.76, 7.07±1.37 and 5.31±0.80 vs. 12.36±1.96); the collagen volume fraction of EAW-H group was lower than that of EAW-L, EAT-L and EAT-H groups, and the differences were statistically significant( q=11.68, 17.78, 9.94, 13.25; 6.10, 7.84, 4.53; all P <0.05). The HAI and Ishak system scores of EAW-H and EAT-H groups were lower than those of CTM group (6.0 (5.5, 7.5) and 7.0 (6.0, 7.5) vs. 13.0 (12.0, 13.0), 1.0 (1.0, 2.0) and 2.0 (1.0, 2.0) vs. 4.0 (3.0, 4.0)), and the differences were statistically significant( ZHAI=3.38, 3.23, Zlshak=3.22, 3.03; all P<0.05). The result of immunohistochemical analysis showed that the expression levels of α-SMA in the mice liver tissues of EAW-L, EAW-H, EAT-L, EAT-H and CTM groups were 4.76±0.36, 2.75±0.29, 3.72±0.34, 5.20±0.79 and 5.98±0.52, respectively. The result of Western blotting showed that the expression levels of α-SMA in the mice liver tissues of CTM, EAW-L, EAW-H, EAT-L and EAT-H groups were 0.96±0.11, 0.67±0.07, 0.22±0.01, 0.78±0.08 and 0.68±0.07, respectively. Two detection methods both showed that the expression levels of α-SMA of EAW-L, EAW-H and EAT-H groups were lower than that of CTM group; the expression level of α-SMA of EAW-H group was lower than that of EAW-L, EAT-L and EAT-H group, and the differences were statistically significant( qimmunohistochemical =6.06, 15.95, 11.18, 9.92, 12.10 and 4.79, qWestern blotting=7.29, 18.34, 6.84, 11.05, 13.97 and 11.49, all P<0.05). The expression levels of MMP2 and ERK1/2 at protein and mRNA levels in the mice liver tissues of EAW-L, EAW-H, EAT-L, EAT-H and CTM groups were 0.18±0.04, 0.16±0.04, 0.28±0.02, 0.21±0.02 and 0.84±0.02, 0.80±0.02, 0.57±0.08, 0.83±0.03, 0.69±0.02 and 0.91±0.04, 18.74±1.90, 10.73±1.24, 24.99±1.84, 7.19±0.48 and 24.68±1.18, 29.44±4.47, 11.96±0.53, 24.75±4.04, 5.30±0.36 and 35.76±0.85, respectively. The expression levels of MMP2 at protein level in EAW-L, EAW-H, EAT-L and EAT-H groups were lower than that in CTM group; the expression levels of ERK1/2 at protein level in EAW-H and EAT-H groups were lower than that in CTM group; the expression level of ERK1/2 at protein level in EAW-H group was lower than that in EAT-H group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAW-H and EAT-H group were lower than those in CTM group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAW-H group were lower than those in EAW-L group; the expression levels of MMP2 and ERK1/2 at mRNA level in EAT-H group were lower than those in EAT-L and EAW-H groups, and the differences were statistically significant( q=22.15, 22.96, 18.87, 21.31; 13.42, 8.53; 4.90; 18.57, 23.29, 16.49, 21.11; 10.66, 12.12; 23.70, 15.38, 13.48, 16.73; all P<0.05). Conclusions:Both EAT and EAW can alleviate carbon tetrachloride-induced liver injury and liver fibrosis in mice, which may be related with inhibiting the expression of ERK1/2 and IL-6 and then affecting the Ras/ERK-MMP2 signaling pathway.

Effects of GSK3β/eEF2K signaling pathway on pulmonary fibrosis in mice / 中国应用生理学杂志

Objective: To investigate the effects of glycogen synthase kinase-3β (GSK3β)/eukaryotic extension factor kinase 2 (eEF2K) signaling pathway on the process of pulmonary fibrosis through in vivo experiments, and find new ideas for clinical treatment of pulmonary fibrosis. Methods: The pulmonary fibrosis model of C57BL/6 male mice was induced by bleomycin with intratracheal injection at the dose of 2 mg/kg. After 14 days of modeling, animals were divided into model group, negative inhibition group and inhibition group (n=5 for each group), and control group was not processed. The inhibition group was treated with TDZD-8 (4 mg/kg) after modeling, the negative inhibition group was given DMSO solution after modeling, and the samples were collected after 28 days. Hematoxylin-eosin staining method was used to detect lung fibrosis in mice and scored according to Ashcroft scale. Expression levels of GSK3β, p-GSK3β, eEF2K, p-eEF2K (Ser70, Ser392, Ser470), precursor protein of matrix metalloproteinase-2 (pro-MMP-2), matrix metalloproteinase-2 (MMP-2), collagen I (Col I), collagen Ⅲ (Col Ⅲ) and α-smooth muscle actin (α-SMA) were detected by Western blot. Results: Compared with control group, the fibrosis score was up-regulated, the expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were increased, while that of eEF2K was decreased in model group (P＜0.05). Compared with model group, the fibrosis score, expression levels of GSK3β, p-GSK3β, p-eEF2K (Ser70, Ser392, Ser470), pro-MMP-2, MMP-2, Col I, Col Ⅲ and α-SMA were decreased, but the expression level of eEF2K was increased in inhibition group (P＜0.05). Conclusion: GSK3β can activate eEF2K by phosphorylation at the sites of Ser70, Ser392 and Ser470, increase the contents of fibrosis indicators, promote the formation of pulmonary fibrosis, and aggravate lung tissue lesions.

The Effect of Doxycycline on the Expression of MMP-2 and MMP-9 in Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in multiple myeloma (MM) patients, and analyze the effect of doxycycline (DOX) on the expression of MMP-2 and MMP-9 in MM cells.@*METHODS@#The peripheral blood and bone marrow samples of MM patients were collected, and the patients were divided into three groups: newly diagnosed group, remission group and relapsed/refractory group, while the peripheral blood samples of 34 health people and the bone marrow samples of 17 IDA patients were selected as normal control and control group. The levels of MMP-2 and MMP-9 were detected by ELISA. The protein levels of MMP-2 and MMP-9 in H929 cells treated by different concentrations of DOX were analyzed by Western blot. After H929 cells was treated by Akt inhibitor MK-2206 2HCl in combination with DOX, Western blot was used to detect the levels of MMP-2 and MMP-9.@*RESULTS@#The levels of MMP-2 and MMP-9 in newly diagnosed MM patients were higher than those in control (P<0.05), while for the patients in the remission group were decreased, but still higher than those in control. The levels of MMP-2 and MMP-9 were increased again for the patients in relapsed/refractory group, and showed no significant difference as compared with those in newly diagnosed group. The levels of MMP-2 and MMP-9 could be inhibited by 10 mg/L and 15 mg/L DOX treated by H929 cell. The protein levels of MMP-2 and MMP-9 showed no altered in H929 cells treated by 5 nmol/L MK-2206 2HCl alone. DOX exerted more profound inhibitory effect to MMP-2 and MMP-9 expression in H929 cells when Akt inhibitor MK-2206 2HCl was combined with DOX.@*CONCLUSION@#The levels of MMP-2 and MMP-9 are increased in MM patients and related to the disease status of MM. DOX can inhibit the expression of MMP-2 and MMP-9 in MM cells, and antagonizing its activation of Akt signaling pathway can further enhance the inhibitory effect.

Effects of Cystatin C on brain edema and microvessels in rats with cerebral ischemia reperfusion injury / 中风与神经疾病杂志

@#Objective　To investigate the morphological changes of Cys C intervention on cerebral cortex AQP4,MMP-2 and BBB ultrastructure after cerebral ischemia reperfusion injury (CIRI) in rats and to explore its underlying mechanisms.Methods　One hundred male SD rats were randomly divided into sham group,IR group，Cys C low,medium and high concentration group.The rat middle cerebral artery embolism model was established by the modified Longa occlusion method.The water content of brain tissue was measured by dry and wet weight method,the content of EB in brain tissue was detected,and the degree of permeability change of the BBB was evaluated by focal ischemia for 2 h with middle cerebral artery occlusion and 24 h reperfusion.TEM was used to observe the ultrastructure changes of BBB.The opening of microvessels was observed under SEM;Western blot was used to detect AQP4 expression in cerebral cortex tissue.MMP-2 was detected expression by immune histochemistry.Results　Compared with IR group,the degree of BBB injury in Cys C low、medium groups was significantly reduced,and the number of capillaries opening increased.The expression of AQP4 protein was decreased,and the OD value of MMP-2 expression was also significantly decreased.In Cys C high group,the BBB was severely damaged.AQP4 protein expression was significantly increased,and the OD value of MMP-2 expression was also significantly increased.Conclusions　The application of Cys C intervention can reduce the damage of the BBB.It is mechanism may be relation to the decrease of AQP4 protein expression and the decrease of OD value of MMP-2 expression.

Nitric oxide regulates adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells

Anoikis is a type of apoptosis that occurs in response to the loss of adhesion to the extracellular matrix (ECM). Anoikis resistance is a critical mechanism in cancer and contributes to tumor metastasis. Nitric oxide (NO) is frequently upregulated in the tumor area and is considered an important player in cancer metastasis. The aim of this study was to evaluate the effect of NO on adhesiveness, invasiveness, and migration of anoikis-resistant endothelial cells. Here, we report that anoikis-resistant endothelial cells overexpress endothelial nitric oxide synthase. The inhibition of NO release in anoikis-resistant endothelial cells was able to decrease adhesiveness to fibronectin, laminin, and collagen IV. This was accompanied by an increase in cell invasiveness and migration. Furthermore, anoikis-resistant cell lines displayed a decrease in fibronectin and collagen IV protein expression after L-NAME treatment. These alterations in adhesiveness and invasiveness were the consequence of MMP-2 up-regulation observed after NO release inhibition. The decrease in NO levels was able to down-regulate the activating transcription factor 3 (ATF3) protein expression. ATF3 represses MMP-2 gene expression by antagonizing p53-dependent trans-activation of the MMP-2 promoter. We speculate that the increased release of NO by anoikis-resistant endothelial cells acted as a response to restrict the MMP-2 action, interfering in MMP-2 gene expression via ATF3 regulation. The up-regulation of nitric oxide by anoikis-resistant endothelial cells is an important response to restrict tumorigenic behavior. Without this mechanism, invasiveness and migration potential would be even higher, as shown after L-NAME treatment.

Photothermal/matrix metalloproteinase-2 dual-responsive gelatin nanoparticles for breast cancer treatment

The chemotherapy combined with photothermal therapy has been a favorable approach for the treatment of breast cancer. In present study, nanoparticles with the characteristics of photothermal/matrix metalloproteinase-2 (MMP-2) dual-responsive, tumor targeting, and size-variability were designed for enhancing the antitumor efficacy and achieving "on-demand" drug release markedly. Based on the thermal sensitivity of gelatin, we designed a size-variable gelatin nanoparticle (GNP) to encapsulate indocyanine green (ICG) and doxorubicin (DOX). Under an 808 nm laser irradiation, GNP-DOX/ICG responded photothermally and swelled in size from 71.58 ± 4.28 to 160.80 ± 9.51 nm, which was beneficial for particle retention in the tumor sites and release of the loaded therapeutics. Additionally, GNP-DOX/ICG showed a size reduction of the particles to 33.24 ± 4.11 nm and further improved drug release with the degradation of overexpressed MMP-2 in tumor. In the subsequently performed

RECK and TIMP-2 mediate inhibition of MMP-2 and MMP-9 by Annona muricata

Up-regulation of MMP-2 and MMP-9 plays a significant role in promoting cancer progression by degradingthe components of the extracellular matrix, thereby enhancing the migration of tumor cells. Although the antiproliferative and apoptotic effect of Annona muricata is well established, its effect on MMP-2 and MMP-9, amajor target in several types of cancers, has not been studied. Powdered samples of various parts of A.muricata like fruit, stem, seed, and twig extracted using aqueous methanol showed significant dose-dependentinhibition of MMP-2 and MMP-9 in a highly metastatic fibrosarcoma cell line, HT1080. Additionally, theseextracts also up-regulated the expression of several endogenous inhibitors of MMP-2 and MMP-9 likeREversion-inducing Cysteine-rich protein with Kazal motifs (RECK) and Tissue Inhibitor of Metalloproteinase-2 (TIMP-2). Furthermore, primary cells developed from tumor tissues obtained from patients notexposed to chemotherapy, also exhibited similar results. Remarkably, the inhibition of MMP-2 and MMP-9observed was tumor specific, with the A. muricata fruit extract showing only 2% inhibition in cells obtainedfrom normal tissues, when compared to 60% inhibition observed in cells obtained from tumor samples. Thepresent study elucidates a novel mechanism by which A. muricata extracts selectively exhibit their anti-canceractivity in tumor cells by down-regulating MMP-2 and MMP-9 that are important biomarkers in cancer.

The long non-coding RNA MIAT/miR-139-5p/MMP2 axis regulates cell migration and invasion in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is a complex disease which is influenced by multiple factors. Recentstudies demonstrated that long non-coding RNA (lncRNA) MIAT was involved in tumor metastasis. However,the underlying mechanism of MIAT in NSCLC remains largely unknown. In this study, MIAT, miR-139-5pand MMP2 expression were measured by quantitative reverse transcriptase PCR (QRT-PCR) or Westernblotting, respectively, and we found the expression of MIAT and MMP2 were elevated, while miR-139-5p wasdecreased in NSCLC tissues and cell lines. Transwell assay showed MIAT and MMP2 functioned as anoncogene to induce cell migration and invasion in NSCLC, but miR-139-5p served as a tumor suppressor inNSCLC to inhibit cell migration and invasion. Besides that, in vivo experiments also indicated MIAT deletioninhibited tumor growth. The relationship between miR-139-5p and MIAT or MMP2 was then confirmed byLuciferase reporter assay, and the results showed that MIAT directly interacted with miR-139-5p and miR-139-5p targetedly suppressed MMP2 in NSCLC cells. Furthermore, expression analysis showed that MIAT indirectly regulated MMP2 by sponging miR-139-5p. Finally, rescue assay suggested that miR-139-5p restorationreversed MIAT-overexpression-induced promotion on the migration and invasion of NSCLC cells. In conclusion, our results demonstrated that lncRNA MIAT modulated the migration and invasion of NSCLC byregulating miR-139-5p and MMP2.

Effect of mebt/mebo on expression of mmp-2 and mmp-9 in chronic refractory wounds / 中国药理学通报

Aim To investigate the mechanism of MEBT/MEBO in promoting chronic wound healing by MMP-2 and MMP-9. Methods Ninety Wistar rats were randomly divided into five groups: MEBO group, rb-bFGF group, chronic wound group, acute wound group and blank group. Eighteen rats in each group were modeled as chronic refractory wounds. The ex-pression of MMP-2 and MMP-9 in wound tissues, on 3rd and 14th day, was detected by ELISA, Western blot and qRT-PCR. Results ( 1 ) Compared with chronic wound group, the time of wound healing was obviously shorter and the rate of wound healing was higher in MEBO group and rb-bFGF group ( P < 0. 05) ; meanwhile, the growth of wounds and the pathological changes of wounds were better in MEBO group and rb-bFGF group. (2) On 3rd day, the ex pression of MMP-2 and MMP-9 in MEBO group and rb-bFGF group was higher than that in chronic wound group (P <0. 05) ; however, on 14th day, the expression of MMP-2 and MMP-9 in MEBO group and rb-bF-GF group also decreased (P < 0. 05). (3) Compared with chronic wound group, the expression of MMP-2 and MMP-9 in MEBO group and rb-bFGF group increased on 3rd day (P <0. 05) , while the expression of MMP-2 and MMP-9 decreased in MEBO group and rb-bFGF group on 14th day (P <0.05). Conclusions MEBT/MEBO promoting chronic wound healing may be associated with regulating MMP-2 and MMP-9 to participate in the degradation and remodeling of ECM.

Effect of baicalin on expression of fibrogenic factors TGF-β1, mmp2 and timp2 in tissue of mice with pulmonary fibrosis / 中国中药杂志

To investigate the effect of baicalin extracted from Qinbai Qingfei Concentrated Pills on the expressions of TGF-β1, mmp2 and timp2 in mice with pulmonary fibrosis induced by bleomycin. The Biacore technique was used to detect the specific binding between Qinbai Qingfei Concentrated Pills and TGF-β1, and the affinity components were enriched, regenerated and recovered by Biacore fishing. Then ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) were used to determine whether the monomer was baicalin. Biacore was used to verify the affinity kinetics of baicalin, which was validated by pharmacodynamics in vivo. Totally 30 BALB/C mice were randomly divided into three groups: baicalin group, blank group and model group. The blank group was given sodium chloride injection(0.08 mL·kg~(-1)), while the model group and the baicalin group were injected with 4 mg·kg~(-1) bleomycin. The localization of TGF-β1, mmp2 and timp2 protein in the cells and the mRNA expressions of TGF-β1, mmp2 and timp2 were detected by RT-PCR 14 days later. The results of Biacore affinity analysis showed that the peak of binding response between Qinbai Qingfei Concentrated Pills and TGF-β1 protein reached 1 524.0 RU, with specific binding. The affinity constant K_D of baicalin and TGF-β1 was 1.620 06 μmol·L~(-1), which was determined by SPR kinetic analysis, suggesting a stable binding between baicalin and TGF-β1, which verified the results of angulation. The results of immunohistochemistry showed that the deposition of cellulose in baicalin group was significantly less than that in model group, the mRNA expressions of TGF-β1, mmp2 and timp2 were decreased in baicalin solution compared with the model group. Baicalin combined with TGF-β1 could inhibit the expressions of mmp2 and timp2 and delay the progress of pulmonary fibrosis.

Effect of fatty acid binding protein 7 （FABP7） on the permeability of blood-brain barrier in rats with cerebral ischemia-reperfusion / 中风与神经疾病杂志

@#Objective　To investigate the effect of fatty acid binding protein 7 （FABP7） on blood-brain barrier （BBB） permeability in cerebral ischemia/reperfusion （I/R） rats and the mechanisms involved. Methods　BBB cell model was established by co-culture of rat brain microvascular endothelial cells and astrocytes. The BBB model of I/R injury was established by using oxygen-glucose deprivation and reoxygenation （OGD/R） method. This model was treated with the FABP7 recombinant protein （rh-FABP7） alone or together with S7155.ENDOHM instrument，Western blotting and flow cytometry were used to determine the transendothelial electrical resistance （TEER），FABP7，TJ and matrix metalloproteinase （MMP） protein levels，and apoptosis. The rat brain I/R model was established，and rh-FABP7 was injected intraperitoneally. The neurological function score，wet and dry weight method，and Evans blue staining were used to determine the neurological function，brain water content，and BBB permeability of rats. Results　FABP7 overexpression reversed the OGD/R-induced down-regulation of TEER，and FABP7 and TJ protein expression，as well as the up-regulation of MMP2/9 expression and apoptosis （P<0.05）. S7155 treatment promoted the effect of FABP7 on MMP2/9 and TJ protein levels，TEER，and apoptosis （P<0.05）. Moreover，FABP7 overexpression reversed the I/R-induced increase of neural function score，brain water content，and BBB permeability and decrease of FABP7 protein level in rats（P<0.05）. Conclusion　FABP7 alleviated the damage of brain I/R to BBB integrity by inhibiting MMP2/9.

Over-expression of Hsp83 in grossly depleted hsrω lncRNA background causes synthetic lethality and l(2)gl phenocopy in Drosophila

We examined interactions between the 83 kDa heat-shock protein (Hsp83) and hsrω long noncoding RNAs (lncRNAs) inhsrω66 Hsp90GFP homozygotes, which almost completely lack hsrω lncRNAs but over-express Hsp83. All +/+; hsrω66Hsp90GFP progeny died before the third instar. Rare Sp/CyO; hsrω66 Hsp90GFP reached the third instar stage butphenocopied l(2)gl mutants, becoming progressively bulbous and transparent with enlarged brain and died after prolongedlarval life. Additionally, ventral ganglia too were elongated. However, hsrω66 Hsp90GFP/TM6B heterozygotes, carrying +/+ or Sp/CyO second chromosomes, developed normally. Total RNA sequencing (+/+, +/+; hsrω66/hsrω66, Sp/CyO; hsrω66/hsrω66, +/+; Hsp90GFP/Hsp90GFP and Sp/CyO; hsrω66 Hsp90GFP/hsrω66 Hsp90GFP late third instar larvae) revealedsimilar effects on many genes in hsrω66 and Hsp90GFP homozygotes. Besides additive effect on many of them, numerousadditional genes were affected in Sp/CyO; hsrω66 Hsp90GFP larvae, with l(2)gl and several genes regulating the centralnervous system being highly down-regulated in surviving Sp/CyO; hsrω66 Hsp90GFP larvae, but not in hsrω66 orHsp90GFP single mutants. Hsp83 and several omega speckle-associated hnRNPs were bioinformatically found topotentially bind with these gene promoters and transcripts. Since Hsp83 and hnRNPs are also known to interact, elevatedHsp83 in an altered background of hnRNP distribution and dynamics, due to near absence of hsrω lncRNAs and omegaspeckles, can severely perturb regulatory circuits with unexpected consequences, including down-regulation of tumoursuppressor genes such as l(2)gl.