Análisis inmunohistoquímico de MMP-1, MMP-2 y MMP-9 en ameloblastoma convencional / Immunohistochemical analysis of MMP-1, MMP-2, MMP-9 in conventional ameloblastoma

Introducción: las metaloproteinasas son enzimas que participan en la remodelación tisular y su función se relaciona con procesos fisiológicos y patológicos, como la invasión y la metástasis. El ameloblastoma convencional (AMC) es una neoplasia epitelial benigna odontogénica intraósea caracterizada por una progresión lenta y localmente invasiva, cuyo crecimiento se ha vinculado con el recambio ósea y la remodelación de la matriz extracelular. El objetivo del presente trabajo fue determinar la presencia inmunohistoquímica de MMP-1, MMP-2 y MMP-9 en el AMC. Material y métodos: se realizó un estudio piloto observacional analítico utilizando cinco muestras de AMC. Los especímenes fueron recolectados aleatoriamente del archivo del Departamento de Patología Oral y Maxilofacial, de la Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. Como grupo control se emplearon dos especímenes de folículo dental, obtenido de pacientes con indicación de su extracción por motivos ortodóncicos. Se realizó la técnica de inmunohistoquímica por peroxidasa, recolectando el nivel y proporción de inmunoexpresión de manera semicuantitativa. Resultados: cuatro pacientes fueron de género masculino y uno femenino, la edad promedio fue de 40.6 ± 14.9 años. Todas las muestras fueron obtenidas de la región mandibular posterior. Se observaron dos especímenes con patrón folicular y tres con plexiforme. Las MMP-2 y MMP-9 se detectaron sólo en uno de los cinco especímenes y únicamente en el parénquima de la lesión, con una proporción de 100%. Conclusión: según nuestro análisis inmunohistoquímico, las MMP-2 y MMP-9 son las metaloproteinasas que presentaron expresión positiva dentro de la patogénesis del AMC comparado a la MMP-1; no obstante, es necesario realizar este tipo de estudios en una población mayor (AU)

Introduction: metalloproteinases are enzymes involved in tissue remodeling and their function is related to physiological and pathological processes, such as invasion and metastasis. These enzymes are capable of degrading components of the extracellular matrix, which may promote tumor progression. Conventional ameloblastoma (CA) is described as a benign intraosseous epithelial odontogenic neoplasm characterized by a slow and locally invasive progression, whose growth has been linked to bone turnover and extracellular matrix remodeling. The aim of the present work was to determine the immunohistochemical presence of MMP-1, MMP-2 and MMP-9 in CA. Material and methods: an analytical observational pilot study was performed using 5 CA, randomly collected from the archive of the Department of Oral and Maxillofacial Pathology, Escuela Nacional de Estudios Superiores (ENES) Unidad León, UNAM. The control group used were two dental follicle samples, obtained from patients with extraction indication for orthodontic treatment. The peroxidase immunohistochemistry assay was performed, collecting semiquantitatively level and proportion of immunoexpression. Results: four patients were male and one female, the average age was 40.6 ± 14.9 years. All specimens were obtained from the posterior mandibular region. Two specimens were observed with follicular pattern and three with plexiform pattern. MMP-2 and MMP-9 were detected only in one of the five specimens, with presence in the parenchyma of the lesion, with a proportion of 100% of the cell analyzed. Conclusion: according to our immunohistochemical analysis, MMP-2 and MMP-9 are the metalloproteinases that presented positive expression within the pathogenesis of CA compared to MMP-1; however, it is necessary to perform this type of studies in a larger population (AU)

Correlation analysis of serum MMP-1 and MMP-2 levels with lower extremity deep venous thrombosis after operation for lower limb fracture / 中国骨伤

OBJECTIVE@#To investigate the relationship between serum matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-2(MMP-2) and the formation of deep venous thrombosis(LDVT) in lower extremity patients after surgery for lower extremity fracture, and to analyze the value of MMP-1 and MMP-2 in predicting the occurrence of LDVT after lower extremity fracture.@*METHODS@#From June 2018 to December 2021, 352 patients who planned to receive surgical treatment of lower limb fracture in our hospital were selected as the research objects. Venous blood was collected at 1, 2 and 3 days after surgery, respectively, and serum MMP-1 and MMP-2 levels were detected. The incidence of LDVT during hospitalization was analyzed, and the risk factors of postoperative LDVT in patients with lower limb fracture surgery and the predictive value of MMP-1 and MMP-2 for LDVT were analyzed.@*RESULTS@#LDVT occurred in 40 patients (LDVT group), the incidence of LDVT was 11.36%, and 312 patients did not occurred(no occurred group). The serum levels of MMP-1 and MMP-2 in LDVT group increased gradually after surgery; the serum levels of MMP-1 and MMP-2 in the no occurred group increased slightly after surgery at 2 days and then decreased at 3 days after surgery (P<0.01);the serum levels of MMP-1 and MMP-2 in LDVT group were higher than those in the no occurred group at 2 days and 3 days after surgery (P<0.05). Serum levels of MMP-1 and MMP-2 were positively correlated with serum levels of interleukin-6 (IL-6), IL-8 and tumor necrosis factor -α (TNF-α) in LDVT patients at 2 days and 3 days postoperatively (P<0.05). Operative time, MMP-1 and MMP-2 postoperative 3 days were related to the occurrence of LDVT after lower limb fracture (P<0.01). The area under the curve(AUC) predicted by MMP-1 and MMP-2 postoperative 3 days for LDVT after lower limb fracture was 0.738 and 0.744 respectively, and the AUC predicted by combined MMP-1 and MMP-2 was 0.910, which was higher than that predicted by single indicator(Z=2.819 and 2.025, P<0.05).@*CONCLUSION@#High levels of MMP-1 and MMP-2 after lower extremity fracture are closely related to the occurrence of LDVT, and 3 d mMP-1 and MMP-2 after surgery maybe used as evaluation indexes for LDVT risk prediction.

Effect of Juanbi Qianggu Formula on biological behaviors of fibroblast-like synoviocytes in rheumatoid arthritis by regulating FGFR1 signaling pathway based on network pharmacology and cell function experiments / 中国中药杂志

This study aimed to explore the molecular mechanism of Juanbi Qianggu Formula(JBQGF), an empirical formula formulated by the prestigious doctor in traditional Chinese medicine, in the treatment of rheumatoid arthritis based on network pharmacology and cell function experiments. The main active components and targets of JBQGF were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Encyclopedia of Traditional Chinese Medicine(ETCM), and the core targets underwent functional enrichment analysis and signaling pathway analysis. Cytoscape 3.6.0 was used to construct a visualized "active component-target-signaling pathway" network of JBQGF. After screening, nine potential pathways of JBQGF were obtained, mainly including G protein-coupled receptor signaling pathway and tyrosine kinase receptor signaling pathway. As previously indicated, the fibroblast growth factor receptor 1(FGFR1) signaling pathway was highly activated in active fibroblast-like synoviocytes(FLS) in rheumatoid arthritis, and cell and animal experiments demonstrated that inhibition of the FGFR1 signaling pathway could significantly reduce joint inflammation and joint destruction in collagen-induced arthritis(CIA) rats. In terms of the tyrosine kinase receptor signal transduction pathway, the analysis of its target genes revealed that FGFR1 might be a potential target of JBQGF for rheumatoid arthritis treatment. The biological effect of JBQGF by inhibiting FGFR1 phosphorylation was preliminarily verified by Western blot, Transwell invasion assay, and pannus erosion assay, thereby inhibiting matrix metalloproteinase 2(MMP2) and receptor activator of nuclear factor-κB ligand(RANKL) and suppressing the invasion of fibroblasts in rheumatoid arthritis and erosive effect of pannus bone. This study provides ideas for searching potential targets of rheumatoid arthritis treatment and TCM drugs through network pharmacology.

Effect of acetylalkannin from Arnebia euchroma on proliferation, migration, and invasion of human melanoma A375 cells / 中国中药杂志

This study aimed to explore the effect and mechanism of acetylalkannin from Arnebia euchroma on the proliferation, migration, and invasion of human melanoma A375 cells. A375 cells were divided into a blank group, and low-, medium-, and high-dose acetylalkannin groups(0.5, 1.0, and 2.0 μmol·L~(-1)). The MTT assay was used to detect cell proliferation. Cell scratch and transwell migration assays were used to detect cell migration ability, and the transwell invasion assay was used to detect cell invasion ability. Western blot was used to detect the protein expression of migration and invasion-related N-cadherin, vimentin, matrix metalloproteina-se-9(MMP-9), and Wnt/β-catenin pathway-related Wnt1, Axin2, glycogen synthase kinase-3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, cell cycle protein D_1(cyclin D_1), and p21. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) was used to detect the mRNA expression of E-cadherin, matrix metalloproteinase-2(MMP-2), N-cadherin, vimentin, β-catenin, snail-1, and CD44. MTT results showed that the cell inhibition rates in the acetylalkannin groups significantly increased as compared with that in the blank group(P<0.01). The results of cell scratch and transwell assays showed that compared with the blank group, the acetylalkannin groups showed reduced cell migration and invasion, and migration and invasion rates(P<0.05, P<0.01) and weakened horizontal and vertical migration and invasion abilities. Western blot results showed that compared with the blank group, the high-dose acetylalkannin group showed increased expression of Axin2 protein(P<0.05), and decreased expression of N-cadherin, vimentin, MMP-9, Wnt1, p-GSK-3β, β-catenin, cyclin D_1, and p21 proteins(P<0.05, P<0.01). The expression of GSK-3β protein did not change significantly. PCR results showed that the overall trend of MMP-2, N-cadherin, vimentin, β-catenin, snail-1, and CD44 mRNA expression was down-regulated(P<0.01), and the expression of E-cadherin mRNA increased(P<0.01). Acetylalkannin can inhibit the proliferation, migration, and invasion of human melanoma A375 cells, and its mechanism of action may be related to the regulation of Wnt/β-catenin signaling pathway.

Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP / 中国中药杂志

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

Effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction / 中国中药杂志

Jiming Powder is a traditional ancient prescription with good therapeutic effect in the treatment of heart failure, but its mechanism lacks further exploration. In this study, a mouse model of coronary artery ligation was used to evaluate the effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction. The study constructed a mouse model of heart failure after myocardial infarction using the method of left anterior descending coronary artery ligation. The efficacy of Jiming Powder was evaluated from multiple angles, including ultrasound imaging, hematoxylin-eosin(HE) staining, Masson staining, Sirius Red staining, and serum myocardial enzyme spectrum detection. Western blot analysis was performed to detect key proteins involved in ventricular remodeling, including transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), wingless-type MMTV integration site family member 3a(Wnt3a), β-catenin, matrix metallopeptidase 2(MMP2), matrix metallopeptidase 3(MMP3), TIMP metallopeptidase inhibitor 1(TIMP1), and TIMP metallopeptidase inhibitor 2(TIMP2). The results showed that compared with the model group, the high and low-dose Jiming Powder significantly reduced the left ventricular internal diameter in systole(LVID;s) and diastole(LVID;d), increased the left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), effectively improved cardiac function in mice after myocardial infarction, and effectively reduced the levels of myocardial injury markers such as creatine kinase(CK), creatine kinase isoenzyme(CK-MB), and lactic dehydrogenase(LDH), thus protecting ischemic myocardium. HE staining showed that Jiming Powder could attenuate myocardial inflammatory cell infiltration after myocardial infarction. Masson and Sirius Red staining demonstrated that Jiming Powder effectively inhibited myocardial fibrosis, reduced the collagen Ⅰ/Ⅲ ratio in myocardial tissues, and improved collagen remodeling after myocardial infarction. Western blot results showed that Jiming Powder reduced the expression of TGF-β1, α-SMA, Wnt3a, and β-catenin, decreased the levels of MMP2, MMP3, and TIMP2, and increased the level of TIMP1, suggesting its role in inhibiting cardiac fibroblast transformation, reducing extracellular matrix metabolism in myocardial cells, and lowering collagen Ⅰ and α-SMA content, thus exerting an anti-myocardial fibrosis effect after myocardial infarction. This study revealed the role of Jiming Powder in improving ventricular remodeling and treating myocardial infarction, laying the foundation for further research on the pharmacological effect of Jiming Powder.

Mechanism of total flavonoids of Ziziphora clinopodioides in improving atherosclerosis by regulating PI3K/Akt/mTOR pathway / 中国中药杂志

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.

Astragali Radix-Curcumae Rhizoma combination inhibits proliferation, migration, and invasion of colon cancer HT-29 cells by regulating EMT / 中国中药杂志

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.

Neutrophil extracellular traps activates focal adhesion kinase by upregulating MMP9 expression to promote proliferation and migration of mouse colorectal cancer cells / 细胞与分子免疫学杂志

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by TranswellTM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.

Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy.@*METHODS@#U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family.@*RESULTS@#Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05).@*CONCLUSION@#Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies / 中国结合医学杂志

OBJECTIVE@#To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF).@*METHODS@#Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting.@*RESULTS@#The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs.@*CONCLUSIONS@#STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.@*METHODS@#Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.@*RESULTS@#Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).@*CONCLUSION@#Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Synergistic effects of dendrosomal nanocurcumin and oxaliplatin on oncogenic properties of ovarian cancer cell lines by down-expression of MMPs

BACKGROUND: Contrary to the advantageous anticancer activities of curcumin (Cur), limited bioavailability and solubility hindered its efficacy. Here, nontoxic dendrosomal nano carrier with Cur was used to overcome these problems. Despite considerable antitumor properties of Oxaliplatin (Oxa), the limiting factors are drug resistance and adverse side-effects. The hypothesis of this study was to evaluate the possible synergism between dendrosomal nanocurcumin (DNC) and Oxa and these agents showed growth regulatory effects on SKOV3 and OVCAR3 cells. METHODS: and materials In the present study, colony formation, wound healing motility, cell adhesion, transwell invasion and migration assay and cell cycle arrest with or without DNC, Oxa and Combination were defined. In addition to, real time PCR and Western blot were used to analyze AKT, PI3K, PKC, JNK, P38 and MMPs mRNAs and proteins expressions. Docking of MMP-2-Cur, MMP-2-DNC and MMP-2-Oxa was performed and the results of all three complexes were simulated by molecular dynamics. RESULTS: Our findings illustrated that DNC had the greatest effect on cell death as compared to the Cur alone. Moreover, the growth inhibitory effects (such as cell death correlated to apoptosis) were more intense if Oxa was added followed by DNC at 4 h interval. However, insignificant effects were observed upon simultaneous addition of these two agents in both cell lines. Besides, a combination of agents synergistically alters the relative expression of MMP-9. CONCLUSIONS: The docking results showed that His70 and Asp100 may play a key role at the MMP-2 binding site. The matrigel invasion as well as cell viability of ovarian cancer cell lines SKOV3 and OVCAR3 by DNC alone or in combination with Oxa was inhibited significantly. The inhibitory effects of these agents were due to the differential expression levels of MMP 2 and MMP 9 regulated by multiple downstream signaling cascades. From the molecular dynamic simulation studies, it was confirmed that DNC established a strong interaction with MMP-2.

Evaluating TIMP-2 and IGFBP-7 as a predictive tool for kidney injury in ureteropelvic junction obstruction

ABSTRACT A major challenge in the management of ureteropelvic junction obstruction (UPJO) is the selection of patients who would benefit from surgical treatment. Tissue inhibitor metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) indicate renal cell stress and are associated with cell cycle arrest. The [TIMP-2] [IGFBP7] ratio (Nephrocheck®) has been recently applied in patients in intensive care units patients to predict the development of acute kidney injury. In this study, we evaluated the performance of these biomarkers performance to distinguishing obstructive hydronephrosis (HN) from non-obstructive HN. Materials and Methods: Consecutive patients with UPJO were enrolled in this study. Urinary [TIMP-2] [IGFBP7] and clinical characteristics (hydronephrosis grade, differential renal function, and drainage half-time) were measured in the following groups: 26 children with obstructive HN at initial diagnosis (group 1A) and after six months of dismembered pyeloplasty (group 1B); 22 children with non-obstructive HN (group 2), and 26 children without any urinary tract condition, as the control group (group 3). Results: Comparing the initial samples, [TIMP-2] [IGFBP7] had higher levels in the HN groups and lower levels in the control group; however, no difference was observed between the HN groups (obstructive vs. non-obstructive). After six months of follow-up, patients who underwent dismembered pyeloplasty showed stability in the urinary concentration of [TIMP-2] [IGFBP7]. All patients with [TIMP-2] [IGFBP7] higher than 1.0 (ng/mL)2/1000 had diffuse cortical atrophy on ultrasonography. Conclusions: We showed that urinary levels of urinary [TIMP-2] [IGFBP7] are higher in children with HN than controls. Nephrocheck® is not reliable in predicting the need for surgical intervention for pediatric patients with UPJO.

Overwork Affects Extracellular Matrix of Arterial Vessel Wall in Rats / 中国医学科学院学报

Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(n=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all P>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all P<0.001) and had no significant difference between each other(P>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(P>0.05) but were higher than that in control group(all P<0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all P>0.05).Groups OW and SD+OW had lower mRNA level(all P<0.001) and protein level(all P<0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(P>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.

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.

Agkistrodon halys venom antitumor component-I inhibits vasculogenic mimicry in triple-negative breast cancer cells in vitro by down-regulating MMP2 / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of agkistrodon halys venom antitumor component-I (AHVAC-I) on vasculogenic mimicry (VM) formation in triple-negative breast cancer MDA-MB-231 cells and explore its possible mechanism.@*METHODS@#CCK8 assay was used to determine the optimal concentration of AHVAC-I for cell treatment based on its halfinhibitory concentration (IC50). MDA-MB-231 cells were treated with different concentrations of AHVAC-I or 5-Fu, and the changes in vasomimetic capacity of the cells were examined using Matrigel assay. The expression levels of matrix metalloproteinase-2 (MMP2) and MMP9 in the treated cells were detected using quantitative PCR and Western blotting.@*RESULTS@#Compared with the control treatment with culture medium, treatment with 5, 10 and 20 μg/mL AHVAC-I significantly reduced vasomimetic ability of MDA-MB-231 cells in a dose-dependent manner (P < 0.01). MMP2 supplementation obviously restored the vasomimetic ability of the cells inhibited by AHVAC-I.@*CONCLUSION@#AHVAC-I inhibits VM formation in triplenegative breast cancer cells in vitro by down-regulating MMP2 production.

Talin1 is highly expressed in the fallopian tube and chorionic villi to promote trophoblast invasion in tubal pregnancy / 南方医科大学学报

OBJECTIVE@#To investigate the expression of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and its role in regulating invasion and migration of trophoblasts.@*METHODS@#Immunohistochemistry and Western blotting were used to detect the localization and expression level of Talin1 in the fallopian tube and chorionic villi in patients with tubal pregnancy and in women with normal pregnancy. In the cell experiment, HTR-8/SVneo cells was transfected with Talin1 siRNA and the changes in cell invasion and migration were assessed using scratch assay and Transwell assay. The expressions of MMP-2, MMP-9, N-cadherin and Snail in the transfected cells were detected by qRT-PCR and Western blotting.@*RESULTS@#Positive expression of Talin1 was detected in both normal fallopian tube tissues and tissues from women tubal pregnancy, and its expression was localized mainly in the cytoplasm of cilia cells. The expression level of Talin1 was significantly higher in both the fallopian tube and chorionic villi in women with tubal pregnancy than in normal fallopian tube and chorionic villi samples (P < 0.01). In HTR-8/SVneo cells, transfection with Talin1 siRNA significantly inhibited cell invasion (P < 0.01) and migration (P < 0.05), down-regulated the expression of N-cadherin, MMP-2 and Snail (P < 0.05), and up-regulated the expression of MMP-9 in the cells (P < 0.05).@*CONCLUSION@#The expression of Talin1 in the fallopian tube and chorionic villi is significantly increased in women with tubal pregnancy, suggesting the association of Talin1-regulated trophoblast cell invasion with the occurrence of tubal pregnancy.

Casticin inhibits the proliferation, migration and invasion of bladder cancer cells by inhibition of TM7SF4 expression / 中华肿瘤杂志

Objective: To explore the effect and mechanism of Casticin (CAS) on the proliferation, migration and invasion of bladder cancer T24 cells. Methods: T24 cells were cultured in vitro and divided into control group, 5, 10, 20 μmol/L CAS groups, si-NC group, si-TM7SF4 group, CAS+ pcDNA group and CAS+ pcDNA-TM7SF4 group. Cell counting kit-8 (CCK-8) was used to detect cell proliferation; Transwell was used to detect cell migration and invasion; western blot was used to detect the protein expressions of cyclin D1, p21, MMP-2, MMP-9 and TM7SF4, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of TM7SF4 mRNA. Results: The inhibition rates of T24 cells in the 5, 10, 20 μmol/L CAS groups were (17.68±1.41)%, (33.54±3.16)% and (61.44±5.50)%, respectively, higher than (0.00±0.00)% of the control group (P<0.001), but the numbers of migration and invasion were 72.83±5.66, 59.13±4.27, 41.25±3.22 and 55.83±5.15, 42.19±3.06, 31.13±3.22, respectively, lower than 86.11±5.16 and 68.82±5.29 of the control group (P<0.001). The protein expression levels of cyclin D1, MMP-2, MMP-9, TM7SF4 and the expression levels of TM7SF4 mRNA in the 5, 10, and 20 μmol/L CAS groups were lower than the control group (P<0.001). However, the protein expression levels of p21 were 0.37±0.03, 0.51±0.04, and 0.66±0.06, respectively, higher than 0.25±0.03 in the control group (P<0.001). The inhibition rate of T24 cells in the si-TM7SF4 group was (50.35±4.67)%, higher than (6.31±0.58)% in the si-NC group (P<0.001), but the numbers of migration and invasion were 53.51±4.18 and 42.92±3.81, lower than 85.26±4.99 and 67.93±4.64 of the si-NC group (P<0.001). The protein expression levels of TM7SF4, CyclinD1, MMP-2, MMP-9 in the si-TM7SF4 group were lower than the si-NC group (P<0.001). However, the protein expression level of p21 in the si-TM7SF4 group was higher than the si-NC group (P<0.001). The inhibitory rate of T24 cells in the CAS+ pcDNA-TM7SF4 group was (21.45±2.46)%, lower than (64.06±4.49)% of the CAS+ pcDNA group (P<0.001), but the number of migration and invasion in the CAS+ pcDNA-TM7SF4 group were 75.66±6.57 and 59.35±5.40, higher than 40.43±3.85 and 30.25±3.32 in the CAS+ pcDNA group (P<0.001). The protein expression levels of TM7SF4, CyclinD1, MMP-2 and MMP-9 in the CAS+ pcDNA-TM7SF4 group were higher than the CAS+ pcDNA group (P<0.001), but the protein expression level of p21 was lower than the CAS+ pcDNA group (P<0.001). Conclusion: CAS may suppress the proliferation, migration and invasion of bladder cancer T24 cells by inhibiting the expression of TM7SF4.

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.