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.
Rats ; Animals ; Synoviocytes ; Matrix Metalloproteinase 2/metabolism* ; Network Pharmacology ; Receptor, Fibroblast Growth Factor, Type 1/therapeutic use* ; Arthritis, Rheumatoid/genetics* ; Signal Transduction ; Fibroblasts ; Drugs, Chinese Herbal/therapeutic use*

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.
Animals ; Mice ; Apolipoproteins E ; Atherosclerosis/genetics* ; Beclin-1 ; Cholesterol, LDL ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Plaque, Atherosclerotic ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/genetics* ; Vascular Cell Adhesion Molecule-1/genetics*

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.
Animals ; Collagen Type I ; Extracellular Matrix/metabolism* ; Matrix Metalloproteinase 1/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA, Messenger/genetics* ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1/metabolism*

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; Blood-Brain Barrier ; drug effects ; enzymology ; immunology ; Brain ; Brain Ischemia ; drug therapy ; enzymology ; genetics ; Caffeic Acids ; administration & dosage ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Lactates ; administration & dosage ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology ; genetics ; immunology ; prevention & control ; Salvia miltiorrhiza ; chemistry ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; immunology

Objective: To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. Methods: The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. Results: miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(P<0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. Conclusion: miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Apoptosis ; genetics ; Cell Proliferation ; genetics ; Cicatrix, Hypertrophic ; Fibroblasts ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; MicroRNAs ; metabolism

BACKGROUND: Macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase-9 (MMP-9), and its specific tissue inhibitor - tissue inhibitor of metalloproteinases-1 (TIMP-1) may play an important role in the pathogenesis and spread of cancer. We investigated the plasma levels of M-CSF, MMP-9, and TIMP-1 in comparison with a commonly accepted tumor marker CA 15-3 in breast cancer patients and in control groups. METHODS: The cohort included 110 breast cancer patients in groups at stages I-IV. The control group consisted of 50 healthy volunteers and 50 benign tumor patients. Plasma levels of M-CSF, MMP-9, and TIMP-1 were determined by using ELISA, while CA 15-3 concentrations were determined by using chemiluminescent microparticle immunoassay (CMIA). RESULTS: The results showed significant differences in concentrations of the analyzed parameters and in levels of CA 15-3 between the groups of breast cancer patients and the two control groups. Diagnosis using these markers was equal to that using CA 15-3 in terms of sensitivity, predictive values of positive and negativetest results (PPV, NPV) and area under the ROC curve (AUC) in the studied groups. The diagnostic specificities of MMP-9, TIMP-1, M-CSF, and CA 15-3 showed equally high values (95%). The combined use of all tested parameters with CA 15-3 resulted in increased sensitivity, NPV, and AUC, especially in the combination of M-CSF with tumor markers (76%, 64%, and 0.8653). CONCLUSIONS: These findings suggest the tested parameters are useful in the diagnosis of breast cancer patients (except stage I), when combined with CA 15-3.
Adult ; Aged ; Area Under Curve ; Biomarkers, Tumor/*blood ; Breast Neoplasms/*diagnosis/genetics/pathology ; Case-Control Studies ; Female ; Humans ; Macrophage Colony-Stimulating Factor/*blood ; Matrix Metalloproteinase 9/*blood ; Middle Aged ; Mucin-1/blood ; Neoplasm Staging ; Poland ; ROC Curve ; Sensitivity and Specificity ; Tissue Inhibitor of Metalloproteinase-1/*blood

BACKGROUNDAs a novel molecular markerof non-small cell lung cancer (NSCLC), PRDI-BF1 and RIZ homology domain containing protein 14 (PRDM14) is over-expressed in NSCLC tumor tissues. Extracellular matrix degradation mediated by the balance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) is one of the most important mechanism in lung cancer metastasis. This study aimed to determine if PRDM14 promoted the migration of NSCLC cells through extracellular matrix degradation mediated by change of MMP/TIMP expression.

METHODSThe expression of PRDM14 was down-regulated in human cell line A 549 after transfection with lentiviral vector-mediated short-hairpin ribonucleic acids (shRNAs) which targeted the PRDM14 promoter. Cellular migration of shRNA-infected cells was detected by a scratch wound healing assay and transwell cell migration assay. Expression levels of MMP1, MMP2, TIMP1, and TIMP2 were measured by quantitative real-time polymerase chain reaction (RT-PCR).

RESULTSMigration of PRDM14-shRNA-infected cells was significantly inhibited relative to control cells as measured by the scratch wound healing (P < 0.05) and transwell cell migration assays (P < 0.01). The expression of MMP1 in A549 cells infected by PRDM14-shRNA was down-regulated significantly (P < 0.01), whereas the expression of TIMP1 and TIMP2 was up-regulated significantly (P < 0.01).

CONCLUSIONSPRDM14 accelerates A549 cells migration in vitro through extracellular matrix degradation. PRDM14 is considered as a potential therapeutic target in metastatic NSCLC.

Carcinoma, Non-Small-Cell Lung ; metabolism ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Extracellular Matrix ; metabolism ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Metastasis ; genetics ; Repressor Proteins ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism

OBJECTIVETo investigate the effect of RbAp48 knockdown on the migration and invasion of human cervical cancer cells and explore the mechanism.

METHODSA small interference RNA (siRNA) was used to knock down the expression of RbAp48 in MS751 cells. The changes in cell migration and invasion were evaluated using wound healing assay and Transwell assay, respectively, and the expressions of RbAp48, vimentin, N-cadherin, E-cadherin, Snail, Twist, MMP-2 and TIMP-2 were determined with Western blotting.

RESULTSAfter siRNA-mediated RbAp48 knockdown, MS751 cells showed a significantly reduced expression of RbAp48 with significantly suppressed cell migration and invasion (P<0.01). RbAp48 knockdown induced obvious down-regulation of the expressions of interstitial cell phenotype proteins vimentin, N-cadherin, and MMP-2 and up-regulation of epithelial cell phenotype proteins E-cadherin and TIMP-2, suggesting the inhibition of epithelial- mesenchymal transition of the cells. The expressions of Snail and Twist were significantly down-regulated in the cells following RbAp48 knockdown.

CONCLUSIONKnockdown of RbAp48 can significantly inhibit epithelial-mesenchymal transition and suppress the migration and invasion of cervical cancer cell line MS751, the mechanism of which may involve the down-regulation of Snail and Twist expressions.

Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Gene Knockdown Techniques ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; RNA, Small Interfering ; Retinoblastoma-Binding Protein 4 ; genetics ; Snail Family Transcription Factors ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transcription Factors ; metabolism ; Twist-Related Protein 1 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vimentin ; metabolism

To investigate the effect of Shenxiong injection on the inflammation injury of ischemia-reperfusion injury senile rats. Totally 84 Sprague-Dawley (SD) rats were randomly divided into six groups: the sham operation group, the model group, the Nimodipine group and the Shenxiong injection(low, middle, and high dosage) groups. The rat cerebral ischemia-reperfusion model was established through intraperitoneal injection for 3 d and middle cerebral artery occlusion (MCAO). Ater the reperfusion for 24 h, efforts were made to give neurological score, collect brains for TTC staining, detect tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) content in serum by enzyme-linked immunosorbent assay (ELISA) method and measure IL-1β, ICAM-1 and MMP-9 mRNA expressions in hippocampal area by Real-time PCR (RT-PCR). According to the results, Shenxiong injection could decrease the cerebral infarction volume, greatly improved the neurological function and reduce IL-1β, TNF-α, ICAM-1 and MMP-9 mRNA expressions and IL-1β and TNF-α contents. In conclusion, Shenxiong injection shows the significant protective effect on ischemia-reperfusion injury in rats. Its mechanism may be related to the inhibition of inflammatory factor expression.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; immunology ; Interferon-alpha ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Male ; Matrix Metalloproteinase 9 ; genetics ; immunology ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; genetics ; immunology