Periodontal disease is an inflammatory disease that affects the destruction of the bone supporting the tooth and connective tissues surrounding it. Periodontal ligament fibroblasts (PDLFs) induce overexpression of matrix metalloproteinase (MMP) involved in periodontal diseaseʼs inflammatory destruction. Osteoclasts take part in physiological bone remodeling, but they are also involved in bone destruction in many kinds of bone diseases, including osteoporosis and periodontal disease. This study examined the effect of baicalin on proteolytic enzymesʼ production and secretion of inflammatory cytokines in PDLFs and RAW 264.7 cells under the lipopolysaccharide (LPS)-induced inflammatory conditions. Baicalin inhibited the expression of the protein, MMP-1 and MMP-2, without affecting PDLFs’ cell viability, suggesting its possibility because of the inhibition of phosphorylation activation of mitogen-activated protein kinase’s p38, and the signal transduction process of nuclear factor κB (NFκB)-related protein. Also, baicalin reduced the expression of MMP-8 and MMP-9 in RAW 264.7 cells. This reduction is thought to be due to the inhibition of the signal transduction process of NFκB-related proteins affected by inhibiting p65RelA phosphorylation. Also, baicalin inhibited the secretion of nitric oxide and interleukin-6 induced by LPS in RAW 264.7 cells. These results suggest that baicalin inhibits connective tissue destruction in periodontal disease. The inhibition of periodontal tissue destruction may be a therapeutic strategy for treating inflammatory periodontal-diseased patients.

Periodontal diseases have been associated with the development of cardiovascular diseases. Accumulating evidences have indicated that Porphyromonas gingivalis , a major periodontopathic pathogen, plays a critical role in the pathogenesis of atherosclerosis. In the present study, we demonstrated that P. gingivalis lipopolysaccharide (LPS) increases the mRNA and protein expression of matrix metalloproteinase-9 (MMP-9) in rat vascular smooth muscle cells. We showed that the MMP-9 expression induced by P. gingivalis LPS is mediated by the activation of signal transducer and activator of transcription 3 (STAT3) in vascular smooth muscle cells. Furthermore, the inhibition of STAT3 activity reduced P. gingivalis LPS-induced migration of vascular smooth muscle cells. Overall, our findings indicate that P. gingivalis LPS stimulates the migration of vascular smooth muscle cells via STAT3-mediated MMP-9 expression.

Flavonoid myricetin, usually found in tea and medicinal plants, has antioxidant and anti-inflammatory effects. Our objectives in this study were to verify the effects of myricetin on periodontal ligament fibroblasts (PDLFs) under inflammatory conditions and to observe its effects on osteoclast generation and on cytokine expression in RAW264.7 cells. To determine the effects of myricetin on PDLFs, we examined the expression and activity of proteolytic enzymes, including MMP-1, MMP-2, and MMP-8, which all play an important role in chronic periodontitis. We observed the effects of myricetin on intracellular signal transduction to verify the molecular mechanism involved. By measuring the formation of TRAP–positive multinucleated cells and the expression and activity of MMP-8, we were able to assess the effects of myricetin on osteoclast generation. In addition, by measuring the secretion of IL-6 and NO, we could evaluate the effects of myricetin on inflammatory mediators. We found that Myricetin had no effect on the viability of the PDLFs in the presence of inflammation, but it did decrease both the expression of MMP-1 and MMP-8 and the enzyme activity of MMP-2 and MMP-8 in these fibroblasts. Myricetin also decreased the lipopolysaccharide-stimulated phosphorylation of JNK, p38 signaling, IKKB, AKT, and p65RelA in the PDLFs. In the RAW264.7 cells, myricetin inhibited both the expression and the activity of MMP-8. Furthermore, Myricetin not only suppressed the generation of LPS-stimulated osteoclasts, but it also slightly inhibited LPS-stimulated degradation of IkB and decreased the release of LPS-induced IL-6 and NO. These findings suggest that myricetin alleviates the tissue-destructive processes that occur during periodontal inflammation.

Identification of specific marker proteins in cells is useful for isolating cells and determining their cellular characteristics and functions. Based on our previous study showing that matrix metalloproteinase 9 (MMP-9) can be used as a marker for porcine spermatogonia, the expression pattern of MMP-9 was determined in both pre- (5-month old) and post-pubertal (11–month old) bovine testes. Histological analysis revealed that spermatogonia were located near the basement membrane in both testes, while spermatozoa were not detected in the 5-month old pre-pubertal bovine testes and epididymides. Mature spermatozoa were observed in the 11-month bovine testes and epididymides, and MMP-9 expression in 11-month old bovine testes was lower than 5-month old testes, according to reverse transcription-PCR and real-time-PCR data. To determine the specific expression sites of MMP-9 in the bovine testes, immunohistochemistry was performed. Expression of MMP-9 was observed in cells near the basement membrane of seminiferous tubules in both 5- and 11-month old testes. Furthermore, MMP-9 positive cells expressed protein gene product 9.5 (PGP9.5) and deleted in azoospermia (DAZL) that are already known as bovine spermatogonial stem cells markers. In the present study, MMP-9 expression was identified in both pre- and post-pubertal bovine spermatogonia expressing PGP9.5 and DAZL, and located near the basement membrane of seminiferous tubules. Thus, MMP-9 can be used as a marker for bovine spermatogonia, and may provide useful platforms for understanding the interaction between germ cells and extracellular matrix during spermatogenesis in the seminiferous tubules.

Background: Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation of the extracellular matrix. MMP-8 has been reported to be involved in the degradation of collagen in progression of dental caries. MMP-13 was found to be expressed in both normal and caries pulp, implying its involvement in the pathogenesis of dental caries. Methods: Four extracted teeth were used. They were categorized into four grades according to caries progression. Three sections were prepared from each separated crown and root. Immunofluorescence of the FITC of the MMP-8 and 13 in coronal and radicular dentin was analyzed by confocal microscopy. Results: Immunofluorescence signals that were indicative of MMP-8 were observed both in radicular and coronal dentin in the sound, C1 and C3 carious teeth. In C2 carious teeth, immunofluorescence signals that were indicative of MMP-8 were observed only in radicular dentin. Immunofluorescence signals that were indicative of MMP-13 were observed both in radicular and coronal dentin in the sound teeth. In C1, C2 and C3 carious teeth, immunofluorescence signals that were indicative of MMP-13 were not observed both in radicular and coronal dentin. Conclusion: Immunofluorescence signals revealed that MMP-8 and 13 were present in coronal and radicular dentin, and was differently expressed as caries progressed.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation of the extracellular matrix.In a previous study, MMP-13 was found to be expressed in pulp implying its involvement in the pathogenesis of dental caries. Two extracted teeth were used. A sound tooth and a tooth with wide range of dental caries were used. Two sections were obtained each from isolated crown and root. Immunofluorescence of the FITC of the MMP-13 in coronal and radicular dentin was analyzed by confocal microscopy. Immunofluorescence signals that were indicative of MMP-13 were observed in coronal dentin of sound teeth and in carious teeth with a wide range of caries. Marked immunofluorescent reaction was observed in the border line of caries infected and affected coronal dentin. MMP-13 expression was not detected in the root dentin. The expressions of MMP-13 in carious dentin imply the roles of MMP-13 in caries progression.

The salivary gland undergoes complex process of growth and differentiation of the branching morphogenesis of ductal system during the prenatal and early postnatal periods which are regulated by various elements in the extracellular matrix. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule. In the present study, localization and expression of EMMPRIN in development and effects of chorda-lingual denervation and cyclosporine A (CsA) treatment on the EMMPRIN expression were investigated. Immunohistochemistry, RT-PCR and Western blot were used to determine expression level. Immunohistochemistry revealed that EMMPRIN was localized specifically in the cytoplasm of ductal cells, not acini of the submandibular gland all the postnatal periods. At prenatal day 18, when the formation of ducts was not definite, no immunoreactivity was observed. Both Western blot and RT-PCR analyses revealed that EMMPRIN expression was maintained up to postnatal day 7, decreased after postnatal day 10. The EMMPRIN expression was upregulated by the surgical denervation of the chorda-lingual nerve in the gland as well as by the CsA treatment. The present study suggests that EMMPRIN is a crucial molecule for maintaining physiological functions of the salivary gland.

Matrix Metalloproteinases (both MMP2 and -9) play a pivotal role of the embryos hatching and implantation. Therefore, the objective of this study was carried out to investigate the influence of MMP2 and MMP9 on embryo development potential and subsequent effect at molecular level. There was no significant difference of cleavage rate among the groups. The development competence of blastocyst was significantly higher (P<0.05) in MMP9 treatment (39.81±16.61) than that to the combined treatment of MMP2 and –9 (23.68±0.27), but there was no significant difference among the control vs. MMP2 vs. MMP9 (35.05±2.74 vs. 32.71±6.18 vs. 39.81±16.61, respectively). On the other hand, the hatching rate of blastocysts was significantly lower (P<0.05) in combined group of MMP2 and –9 (12.55±0.09) (Table1). The expression level of MMP2 and MMP9 was significantly lower (P<0.05) in the entire treatment groups than that in the control group. But the expression of MMP9 was significantly higher (P<0.05) when compared in the entire treatment groups. The relative expression embryonic developmental gene, IFNt expression level significantly lower (P < 0.05) in the MMP9 embryos. The placenta establishment genes, PLAC8 and SSLP1, expression were significantly higher (P < 0.05) in the MMP2 embryos compared to other groups. Transcription regulation gene, HNRNPA2B1, was higher (P < 0.05) in the combined group of MMP2+MMP9 than that in the other groups. In conclusion, our results suggest that MMPs to culture medium improves the blastocyst development rate and further impact on target gene expression analysis.

Matrix metalloproteinases (MMPs) have been known to affect to cell migration, proliferation, morphogenesis and apoptosis by degrading the extracellular matrix. In the previous studies, undifferentiated mouse embryonic stem cells (ESCs) were successfully proliferated inside the extracellular matrix (ECM) analog-conjugated three-dimensional (3D) poly ethylene glycol (PEG)-based hydrogel. However, there is no report about MMP secretion in ESCs, which makes it difficult to understand and explain how ESCs enlarge space and proliferate inside 3D PEG-based hydrogel constructed by crosslinkers containing MMP-specific cleavage peptide sequence. Therefore, we investigated what types of MMPs are released from undifferentiated ESCs and how extracellular signals derived from various niche conditions affect MMP expression of ESCs at the transcriptional level. Results showed that undifferentiated ESCs expressed specifically MMP2 and MMP3 mRNAs. Transcriptional up-regulation of MMP2 was caused by the 3D scaffold, and activation of integrin inside the 3D scaffold upregulated MMP2 mRNAs synergistically. Moreover, mouse embryonic fibroblasts (MEFs) on 2D matrix and 3D scaffold induced upregulation of MMP3 mRNAs, and activation of integrins through conjugation of extracellular matrix (ECM) analogs with 3D scaffold upregulated MMP3 mRNAs synergistically. These results suggest that successful proliferation of ESCs inside the 3D PEG-based hydrogel may be caused by increase of MMP2 and MMP3 expression resulting from 3D scaffold itself as well as activation of integrins inside the 3D PEG-based scaffold.

Pleomorphic adenoma (PA) is the most common benign salivary gland tumor. It is biphasic and is characterized by an admixture of epithelial and spindle-shaped myoepithelial cells in a variable background stroma. Epithelial-myoepithelial carcinoma (EMC) is a malignant biphasic salivary gland tumor typically composed of clear myoepithelial cells that surround epithelial-lined ducts resembling intercalated ducts. The differential diagnosis between the two tumor may be occasionally encountered because of the shared histophatologic feature. And then, it would be more reliable to differentiate the tumors based on biological behavior such as the expression of distinct intermediate filaments such as cytokeratin, invasiveness- related molecules, and the growth factor receptor to aberrantly facilitate the tumor growth, and the growth fraction of tumors. Therefore, from the 10 cases of PA and 6 cases of EMC, we immunohistochemically examined the differential expression of the cytokine 7 and 14, matrix metalloproteinase-9, C-KIT, and Ki-67 between the two tumor. At the results, there were significant differences of CK7 expression in non-luminal cells (P = 0.000) and CK14 expression in luminal and non-luminal cells of the both tumors (P = 0.025 and P = 0.000, respectively). In the comparison of the biologic behavior, a significantly increased expression of MMP-9, C-KIT and Ki-67 was found in the cases of EMC when compared to those of PA (P = 0.043, P = 0.011, and P = 0.000, respectively). In conclusion, the differences of CK expression in luminal and non-luminal cells between PA and EMC seem to reflect the difference of the origin and the level of the maturation of the tumor cell. Increased expression of MMP-9, C-KIT, and Ki-67 in EMC may represent more aggressive biologic behavior of the tumor compared with benign salivary tumor such as PA. Our results may be helpful to understand the histiogenesis of the two tumors and the difference of biologic behavior and to differentiate them when the limited specimen was submitted. Further study of many more cases of EMC is needed to validate the usefulness of these molecules as the diagnostic aid.

Gingival overgrowth can cause dental occlusion and seriously interfere with mastication, speech, and dental hygiene. It is observed in 25 to 81% of renal transplant patients treated with cyclosporine A (CsA). CsA-induced gingival overgrowth (CIGO) is caused by quantitative alteration of the extracellular matrix components, particularly collagen. However, the molecular mechanisms involved in the pathogenesis of CIGO remain poorly understood, despite intense clinical and laboratory investigations. The aim of the present work is to identify differentially expressed genes closely associated with CIGO. Human gingival fibroblasts were isolated by primary explant culture of gingival tissues from five healthy subjects (HGFs) and two patients with the CIGO (CIGO-HGFs). The proliferative activity of CsA-treated HGFs and CIGO-HGFs was examined using the MTT assay. The identification of differentially expressed genes in CsA-treated CIGO-HGF was performed by differential display reverse transcriptase-polymerase chain reaction (RT-PCR) followed by DNA sequencing. CsA significantly increased the proliferation of two HGFs and two CIGO-HGFs, whereas three HGFs were not affected. Seven genes, including the beta subunit of prolyl 4-hydroxylase (P4HB) and testican 1, were upregulated by CsA in a highly proliferative CIGO-HGF. The increased P4HB and testican-1 mRNA levels were confirmed in CsA-treated CIGO-HGFs by semiquantitative RT-PCR. Furthermore, CsA increased type I collagen mRNA levels and suppressed MMP-2 mRNA levels, which are regulated by P4HB and testican-1, respectively. These results suggest that CsA may induce gingival overgrowth through the upregulation of P4HB and testican-1, resulting in the accumulation of extracellular matrix components.

Human gingival fibroblasts are necessary for oral homeostaslS These cells are fundamental in tissue healing and tissuc remodeling processes under a response to physiological actions such as mastication, Collagen and elastin, that are extracell ular glycoprotein of gingival fibroblast, are found in all animals, '1γpe 1 collagen is most dominant protein found in human gingival fibroblasts , Matrix metalloproteinase-1(MMP-1) has a role play in destruction of metabolism of extracellular matrix(ECM) and MMP-1 can destroy many ECMs as well as non-ECM molecules MMP-1’s local activation is conytolled by tissue inhibitor of metalloproteinase-1(TIMP-1) , Therefore, it is important to have a balance between in both s ituations MMPs and TIMPs of increased 0 1' decreased extracelluar matrix molecules, The purpose of trus study is to find out the effect of physical stimulus to human gingival fibroblast on mRNA, proteins of collagen 1, elastin, MMP-1, and TIMP-1 Healthy human gingival fibroblasts were separated and cultur때 in DMEM(Dulbeco’s Modified Eagle’s Medium) , When the sample reached to confluence state, it was separated with 0,25% t rypsin and 0‘ 53mM ethylendiaminetetraacetic aCld Separated cells were centrifuged in a cell culturing flask at 1000rpm for 30, 60, 120 mmutes Then it was forced by 35g/cm' continuously, The obtained results that expression of mRNA using histological study and Reverse Transcription Polymerase Chain Reaction (RT-PCR) , expression of protein using Enzyme-Linked Immunosorbent Assay(ELISA) for this study, At 30minutes after cen trifuging, there were s pindl e shaped gingival fibroblasts with long processes parallel to other cells in the control group , However the cell density was simil ar to compared group, At 60minutes after centrifuging, spindle shaped human gingival fibroblast with relatively long process, less densely packed, At 120minutes after centrifuging, cell processes were lengthened 2-3 times‘ and cell density was lower, At 30-60 minutes after centrifuging, it was increased by 1,3-1,7 times in expressoin of collagen 1 mRNA as compared with comparison group, However, there was no change in elastin, TIMP-l, and MMP-1, At 120 minutes after centrifuging, The revealed collagen 1 mRNA was increased 3 times as compared with comparison group, It was increased 2 times in elastin , 12 times in TIMP-1 as compared with comparison group, However, there was no change in MMP-l. At 30-60 minutes after centrifuging, it was increased by 1.1 times in revealing of protein revealing in collagen 1, TIMP-1 But there was no cha nge in elastin, MMP-1 At 120 minutes after centrifuging, it was increased by 1,2 times in revealing collagen 1 protein, 11 times in elastin, 12 times in TIMP-l, but there was no change in MMP-l. ln conclu s ion, it increased in revelation of collagen 1 ,elastin and TIMP-1 by continous stimulus in human gi ngival fibroblast, But there was no change in revelation of MMP-l Therefore, th is type of pressu re is one of the components for healing of gingiva l fibroblast

포유동물의 암컷 생식기관에 존재하는 다양한 종류의 matrix metallo-proteinase (MMP)는 난소와 자궁의 구성성분의 주기적인 변화를 조절하며 이중 난소의 MMP는 난포의 성장과 배란 그리고 퇴화 동안 조직재구성에 매우 중요한 역할을 한다고 알려져 있다. 본 실험에서는 근래에 새로 발견된 사람의 난포액에 존재하는 분자량 약 110kDa인 MMP-2 isoform GA110을 동정하고 자 하였다. 난포액으로부터 GA110 단백질을 분리하기 위하여 난포액에 5mM ethylenediaminetetraacetic acid(EDTA)를 처리한 후 DEAE Sepharose Fast Flow를 이용한 chromatography를 시행하였다. 그 결과, 난포액 단백질들은 0.2M NaCl 의 분획에서 GA110 활성을 나타내었고 anti-human MMP-2 antibody에 대한 면역반응도 뚜렷이 나타났다. DEAE Sepharose Fast Flow에서 얻은 분획 중 GA110의 활성과 면역반응을 모두 나타내는 분획만을 모아 Gelatin Sepharose 4B affinity chromatography로 다시 분리하였다 분리한 결과 resin에 흡착된 단백질 (eluate) 분획들에서 매우 뚜렷한 GA110 gelatinase 활성을 나타내었으며 면역반응 또한 관찰되었다. 이 분획들의 단백질을 농축한 후 zymography를 시행하여 나타난 GA110 단백질 band를 잘라 내었으며 이로부터 단백질을 electroelution하여 농축한 후 reducing agent인 2-mercaptoethanol를 처리하였다. 이를 전기영동 후 MMP-2 (propeptide region) antibody를 사용하여 immunoblotting 한 결과 70-72kDa의 단백질만이 면역반응을 나타내었다. 마지막으로 위와 같이 준비된 70-72kDa 단백질의 아미노산 서열을 Edman degradation 방법으로 분석하였다. 그 결과 이 단백질의 N 말단의 10개의 아미노산 배열 순서가 알려진 사람의 proMMP-2의 전체 배열순서 중 propetide domain의 N 말단에서부터 다섯 번째에서 시작하여 10개의 아미노산의 서열과 정확하게 일치하였다. 위 결과들로 미루어 사람의 난포액에 존재하는 MMP-2의 새로운 isoform인 GA110은 70-72kDa의 ProMMP-2가 disulfide bond를 통해 homodimer 구조를 이루고 있는 것으로 여겨진다.

건조한 아선약을 80% 메탄올로 침지 추출하여 얻어진 추출물에 대해 Diaion HP-20 수지를 충진제로 사용한 칼럼크로마토그래피를 수해하여 6종의 용리액을 얻었으며, 얻어진 추출물에 대하여 MMP-1 저해 및 type-1 procollagen 합성 촉진 활성을 평가하였다. 먼저 MMP-1 저해활성은 총페놀성 화합물의 함량이 상대적으로 높은 40% 메탄올 용리액에서 IC50값이 15.6±1.3 μg/mL으로 positive control로 녹차의 주름개선 성분인 (-)-EGCG의 IC50값인 38.4±2.3 μg/mL 보다 우수한 활성이었다. 또한 type-1 procollagen 합성 촉진 활성은 총페놀성 화합물 함량이 가장 높은 40% 메탄올 용리액에서 우수한 EC50값이 6.9±0.7 μg/mL을 나타내었으며, 양성대조구인 (-)-EGCG의 효능과 동등한 활성이었다. 본 실험에 사용된 아선약 시료의 경우 다양한 화합물이 공존하는 추출물 및 용리액 상태이며 이들 시료에 우수한 효능의 성분의 존재 및 추출물에 존재하는 화합물의 우수한 상승효과의 가능성을 시사하였다. 향후 각종 칼럼크로마토 그래피를 활용한 이들 주름개선 효능 관련 물질의 분리를 통한 활성물질의 구조 결정 및 활성 기작에 대한 연구를 수행할 예정이며, 본 연구결과는 천연물 유래의 우수한 MMP-1 저해 및 type-1 procollagen 합성 촉진 활성을 가지는 새로운 천연 소재 발굴을 위한 기초자료로 활용 가능할 것으로 사료된다.

자외선은 피부 광노화의 주요 요인이며, 효과적인 자외선 차단제가 피부의 건강과 아름다움을 위해 필요하다. 본 연구는 세포 실험을 통하여 자외선에 의해 유도된 세포 사멸, 산화적 스트레스, matrix metalloproteinase 1 발현에 미치는 죽여추출물의 영향을 알아보고자 수행하였다. HaCaT 인간 각질세포를 여러 농도의 죽여추출물 유무 조건에서 자외선을 조사하고 세포의 생존율과 생화학적 과정들의 변화를 분석하였다. 죽여추출물은 자외선을 조사한 세포의 생존율을 증진시켰고, procaspase 3가 활성화 형태로 절단되고, Bax/Bcl-2 비율이 증가하는 세포 자살 과정을 완화시켰다. 죽여추출물은 자외선에 노출된 세포에서 활성산소의 발생과 지질 과산화도 감소시켰다. 또한 죽여추출물은 자외선에 의해 자극된 matrix metalloproteinase 1의 발현과 c-Jun N-terminal kinase의 인산화를 억제하였다. 본 연구는 죽여추출물이 자외선에 의한 세포 사멸, 산화적 스트레스, 그리고 matrix metalloproteinase 1 발현을 억제함을 보여 주었으며, 이 추출물이 피부 광노화의 일부 현상을 억제하는 화장품 원료로 유용함을 시사하였다.

We previously reported that purified hepatocyte-like cells derived from human embryonic stem cell (hESC) promoted the liver tissue recovery not only by cell replacement, but also by delivering proteins (secretome) that enhance endogenous host liver regeneration. In this study, we investigated possible therapeutic effects of secretomes obtained from undifferentiated hESC and mesenchymal stem cell (hMSC), and explored the underlying mechanism in a mouse model of chronic liver injury. Mice pre-intoxicated with dimethylnitrosamine (DMN) were treated with single intraperitoneal injection of secretome or medium used to support the growth of hESCs or hMSCs. Both hESC- and MSC-secretomes induced robust host liver regeneration, as determined by biochemical and histological analyses. The expression of MMP2 was significantly increased in the liver that received hESC- or hMSC-secretome, compared to control groups. In contrast, expression of α-SMA, a hallmark of activated hepatic stellate cells, was profoundly decreased after administration of both secretomes. These results suggest that hESCs and MSCs may release soluble factors that support the host tissue regeneration of chronically injured liver.